Nicotiana tabacum is an important economic crop. Topping, a common agricultural practice employed with flue-cured tobacco, is designed to increase leaf nicotine contents by increasing nicotine biosynthesis in roots. Many genes are found to be differentially expressed in response to topping, particularly genes involved in nicotine biosynthesis, but comprehensive analyses of early transcriptional responses induced by topping are not yet available. To develop a detailed understanding of the mechanisms regulating nicotine biosynthesis after topping, we have sequenced the transcriptomes of Nicotiana tabacum roots at seven time points following topping.Differential expression analysis revealed that 4830 genes responded to topping across all time points. Amongst these, nine gene families involved in nicotine biosynthesis and two gene families involved in nicotine transport showed significant changes during the immediate 24 h period following topping. No obvious preference to the parental species was detected in the differentially expressed genes (DEGs). Significant changes in transcript levels of nine genes involved in nicotine biosynthesis and phytohormone signal transduction were validated by qRT-PCR assays. 549 genes encoding transcription factors (TFs), found to exhibit significant changes in gene expression after topping, formed 15 clusters based on similarities of their transcript level time-course profiles. 336 DEGs involved in phytohormone signal transduction, including genes functionally related to the phytohormones jasmonic acid, abscisic acid, auxin, ethylene, and gibberellin, were identified at the earliest time point after topping.N. tabacum, and identifies excellent candidates for further detailed studies concerning the regulation of nicotine biosynthesis in tobacco roots.Our research provides the first detailed analysis of the early transcriptional responses to topping in  Nicotiana tabacum L.) plants, topping  is an essential cultivation practice. Topping switches the plant from a seed reproductive to a leaf vegetative phase, and this significantly increases leaf nicotine contents [For tobacco  technologies. Of the sixty-nine TFs, six (from three TF families) affect nicotine metabolism, with NbbHLH1 and NbbHLH2 (basic helix-loop-helix) genes positively regulating the jasmonate activation of nicotine biosynthesis, as evidenced by overexpression [A recent report  employedpression .N. tabacum, formed through the hybridization of N. sylvestris (S-subgenome) and N. tomentosiformis (T-subgenome), and how these two subgenomes respond to topping is unclear. In this study, we have sequenced tobacco root transcriptomes at seven different time points  after topping. These time points were chosen to identify candidate genes associated with the regulation of nicotine biosynthesis at the earliest stages, as well as to allow discovery of upstream regulators of nicotine synthesis through clustering of the time-course profiles of TF gene expression, and to compare the responses of the two subgenomes to topping. This comprehensive approach to characterization of the transcriptional responses of tobacco, especially focusing on the early regulation of nicotine biosynthesis, should serve to advance genetic improvement in this crop.Although specific genes regulating nicotine synthesis after tobacco topping have been identified, a detailed description of the transcriptional regulatory network that responds to topping is not available. The situation is further complicated by the allotetrapoid status of Total RNA of tobacco roots, isolated separately from 18 individual plants, was employed for RNA sequencing (RNA-Seq) library construction. The 18 RNA-Seq libraries were sequenced using the Illumina platform. After filtering out low quality sequences , 105 Gb of cleaned data was obtained, representing approximately 6 Gb per sample. The cleaned sequence GC content varied from 42.1 to 42.7% . The values of Pearson’s Correlation Coefficient across biology replicates exceeded 0.82. In terms of the correlation between samples from different time points, some samples displayed higher values with those from other time points. For example, BWR3-2A showed a correlation coefficient of 0.96 as compared to BWR24-1A, and 0.95 with BWR5-2A . These results confirm that the alterations in gene expression detected by RNA-Seq accurately reflect transcript differences at the different time points after topping.To validate the transcriptional results obtained by RNA-Seq, we selected nine genes related to nicotine biosynthesis and phytohormone signal transduction, and examined their transcriptional responses by qRT-PCR. The expression trends of these genes analyzed by qRT-PCR were consistent with the RNA-Seq analysis performed at the corresponding time points Fig. . The cha4830 DEGs showing significant variation at the different time points after topping were selected for further analysis. Based on their relative expression levels, the DEGs were divided into different categories using hierarchical clustering, being distinguishable in terms of the temporal patterns of the transcriptional responses of the roots at the various time points after topping , carbohydrate metabolism (212), biosynthesis of other secondary metabolites (166), and metabolism of terpenoids and polyketides (69)  , as identified by showing at least 93% identities with the primary sequences of previously reported enzymes, were found in the DEG dataset  at the corresponding time points  Fig. , and twoTo investigate the upstream regulatory mechanisms of nicotine biosynthesis after topping, we next focused on the types of TFs represented in the DEGs from the tobacco root transcriptome. In our study, a total of 549 DEGs encoding TFs were identified , abscisic acid (ABA), ethylene, gibberellin (GA), and jasmonic acid (JA) , and reduced by 23.5 and 18.9% at t = 8 h and t = 24 h  as compared with untreated plants. The levels of IAA significantly increased at t = 3 h , whilst declining to initial levels at t = 24 h , and to 1.6-fold at t = 24 h , as compared to the untreated plants. We also measured nicotine levels after topping. Our analyses indicated the nicotine levels significantly increased to 1.5-fold at t = 24 h  after topping , pyrrolidine ring synthesis , and the coupling of the two nicotine rings (A622 and BBL). Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine. Down-regulation of ODC transcript levels using RNAi led to lower leaf levels of nicotine in N. tabacum [ODC gene was significantly up-regulated at t = 8 h and t = 12 h. A second example involves PMT, which converts putrescine into N-methylputrescine [PMT genes were investigated . Transcripts derived from NtPMT2 and NtPMT1b showed the greatest increase in abundance (about 3-fold) during the first 24 h after topping [SAMS genes were significantly up-regulated at early time points after topping (H) synthesis [QPT genes (designated QPT1 and QPT2). QPT1 is expressed at a constitutive basal level in all plant tissues, with somewhat higher levels of expression within the apical meristem. In comparison, QPT2 is expressed exclusively in the tobacco root and is regulated coordinately with other structural genes for nicotine biosynthesis [QPT1 and QPT2 were not present in our list of DEGs, the gene of QPT2 (Nitab4.5_0000742g0010) showed significant up-regulation at 3 h and 24 h in our qPCR assay (data not shown).The enzyme QPT plays a critical role in the synthesis of the pyridine moiety of nicotine in ynthesis . The tobynthesis , 34. AltA622, which is expressed in the root, and may be involved in the final condensation reaction of nicotine biosynthesis [N. glauca to produce anabasine was markedly reduced when an RNAi approach was used to down-regulate gene expression, thereby decreasing A622 protein levels. This resulted in plants having almost undetectable levels of pyridine alkaloids in their leaves, even after suffering damage to apical tissues [A622 was significantly up-regulated at t = 8 h and t = 24 h after topping . N. tomentosiformis displayed much lower nicotine levels than N. sylvestris in both roots and leaves [d leaves . All DEGERF1, ERF189, and ERF32, have been recently identified as direct regulators of the structural genes of nicotine biosynthesis [Nicotiana is the MYC2-like bHLH family. MYC2, belonging to the bHLH-family of TFs, is a key component in conserved jasmonate signaling [ERF genes [NtMYC2a led to great enhancement, under field testing, of nicotine levels in the transgenic lines. Although the mechanism by which ARF1 regulates nicotine synthesis remains unclear, VIGS (virus-induced gene silencing) of bHLH3 and ARF1 results in a significant increase in nicotine content as compared to control plants [WRKY-R1, the Group II member of the WRKY family, was specifically and highly expressed in tobacco roots. This suggests it regulates the expression of genes related to nicotine synthesis, like PMT [The pattern of expression changes induced by topping for the structural genes of nicotine biosynthesis was initially derived from bioinformatics analyses of sequence data, but was confirmed by the qRT-PCR experiments. It was therefore reasonable to speculate that the sequence data could also be used to identify the TF(s) that actively regulate the production of nicotine at the early time points after topping. As reported previously, many TFs play important roles in regulating nicotine biosynthesis, including members of the AP2/EREBP, bHLH, ARF, and WRKY families , 38. Theynthesis , 33, 40.ignaling . It posiRF genes , 43. WanRF genes  found thl plants . In addilike PMT .AP2/EREBP (75), bHLH (44), WRKY (23), and ARF (4) families.  