The correct names are: Minh Duc Pham, Van Hoang Pham and Thang Chien Nguyen. The correct citation is: Pham MD, Byrkit M, Pham VH, Pham T, Nguyen TC (2013) Improving Pharmacy Staff Knowledge and Practice on Childhood Diarrhea Management in Vietnam: Are Educational Interventions Effective? PLoS ONE 8(10): e74882.


Management of patients with an advance decision and suicidal behaviour: a systematic review. BMJ Open 2019;9:e023978. doi: 10.1136/bmjopen-2018-023978Nowland R, Steeg S, Quinlivan LM, This article was previously published with an error.In second affiliation, NHIR should be NIHR. The correct affiliation number 2 is:NIHR Greater Manchester Patient Safety Translational Research Centre, University of Manchester, UK


AbstractGracixalus, Gracixalusyunnanensissp. n., is described based on a series of specimens collected from southwestern and southern Yunnan, China. This species is distinguished from all other known congeners by a combination of the following characters: relatively small body size in adult males (SVL 26.0–34.2 mm); dorsal surface yellow brown or red brown; distinctive conical asperities on dorsum; males with an external subgular vocal sac and linea masculina; throat, chest, and belly nearly immaculate; venter surface orangish with yellow spots, semi-transparent; snout rounded; supratympanic fold distinct; iris bronze; lack of white patch on temporal region; tibiotarsal projection absent; sides of body nearly smooth with no black blotch; finger webbing rudimentary; and toe webbing formula I1.5–2II1.5–2.7III.5–3IV2.5–1.5V. Genetically, the new species diverges from its congeners by 2.2%–14.1% (uncorrected p-distance) and is closest to G.guangdongensis. However, the new species can morphologically be separated from G.guangdongensis by distinctive conical tubercles on dorsum (versus absent), lateral surface nearly smooth with no black blotches on ventrolateral region , snout rounded (versus triangularly pointed), iris bronze (versus iris brown), and ventral surface orangish (versus throat and chest creamy white and belly light brown).A new species of the genus  Gracixalus Delorme, Dubois, Grosjean & Ohler, 2005 is known from southern and southwestern China, Vietnam, Laos, Thailand, and Myanmar and contains 16 species including G.ananjevae , G.carinensis , G.gracilipes , G.guangdongensis Wang, Zeng, Liu & Wang, 2018, G.jinggangensis Zeng, Zhao, Chen, Chen, Zhang & Wang, 2017, G.jinxiuensis , G.nonggangensis Mo, Zhang, Luo, Zhou & Chen, 2013, G.quangi, Rowley, Dau, Nguyen, Cao & Nguyen, 2011, G.quyeti , G.sapaensis Matsui, Ohler, Eto & Nguyen, 2017, G.seesom Matsui, Khonsue, Panha & Eto, 2015, G.supercornutus , G.tianlinensis Chen, Bei, Liao, Zhou & Mo, 2018, and G.waza Nguyen, Le, Pham, Nguyen, Bonkowski & Ziegler, 2013  and outer (third and fourth) fingers non-opposable, and an inversed Y-shaped dark brown marking on dorsum , Chinese Academy of Sciences and Guangxi Normal University (GXNU).Specimens were collected during fieldworks in Menghai County, Lancang County, and Lvchun County of Yunnan, China in 2014 to 2018 ;HL head length (from tip of snout to rear of jaws);HW head width (width of head at its widest point);SL snout length (from tip of snout to anterior border of eye);IND internarial distance (distance between nares);IOD interorbital distance (minimum distance between upper eyelids);UEW upper eyelid width (maximum width of upper eyelid);ED eye diameter ;TD tympanum diameter;FHL forearm and hand length (from elbow to tip of third finger);THL thigh length (from vent to knee);TL tibia length (distance from knee to heel);FL foot length ;TFL length of foot and tarsus .Gracixalus species were taken from their original descriptions or re-descriptions  were selected as outgroups according to Total genomic DNA was extracted from liver tissues. Tissue samples were digested using proteinase K, and subsequently purified following a standard phenol/chloroform isolation and ethanol precipitation. A fragment encoding partial 16S rRNA gene was amplified and sequenced following AICc) in jMODELTEST version 2.1.10  was performed in MRBAYES version 3.2.6 (BPPs). Secondly, maximum likelihood (ML) analysis was conducted in RAXML-HPC version 8.2.10  that were sequenced by previous studies  with weak support  to 14.1% (G.lumarius) , Nghe An (AMS R173454) and Lao Cai  of Vietnam, and Jinping of Yunnan , lateral surfaces nearly smooth with no black blotches on ventrolateral region , snout rounded (versus triangularly pointed), iris bronze (versus iris brown), and ventral surface orangish (versus throat and chest creamy white and belly light brown). These specimens also differ from other members of Gracixalus in a series of characters. Herein we describe these specimens as a new species.Morphologically, these newly collected specimens can be distinguished from Taxon classificationAnimaliaAnuraRhacophoridaehttp://zoobank.org/1D19A62E-B4B2-4EDA-975D-4DCFD58DEDADHolotype.KIZ 20160222, an adult male, collected at 21:05 on 1 June 2017 by Hong Hui from Xuelin Township, Lancang County, Yunnan Province, China .Paratypes. Seven adult males: KIZ 20160223 collected at 21:05 on 1 June 2017 by Hong Hui from the type locality; KIZ 20160216 collected at 21:00 on 7 June 2014 by Hong Hui from Bada Township, Menghai County, Yunnan Province, China ; KIZ 20160226 collected at 21:50 on 27 May 2017 by Hong Hui from Fudong Township, Lancang County, Yunnan Province, China ; KIZ 20160228–20160230 collected at 21:40–22:15 on 10 June 2017 by Hong Hui from Fazhanhe Township, Lancang County, Yunnan Province, China ; and GXNU YU000060 collected at 21:00 on 7 June 2018 by Jian Wang from Mt. Huanglian, Lvchun County, Yunnan Province, China .yunnanensis refers to the distribution of this species in China, Yunnan Province.The specific epithet Gracixalus based upon molecular data and the following morphological characters: the presence of intercalary cartilage between terminal and penultimate phalanges of digits, tips of digits enlarged to discs bearing circummarginal grooves, vomerine teeth absent, inner (first and second) and outer (third and fourth) fingers non-opposable, and an inversed Y-shaped dark brown marking on dorsum  dorsal surface yellow brown or red brown; 3) distinctive conical tubercles on dorsum; 4) males with an external subgular vocal sac; 5) throat granular; 6) finger webbing rudimentary; 7) linea masculina, a band of connective tissue between the rectus abdominus muscle and oblique abdominus muscle, present in males; 8) tibiotarsal articulation reaching eye; 9) snout rounded; 10) white patch absent on temporal region; 11) tibiotarsal projection absent; 12) supratympanic fold distinct; 13) ventral surface orangish, nearly immaculate, and semi-transparent; 14) nuptial pads present on finger I; 15) heels overlapping when legs at right angle to body; 16) iris bronze; and 17) body sides nearly smooth with no black blotch.The new species is assigned to genus n dorsum . The newSVL 29.7 mm); head wider (HW 10.9 mm) than long (HL 9.5 mm); snout rounded, slightly projecting beyond margin of lower jaw in ventral view, rounded in profile; canthus rostralis rounded; loreal region oblique, slightly concave; nostril oval, protuberant, closer to tip of snout than eye; IND (2.9 mm) slightly narrower than IOD (3.0 mm) and wider than UEW (2.5 mm); eye large, horizontal diameter (ED 4.2 mm) equal to snout length (SL 4.2 mm); pupil oval, horizontal; pineal ocellus absent; tympanum distinct, diameter (TD 1.5 mm) smaller than half of ED; supratympanic fold distinct, extending from posterior corner of eye to above insertion of arm; vomerine teeth absent; tongue notched posteriorly; a pair of vocal sac slits on floor of mouth at both corners; an external subgular vocal sac.Adult male (FHL 14.1 mm) 47% of SVL; relative length of fingers I < II < IV < III; tips of all fingers expanded into discs with circummarginal grooves; disc of third finger large, slightly wider than tympanum; nuptial pads present on base of finger I; webbing between fingers rudimentary; subarticular tubercles prominent, rounded, single, formula 1, 1, 2, 2; supernumerary tubercles present; an inner metacarpal tubercle, oval; one outer metacarpal tubercle, rounded.Forelimb relatively robust; length of forearm and hand  is yellow brown, and dorsal ground color of remaining paratypes  is red brown. Ventral surface of all specimens is nearly immaculate with the exception of paratype GXNU YU000060, which has dark marbling on throat, chest, and belly . No eggs and tadpoles were found.In China, the new species is known from Yunnan . In addition, the new species also occurs in Laos (Houapan) and Vietnam (Lao Cai and Nghe An) because our molecular analyses revealed that samples from Houapan KUHE 32453), Lao Cai , and Nghe An (AMS R173454) that were sequenced by previous studies also belong to the new species , sides of body smooth (versus coarsely granular), skin of throat granular (versus plain), and snout rounded (versus slightly pointed); from G.carinensis by having smaller body size in males (SVL 26.0–34.2 mm versus 30.2–38.1 mm), having distinctive conical tubercles on dorsum (versus absent), having an external vocal sac in males , ventral surface orangish (versus white), and less developed toe webbing , distinctive conical tubercles present on dorsum (versus absent), dorsal surface yellow brown or red brown (versus greenish), males with an external vocal sac , throat granular (versus