whose transcription is regulated by JAs [Plant hormones play pivotal roles in regulating numerous aspects of plant growth and development, including secondary metabolism. Five major classes of plant hormones are known to be involved in the regulation of nicotine biosynthesis, including JA, IAA, GA, ethylene, and ABA , 48–51.  and PMT , 52, 53. and PMT  reportedd by JAs .In the present study, we propose that topping, as a form of mechanical wounding, activates JA signaling as one of the earliest events. JA-Ile, as the only JA derivative known to be involved in JA signaling, displayed continuously increasing levels over the first 24 h after topping, indicating a vital role in the regulation of nicotine biosynthesis. The expression levels of the two JAZ-encoding genes were significantly up-regulated at t = 0.5 h and t = 8 h, respectively, which is consistent with the observed increases in JA and JA-Ile levels. Our experimental data also indicated similar patterns of changes in concentrations of JA and IAA following topping. This is consistent with the report that JA and auxin have very similar signal transmission mechanisms. However, how JA and IAA collaboratively respond to topping stimulation and regulate nicotine biosynthesis remains unclear , and wilPYL expression affects the JA response to nicotine biosynthesis in tobacco. Finally, in our study, we found that a large number of regulatory genes associated with auxin signaling, ABA signaling, ethylene signaling, and gibberellin signaling showed significantly altered expression levels at t = 0.5 h after topping, indicating the complex networks with crosstalk are promptly constructed across these phytohormonal signaling pathways as an early response to topping . All the materials were grown for 8 weeks in commercial potting soil in a Percival PGC-10 incubator set for a 16 h day/8 h night cycle at 28 °C. Individual plants with the most similar morphology were selected for topping by removing the apices above the youngest unfolded leaf. Roots were washed with water, and tissues excised using a surgical blade, 18 samples being taken 0, 0.5, 1, 3, 5, 8 and 24 h after topping, with 2–3 replicates at each time point  and Btrim [Nicotiana tabacum genome [ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/edwards_et_al_2017/assembly/Nitab-v4.5_genome_Chr_Edwards2017.fasta.gz). Gene expression levels were estimated as FPKM (fragments per kilobase of exon per million fragments mapped reads) values [Before further analysis, the raw reads were filtered to remove adaptor sequences, low-quality reads, and reads containing poly-N, using CutAdapt  values  using Cu) values .2 fold changes (FC) ≥ 1 and an adjusted p-value  ≤ 0.05 were defined as differentially expressed genes (DEGs). The FPKM values of the DEGs were normalized and clustered by pheatmap with parameters “scale=row, cluster_rows=T, cutree_rows=8”, where cutree function in R was used to estimate category number and eight categories were obtained when h = 4.8.Differential expression of genes between the t = 0 h sample and other samples at different time points were calculated with the cuffdiff program in Cufflinks . Gene abp-value  ≤ 0.05 was chosen as the threshold for significantly enriched KEGG pathways and GO terms.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to identify the enrichment of DEGs in GO terms and metabolic pathways, respectively. For functional categorization and pathway visualization of DEGs, the metabolic pathways of DEGs were predicted using the KEGG Automated Annotation Server (KAAS) with default parameters. The DEGs in the KEGG pathways and GO analysis were enriched using ClusterProfile R and WEGO , 61. A cClustering of differentially expressed TFs and the DEGs involved in nicotine biosynthesis was performed using R package mfuzz with the cluster number assigned to 15 using the Dmin function and others as defaults on the profiles of FPKM at the different time points after topping.N. tomentosiformis and N. sylvestris. Subgenomes were assigned based on the first hit species if the first hit had at least 90% identity and the second hit showed a lower identity value than the first hit. For the DEGs involved in nicotine biosynthesis and transport, a manual check was performed based on previous reports.All DEGs were used as queries to search against all the annotated proteins in the genomes of Actin gene as an internal standard for normalization, and SYBR Green as the fluorochrome. A three-step PCR process was performed with a pre-denaturation at 95 °C for 30 s, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at the optimal temperature of each primer pair for 20 s, and elongation at 72 °C for 20 s, and finally for melting point curve analysis  to test amplicon specificity. Relative quantification of gene expression level was carried out using the 2-△△Ct method. The specific primer sequences of the selected genes for qRT-PCR validation were designed based on the divergent regions among the orthologous genes and listed in Additional file To confirm the gene expression levels of the RNA-Seq assay, 1 μg total RNA mixed equally from 3 plants for each RNA-Seq sample was used for the qRT-PCR assay as previously described . The RNAo C for 30 min and at 60o C for 3d. Each sample (100 mg) equally mixed from 4 individual plants was mixed with 1 mL of extraction solution (40% methanol containing 0.5% acetic acid (v/v)), and nicotine was quantified by HPLC as previously described [13C2-JA, 13C2-JA-Ile, D4-SA, D6-ABA and D5-IAA were used as the internal standards for JA, JA-Ile, SA, ABA and IAA, respectively. Phytohormone extraction and quantification were described as previously by HPLC-MS/MS  system [For measurement of nicotine content, roots were dried at 105escribed . Three b) system . Three bAdditional file 1: Figure S1. The FPKM density of each sample after topping in tobacco. The horizontal axis indicates the sample log10(FPKM) and the vertical axis indicates the corresponding probability density. The different colors denote different samples.Additional file 2: Figure S2. Correlation of different biological replicates across all samples.Additional file 3: Figure S3. Hierarchical cluster analysis of 4830 DEGs from different time points after topping in tobacco. The samples and treatments are indicated below each column. DEGs are defined by different colors, with the normalized expression levels employing a color gradient from low (blue) to high (red).Additional file 4: Figure S4. Classification statistics of DEGs according to the GO annotations. The GO classification is indicated on the abscissa, with the ordinate providing the percentage of the number of genes (left) and the number of genes (right).Additional file 5: Figure S5. KEGG overview of DEGs after topping in tobacco. The numbers in the picture represent the DEGs that were annotated to the metabolic pathway, with the percentage representing the ratio of this count to the number of DEGs annotated to the total metabolic pathway.Additional file 6: Table S1. Quality and mapping rates of RNA-Seq data.Additional file 7: Table S2. List of DEGs in roots after topping.Additional file 8: Table S3. DEGs involved in nicotine biosynthesis and transport after topping.Additional file 9: Table S4. Differentially transcribed transcription factors after topping.Additional file 10: Table S5. Clustering TFs and up-regulated gene families in nicotine biosynthesis according to expression pattern after topping.Additional file 11: Table S6. DEGs involved in phytohormone signal transduction after topping.Additional file 12: Table S7. Sequence-specific primers used in qRT-PCR.


Mysticellus franki gen. et sp. nov. and its close evolutionary relationship with the Southeast Asian genus Micryletta also provide insights on the biogeography of Microhylinae. Genus-level divergences within the subfamily suggest multiple Cenozoic biotic exchange events between India and Eurasia, particularly through postulated Eocene land bridges via Southeast Asia prior to accretion of the two landmasses.Anurans in Peninsular India exhibit close biogeographical links with Gondwana as well as Laurasia, often explainable by the geological history of the Indian subcontinent; its breakup from Gondwanan landmasses followed by long isolation that resulted in diversification of endemic lineages, and subsequent land connections with Asia that enabled dispersal of widespread groups. Although widely distributed, the frog subfamily Microhylinae mostly comprises of geographically restricted genera found either in Southeast and East Asia or Peninsular India and Sri Lanka. Here we report a previously unknown microhylid from the Western Ghats in Peninsular India with closest relatives found over 2,000 km away in Southeast Asia. Based on integrated evidence from mitochondrial and nuclear DNA, adult and tadpole morphology, hand musculature, male advertisement call, and geographical distance, we recognize this enigmatic frog as a distinct new species and genus endemic to the Western Ghats. The discovery of  This region is also considered as a distinct biogeographical unit, a recognition chiefly garnered due to the role of various events in the geological history of the Indian subcontinent13. After the initial break-up of Madagascar-Seychelles-India from Africa and Australia-Antarctica blocks (∼140–130 Mya), followed by separation of the Indian subcontinent from Madagascar (∼88 Mya) and subsequently the Seychelles (∼65 Mya), the northward-drifting Indian plate is considered to have been a “biotic ferry”14 during its course to unite with the Eurasian plate (starting ∼55 Mya)16. The biotic elements that moved along with the erstwhile Indian landmass during the Late Cretaceous or Palaeogene experienced long periods of isolation resulting in the origin of several endemic anuran lineages in the Western Ghats15. The biogeographical links of ancient frog lineages such as Nasikabatrachidae have provided crucial evidence for tracing back the evolutionary history of present day amphibians in the Western Ghats10.The Western Ghats is a chain of mountains that stretches over 1,600 km in southwest India. It is part of a global biodiversity hotspot with remarkable amphibian diversity and endemism18. Despite the constantly changing landscape and climatic conditions that created physical barriers for the movement of anurans20, several recent radiations  acquired wider distribution patterns in India as well as South and Southeast Asia. In this context, the Northeast Indian region is often considered as a ‘gateway’22 bridging the Indian subcontinent with the rest of Asia, particularly before the rise of the Himalaya and upliftment of the Tibetan plateau. These present day physical features are known to have played a significant role in determining the distribution patterns of anurans in South and Southeast Asia25. Currently, four biogeographical regions encompassing the Indian subcontinent—Himalaya, Indo-Burma, Sundaland and Western Ghats-Sri Lanka—are recognized as global biodiversity hotspots26.While the isolation of the Indian subcontinent is regarded as a unique event, the union of the Indian plate with Eurasia is an equally intriguing juncture that provided opportunity for faunal exchanges between these landmasses27. Although higher-level taxonomic placement of the recognized subfamilies and their phylogenetic relationships are still disputed33, based on the more widely used classification by Frost et al.29 as revised by Peloso et al.33, five microhylid subfamilies are found in Asia , of which only two are known to occur in the Indian peninsula (Melanobatrachinae and Microhylinae). Melanobatrachinae is monotypic with a single known species and restricted to the southern Western Ghats of India. On the other hand, Microhylinae is widely distributed in South, Southeast and East Asia. It is a large radiation comprising of 87 species in seven genera34. Phylogenetically, Microhylinae is sister to Dyscophinae, and the split between the two subfamilies has been a significant point of interest for biogeographical interpretations, largely due to the congruence of geological events such as the break-up of India–Seychelles–Madagascar landmass during the Late Cretaceous13 with the restricted geographical distribution of members of these subfamilies to Asia and Madagascar, respectively. Phylogenetic relationships among members of the subfamily Microhylinae have been discussed in several studies35. A few have also provided insights on origin, possible routes of colonization, and patterns of distribution, however, in larger contexts of either global or selected Asian microhylids37. Hence, a detailed phylogenetic investigation and divergence ages within the subfamily Microhylinae are still lacking.The Narrow-mouthed anuran family Microhylidae is distributed throughout the tropics and currently comprises of 653 species in 53 genera and 13 subfamiliesMysticellus franki gen. et sp. nov. and discuss new phylogenetic insights highlighted through this finding, along with their biogeographical implications.Our recent explorations in Peninsular India have led us to the discovery of a previously unknown microhylid frog that could not be assigned to any of the known microhylid members of this region. Evidence from multiple approaches concordantly suggests the newly discovered microhylid to be a distinct species, which also warrants recognition of a new genus within the subfamily Microhylinae. This novel evolutionary lineage is endemic to the Western Ghats, with its closest known relatives found over 2,000 km (as the crow flies) in the Indo-Burma and Sundaland hotspots. Here, we provide a formal description of Amphibia Linnaeus, 1758Anura Fischer von Waldheim, 1813Microhylidae Günther, 1858 (1843)Microhylinae Günther, 1858 (1843)urn:lsid:zoobank.org:act:6145F26C-E140-4BBF-9322-550047926EA2Figures –S4.FigurMysticellus franki sp. nov.Mysticellus, is a masculine noun derived from the Latin mysticus (meaning mysterious) + ellus (a diminuitive), highlighting the ability of this small frog to remain out of sight despite its occurrence in wayside areas surrounding human settlements.The genus name, Mysterious Narrow-mouthed Frog.Mysticellus differs from other Microhylinae genera by the combination of following characters: small adult snout-vent size , slender body; snout longer than eye length, male SL 2.8–3.0 mm, 2.9 ± 0.1 mm, N = 5, female SL 3.0–3.2 mm, 3.1 ± 0.1 mm, N = 2 vs. male EL 2.4–2.6 mm, 2.5 ± 0.1 mm, N = 5; female EL 2.5–2.7 mm, 2.6 ± 0.1 mm, N = 2; absence of maxillary and vomerine teeth; finger and toe tips rounded, with small discs; presence of well-developed subarticular tubercles on all fingers and toes, rounded, alternating with additional smaller tubercles; prominent inner metatarsal tubercle and a small outer metatarsal tubercle on foot; webbing between fingers absent; rudimentary webbing between toes; lateral surfaces from tip of the snout up to the groin prominently dark blackish-brown; two prominent dark blackish-brown ‘false-eye’ like spots on either side of the groin extending just above the hind legs; a thin mid-dorsal line invariably extending from tip of the snout up to the vent; ventral surfaces of throat, belly, arms and legs dark brown with a violet tinge and various sized greyish-white blotches and speckles. This new genus can also be distinguished from other members of the subfamily by its hand musculature in having the m. flexor teres digiti III ventral to both slips of the m. transversus metacarpus 138; and the dorsal surface with two previously unreported flexor muscles on digits III and IV, one lateral to the m. lumbricalis brevis digiti III, and the other medial to the medial slip of the m. lumbricalis brevis digiti IV , absence of maxillary and vomerine teeth (vs. present), horizontal pupil , and well-developed tubercles on the hand (vs. not enlarged); differs from Kaloula Gray, 1831 by its smaller adult snout-vent size, SVL < 30 mm (vs. SVL > 30 mm), slender body (vs. robust), absence of ridge on posterior sides of choanae (vs. present), supratympanic fold absent (vs. present), finger and toe tips slightly enlarged (vs. enlarged with prominent discs), and inner metatarsal tubercle not enlarged (vs. enlarged and spatulate); differs from Metaphrynella Parker, 1934 by absence of ridge on posterior sides of choanae (vs. present), finger and toe tips slightly enlarged (vs. enlarged with prominent discs), webbing absent between fingers (vs. present), terrestrial habitat , and behavior of breeding around temporary water puddles (vs. tree holes); differs from Microhyla Tschudi, 1838 by its body relatively linear in shape (vs. triangular), lateral surfaces of the body prominently dark blackish-brown from tip of the snout up to the lower abdomen, tapering close to the groin and extending towards the dorsal surface just above the hind legs in the form of two prominent blackish-brown ‘false-eye’ like spots on either side , and prominent subarticular tubercles alternating with additional smaller tubercles ; differs from Micryletta Dubois, 1987 by its tympanum externally indistinct (vs. distinct), and lateral surfaces of the body prominently dark blackish-brown from tip of the snout up to the lower abdomen, tapering close to the groin and extending towards the dorsal surface just above the hind legs in the form of two prominent blackish-brown ‘false-eye’ like spots on either side ; differs from Phrynella Boulenger, 1887 by its finger and toe tips slightly enlarged (vs. enlarged with prominent discs), metatarsal tubercles separate (vs. united), and rudimentary webbing between toes (vs. nearly fully webbed); and differs from Uperodon Duméril and Bibron, 1841 by its slender body (vs. robust and globular), absence of ridge on posterior sides of choanae (vs. present), prominent subarticular tubercles alternating with additional smaller tubercles , lateral surfaces of the body prominently dark blackish-brown from tip of the snout up to the lower abdomen, tapering close to the groin and extending towards the dorsal surface just above the hind legs in the form of two prominent blackish-brown ‘false-eye’ like spots on either side (vs. absent), finger and toe tips slightly enlarged (vs. enlarged with prominent discs except in U. systoma and U. globulosus), inner metatarsal tubercle not enlarged (vs. enlarged or spatulate), and rudimentary webbing between toes .The new genus Mysticellus franki sp. nov.The new genus currently contains a single species urn:lsid:zoobank.org:act:B1FBD56B-B6B3-412A-8988-E527A082C4C5Figures –S4.franki, is a Latin genitive honoring evolutionary biologist Prof Franky Bossuyt (Vrije Universiteit Brussel), recognizing his role in global amphibian research and education, and particularly for his contribution to the study of Indian amphibians.The species name, Franky’s Narrow-mouthed Frog.ZSI/WGRC/V/A/966, an adult male, SVL 23.0 mm, from Suganthagiri , Wayanad district, Kerala state, India, collected by Sonali Garg and SD Biju on 05 June 2015.ZSI/WGRC/V/A/967–970, four adult males, and ZSI/WGRC/V/A/971–972, two adult females, collected along with the holotype.I 2.5, FLII 3.1, FLIII 4.8, FLIV 2.7); finger tips rounded, slightly enlarged with small discs, fingers without dermal fringes, webbing absent between fingers; subarticular tubercles prominent, rounded, all present, alternating with additional smaller tubercles; three prominent palmar tubercles, middle tubercle rounded (PTL 0.5), outer tubercle oval (OPTL 0.7), inner tubercle rounded (IPTL 0.4); supernumerary tubercles present. Hind limbs long, thigh (THL 10.3) slightly shorter than shank (SHL 10.5) and shorter than foot (FOL 11.7); distance from the base of tarsus to the tip of toe IV (TFOL 17.0); toes long, relative length of toes I < V < II < III < IV; toe tips rounded, slightly enlarged with small discs, toes with weakly developed dermal fringes, rudimentary webbing between toes; subarticular tubercles prominent, oval, all present, alternating with additional smaller tubercles; inner metatarsal tubercle prominent (IMTL 0.8), oval; outer metatarsal tubercle small (OMTL 0.4), rounded; supernumerary tubercles present on toes , rather slender; head wider than long , flat above; snout nearly truncate in dorsal and ventral view, acute in lateral view, snout length (SL 2.9) longer than horizontal diameter of eye (EL 2.4); loreal region acute, rounded canthus rostralis; interorbital space flat, twice (IUE 3.2) as wide as upper eyelid (UEW 1.6) and internarial distance (IN 1.6); nostril oval, closer to snout (NS 0.7) than eye (EN 1.3); pupil oval; vomerine ridge absent; tongue small, not emarginated, spatulate, bearing no median lingual process. Arms long, forearm length (FAL 5.5) shorter than hand length (HAL 7.0); relative length of fingers I < IV < II < III  reddish-brown; dorsal surface of hind limbs (including toes) brick red to reddish-brown with faint greyish-brown transverse bands; posterior parts of thigh light brown, shank and tarsus reddish-brown. Ventral surfaces of throat, belly, fore- and hind limbs dark brown with a violet tinge and various sized greyish-white blotches and speckles Fig. .Mysticellus gen. nov.As for 2 area). The specific site was located close to a secondary forest. Breeding activities were observed only for four to five days, after which the animals disappeared and no individuals could be located despite repeated visits. Tadpoles (stage 34) were observed at the same site towards the end of July  are as follows: call duration, 1771.4 ms; call rise time, 91.4 ms; call fall time, 1681.9 ms; number of pulses, 141; pulse rate, 80.2 pulses per second; call spectrum with a single broad peak and mean dominant frequency of 3.7 kHz  as a distinct and well-supported sister lineage to genus Micryletta . The new lineage was also well differentiated in independent analyses of nuclear and mitochondrial gene datasets. Pairwise distances for the mitochondrial 16S rRNA gene sequences showed considerable genetic differentiation (minimum 8.2%) between the new species and members of its closest generic relative Micryletta. This was also concordant with genetic divergences observed among various known genera in the subfamily Microhylinae , except ted Fig. . Our phynae Fig. –S3. HencMysticellus gen. nov. diverged from its sister lineage Micryletta ~39.7 (40.6–38.8) Mya during the Eocene  and China, including Taiwan27. Even though molecular data have supported monophyly of Micryletta, the phylogenetic position of this clade has remained confusing37. In our phylogenetic analyses  split from Dyscophinae (Madagascar), an event that coincides with the separation between Madagascar and Indian subcontinent, subsequent to which the latter moved northwards , topographical changes (connection vs. isolation) and climatic fluctuations (favorable vs. unfavorable) that resulted after India’s collision47. These massive changes had important consequences to the movement of amphibians due to their limited dispersal abilities48.In general, the dispersal route that formed following the collision of India with Asia has often been considered instrumental in faunal exchange between these landmasses51. Our discovery of a new microhylid genus from the Western Ghats with closest links in Southeast Asia provides evidence for at least one or more dispersal events between India and Southeast Asia  and the MRCA of two large radiations of Microhyla seem to have utilized Early Eocene land bridges (~45.1 Mya) to colonize Southeast Asia through direct land connection with Sundaic regions such as Sumatra (believed to have been a single landmass). Whereas, MRCAs of microhyline radiations such as Uperodon +  (~40.8 Mya) and Micryletta + Mysticellus (~39.7 Mya) possibly dispersed through a second land bridge with Myanmar–Malay Peninsula later during the Eocene53  forming more prolonged connections . The subsequent period starting around Oligocene is largely represented by diversification within the genus-level endemic radiations , could have then utilized the newly created dispersal opportunities to move into Asia. At the same time, Southeast Asian taxa in groups such as Microhyla, Micryletta and Kaloula probably also recolonized parts of Northeast India through these connections  prolonged isolation of Indian Peninsula by the Deccan traps (since the K/Pg boundary) along with intermitted periods of isolation during Eocene–Oligocene, and (3) limited Miocene exchange between northern Indian regions  and Asia due to complex geological and paleoclimatological events associated with Indo-Asia collision—explains early colonization of certain faunal groups from India to Southeast Asia, as well as the observed patterns of regional endemism and widespread distributions. Future research would benefit from wider taxon sampling across faunal groups with Indo-Southeast Asian affinities, and a better understanding of the exact timings and positions of land bridges, which remain debatable till date.Phylogenetic evidence from the study of Microhylinae is suggestive of multiple dispersal events between India and Asia through postulated land bridges right from Eocene up to Miocene. While the more widely discussed single Miocene exchange after India’s final accretion with Asia explains the affinities between Indian and Southeast Asian faunal elements, it does not provide a convincing justification for early genus-level divergences and the parallel occurrence of endemism as well as widespread distribution patterns observed in Microhylinae. Hence, a trichotomy of (1) Eocene land bridges between India and Southeast Asia prior to the final Indo-Asia accretion, also termed as the “Eocene exchange hypothesis”Field surveys were conducted during the monsoon season in the months of June and July (2015). Sampled individuals were photographed and euthanized in aqueous solution of Tricaine methanesulfonate (MS-222). Adults were fixed in 4% formalin and preserved in 70% alcohol. Tadpoles were preserved in 10% neutral-buffered formalin. Prior to preservation, tissue samples from the tadpole (tail muscle) and adults (thigh muscle) were taken in absolute alcohol for molecular studies and stored at −20 degree in Systematics Lab, Department of Environmental Studies, University of Delhi (SDBDU). Male advertisement calls were recorded using a Marantz PMD620 solid-state digital recorder  with a handheld Sennheiser ME 66 unidirectional microphone. Air temperature (dry bulb and wet bulb) at the calling site was recorded to the nearest 0.1 °C. Type specimens are deposited in Zoological Survey of India–Western Ghats Regional Centre (ZSI-WGRC), Kozhikode and referred specimens are available in the SDBDU collection. The study was conducted with permission and following the guidelines by the concerned authorities in the State Forest Department, Government of Kerala (Permit No. WL10-25421/2014).55 and cytochrome oxidase I 56, and four nuclear (nu) genes—brain-derived neurotrophic factor 36, histone H3 57, seven in absentia homolog 1 58 and tyrosinase 15. Sequencing was performed on both strands using the BigDye terminator cycle sequencing kit on ABI 3730 automated DNA sequencer (Applied Biosystems). Nucleotide sequences were checked and assembled in ChromasPro v1.34 (Technelysium Pty Ltd.), and deposited in the Genbank under accession numbers MK285340–MK285351.Total genomic DNA was extracted from adult and tadpole tissue samples using Qiagen DNeasy tissue kit, following the manufacturer’s protocols. Based on availability of GenBank data, the following gene fragments were PCR-amplified using previously published primers: two mitochondrial (mt) genes—ribosomal subunit 16 S rRNA 59. For non-coding DNA, ambiguous portions were identified manually and excluded from the analyses. A combined mitochondrial and nuclear DNA dataset of 3,088 basepairs was assembled and phylogenetic estimations were made based on Maximum Likelihood (ML) and Bayesian analyses. The data matrix was partitioned by genes and appropriate likelihood models were estimated independently using Modeltest 3.460 that yielded GTR + G + I as the best-fitting model for each gene. We used RAxML 7.3.061 as implemented in raxmlGUI 1.162 to perform ML searches with GTRGAMMA model recommended in RAxML61. The rapid bootstrap algorithm63 was executed with 1000 replicates and a thorough ML search based on 200 independent runs (20 percent of bootstrap replicates). Bootstrap support (BS) was computed on the resulting majority-rule consensus tree. Bayesian analysis was performed in MrBayes 3.1.264 using GTR + G + I model across all gene partitions. Two parallel runs of four Markov Chain Monte Carlo (MCMC) chains