smooth), finger webbing rudimentary (versus absent), tibiotarsal articulation reaching to eye (versus reaching to between eye and nostril), snout rounded (versus triangular pointed), white patch absent on temporal region (versus present), tibiotarsal projection absent (versus present), and iris bronze (versus brown); and from G.guangdongensis by having distinctive conical tubercles on dorsum (versus absent), dorsal surface yellow brown or red brown (versus brown), flanks nearly smooth with no black blotches on ventrolateral region , snout rounded (versus triangularly pointed), ventral surface orangish (versus throat and chest creamy white and belly light brown), and iris bronze (versus iris brown).A summary of morphological comparisons presents in Table ing Fig. ; from G.Gracixalusyunnanensis sp. n. differs from G.jinggangensis by having distinctive conical tubercles on dorsum (versus absent), flanks nearly smooth (versus rough with tubercles), snout rounded (versus triangularly pointed), ventral surface orangish and immaculate , nuptial pads present only on finger I , heels overlapping when hindlimbs held at right angles to the body (versus just meeting), and iris bronze (versus iris golden); from G.jinxiuensis by larger body size in males (SVL 26.0–34.2 mm versus 23.5–26.3 mm), males with an external vocal sac , flanks nearly smooth (versus rough with tubercles), linea masculina present (versus absent), ventral surface orangish and immaculate , and sole of feet and palms smooth (versus rough with dense large tubercles); and from G.lumarius by smaller body size in males (SVL 26.0–34.2 mm versus 38.9–41.6 mm), dorsal surface yellow brown or red brown (versus yellow), and venter orangish and semi-transparent (versus venter opaque pink), supratympanic fold distinct (versus indistinct), and iris bronze (versus dark gold).Gracixalusyunnanensis sp. n. can be distinguished from G.medogensis by having distinctive conical tubercles on dorsum (versus absent), dorsal surface yellow brown or red brown (versus grass green), males with an external vocal sac , finger webbing rudimentary (versus absent), and venter orangish ; from G.nonggangensis by having conical tubercles on dorsum (versus absent), dorsum yellow brown or red brown with a dark brown marking (versus yellowish-olive with a dark-green marking), males with an external vocal sac , flanks smooth (versus rough with tubercles), finger webbing rudimentary (versus absent), linea masculina present in males (versus absent), tibiotarsal articulation reaching to eye (versus reaching to tip of snout), ventral surface immaculate , nuptial pads present on finger I (versus absent), and iris bronze (versus olive); from G.quangi by having bigger body size in males (SVL 26.0–34.2 mm versus < 25 mm), dorsal surface yellow brown or red brown (versus olive-green), black spots absent on flanks and ventral surface of thighs (versus present), throat granular (versus smooth), finger webbing rudimentary (versus absent), snout rounded (versus triangular pointed), white patch absent on temporal region (versus present), tibiotarsal projection absent (versus present), and ventral surface orangish ; and from G.quyeti by dorsal surface yellow brown or red brown (versus brownish to moss-green), flanks nearly smooth (versus rough with sharp tubercles), throat granular (versus smooth), tibiotarsal articulation reaching to eye (versus reaching to snout), supratympanic fold distinct (versus indistinct), and throat and chest immaculate .Gracixalusyunnanensis sp. n. differs from G.sapaensis by having distinctive conical tubercles on dorsum (versus absent) and sides of body nearly smooth (versus coarsely scattered with large round tubercles); from G.seesom by bigger body size in males (SVL 26.0–34.2 mm versus 21.6–23.0 mm), conical tubercles present on dorsum (versus absent), dorsal surface yellow brown or red brown (versus tan), flanks nearly smooth with no white blotches (versus flanks with large tubercles and white blotches), throat granular (versus smooth), snout rounded (versus triangular pointed), nuptial pads present on finger I (versus absent), and iris bronze (versus golden); and from G.supercornutus by bigger body size in males (SVL 26.0–34.2 mm versus 22.0–24.1 mm), conical tubercles on dorsum small , dorsal surface yellow brown or red brown (versus greenish), snout rounded (versus triangular pointed), white patch absent on temporal region (versus present), and tibiotarsal projection absent (versus present).G.tianlinensis by smaller body size in males (SVL 26.0–34.2 mm versus 30.3–35.9 mm), distinctive conical tubercles present on dorsum (versus absent), dorsal surface yellow brown or red brown (versus brown to beige), finger webbing rudimentary (versus absent), ventral surface orangish, immaculate, and semi-transparent , and nuptial pads present on finger I (versus on fingers I and II); and from G.waza by having distinctive conical tubercles on dorsum (versus absent), dorsal surface yellow brown or red brown (versus greyish-green to moss-green), throat granular (versus smooth), finger webbing rudimentary (versus absent), and ventral surface immaculate (versus throat and chest with dark marbling).The new species can be distinguished from G.yunnanensis sp. n. only diverges from G.guangdongensis by a distance of 2.2%, it can be morphologically separated from G.guangdongensis by a series of characters including distinctive conical tubercles on dorsum (versus absent), dorsal surface yellow brown or red brown (versus brown), flanks nearly smooth with no black blotches on ventrolateral region , snout rounded (versus triangularly pointed), ventral surface orangish (versus throat and chest creamy white and belly light brown), and iris bronze (versus iris brown)  Table . In addin) Table , whereasina Fig. , althougort Figs . TherefoG.yunnanensis sp. n. was once confused with G.jinxiuensis in that the Jinping specimen (KIZ 060821126), Houapan specimen (KUHE 32453), Nghe An specimen (AMS R173454), and Lao Cai specimens  were originally identified as G.jinxiuensis by Yu et al. (2008), G.jinxiuensis by having bigger body size, an external vocal sac, and linea masculina in males , one consisting of G.seesom, G.quyeti, G.quangi, G.supercornutus, and G.gracilipes (Clade II), and one consisting of all other species (Clade III)  belongs to G.ananjevae or not. We found that they are sister to each other with strong support values  Figs , 3. This


MWASTools is an R package designed to provide an integrated pipeline to analyse metabonomic data in large-scale epidemiological studies. Key functionalities of our package include: quality control analysis; metabolome-wide association analysis using various models ; visualization of statistical outcomes; metabolite assignment using statistical total correlation spectroscopy (STOCSY); and biological interpretation of metabolome-wide association studies results.https://bioconductor.org/packages/MWASTools/.The MWASTools R package is implemented in R (version  > =3.4) and is available from Bioconductor: Bioinformatics online. Owing to sustained developments in high-throughput platforms [i.e. nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS)], metabolic phenotyping (metabotyping) is now used for large-scale epidemiological applications such as metabolome-wide association studies (MWAS)  QC analysis; (ii) MWAS analysis; (iii) visualization of MWAS results; (iv) metabolite assignment using correlation analysis. For demonstration purposes, the MWASTools package was used to analyse plasma MWASTools performs essential QC analyses via Principal Component Analysis (PCA) and by computing the coefficients of variation (CV) (ratio of standard deviation to the mean) of individual metabolic features across the QC samples . The resP-values are corrected for multiple-testing with several possible methods, such as Benjamini–Hochberg (BH) procedure ] and each metabolic feature (or metabolite). Depending on the nature of data to be modeled, the user can choose among the following association methods: linear/logistic regression or Pearson/Spearman/Kendall correlation. The models can be adjusted for confounder factors, including age, gender or other clinical covariates (e.g. medication). The rocedure . MWASTooP-values (sign adjusted for the direction of the association) are displayed on the y-axis  analysis . In ordeAltogether, the MWASTools R package provides an integrated pipeline with efficient analysis and visualization tools for: (i) performing QC analysis; (ii) conducting robust MWAS analysis with efficient handling of epidemiological confounders; (iii) structural assignment of metabolic features of interest; (iv) biological interpretation of MWAS results. The MWASTools package can be applied to both targeted and untargeted metabonomic datasets, acquired with different analytical platforms. The open nature of R allows for integration of MWASTools with other packages for the analysis of metabonomic data.This work was supported by Medical Research Council Doctoral Training Centre scholarship (MR/K501281/1), Imperial College scholarship (EP/M506345/1), La Caixa studentship to A.R.M.; FCT(BD/52036/2012 to A.L.N.); British Heart Foundation program grant (RG/15/5/31446) to C.E. and E.P.; BHF Chair to CE (CH/15/31199); European Commission (FGENTCARD, LSHG-CT-2006-037683 to D.G., J.K.N. and M.E.D.Conflict of Interest: none declared.Supplementary DataClick here for additional data file.