were executed for 10 million generations with sampling at every 1000th generation. Convergence of the parallel runs was determined by split frequency of <0.01 standard deviations and potential scale reduction factors of ∼1.0. Stationarity of the likelihood scores and effective sample sizes (ESS) for all parameters were checked in Tracer v1.665. Trees were summarized with a burn-in value of 10 percent and Bayesian Posterior Probabilities (BPP) were used to estimate clade credibility. Additionally, ML trees were generated for each nuclear and mitochondrial gene in order to independently assess distinctness of the new lineage. Uncorrected intra- and interspecific pairwise genetic distances were computed for the 16S mitochondrial gene sequences using PAUP* 4.0b1066. Pairwise identity was graphically visualized using the Sequence Demarcation Tool v1.267.DNA sequences for 43 taxa representing all the recognized genera in subfamily Microhylinae and five outgroup taxa (Asterophryinae and Dyscophinae) were retrieved from the GenBank  the split between Dyscophinae and Microhylinae representing the most recent common ancestor (MRCA) of Microhylinae was calibrated at a mean age of 68 Mya and standard deviation of 3.5 Mya, (2) the MRCA of Kaloula and Microhyla was calibrated at a mean age of 62 Mya and standard deviation of 3.5 Mya, and (3) the MRCA of Glyphoglossus and Microhyla was calibrated at a mean age of 48 Mya and standard deviation of 2.5 Mya. Four independent runs with MCMC chain length of 50 million each were executed in BEAST v2.4.468 and trees were sampled at every 500th generation. Convergence of the runs was assessed in Tracer v1.665 and the initial 10 percent trees were discarded as burn-in. The results were combined in LogCombiner68 and ESS values of over 200 were largely achieved for all parameters, except posterior (ESS = 126). In order to avoid overparameterization, an additional analysis of 100 million generations with 10 percent burn-in was performed using only the HKY model for each locus, which yielded ESS values over 1000 for all parameters.For estimation of divergence times in the subfamily Microhylinae, an uncorrelated lognormal relaxed-clock analysis was performed in the software package BEAST v2.4.4et al.72; for abbreviations see electronic supplementary material. All measurements provided in the taxonomy section are in millimeters. Webbing formulae and the degree of webbing is described following Biju et al.73. Measurements and photographs were taken for the right side of the specimens, except when a character was damaged, in which case they were taken for the left side.Sex and maturity of specimens were determined either by the presence of secondary sexual characters  or by examining the gonads through a small ventral incision. Only adult specimens were used for morphometric studies. Measurements were taken to the nearest 0.1 mm by using a digital slide-caliper or a binocular microscope with a micrometer ocular. Measurements and associated terminologies follow Biju 74. Descriptions are based on stage 34 tadpoles (N = 3). Measurements were taken to the nearest 0.01 mm using a stereomicroscope with a micrometer ocular or a digital slide-caliper. Measurements and larval terminologies were adapted from McDiarmid and Altig75. Oral morphology and margins of papillae were visualized using blue ink.The tadpoles were morphologically examined and staged according to Gosner76 and Blotto et al.77.Palmar musculature was examined under stereomicroscope after dissection and removal of superficial layers . Dissection procedures, character sampling, terminologies and abbreviations follow Burton78. Dominant frequency was measured after averaging spectrum over the entire call. Terminologies and graphical representation of the analysed call properties follow Bee et al.79.Temporal and spectral properties were measured for a single call using Raven Pro 1.4http://zoobank.org/’. The LSID for this publication is urn:lsid:zoobank.org:pub:522D72B4-87B3-4A9B-B533-EA794695755D; LSID for Mysticellus gen. nov. is urn:lsid:zoobank.org:act:6145F26C-E140-4BBF-9322-550047926EA2; LSID for Mysticellus franki sp. nov. is urn:lsid:zoobank.org:act:B1FBD56B-B6B3-412A-8988-E527A082C4C5.This article is published in an electronic journal with an ISSN (2045–2322), and has been archived in PubMed Central. Taxonomic nomenclature published in this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence is available under ICZN. This publication and the nomenclatural acts it contains have been registered in ZooBank, the proposed online registration system for the ICZN. The ZooBank LSID (Life Science Identifier) for this publication can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ‘Electronic Supplementary Material


Bordetella pertussis detection and diagnosis in patients with severe respiratory illness (SRI), influenza-like illness (ILI), and asymptomatic controls.We assessed the utility of a multi-target, real-time PCR assay for 481, pIS1001, hIS1001 and ptxS1 was performed on nasopharyngeal specimens  and induced sputum (SRI) collected from June 2012 to May 2016 through respiratory illness surveillance. Using PCR cycle threshold (Ct) value cut-offs, IS481 positive cases were classified as confirmed (Ct < 35) or possible (Ct 35–39) pertussis disease.Real-time PCR detection of IS481 positive of which 62% (90/146) were classified as confirmed. The attributable fraction (AF) was 92.2%  and 90.5%  amongst SRI and ILI PCR-confirmed pertussis cases, respectively. Amongst possible pertussis cases, AF was 36.9%  and 67.5%  in the SRI and ILI groups, respectively.Among 12,922 samples, 146 (1.1%) were IS481 positive specimens could be considered as B. pertussis infection, and potentially pertussis disease with supportive clinical information.All ISThe online version of this article (10.1186/s12879-019-3869-7) contains supplementary material, which is available to authorized users. Bordetella pertussis ) to severe (SRI) (including only patients that tested positive on NP specimens) or mild (ILI) illness using unconditional logistic regression by comparing the B. pertussis detection rate among SRI or ILI cases to those of controls. All models were adjusted for HIV status and age. The AF was estimated from the odds ratio (OR) obtained from the regression models using the following equation: AF = (OR-1)/(OR) × 100 as previously described  compared to NP specimens  (p = 0.002).Among SRI cases with both specimen types collected, the detection rate was 0.2% (6/2678) for NP specimens (using NP specimens only) and 1.2% (31/2678) for induced sputum (using induced sputum only)  of NPs were culture positive for B. pertussis. All isolates were from IS481-positive samples with Ct values < 35.Of 3104 induced sputum samples, 3 (0.1%) were culture positive for B. pertussis individuals (patients positive on either NP or induced sputum) were identified in all three surveillance groups, of which 62% (90/146) were categorised as confirmed .Overall, 146 PCR-positive t value was 23 ± 7 for confirmed cases and 37 ± 2 for possible cases  samples from confirmed and 6/14 (43%) samples from possible cases were confirmed through BLAST analysis. IS481 amplicons from one confirmed (Ct = 34) and eight possible (Ct range 35–39) cases could not be confirmed by sequencing although PCR amplicons of the expected size were confirmed using agarose gel electrophoresis.The mean CB. pertussis detection to respiratory disease was 72.4% ; with 92.2%  and 36.9%  for confirmed and possible cases, respectively. For ILI cases compared to controls, the overall AF was 77.3% ; with 90.5%  and 67.5%  for confirmed and possible cases, respectively.For SRI patients compared to controls, the overall AF of B. pertussis, of which 60% (56/94) were categorised as confirmed B. pertussis cases. Possible cases were more likely to occur among patients aged 45–64 years than among patients aged < 1 year   were PCR positive for B. pertussis, of which 68% (32/47) were confirmed cases. There was no difference in the demographic and clinical characteristics between confirmed and possible cases in this group  were PCR positive for ich 68% 3/47 were B. pertussis  compared to previous months. The increase occurred in samples collected at one geographical site (KTHC and Jouberton Clinic). An evaluation of all laboratory systems and testing of 32 environmental samples collected from the affected hospital and clinic excluded facility and laboratory contamination, indicating a true increase in B. pertussis infection. In addition, 17 PCR-positive patients [9/17 (53%) SRI and 8/17 (47%) ILI], enrolled during the period when the increase in B. pertussis detection was observed, were retrospectively interviewed  and mild (ILI) cases. However, for PCR-possible cases (both SRI and ILI) there was no significant association with B. pertussis disease.In the absence of pertussis-specific surveillance in South Africa, we evaluated the utility of real-time PCR detection of ividuals –29; howet values (> 35), low bacterial loads may represent asymptomatic colonisation, or residual DNA post disease, or pertussis disease [t value cut-offs may still result in the misdiagnosis of some true pertussis cases given that adults and adolescents have been shown to harbor lower bacterial loads and may present with milder disease or atypical symptoms [B. pertussis PCR-positive cases had low bacterial loads (Ct > 35) and were defined as possible pertussis cases. This is potentially due to the case definition that was not specific for pertussis and the enrollment of individuals of all ages. We also showed that in hospitalised patients, pertussis-possible individuals were more likely to be adults (45–64 years of age) than infants (< 1 year of age) compared with pertussis-confirmed individuals.In individuals with high C disease , 30. Thesymptoms , 30, 31.B. pertussis (confirmed and possible cases) from 2014 were retrospectively interviewed. The majority of confirmed cases reported clinical symptoms consistent with pertussis; however, 50% (4/8) of possible cases also had clinical pertussis. Since this was only a small group of individuals, we are prospectively conducting active case follow-up to understand the association between PCR Ct values and pertussis symptoms. In Canada, Public Health Ontario Laboratories determined the association between PCR Ct with pertussis severity and symptoms, and showed that the proportion of patients with pertussis symptoms did not differ between individuals