Acrochordus andadvanced colubroidean snakes that radiated mainly during the Neogene. Althoughcaenophidian snakes are a well-supported clade, their inferred affinities, basedeither on molecular or morphological data, remain poorly known or controversial.Here, we provide an expanded molecular phylogenetic analysis of Caenophidia anduse three non-parametric measures of support–Shimodaira-Hasegawa-Like test(SHL), Felsentein (FBP) and transfer (TBE) bootstrap measures–to evaluate therobustness of each clade in the molecular tree. That very different alternativesupport values are common suggests that results based on only one support valueshould be viewed with caution. Using a scheme to combine support values, we find20.9% of the 1265 clades comprising the inferred caenophidian tree areunambiguously supported by both SHL and FBP values, while almost 37% areunsupported or ambiguously supported, revealing the substantial extent ofphylogenetic problems within Caenophidia. Combined FBP/TBE support values showsimilar results, while SHL/TBE result in slightly higher combined values. Weconsider key morphological attributes of colubroidean cranial, vertebral andhemipenial anatomy and provide additional morphological evidence supporting theclades Colubroides, Colubriformes, and Endoglyptodonta. We review and revise therelevant caenophidian fossil record and provide a time-calibrated tree derivedfrom our molecular data to discuss the main cladogenetic events that resulted inpresent-day patterns of caenophidian diversification. Our results suggest thatall extant families of Colubroidea and Elapoidea composing the present-dayendoglyptodont fauna originated rapidly within the early Oligocene–betweenapproximately 33 and 28 Mya–following the major terrestrial faunal turnoverknown as the “Grande Coupure” and associated with the overall climate shift atthe Eocene-Oligocene boundary. Our results further suggest that the caenophidianradiation originated within the Caenozoic, with the divergence betweenColubroides and Acrochordidae occurring in the early Eocene, at ~ 56 Mya.Caenophidian snakes include the file snake genus  Determining the phylogenetic affinities within snakes was viewed by manyherpetologists in the past as an insurmountable challenge. Underwood  expresseMolecular phylogenies ultimately provided strong support for the monophyly ofCaenophidia, and further corroborated more controversial morphological hypotheses,such as the polyphyly of solenoglyphous  and protThree large-scale molecular phylogenies of snakes were published recently ,27,28. HLarge-sample comparative morphological studies are often difficult to achieve due tothe need for destructive investigative procedures on limited museum specimens and bythe lack of comprehensive taxonomic coverage in skeletal collections all over theworld. Morphological information on caenophidian snake anatomy is still very limitedcompared to the diversity within the group, with only a few detailed analyses oflarger clades being available in the literature and mainly focused on the cranialand hemipenial complexes ,29. A noThe paleontological record for caenophidian snakes is largely biased towardsdisarticulated postcranial  materials ,32. AlthHere, we provide an expanded molecular phylogenetic tree of Caenophidia, highlightingstrongly and weakly supported hypotheses of relationships that need furtherinvestigation. We evaluate alternatively three non-parametric supportvalues–Shimodaira-Hasegawa-Like test (SHL), Felsentein bootstrap proportions (FBP),and transfer bootstrap expectation metrics (TBE)–and combine two of these (FBP andSHL) to give a seven-category classification of the robustness of clades in themolecular tree . ContradCyclocorus and Oxyrhabdium, which alongwith Hologerrhum and Myersophis, wereretrieved in their analysis as a well-supported elapoid clade of endemicPhilippine snakes. Weinell and Brown . En. EnXylopl bodies  [23].Causus and Azemiops.Causus retains an insipient articular facet thatprobably evolved independently from higher endoglyptodonts [Causus,Atheris retains a simple contact between theseptomaxillae and frontals [Oxyrhabdium, the atractaspididsMacrelaps, Aparallactus andAtractaspis, the lamprophiidsChamaelycus and Lycophidion, thenatricids Atretium and Aspidura, and thecalamariids Macrocalamus and Oreocalamus and Weinell and Brown [Cyclocorus and Oxyrhabdium as twounrelated elapoid lineages with uncertain affinities, while the molecularanalysis of Weinell and Brown [Hologerrhum and Myersophis in awell-supported, entirely endemic elapoid clade of Philippine snakes. Weinelland Brown [Cyclocorus and Oxyrhabdium. Althoughmonophyly of cyclocorids seems to be now well established, theirsister-group relationship within Elapoidea remains unresolved and in need offurther investigation [Recently, Weinell and Brown  resolvedn et al. , and itsn et al. ,23,26.Cnd Brown . Howevernd Brown  retrievend Brown accommodtigation .Buhoma,Micrelaps, Psammodynastes, andOreocalamus remain as rogue taxa in all recentmolecular analyses , have lobes ornamented by distinct diminute spinulesthat are often densely packed . Both Gongylosoma andLiopeltis have been referred to the family Colubridaerecently (ex Colubrinae) without further clarification [Gongylosoma balioderum andLiopeltis frenatus . However, they are strikingly distinct from eachother and do not offer a clue about their close affinities.Cyclocorus has an extremely long and narrow, unilobedhemipenis that lacks any specialized ornamentation, being sparcely coveredby spinules and retaining a single sulcus spermaticus. In contrast,Oxyrhabdium modestum has deeply bilobed hemipenes, witha centrolineal sulcus spermaticus, and is covered by uniform and sparcelydistributed small spines. Cyclocorus lineatus has a typicalcolubriformes precloacal vertebral morphology, with well-developedhypapophyses, laterally tapering prezygapophyseal processes, a low uniformlythin, blade-like neural spine that reaches the zygosphenal tectum, distinctpara- and diapophyseal articular facets projecting ventro-laterally, roundcondyle, and well-defined cotylar ventrolateral processes. The presence ofexpanded blade-like hypapophyses conforms with the generalized elapoidmorphotype. Vertebrae of Oxyrhabdium modestum were notavailable for study.nd Brown  clarifieOreocalamus—We provide the first assessment ofthe skull and hemipenis of this genus. Oreocalamus has apeculiar hemipenis that ressembles the organ of Calamariain its overall morphology, with a pair of short, but deeply bilobed androunded lobes ornamented by symmetrically disposed calyces, centrifugalsulcus spermaticus, and spineless (mostly nude) hemipenial body. However, itdiffers from Calamaria by the presence of a large pair ofpockets on the asulcate surface of the hemipenial body and deeply definedcapitular grooves at the base of the lobes (lobes are bicapitulated). BothOreocalamus and Calamaria havetypically colubroid vertebral morphologies with an elongate centrum thatlacks hypapophyses  [Nebraskophis from the upper Eocene of HardieMine, USA (34.2 Mya) [Vectophis wardiand Headonophis harrisoni from the upper Eocene of the Isleof Wight, England (33.9 Mya) [Our time calibrated tree places the divergence of stem-colubroideans at ~ 56Mya, near the Paleocene/Eocene boundary, while stem-colubriforms diverged at~ 53 Mya within the Ypresian . Early fs sahnii  from thes sahnii ,71,72.Ts sahnii ; an unnas sahnii ; Renenut(37 Mya) ; a verte4.2 Mya) ; and an 4.2 Mya) . Because3.9 Mya) ,173 retaRenenutet enmerwer in the Upper Eocene of Egypt[Molecular evidence supports an early Paleogene divergence of colubroideans inAsia ,134, butof Egypt along wiof Egypt,indicatof Egypt. In thatof Egypt, whicheof Egypt. HoweverColuber cadurci [The early Oligocene was marked by a much cooler and more temperate global climatethan the warm "greenhouse" conditions that characterized