with low (Ct < 36) and high IS481 Ct values [In our study, a subset of 17 patients that were PCR positive for t values .481 is a multi-copy target with up to 250 copies per B. pertussis genome which increases assay sensitivity, but also increases the risk of false positives due to environmental or laboratory contamination [t value cut-offs have been recommended when interpreting PCR data and these vary between studies. In a Tunisian study, B. pertussis cases were defined as PCR positive for IS481 and ptxS1 with a Ct < 45, or as Bordetella spp. if they were positive for IS481 only with a Ct < 45 [B. pertussis at an average Ct of 33.3 [481 Ct cut-off (< 35) [t values could still indicate positivity and, in a setting where testing procedures are standardised, should not be ignored. Also, Ct values in clinical specimens may vary due to specimen composition or may be affected during storage or transit, and thus may not directly emulate experimental sensitivity in a controlled laboratory environment.ISmination , 18, 32. Ct < 45 . The ass of 33.3 . This, tf (< 35) . However481 gene from a subset of PCR-positive samples, ruling out non-specific amplification, primer dimer formation and probe degradation, however, one B. pertussis confirmed, and eight possible samples could not be confirmed by sequencing likely due to insufficient DNA concentration for Sanger sequencing. Although only 7% of possible B. pertussis cases tested positive for the ptxS1 gene due to the lower sensitivity of the assay compared to IS481, we excluded B. holmesii from all IS481 PCR-positive samples, suggesting that these were positive for B. pertussis.Sequencing confirmed the presence of the ISB. pertussis [B. pertussis detection has been suggested by WHO [B. pertussis 5-fold among hospitalised SRI patients. This is similar to another South African study where the B. pertussis detection rate was 3.7% when testing NP specimens and increased to 7% when induced sputum specimens were additionally included [Earlier studies have indicated that NP is the preferred specimen type for laboratory detection of ertussis , 33, 34.included .B. pertussis by PCR in 1.1% of patients with either mild or severe respiratory illness, which correlates with another South African study that enrolled an infant cohort from 2012 to 2014 as part of the Drakenstein Child Health Study in the Western Cape and found an overall pertussis prevalence of 2% among infants presenting with pneumonia [The World Health Organization has recommended that all countries perform pertussis surveillance and have suggested that for resource-limited settings it can be linked to surveillance for other respiratory illnesses. Due to the lack of pertussis data in South Africa, our aim was to establish pertussis surveillance by linking it to an already established syndromic SRI and ILI surveillance platform. While not pertussis-specific surveillance, this enabled us to obtain pertussis data for the country at a low cost by leveraging systems and staff already in place. An additional benefit of this surveillance is that children and adults with mild and severe respiratory illness that do not necessarily present with typical pertussis symptoms were included, adding to our understanding of pertussis disease in individuals that are not clinically suspected to have pertussis. However, use of syndromic surveillance platforms for pertussis surveillance also has several drawbacks, which need to be acknowledged. As the case definition for ILI cases and SRI adult cases included fever, it is likely that some pertussis cases were missed, and the data likely represents an underestimation of disease prevalence. In addition, the interpretation and clinical relevance of PCR-positive individuals that do not present with typical pertussis symptoms is not well understood. However, despite these limitations, we detected neumonia .t value could not be determined.The study has limitations that need to be considered. The surveillance case definition was not specific for pertussis therefore the time to swabbing following disease onset could not be determined. Overall, there was a small number of pertussis cases, which limited our statistical power for analyses, and as a result, differences between the characteristics of confirmed and possible cases which may exist, may not have been detected. Pertussis vaccination history was not available for adults, and for children < 5 years of age the collected data were poor, so the impact of vaccination on pertussis disease and C481-positive specimens with Ct values < 40 should be considered indicative of B. pertussis infection. Low IS481 Ct values (< 35) are more likely to represent true cases of pertussis disease, whereas specimens with high Ct values may represent B. pertussis carriage , residual B. pertussis DNA following disease, or active disease especially in older individuals who may present atypically. As such, in a respiratory surveillance setting and for public health reporting, low bacterial loads may not reflect true cases of disease. However, for individual patient management, possible cases should be interpreted in the context of other clinical and epidemiological factors including patient symptoms, age, immune status and epidemiological links with laboratory-confirmed pertussis cases. B. pertussis carriage is not well understood, and further research is required to better understand the significance of PCR detection of B. pertussis in asymptomatic individuals and individuals lacking typical pertussis symptoms.In a controlled setting with standardised procedures and when contamination can conclusively be excluded, we suggest that ISIn conclusion, based on the results presented in this manuscript, we believe that in the absence of a pertussis-specific surveillance, leveraging of an already existing respiratory illness surveillance system as recommended by the WHO will allow countries to monitor the trend of infection over time, detect outbreaks and allow further research into cases that present with atypical pertussis symptoms.Additional file 1:N = 38) and possible (N = 22) pertussis cases  in hospitalized patients with severe respiratory illness, South Africa, June 2012 – May 2016 (N = 60). (DOCX 18 kb)Comparison of confirmed  pertussis cases  in patients with influenza-like illness, South Africa, June 2012 – May 2016 (N = 47). (DOCX 17 kb)Comparison of confirmed (N = 32) and probable (


TREM2, PLCG2, and ABI3 were recently associated with the susceptibility to Alzheimer’s disease (AD) in Caucasians. Frequencies and AD-associated effects of variants differ across ethnicities. To start filling the gap on AD genetics in South America and assess the impact of these variants across ethnicity, we studied these variants in Argentinian population in association with ancestry. TREM2 (rs143332484 and rs75932628), PLCG2 (rs72824905), and ABI3 (rs616338) were genotyped in 419 AD cases and 486 controls. Meta-analysis with European population was performed. Ancestry was estimated from genome-wide genotyping results. All variants show similar frequencies and odds ratios to those previously reported. Their association with AD reach statistical significance by meta-analysis. Although the Argentinian population is an admixture, variant carriers presented mainly Caucasian ancestry. Rare coding variants in TREM2, PLCG2, and ABI3 also modulate susceptibility to AD in populations from Argentina, and they may have a European heritage.Rare coding variants in  Over the last decade, more than 20 loci containing common genetic variants  >5%) have been associated with AD2. The advent of new genetic sequencing technologies has enabled the identification of several rare variants (MAF <1%) with moderate effects on AD susceptibility3. In 2017, the International Genomics of Alzheimer’s Project (IGAP) reported four rare coding variants significantly associated with AD4, all of them being involved in microglial-mediated innate immunity. Two of them are novel nonsynonymous variants: a protective one in PLCG2 (rs72824905) and a risk one in ABI3 (rs616338). The other two were previously reported in the susceptibility gene TREM2 (rs143332484 and rs75932628), and are responsible for p.R62H and p.R47H substitutions, respectively. The findings in TREM2 have been consistently replicated in Caucasian6 and African-American populations7. However, the association of TREM2 p.R47H with AD could not be found in East Asian population, because its frequency is extremely low9. This latter observation suggests ethnic variability fostering investigation of these variants in different ethnic groups. Given the increasing population diversity observed in countries all over the world, understanding population-shared and -specific risk factors of AD will translate into improved and specific prevention and/or treatment for people.Alzheimer’s disease (AD) is the most common form of dementia, and has an estimated genetic component of 60–80%APOE-ε4. In this report, we provide the first evidence for an association between TREM2, PLCG2, and ABI3 rare variants and AD in the Argentinian population.Latin America is a vast territory, with a wide diverse admixture of European, Native American, and African ancestral populations. The genetic architecture of sporadic AD has not been studied in this population beyond Individuals with AD and without cognitive impairment older than 60 years were recruited from outpatient Neurology Departments of the following hospitals: Instituto de Investigaciones Médicas “Alfredo Lanari” and Hospital de Clínicas (Buenos Aires City), Hospital Interzonal General de Agudos “Eva Perón” , Hospital El Cruce “Dr. Néstor Kirchner” (Florencio Varela county), and several assistance centers located across Jujuy province. Only samples from people born in Argentina or in South America were included in the analysis. This study was approved by the ethical committee “Comité de Bioética Fundación Insituto Leloir ” by the approval of protocol CBFIL #22. All participants and/or family members gave their informed consent.10. The diagnosis of AD includes a clinical examination to evaluate functionality and activities of daily living that should be compromised; a complete panel of neurocognitive tests to evaluate memory, attention, language, and executive function, of which one or more should be altered; a computerized tomography and/or magnetic resonance imaging to assess cortical–hippocampal atrophy and vascular events, and a blood test analysis to exclude metabolic or