most of the Cretaceousand early Cenozoic ,177. The cadurci ,110,109. cadurci ,178,179.Thaumastophis missiaeni approaches the xenodermidvertebral morphology in having vertically oriented blade-like prezygapophysealaccessory processes and a lightly built and elongate vertebral morphology [Among “basal” colubroidean lineages estimated to have diverged within the Eocene, theXenrphology . Howeverrphology . The lacrphology ,180–183.Provipera boettgeri, describedby Kinkelin [Although viperids are abundant in the fossil record, most are confined to theNeogene, consist of isolated vertebrae, and are assigned to extant taxa,89. The Kinkelin based onKinkelin for the Kinkelin  and NortKinkelin ,early oKinkelin , and midKinkelin . The firKinkelin  from theKinkelin ,85 proviKinkelin ,85, as sOur time calibrated tree suggests that the origin of crown-Viperidae occurredin the early Oligocene, at ~ 30.7 Mya, while the basal split betweenviperine and causine subfamilies on the one hand, and crotaline andazemiopine subfamilies, on the other hand, occurred within the lateOligocene, at approximately 26 and 25 Mya, respectively . As suchDuberria also exhibits a similar condition, being theonly known pseudoxyrhophiid snake so far that lacks hemapophyses but retainsa well-developed hemal keel. Although Duberria's caudalmorphology ressembles the caudal vertebra described by McCartney et al.[Within Elapoidea, only the Elapidae and, possibly, Pseudoxyrhophiidae have afossil record ,32,94.TLaticauda. However, these authors refrain in allocatingit to the latter genus given the limited information afforded by oneisolated vertebra.The oldest record of an Australian elapid consists of a vertebra attributedto a hydrophiine found in sediments of Riversleigh dated from the upperOligocene or lower Miocene (24–23 Mya) . AccordiNaja. This genus is also recorded in the middle Mioceneof northern Africa . Elapidya; MN70 ,191). Th70 [Naja ,190,192.Micrurus [Micrurus sp.and Micrurus gallicus, respectively. Referral to the familyElapidae is based on having a low and recurved hypapophysis, a lowanteroposteriorly elongated neural spine, and a poorly vaulted neural arch.However, these characters may correlate well with fossorial habits [Micrurus is questionable. No compellingevidence distinguishes the vertebral morphology of Micrurusfrom other Asian and Neotropical coral snake genera. According to Rage andHolman [Micrurus from the Miocene of Nebraska(USA) and la Grive (France) are comparable to extant Micrurusfulvius, and thus should be referred to this genus. Ourobservations of the vertebral morphology of South AmericanMicrurus shows a very distinctive morphology from thatof Micrurus fulvius, suggesting that the vertebralmorphology of the genus is much more diverse than previously thought. Adetailed description of the vertebral morphology of speciose New WorldMicrurus and its closely related North American andAsiatic genera Micruroides, Leptomicrurus,Calliophis, and Sinomicrurus isnecessary to confidently support the assignment of these Miocene records toany known extant genus, and especially to Micrurus.Isolated posterior trunk vertebrae from the middle Miocene of North America(upper Barstovian) and Europe  were assigned to extantMicrurus , as Micrl habits , and, thdHolman , the fewSub-Saharan elapids from the end of the Paleogene raise doubts about thewell-accepted hypothesis of an Asian origin for the group ,22,103. Our time calibrated tree suggests an early divergence of stem-elapids withinthe early Oligocene at ~ 30.5 Mya, with the main crown-Elapidae lineagesdiversifying during the late Oligocene at ~ 26.5 Mya ; S4Fig.Naja is also well documented [Micrurus in the Miocene of France isquestionable due to the absence of vertebral diagnostic features thatdistinguish members of this genus from the Asiatic coral snake radiation.Its record in the Miocene of North America also needs further corroborationsince Holman [Micrurus(M. fulvius and M.affinis) and Micruroides euryxanthusfor comparison.Apart from the uncertainties involving the debate on an Asian or Africanorigin, it has been commonly thought that the family dispersed to the WestPaleartic, Australian (via Melanesia), and Neartic/Neotropical (via theBering strait) regions independently ,183,194.cumented ,190–192.e Holman used onlOur time calibrated tree places the early divergence of stem-hydrophiines at~ 23 Mya, at the Oligocene–Miocene boundary, and the ancestor of the NewWorld radiation of coral snakes (stem-micrurines) diverged from the OldWorld coral snakes at ~ 21 Mya in the early Miocene. These results supportthe hypotheses of a late Oligocene over-sea dispersal and colonization ofthe Australian continent by the ancestor of hydrophiines  and earlThe fossil record of Colubroidea is much more extensive than that of elapoidsbut is mostly confined to the Neogene. Their vertebral morphology remainspoorly known, and an accurate evaluation of the fossils assigned to thissuperfamily or to specific colubroid families remains far from beingresolved. Pseudoxenodontids, calamariids, sibynophiids, and grayiids havenot been recorded so far in the fossil record. In contrast, “colubrid” andnatricid fossils are abundant and have been recorded throughout the Neogeneof North America and Europe ,175,179.Although Miocene and Pliocene records of colubroids are straightforward,Paleogene records are more elusive and most of them are of uncertainassignment. Here we follow Smith  in assigColuber cadurcifrom the Phosphorites of Quercy .Colubrid precloacal vertebrae can be distinguished from all othercolubroidean family by the presence of the following combination of derivedfeatures: an elongated centrum, long prezygapophyseal accessory processes,distinct epizygapophyseal spines, and an uniformly narrow haemal keel(lacking hypapophyses). Slightly younger records of putative natricids fromthe Phosphorites of Quercy are difficult to allocate due to theirfragmentary condition and their overall similarities with the vertebralmorphology of several extant elapoid families. Holman [The divergence between Colubroidea and Elapoidea in our time calibrated treeis estimated at ~ 36 Mya, lying close to the boundary between the Eocene andOligocene  andin a. Holman  and Szyn. Holman  detailedThe traditional meaning of the superfamily Colubroidea ,26,26,28Procerophis sahni–is here placed as a calibration pointfor the early divergence between colubriformes and xenodermids instead of thetraditional divergence between xenodermids and acrochordids). By reviewing andreinterpreting the relevant fossil record, our treePL analysis was able to highlightpreviously unnoticed correlation between the early diversification of colubroid andelapoid major lineages and the Eocene-Oligocene transition. Our divergence dates arein general younger than most previous studies Click here for additional data file.S2 TableGraphic illustration for combined clade support values when comparing FBP,SHL, and TBE metrics, classified in seven categories, as follows: 1) Red,unambiguously supported; 2) Orange, robustly supported; 3) blue, stronglysupported; 4) green, moderately supported; 5) dark grey, ambiguouslysupported; 6) dark grey, poorly supported; 7) light grey, unsupported (seetext for discussion).(XLSX)Click here for additional data file.S3 TableList of all the terminal taxa and sequences by gene used in the presentstudy, including best partitions, accession numbers of sequences retrievedfrom GenBank and new sequences produced for this study.(XLSX)Click here for additional data file.S4 TableComparisons between the number of families, genera and species of Colubroidesused in the present study and by Figueroa et al.  (2016).(DOCX)Click here for additional data file.S5 TableNumber of species by genus of Colubroides sampled in this study; and a listof all species of Colubroides following Uetz et al. , indicat(XLSX)Click here for additional data file.S6 TableList of accession numbers, with genes names, current identification inGenBank, and probable correct identification for questionable and/orproblematic sequences of snakes available in GenBank.(DOC)Click here for additional data file.S7 TableList with rationale for the species recognized in the present study, but notlisted in Uetz et al. .(DOCX)Click here for additional data file.S8 TableList with sequences for the pairs of primers used to amplify the genefragments used in the present study.(XLSX)Click here for additional data file.S9 TableClade support values based on the combination of (A) FBP/SHL, (B) FBP/TBE,(C) and TBE/SHL, classified in seven categories, as follows: red,unambiguous support ; orange, robustsupport ; blue, strong support ; green, moderate support; dark grey, ambiguous support  or poor support ; light grey,unsupported .(XLSX)Click here for additional data file.S10 TableList of fossil snakes from the Paleogene and Neogene with authorship,stratigraphic occurrence and locality.(XLSX)Click here for additional data file.S1 FigMaximum likelihood tree of Colubroides containing 1263 terminals. Color ofthe squares follow the categories of combined clade support as described in(PDF)Click here for additional data file.S2 FigData matrix and calibrated tree resulting from the treePL analysis ofColubroides, including the outgroups and containing 1278 terminals (1263Colubroides and 15 outgroups).(TRE)Click here for additional data file.S3 FigMaximum likelihood species-level phylogeny of Colubroides includingcomparisons among values of FBP, SHL, and TBE support metrics. Numbersinside de squares on the nodes of the full tree represent the TBE valuesretrieved.(PDF)Click here for additional data file.S4 FigZoomed, large-scale calibrated tree resulting from the treePL analysisshowing the pattern of cladogenic events through time.(PDF)Click here for additional data file.S1 AppendixTropidophis nigriventris (AMNH 81182); Acrochordidae:Acrochordus granulatus (ZMB 9444). Figure B,Xenodermidae: Achalinus spinalis (AMNH 34621),Fimbrios klossi (BMNH 1946.1.15.88). Figure C,Xenodermidae: Xenodermus javanicus (FMNH 158613);Xylophiidae: Xylophis perroteti (BMNH 1955.1.3.10). FigureD, Pareidae: Pareas moellendorffi (AMNH 27770),Apopeltura boa (BMNH 47.12.30). Figure E, Viperidae:Azemiops kharini (ZMB 69985), Bothropsneuwiedi (MZUSP 1476), Causus rhombeatus (FMNH74241), Vipera ursinii (MZUSP 8230). Figure F,Homalopsidae: Bitia hydroides (FMNH 229568),Brachyorrhos albus (FMNH 142322), Enhydrischinensis (AMNH 33870), Fordonia leucobalia(AMNH 107179). Figure G, Homalopsidae: Homalopsis buccata(MNHN 1963.728); Psammophiidae: Malpolon monspessulanus(AMNH 140768), Mimophis mahfalensis (UMMZ 209653). FigureH, Psammophiidae: Psammophylax variabilis (AMNH 73213),Rhamphiophis oxyrhynchus (AMNH 16890),Psammophis phillipsi (AMNH 67750). Figure I,Cyclocoridae: Cyclocorus lineatus (MNHN 1900.413),Oxyrhabdium modestus (FMNH 53386); Atractaspididae:Aparallactus modestus (AMNH 50545). Figure J,Atractaspididae: Atractaspis bibronii (AMNH 82073),Homoroselaps lacteus (LSUMZ 57229), Macrelapsmicrolepidotus (FMNH 205860), Polemon christyi(FMNH 219913). Figure K, Lamprophiidae: Bothrolycus ater(AMNH 11976), Chamaelycus fasciatus (BMNH 1909.4.29.3),Dipsadoboa weileri (AMNH 12472), Lamprophisolivaceus (AMNH 12003). Figure L, Lamprophiidae:Lycodonomorphus rufulus (AMNH 140284),Lycophidion capense (AMNH 63771), Gonionotophiscapensis (AMNH 73208), Pseudoboodonlemniscatus (MNHN 1905.179). Figure M, Pseudoxyrhophiidae:Alluaudina bellyi (UMMZ 201605), Dromicodryasquadrilineatus (UMMZ 209290), Duberria lutrix(UMMZ 154361), Heteroliodon occipitalis (UMMZ 218178).Figure N, Pseudoxyrhophiidae: Ithycyphus miniatus (UMMZ201615), Langaha madagascariensis (UMMZ 218193),Liophidium torquatum (UMMZ 209437),Pseudoxyrhopus tritaeniatus (UMMZ 203649). Figure O,Elapidae: Bungarus caeruleus (AMNH 87483);Calliophis intestinalis (BMNH 1880.9.10.15),Micrurus narduccii (MZUSP 8370), Najanaja (AMNH 86912). Figure P, Elapidae: Notechisscutatus (ZMB 7930), Toxicocalamus loriae(AMNH 95581); Pseudoxenodontidae: Pseudoxenodonstricticaudatus (AMNH 34674). Figure Q, Natricidae:Afronatrix anoscopa (MNHN 1986.1618), Aspiduratrachyprocta (AMNH 120251), Atretiumschistosum (AMNH 85509), Lycognathophisseychellensis (UMMZ 195836). Figure R, Natricidae:Natriciteres fuliginoides (MNHN 1987.1552),Natrix maura (AMNH 115697), Sinonatrixannularis (AMNH 115693), Xenochrophiscerogaster (AMNH 89276). Figure S, Dipsadidae:Apostolepis cf. nelsonjorgei (MZUSP20636), Atractus maculatus (IB 40003), Conophispulcher (AMNH 117934), Contia tenuis (UMMZ133519–1). Figure T, Dipsadidae: Farancia abacura (KU214419), Geophis hoffmanni (AMNH 113561), Helicopspastazae (AMNH 49143), Heterodon nasicus (MNHN1993.1625). Figure U, Dipsadidae: Philodryasmattogrossensis (AMNH 141377), Sibon sartorii(LSUMZ 23243), Tachymenis peruviana (KU 135193),Urotheca multilineata (AMNH 98284). Figure V,Dipsadidae: Xenopholis scalaris (AMNH 60799);Sibynophiidae: Scaphiodontophis annulatus (MZUSP 5971),Sibynophis subpunctatus (AMNH 96073). Figure W,Sibynophiidae: Colubroelaps nguyenvansangi (ZISP/IEBR25682). Figure X, Calamariidae: Calamaria gervaisi (AMNH36744), Macrocalamus lateralis (LSUMZ 45407);Oreocalamus hanitschi (BMNH 1929.12.22.106). Figure Y,Grayiidae: Grayia smithii (AMNH 140428); Colubridae:Boiga dendrophila (AMNH 116014), Coluberconstrictor (FMNH 135284). Figure Z, Colubridae:Dendrelaphis papuensis (AMNH 107175), Ptyasmucosus (AMNH 83993), Scaphiophisalbopunctatus (AMNH 104101), Senticolistriaspis (AMNH 110625). Figure AA, Colubridae: Spilotespullatus (IBSP 4955); Colubridae incertae sedis:Iguanognathus werneri (BMNH 1946.1.6.34). Figure AB,Elapoidea incertae sedis: Buhoma depressiceps (BMNH1907.5.22.10), Micrelaps muelleri (HUJR 8009). Scale bar =1 mm.Skull morphology of representatives of colubroidean families illustrating thenaso-frontal joint and optic foramen/fenestra. Figure A, Tropidophiidae:(PDF)Click here for additional data file.S2 AppendixAcrochordus javanicus(USNM 297404), scale bar = 2 mm; Xenodermidae: Achalinusrufescens (BMNH 1946.1.12.37), scale bar = 1 mm;Fimbrios klossi (BMNH 1946.1.15.88), scale bar = 1 mm;Pareidae: Pareas sp. (MZUSP 12186), scale bar = 1 mm.Figure B, Viperidae: Causus difilippi (MZUSP 18668), scalebar = 5 mm; Vipera ursinii (MZUSP 8230), scale bar = 5 mm;Azemiops feae (ROM 36976), scale bar = 1mm;Bothrops jararaca (MZUSP 14425), scale bar = 2mm.Figure C, Homalopsidae: Cerberus rynchops (MZUSP 9569),scale bar = 2mm; Homalopsis buccata (MZUSP 11483), scalebar = 1mm. Psammophiidae: Psammophis lineolatus (MZUSP8221), scale bar = 1mm; Mimophis mahfalensis (MZSUP 12188),scale bar = 2mm. Figure D, Pseudoxyrhophiidae: Madagascarophiscolubrinus (BMNH 89.8.28.23), scale bar = 2mm;Ditypophis vivax (BMNH_99.12.5.125), scale bar = 1mm;Lamprophiidae: Boaedon fuliginosus (MZUSP 8167), scale bar= 2mm; Crotaphopeltis hotamboeia (MZUSP 19602), scale bar =1mm. Figure E, Atractaspididae: Atractaspis irregulares(MZUSP 10826), scale bar = 1mm; Homoroselaps lacteus (LSUMZ57229), scale bar = 1mm. Elapidae: Sinomicrurusmacclellandi (ROM 37113), scale bar = 1mm; Najanaja (UMMZ 181137), scale bar = 1mm. Figure F, Elapidae:Micrurus corallinus (MZUSP 13112), scale bar = 1mm;Cyclocoridae: Cyclocorus lineatus (BMNH 96.3.30.78), scalebar = 1mm; Natricidae: Natrix natrix (MZUSP 2514), scalebar = 2mm; Natriciteres olivacea (MZUSP 2083), scale bar =1mm. Figure G, Sibynophiidae: Scaphiodontophis annulatus(MZUSP 5971), scale bar = 2mm; Grayiidae: Grayia smithii(MZUSP 8130), scale bar = 1mm; Grayia tholloni (MZUSP8135), scale bar = 2mm; Calamariidae: Oreocalamus hanitschi(BMNH 1929.12.22.106), scale bar = 1mm. Figure H, Colubridae:Chironius bicarinatus (MZUSP 13860), scale bar = 2mm;Spilotes pullatus (MZUSP 13845), scale bar = 