infectious causes of dementia. Individuals were included as controls if neurocognitive and clinical assessments were normal.Diagnosis of AD followed diagnostic criteria from the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s disease and Related Disorders Association (NINCDS-ADRDA)TREM2 (rs143332484 and rs75932628), PLCG2 (rs72824905), and ABI3 (rs616338) variants were genotyped using custom-designed TaqMan assays (Thermo Fisher). Assay accuracy was checked by including positive and negative controls in each experiment. APOE alleles were determined by genotyping rs429358 and rs7412. Association with AD was calculated using Fisher’s exact test with statistical significance of p < 0.05. All variants were in Hardy–Weinberg equilibrium . Power calculations were performed using Genetic Power Calculator for discrete traits (http://zzz.bwh.harvard.edu/gpc/cc2.html). Meta-analysis for the effect of rare variants in association with AD was conducted using beta and standard error with Metafor R-package11. Populations from France (n = 8514), Italy (n = 2306), Spain (n = 3966), and Sweden (n = 2286) from the European Alzheimer's Disease Initiative (EADI)4 were included in the meta-analysis.Genomic DNA was isolated using standard procedures from whole blood or saliva samples. n = 85), Yorubas African , and Native American  ancestral populations were obtained from 1000 Genomes . Argentinian samples (ARG) were subjected to genome-wide genotyping using the Infinium Global Screening Array (GSA) v.1.0+GSA shared custom content (Illumina). Quality controls (QC) were performed as described before12, using PLINK v1.913 and R v3.4.414. After QC, remaining samples (n = 834) have <5% of missing genotypes and passed sex-check and identity-by-state filters. Remaining single nucleotide polymorphisms (SNPs) have >95% call rate, MAF >1%, are in HWE (p > 10−6), and without differences in call rate between cases and controls (p < 1 × 10−5).European , which were specifically selected to estimate ancestry in Latin America4 with AD in an Argentinian population sample (ARG), 905 participants were recruited from different regions of the country. Demographic and clinical information of the 419 AD cases and 486 controls is summarized in Table APOE-ε4 allele on AD susceptibility confirming thereby previous reports (odds ratio (OR) = 3.14, p < 0.0001)17. For APOE-ε2, we observed the expected protective effect, although it did not reach statistical significance . Next, we genotyped the recently described rare variants4, i.e., TREM2 p.R47H (rs75932628) and p.R62H (rs143332484), PLCG2 p.P522R (rs72824905) and ABI3 p.S209F (rs616338). All of them were detected in ARG with MAFs similar to those reported by IGAP 4. Despite larger sample size, statistical power associated with EADI does not allow to reach nominal significant association with AD risk. However, when meta-analyzing both EADI and ARG samples, this gain in power is enough to help in reaching statistical significance for the variants analyzed, in particular the TREM2 p.R47H variant , see Table To evaluate the association of the four rare variants recently reported by IGAPnt Table . Unfortu18. To estimate the ancestry of ARG, we used a panel of 446 SNPs, reported to be precisely balanced to study Latin American populations15. ARG showed to be an admixture of mainly CEU and NAM, and to a lesser extent of AFR , its frequency in AD cases was lower . This lower frequency is in agreement with data previously reported in other Latin American countries21. Interestingly, it is among the lowest worldwide together with that of Mediterranean basin and Native Americans23, which are the main contributors to Argentinian admixture.Here we show information from a case–control study performed in Argentina, being the first study on sporadic AD genetics in South America, beyond 4. Unfortunately, since there are no reports in Amerindians, we could not compare their MAFs. However, TREM2 p.R47H is almost absent in East Asians9, suggesting that it might also be extremely rare in Native Americans. For the rest of the variants, we performed a search in ExaC database (exac.broadinstitute.org), and found that PLCG2 p.P522R, and TREM2 p.R62H were not detected in approximately 4300 East Asian individuals. Unfortunately, results for ABI3 p.S209F are not reliable due to low quality. Notwithstanding, these observations, together with our results showing that the chromosomes containing these rare variants have mainly Caucasian ancestry in the identified carriers, strongly suggest a European origin for these variants. However, the possibility still remains open that the loci containing the studied rare variants might have an ancestry other than Caucasian. To answer this question, additional studies comparing SNPs among ancestral populations are needed to identify AIMs that better explain the ancestry of these loci.It is of note that the rare variants studied here showed MAFs similar to those reported by IGAP in CaucasiansIn conclusion, we report the first genetic data from Argentinian population, which support the contribution of rare coding variants to AD susceptibility. Although this population size is not enough to reach statistical significance for the rare variants studied here, it is a relevant opportunity to start filling the gap on AD genetic architecture in Latin American admixed populations. Our analysis fosters further analysis of these rare variants in other Latin populations to confirm our initial observation. Importantly, expanding research to admixed populations, like this one, will help to identify potential population-specific effects on the genetic structure of AD, in addition to better define conserved relevant pathways involved in the disease.Supplemetal Figures


Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both animals and humans. Here, multilocus sequence typing (MLST) was used to characterize the genetic diversity of Listeria monocytogenes strains isolated from the natural environment and animal clinical cases in Europe. The prevalence of clonal complexes (CCs) obtained was compared according to (i) the origin of isolation – clinical cases vs. natural environment – and (ii) the clinical form of animal listeriosis – rhombencephalitis vs. abortion. To this aim, two datasets were constructed. The clinical dataset consisted of 350 animal clinical isolates originating from France and Slovenia and supplemented with isolates from Switzerland and Great Britain. The natural environment dataset consisted of 253 isolates from the natural environment originating from Slovenia and supplemented with isolates from nine other European countries.For the clinical cases, CC1, CC4-CC217 and CC412 were the most prevalent in rhombencephalitis and CC1, CC37 and CC4-CC217 in abortion. The hypervirulent CC1 and CC4-CC217 prevailed in both datasets. These results indicated that livestock is constantly exposed to hypervirulent CCs. CC1 was significantly associated with a clinical origin, whereas CC9, CC29 and CC14 were associated with the natural environment. CC1 was predominant among rhombencephalitis cases both in cattle and small ruminants, and its prevalence did not differ significantly between these two groups. A novel association of CC37 and CC6 with abortion cases was revealed.L. monocytogenes isolates from the natural environment and animal clinical cases. Moreover, it provides a basis for future studies aiming to determine the underlying mechanisms of phenotypic traits of interest.Here, we show that CC1 and CC4-CC217 are prevalent in isolates of environmental and animal clinical origin, suggesting that ruminants are frequently exposed to hypervirulent CCs. The presence of CC4 in two mastitis cases calls for further attention due to direct threat to the consumer. We showed several associations between CCs and the origin of isolation or clinical form of listeriosis, e.g. CC37 and CC6 with abortion. This study improves our understanding of the population structure of  Listeria monocytogenes is a Gram-positive bacterium that is ubiquitously present in the natural environment , but can also cause life-threatening infections in humans and animals [L. monocytogenes asymptomatically into the environment through feces [L. monocytogenes by animals and may be an additional source of infection in animals [ animals . Among fgh feces . Impropegh feces . The nat animals .In animals, listeriosis can manifest itself in different clinical forms, ranging from a localized infection of the udder (mastitis), the eye  or gastroenteritis to the more severe, invasive forms of listeriosis including septicemia, rhombencephalitis and infection of the gravid uterus, often leading to premature birth or abortion , 5. Of tIn Slovenia, the surveillance system of animal listeriosis cases is passive and relies on reporting the neurological symptoms of the disease. The average reported incidence rate of listerial rhombencephalitis in ruminants during the 2006–2016 period was 5.3 cases per 100,000 small ruminants and 1.0 case per 100,000 cattle, with no significant increase in recent years , 8. ThisL. monocytogenes isolates, including EcoRI ribotyping, serotyping, multilocus genotyping (MLGT), multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome sequencing (WGS) [L. monocytogenes, MLST and WGS-based typing such as core genome MLST (cgMLST) have proved particularly suitable for studies on the population structure and epidemiology of L. monocytogenes [A variety of typing methods have been employed to differentiate ng (WGS) , 11–14. ytogenes –16.L. monocytogenes genotypes with different clinical forms of animal listeriosis, but did not use MLST [Until now, MLST-based population studies have mainly been conducted on isolates from human clinical cases and food , 17–21. use MLST , 23. Twouse MLST , 25 inclL. monocytogenes population structure in animal clinical cases and in the natural environment. For this purpose, two large datasets were constructed. The clinical dataset consisted of strains isolated from animals with different clinical forms of listeriosis – primarily ruminant abortion and rhombencephalitis cases. The second dataset consisted of strains from the natural environment. The rhombencephalitis isolates, the isolates from cases with other pathology and the environmental isolates primarily originated from Slovenia and were obtained within the framework of the passive surveillance. The abortion isolates primarily originated from one French department and were obtained in the framework