2mm;Oxybelis aeneus (MZUSP 13028), scale bar = 2mm;Mastigodryas boddaerti (MZUSP 13052), scale bar = 2mm.Figure I, Colubridae: Simophis rhinostoma (MZUSP 13858),scale bar = 2mm; Dipsadidae: Heterodon platirhinos (MZUSP2991), scale bar = 2mm; Farancia abacura (MZUSP 2953),scale bar = 2mm; Carphophis amoenus (MZUSP 8183), scale bar= 1mm. Figure J, Dipsadidae: Synophis lasallei (MZUSP7713), scale bar = 1mm; Nothopsis rugosus (MZUSP 7490),scale bar = 1mm; Dipsas indica (IBSP 40137), scale bar =1mm; Atractus serranus (MZUSP 17937), scale bar = 1mm.Figure K, Dipsadidae: Boiruna maculata (MZUSP 703), scalebar = 2mm; Helicops angulatus (MZUSP 14234), scale bar =2mm; Philodryas nattereri (MZUSP 13039), scale bar = 2mm;Oxyrhopus clathratus (MZUSP 14010), scale bar =2mm.Posterior trunk vertebral morphology of representatives of colubroideanfamilies. Figure A, Acrochordidae: (PDF)Click here for additional data file.S3 AppendixAcrochordus javanicus (LSUMZ 34406)completely everted and filled, scale bar = 5 mm; Xenodermidae:Xenodermus javanicus (FMNH 138678) partially everted,partially filled, and dyed with alizarin red, scale bar = 1 mm. Figure B,Xenodermidae: Achalinus rufescens (BMNH 1983.193)completely everted and partially filled, scale bar = 2 mm; Fimbriosklossi (BMNH 1965.2.639) opened through a longitudinal slit,one lobe partially filled, scale bar = 1 mm; Pareidae: Pareasmonticola (BMNH 1909.3.9.19) completely everted and filled,scale bar = 1 mm. Figure C, Pareidae: Asthenodipsasmalaccanus (BMNH 1924.10.23.7) completely everted and filled;Aplopeltura boa (BMNH 94.6.30.63) completely evertedand filled; scale bars = 2 mm. Figure D, Xylophiidae: Xylophisperroteti (BMNH 1955.1.3.10) opened through a longitudinalslit, spread flat, and dyed with alizarin red, scale bar = 5 mm. Figure E,Viperidae: Porthidum nasutum (MZUSP 7480) completelyeverted and filled; Vipera ammodytes (MZUSP 8223)completely everted and filled; scale bars = 5 mm. Figure F, Viperidae:Bothrops neuwiedi (MZUSP 11851) completely everted andfilled; Causus bilineatus (MNHN 1993.5992) completelyeverted and filled; scale bars = 5 mm. Figure G, Homalopsidae:Homalopsis buccata (MNHN 1963.728) completely evertedand filled; Brachyorrhos albus (FMNH 142324) completelyeverted and filled; scale bars = 5 mm. Figure H, Homalopsidae:Fordonia leucobalia (AMNH 107179) completely everted,filled, and dyed with alizarin red; Bitia hydroides (FMNH229568) completely everted and filled; scale bars = 5 mm. Figure I,Homalopsidae: Erpeton tentaculatum (AMNH 8850) completelyeverted and filled, scale bar = 5 mm. Psammophiidae: Mimophismahfalensis (UMMZ 209646) completely everted and partiallyfilled, scale bar = 2 mm; Atractaspididae: Polemon christyi(FMNH 219912) completely everted and filled, scale bar = 5 mm. Figure J,Atractaspididae: Atractaspis fallax (AMNH 102298)completely everted and filled, scale bar = 10 mm; Macrelapsmicrolepidotus (FMNH 205863) completely everted and filled,scale bar = 5 mm. Figure K, Cyclocoridae: Cyclocoruslineatus (MNHN 1900.411) opened through a longitudinal slit,spread flat, and dyed with alizarin red, scale bar = 5 mm;Oxyrhabdion modestum (FMNH 68907) completely everted,filled, and dyed with alizarin red, scale bar = 5 mm. Figure L,Lamprophiidae: Lamprophis fuliginosus (MNHN 1994.8111)completely everted and filled, scale bar = 5 mm; Chamaelycusfasciatum (BMNH 1909.4.29.2–3) completely everted and filled,scale bar = 2 mm; Lycodonomorphus rufulus (AMNH 140283)completely everted and filled, scale bar = 5 mm. Figure M, Lamprophiidae:Lycophidion semicinctus (MNHN 1995.3474) completelyeverted, filled, and dyed with alizarin red; Mehelyacapensis (AMNH 73208) completely everted and filled;Pseudoboodon lemniscatus (MNHN 1905.185) completelyeverted and filled; scale bars = 5 mm. Figure N, Pseudoxyrhophiidae:Dromicodryas bernieri (UMMZ 218166) completely evertedand filled, scale bar = 5 mm; Duberria lutrix (AMNH 115639)completely everted, filled, and dyed with alizarin red, scale bar = 3 mm;Alluaudina bellyi (UMMZ 209239) completely everted andfilled, scale bar = 2 mm. Figure O, Pseudoxyrhophiidae:Pseudoxyrhopus tritaeniatus (UMMZ 195854) completelyeverted and filled; Liophidium torquatum (UMMZ 209430)completely everted and filled; scale bars = 5 mm. Figure P, Elapidae:Naja melanoleuca (BMNH 1959.1.7.69) completely evertedand filled; Micrurus frontalis (IBSP 44331) completelyeverted and filled; scale bars = 5 mm. Figure Q, Elapidae:Austrelaps superbus (BMNH 1926.12.25.113) completelyeverted and filled; Bungarus candidus (BMNH 1937.11)completely everted and filled; scale bars = 5 mm. Figure R, Natricidae:Atretium schistosum (AMNH 85505) completely everted,filled, and dyed with alizarin red; Lycognathophisseychellensis (UMMZ 167994) completely everted, filled, anddyed with alizarin red; Afronatrix anoscopus (AMNH 142404)completely everted, filled, and dyed with alizarin red; scale bars = 2 mm.Figure S, Natricidae: Xenochrophis vittatus (BMNH71.7.20.195–6) completely everted and filled; Natriciteresolivacea (AMNH 11905) completely everted and filled;Sinonatrix annularis (AMNH 84530) completely everted,filled, and dyed with alizarin red; scale bars = 2 mm. Figure T, Natricidae:Aspidura trachyprocta (AMNH 120248) completely evertedand partially filled ; Elapoidis fusca (MNHN1895.55) completely everted and filled, scale bar = 2 mm. Figure U,Pseudoxenodontidae: Pseudoxenodon macrops (AMNH 34649)completely everted and filled; Dipsadidae: Conophis pulcher(MNHN 5981) completely everted and filled; scale bars = 5 mm. Figure V,Dipsadidae: Contia tenius (UMMZ 133370) completely evertedand filled, scale bar = 2 mm; Urotheca decipiens (KU103892) completely everted and filled, scale bar = 5 mm. Figure W,Dipsadidae: Oxyrhopus occipitalis (AMNH 129255) completelyeverted, filled, and dyed with alizarin red; Faranciaerythrogramma (KU 197245) completely everted, filled, and dyedwith alizarin red. Scale bars = 5 mm. Figure X, Dipsadidae:Tachymenis chilensis (MZUSP 8239) completely everted,filled, and dyed with alizarin red, scale bar = 2 mm; Heterodonnasicus (MNHN 3636) completely everted and filled, scale bar =5 mm; Philodryas olfersii (IBSP 63455) completely everted,filled, and dyed with alizarin red, scale bar = 5 mm. Figure Y,Sibynophiidae: Sibynophis chinensis (AMNH 34102) completelyeverted and filled, scale bar = 2 mm; Scaphiodontophisannulatus (KU 191073) completely everted and filled, scale bar= 2 mm; Calamariidae: Pseudorabdion longiceps (BMNH1969.1866) completely everted and partially filled, scale bar = 5 mm. FigureZ, Calamariidae: Calamaria lumbricoidis (BMNH 1928.2.18.26)completely everted and partially filled, scale bar = 5 mm; Calamarialinnaei (AMNH 31943) completely everted and partially filled,scale bar = 1 mm; Oreocalamus hanitschi (BMNH1929.12.22.106) completely everted and partially filled, scale bar = 5 mm.Figure AA, Grayiidae: Grayia ornata (BMNH 98.3.25.3)completely everted and filled; Colubridae: Pantherophisguttatus (USNM 523605) completely everted and filled;Spilotes sulphureus (IBSP 68260) completely everted andfilled. Scale bars = 10 mm. Figure AB, Colubridae: Dispholidustypus (AMNH 23110) completely everted and filled;Hierophis viridiflavus (MNHN 1978.414) completelyeverted and filled; Boiga pulverulenta (MNHN 1967.437)completely everted and filled; scale bars = 10 mm. Figure AC, Colubridae:Ptyas korros (AMNH 84460) completely everted andfilled, scale bar = 10 mm; Gongylosoma baliodeirus (MNHN1989.199) completely everted and filled, scale bar = 3 mm; Liopeltisfrenatus (MNHN 1928.75) completely everted and filled, scalebar = 5 mm. Figure AD, Elapoidea Incertae sedis: Buhomadepressiceps (MNHN 1991.1740) completely everted, partiallyfilled, and dyed with alizarin red, scale bar = 1 mm.Hemipenial morphology of representatives of colubroidean families. Figure A,Acrochordidae: (PDF)Click here for additional data file.


The correct name is: Amare Desalegn Wolide. The correct citation is: Desalegn Wolide A, Mossie A, Gedefaw L (2014) Nutritional Iron Deficiency Anemia: Magnitude and Its Predictors among School Age Children, Southwest Ethiopia: A Community Based Cross-Sectional Study. PLoS ONE 9(12): e114059.


The correct citation is: Nguyen Quang M, Rogers T, Hofman J, Lanham AB (2019) New framework for automated article selection applied to a literature review of Enhanced Biological Phosphorus Removal. PLoS ONE 14(5): e0216126.


This article has been corrected: The correct affiliation is given below:2 Department of Oncology and Hematology, China-Japan Union Hospital of Jilin University, Changchun 130012, People’s Republic of Chinahttps://doi.org/10.18632/oncotarget.19856Original article: Oncotarget. 2017; 8:90308-90314.


Background: Experimental and clinical studies have shown that Tao-Hong-Si-Wu decoction (THSWD) improved neurological deficits resulting from Middle Cerebral Artery Occlusion (MCAO). However, the mechanisms of action of THSWD in MCAO have not been characterized. In this study, the mRNA transcriptome was used to study various therapeutic targets of THSWD.Methods: RNA-seq was used to identify differentially expressed genes (DEGs). MCAO-induced up-regulated genes (MCAO vs. control) and THSWD-induced down-regulated genes (compared to MCAO) were identified. Intersection genes were defined as up-regulated differentially expression genes (up-DEGs) identified as MCAO-induced gene expression that were reversed by THSWD. Genes down-regulated by MCAO and up-regulated by THSWD were grouped as another series of intersections. Biological functions and signaling pathways were determined by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses. In addition, several identified genes were validated by RT-qPCR.Results: A total of 339 DEGs were filtered based on the 2 series (MCAO vs. control and MCAO vs. THSWD), and were represented by genes involved in cell cycle (rno04110), ECM–receptor interaction (rno04512), complement and coagulation cascades (rno04610), focal adhesion (rno04510), hematopoietic cell lineage (rno04640), neuroactive ligand–receptor interaction (rno04080), cocaine addiction (rno05030), amphetamine addiction (rno05031), nicotine addiction (rno05033), fat digestion and absorption (rno04975), glycerophospholipid metabolism (rno00564), and others. The protein–protein interaction (PPI) network consisted of 202 nodes and 1,700 connections, and identified two main modules by MOCDE.Conclusion: Cell cycle (rno04110), ECM–receptor interaction (rno04512), complement and coagulation cascades (rno04610), focal adhesion (rno04510), hematopoietic cell lineage (rno04640), and neuroactive ligand–receptor interactions (rno04080) are potential therapeutic targets of THSWD in MCAO. This study provided a theoretical basis for THSWD prevention of neurological deficits resulting from intracerebral hemorrhage. Angelicae sinensis , Paeonia lactiflora , and Rhizoma ligustici Chuanxiong   herbs have been widely used in China and other Asian countries to combat various diseases for thousands of years. It is extremely important to elucidate the molecular mechanisms of TCM using modern advanced technologies . Fortunaese, CX) . In prevese, CX) . AnotherIn recent decades, the expression of mRNA performed by microarray or high-throughput RNA-seq has been used to reveal molecular mechanisms and explore biomarkers for diagnosis and prediction, especially in complex diseases such as cancer, diabetes and stroke [10–12].To comprehensively elucidate potential molecular mechanisms, our present study applied an RNA-seq strategy to characterize the key molecular mechanisms of THSWD. At first, we performed RNA-seq in rat brain hemispheres of control, Middle Cerebral Artery Occlusion (MCAO), and THSWD groups. EdgeR and venny were applied to identify up-DEGs and down-DEGs. And then, clusterProfiler, GO, and pathway annotation of up-DEGs and down-DEGs was performed. PPI networks were structured based on STRING database, visualization was carried out using Cytoscape, and modules were identified by MCODE. Finally, key DEGs were verified by qPCR.The proportion of THSWD compound was TR: HH: SD: DG: BS: CX = 3: 2: 4: 3: 3: 2 . Each coEleven control compounds were obtained, including gallic acid, rehmannioside D, protocatechuic acid, P-hydroxybenzoic acid, hydroxysafflor yellow A, caffeic acid, amygdalin, chlorogenic acid, paeoniflorin, ferulaic acid, and benzoic acid Table . The 11 E was used for MS/MS analysis with a low collision energy of 6 V and a high collision energy of 20–80 V. The scan range was m/z 50–1200. MassLynxTM v4.1 (Waters Co.) and UNIFI®Scientific Information System v1.7 (Waters Co.) were used for data acquisition and analysis.The aqueous extract of THSWD was separated using a Waters Acquity UPLC BEH C18 column . Mobile phases consisted of 0.1% aqueous formic acid (A) and acetonitrile (B). Chromatographic separation was performed at 35°C. Gradient elution with the flow rate of 0.3 mL/min was performed as follows: 3% B at 0–2 min, 3–8% B from 2 to 8 min, 8 to 25% B from 8 to 12 min, 25% B from 12 to 15 min, 25 to 45% B from 15 to 16 min, 45 to 90% B from 16 to 22 min, 90 to 100% B from 22 to 26 min, and 100% B from 26 to 28 min. A Waters SYNAPTG2-Si MS  with an ESI source in negative ion mode was used for MS analysis, and leucine enkephalin was used as the accurate mass calibration solution. The desolvation gas temperature was 350°C. The flow rates of the cone and desolvation gases were set at 50 and 600 L/h, respectively. The capillary, cone, and extraction cone voltages were set to –2.5 kV. MSMale Sprague-Dawley (SD) rats  were obtained from the Experimental Animal Center of Anhui Medical University. Rats were randomly divided into normal group, MCAO group and THSWD group with 10 rats in each group. All rat anesthetized using sodium pentobarbital during all surgeries to minimize suffering, and preparation of the MCAO model was performed following our previous study , which aTM miRNA Isolation kit  following the manufacturer’s instructions. Quantity and quality of RNA samples were determined using a NanoDrop 2000  and Agilent 2100 Bioanalyzer . TruSeq®Stranded Total RNA Sample Preparation kit was used to prepare libraries following the manufacturer’s instructions, and purified libraries were quantified using a Qubit 2.0®Fluorometer  and Agilent 2100 Bioanalyzer . Clusters were generated by cBot library and sequenced using an Illumina HiSeq 2500 . Sequencing was performed at Origin-Biotech Inc. .Total RNA was extracted using mirVana1. Trimming was performed by seqtk for known Illumina TruSeq adapter sequences, poor reads, and ribosomal RNA reads2. The trimmed reads were then mapped to the Rattus norvegicus reference genome (rno6) using Hisat2 (version: 2.0.4)  of RNA-Seq reads (version. 0.11.3): 2.0.4) . Stringt: 2.0.4) , 2016. G: 2.0.4) , and FPK: 2.0.4) . EdgeR w: 2.0.4) , with siange > 1 . Venny wGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched using R package to better understand the functions of DEGs . In our STRING is a database that provides comprehensive information about interactions between proteins, including prediction and experimental interaction data . In our -ΔΔCT method. The following six genes were analyzed: Cdk1, Ccna2, Cdc20, Ndc80, Th, and Calb2. Brain tissues from control, MCAO, and THSWD groups were analyzed (15 samples).Real-time PCR was performed to verify the RNA-seq results, and GAPDH was used as the internal control. Relative expression of mRNAs was determined using the 2p-value less than 0.05 was considered to be statistically significant.Raw data are shown as mean ± standard deviation (SD). Statistical calculations were carried out using GraphPad Prism 5 . A Rt≈1.72 min, MW = 170.12), protocatechuic acid , hydroxysafflor yellow A , amygdalin , and paeoniflorin  by comparing their fingerprints with standards . Figure To further understand the multifaceted mechanisms of THSWD in MCAO, we performed RNA-seq to obtain the transcriptomes of control, MCAO, and MCAO+THSWD samples. Using bioinformatics analysis, 339 DEGs were filtered | > 1 and To further understand the DEGs associated with THSWD treatment of MCAO, GO enrichment analysis was performed with 63 down-DEGs and 276 up-DEGS by clusterProfiler.p < 0.05. The top10 enriched GO biological processes were locomotor behavior (GO:0007626), neuron fate specification (GO:0048665), behavior (GO:0007610), cell fate specification (GO:0001708), monoamine transport (GO:0015844), ammonium transport (GO:0015696), amine transport (GO:0015837), neuropeptide signaling pathway (GO:0007218), adult behavior (GO:0030534), and single-organism behavior (GO:0044708). Neuron projection (GO:0043005), axon (GO:0030424), terminal bouton (GO:0043195), cell projection (GO:0042995), synapse (GO:0045202), neuronal cell body (GO:0043025), axon terminus (GO:0043679), cell body (GO:0044297), neuron projection terminus (GO:0044306), and perikaryon (GO:0043204) were the top10 enriched cellular components. The enriched GO molecular functions were G-protein coupled peptide receptor activity (GO:0008528), ammonium transmembrane transporter activity (GO:0008519), neuropeptide receptor activity (GO:0008188), symporter activity (GO:0015293), sodium ion transmembrane transporter activity (GO:0015081), secondary active transmembrane transporter activity (GO:0015291), ion transmembrane transporter activity (GO:0015075), substrate-specific transmembrane transporter activity (GO:0022891), cation transmembrane transporter activity (GO:0008324), and transmembrane transporter activity (GO:0022857). The top30 GO-terms with highest enrichment factor are shown in Figure Enrichment analysis showed that a total of 196 GO-terms were significantly enriched with 63 down-DEGs, 30 of which were BP, 40 of which were CC, and 60 of which were MF with p < 0.05. The top10 enriched GO biological processes were mitotic cell cycle process (GO:1903047), mitotic cell cycle (GO:0000278), mitosis (GO:0007067), cell division (GO:0051301), chromosome segregation (GO:0007059), cell cycle (GO:0007049), mitotic sister chromatid segregation (GO:0000070), sister chromatid segregation (GO:0000819), chromosome organization (GO:0051276), and regulation of cell cycle process (GO:0010564). Meanwhile, we found that many neuron death related GO-terms were significantly enriched such as negative regulation of neuron apoptotic process , negative regulation of neuron death , and regulation of neuron apoptotic process . Chromosome (GO:0005694), nucleosome (GO:0000786), spindle (GO:0005819), protein-DNA complex (GO:0032993), chromosome, centromeric region (GO:0000775), nuclear chromosome (GO:0000228), kinetochore (GO:0000776), condensed chromosome kinetochore (GO:0000777), chromatin (GO:0000785), and microtubule cytoskeleton (GO:0015630) were the top10 enriched cellular components. The enriched GO molecular functions were protein binding (GO:0005515), protein heterodimerization activity (GO:0046982), microtubule motor activity (GO:0003777), microtubule binding (GO:0008017), protein dimerization activity (GO:0046983), motor activity (GO:0003774), tubulin binding (GO:0015631), extracellular matrix structural constituent (GO:0005201), cytoskeletal protein binding (GO:0008092), and protein complex binding (GO:0032403). The top30 GO-terms with highest enrichment factors are shown in Figure The 276 up-DEGs were significantly enriched into 619 GO-terms, 482 of which were BP, 61 of which were CC, and 54 of MF with Pathway enrichment analysis could provide further information regarding functions of genes and their interactions. ClusterProfiler was applied to GO enrichment analysis with 63 down-DEGs and 276 up-DEGs, which were associated with THSWD treatment of MCAO.p < 0.05. The top10 enriched pathways were neuroactive ligand–receptor interaction (rno04080), cocaine addiction (rno05030), amphetamine addiction (rno05031), nicotine addiction (rno05033), fat digestion and absorption (rno04975), glycerophospholipid metabolism (rno00564), pancreatic secretion (rno04972), cholinergic synapse (rno04725), and amyotrophic lateral sclerosis (ALS) (rno05014). The top30 pathway-terms with highest enrichment factors are shown in Figure KEGG enrichment analysis showed that a total of 45 pathway terms were enriched with 63 down-DEGs, 15 of which were significant with p-value < 0.05. Top10 pathways with the greatest enrichment were cell cycle (rno04110), ECM–receptor interaction (rno04512), complement and coagulation cascades (rno04610), microRNAs in cancer (rno05206), focal adhesion (rno04510), Staphylococcus aureus infection (rno05150), hematopoietic cell lineage (rno04640), systemic lupus erythematosus (rno05322), malaria (rno05144), and oocyte meiosis (rno04114). The top30 enriched pathways are presented in Figure We performed KEGG pathway enrichment analysis for up-DEGs, and found 136 pathway terms including 29 pathway terms with Significant DEGs were used to construct a PPI network based on the STRING database. The PPI network consisted of 202 nodes and 1,700 interactions, as shown in Figure A total of 10 modules were obtained using default criteria by MCODE. Modules were listed in descending order by MCODE score. Two modules with MCODE score ≥ 3 and nodes ≥ 10 were named as module 1 and module 2. Two modules were selected for module network visualization Figure . Most DEAs shown in Figure We analyzed the protein-coding mRNA expression profile in cerebral hemispheres from experimental and control rats by high throughput RNA-seq. 1,007 DEGs were identified, and GO, KEGG pathway, PPI, and PPI module analyses were performed, providing a better understanding of the pathological mechanisms of MCAO.Using pathway enrichment analysis, 15 DEGs were enriched to cell cycle: Cdk1, Cdc20, Cdc6, Ttk, Ccnb1, Bub1, Mcm6, Pttg1, Bub1b, Mcm3, Espl1, Mcm5, Ccna2, Plk1, and Ccnd1 Figure . Nearly Using pathway enrichment analysis, 10 DEGs were enriched into the complement and coagulation cascades, such as C3ar1, Plau, C1qb, C1qc, Vwf, Kng1, Cfd, Vsig4, and C5ar1 Figure . PreviouUsing pathway enrichment analysis, we found 12 DEGs involved in neuroactive ligand–receptor interactions: Brs3, Mc3r, Calcr, Chrna6, Glra1, Galr1, Ntsr1, Cckar, Gabre, C3ar1, Htr2b, and C5ar1 Figure . Lan et E analysis. XD, LX, and QB performed the animal experiments. XD, WC, and CP analyzed the data and pre-viewed the manuscript. All authors read and approved the final manuscript.XD and DP conceived and designed the study. XD and LH performed the UPLC-QTOF/MSThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.