of a targeted research project. To increase their size, both datasets were supplemented with other recent European isolates from animal pathology and the natural environment. These data were obtained from the literature and publicly available databases. Based on the constructed datasets, the second objective of the present study was to identify statistically significant associations of L. monocytogenes CCs and the clinical forms of listeriosis. The third objective of the study was to identify the most prevalent CCs in the natural environment to which animals are constantly exposed, with emphasis on the isolates from animal feed.The first objective of this study was to improve our knowledge on n = 350), having frequency of n > 10 and comprising 66.9% of the total database: CC1 , CC4-CC217 , CC37 , CC6  and CC412   in both datasets was compared. CC1 had the highest clinical frequency/environmental frequency ratio of all clones  of CC4 and CC217 could not be reliably separated because they clustered together according to the 85% similarity rule; therefore, a merged clone CC4-CC217 was constructed for MLST prediction. Considering the results of in silico MLST, the merged CC4-CC217 cluster (n = 45) was mainly composed of CC4 isolates (43/45) and a few CC217 isolates (2/45); therefore, the majority of the merged CC4-CC217 isolates most likely belong to CC4.The following CCs were the most prevalent clones in the complete animal clinical dataset n = 50, havinn = 194), CC1 , CC4-CC217  and CC412  were the most prevalent CCs, comprising 74.2% of all rhombencephalitis isolates . Among the isolates obtained from abortion cases (n = 128), CC1 , CC37  and CC4-CC217  prevailed, comprising 46.9% of all abortion isolates  due to their low frequency and high genetic diversity , the three most prevalent CCs were CC1 , CC37  and CC4-CC217  . Because the FR clinical dataset consisted mostly of abortion isolates , the prevalence of CCs among the FR abortion isolates was compared with their prevalence among all abortion isolates in the complete clinical dataset. The prevalence of all CCs with the frequency of n > 10  did not differ significantly between the compared groups (p ≥ 0.8406). The FR isolates belonged to lineage I  and lineage II ; lineage III and lineage IV isolates were not detected.In the French (FR) subset of the animal clinical dataset (n = 55), which was mostly composed of rhombencephalitis isolates (45/55), the three most prevalent clones were CC1 (24/55), CC4-CC217 (7/55) and ST184 (6/55)  did not differ significantly from their prevalence in the SI subset (p ≥ 0.0935). CC1 was significantly over-represented among the SI animal clinical isolates in comparison with the SI environmental isolates (p = 0.0083). CC1 also prevailed among the SI rhombencephalitis isolates (23/45). Because the SI dataset consisted mostly of rhombencephalitis isolates (45/55), the prevalence of CCs among the SI rhombencephalitis isolates was compared with their prevalence among all rhombencephalitis isolates in the complete clinical dataset. The prevalence of both CCs with a frequency of n > 10 (CC1 and CC4-CC217) did not differ significantly between the compared groups (p ≥ 0.5099). All but one isolate (54/55) had a corresponding lineage defined in the MLST scheme. The SI isolates belonged to lineage I (35/54) and lineage II (19/54); lineage III and lineage IV isolates were not detected.In the Slovenian (SI) subset of the animal clinical dataset , having frequency of n > 10 and comprising 51.8% of the complete database: CC1 , CC4-CC217 , CC37 , CC6 , CC14 , CC9  and CC29  , the most prevalent CC was CC1 (7/40), followed by CC5, CC6, CC29 and CC31   did not differ significantly from their prevalence in the SI subset of the environmental dataset (p ≥ 0.1406). The SI isolates belonged to lineage I (21/40) and lineage II (19/40); lineage III and lineage IV isolates were not detected.In the SI subset of the environmental dataset (p < 0.0001) and lineage II clones with the natural environment (p < 0.0001). CC1 was significantly associated with a clinical origin (p < 0.0001) , CC29 (p = 0.0013) and CC14 (p = 0.0185) were significantly associated with the natural environment . Therefore, for the purpose of statistical comparison, the clinical isolates were not stratified according to the animal species but rather according to the clinical form of the disease. Among the isolates of clinical origin, CC1 was significantly (p < 0.0001) associated with rhombencephalitis, whereas CC37 (p < 0.0001) and CC6 (p < 0.0001) were significantly associated with abortion  was identified between small ruminants and cattle for the two major clinical forms of listeriosis (n = 603), the MLST data showed high genetic diversity (45 different CCs and seven different singleton STs) and non-homogeneous distribution of CCs , 35 different CCs  and three different singleton STs were identified. The Simpson’s index of diversity was 0.820; 95% confidence interval (CI): 0.783–0.856. In the natural environment dataset (n = 253), 36 different CCs  and six different singleton STs were identified. The Simpson’s index of diversity was 0.950 (95% CI: 0.941–0.960) and was significantly higher than in the clinical dataset. The rarefaction curves of CCs showed that at the sampling effort of 253 isolates, the observed taxonomic richness is higher in the natural environment dataset, although the 95% CIs overlap. However, a greater sampling effort would be needed in the both analyzed datasets to better describe their diversity because neither of the curves approached the asymptote  were typed by MLST. Then, the data obtained was supplemented with the MLST data from 398 European isolates obtained from five publications [L. monocytogenes dataset were constructed for the MLST-based population analyses, which until now have mainly focused on isolates of human clinical and food origin. The present study is the first report available on diversity of L. monocytogenes strains isolated from ruminants in France and Slovenia.In the present study, MLST was used to describe the genetic diversity of a panel of 603 ications , 24–27 aThis study showed that distribution of CCs in ruminants, as in humans, is disparate between clinical cases and feed/natural environment (ruminants) or food (humans) Figs.  and 2. IL. monocytogenes isolates typed in the present study were regionally limited to the French department Ille-et-Vilaine (35) and Slovenia and different clinical forms of listeriosis were prevalent in SI and FR subsets, a similar distribution of major CCs was also observed in both complete datasets  consisting of 11 European countries. Further studies are currently undertaken in Europe within the framework of EU One Health European Joint Programme ListAdapt to confirm the data presented herein on a larger European-wide scale [In the present study, the previously described hypervirulent clones CC1, CC4 and CC6 were among the four most prevalent CCs in both analyzed datasets, which is in accordance with the well-established association between feed or immediate ruminant environment and listeriosis in ruminants , 4. Althde scale .L. monocytogenes in bulk tank milk, in which CC29 was frequently detected in a dairy farm environment [In this study, CC1 was over-represented among ruminant clinical cases and significantly associated with rhombencephalitis, whereas CC9, CC29 and CC14 were significantly associated with the natural environment. A similar association of CC1 with a clinical origin was reported in humans . Conversironment .n = 24) belonged to five different STs . Previous studies showed CC14 isolates with indistinguishable PFGE profile belong to different STs, which are genetically relatively distant [L. monocytogenes groups, further studies should focus on ST rather than CC classification.Although a significant association was observed between CC14 and the natural environment, the analyzed CC14 isolates  listerial mastitis presents a direct threat to consumers or suckling animals. The presence of the hypervirulent clones CC1, CC4 and CC6 in milk and milk products has been previously reported [The observed high prevalence of previously reported hypervirulent clones CC4 and CC6 among the animal clinical isolates was similar to that observed in humans , 29, 33.reported , 31.Due to the low number of isolates obtained from animals with rare forms of listeriosis, the prevalence of different CCs in these cases could not be reliably estimated in the present study. However, the observed high genetic diversity of CCs suggests that other risk factors such as feedstuff quality and storage, farm management practices, animal health and hygiene are the major contributors to the disease in these cases, rather than the virulence of the pathogen .L. monocytogenes isolates. Its distribution did not differ significantly between the animal clinical  and natural environment  datasets. This CC was also not encountered among the SI and FR clinical and environmental isolates typed in the present study. Similarly, low prevalence of CC2 has been previously reported in isolates from the farm environment and animal listeriosis cases [CC2, another previously reported hypervirulent CC, was poorly represented in the analyzed European-wide datasets of onment 8/3; 3.2% dis cases , 21 and We identified CC6 as an abortion-associated CC. Interestingly, this CC has not been reported as abortion-associated in humans , which sIn correspondence to the previous findings , the linIn the present study, CC1 was the most prevalent CC among the isolates from animal feed , followed by ST36 and CC4-CC217. This finding, in addition to the generally high prevalence of CC1 and CC4-CC217 in the natural environment dataset, suggests that animals are mostly exposed to the hypervirulent CCs such as CC1 and CC4.L. monocytogenes [L. monocytogenes in the farm environment can help to implement improved control measures to limit the disease burden in animals and the introduction of L. monocytogenes into the food chain. In the present study, CC1, CC4-CC217, CC37 and CC6 were the most prevalent CCs in the natural environment dataset. These results underline the risk of ruminant contamination from the natural/farm environment.Several studies have shown that farm-level management and hygiene practices are associated with the prevalence of ytogenes , 38–40. L. monocytogenes between continents as well as different monitoring and sampling criteria. Further studies of prevalence of L. monocytogenes in Europe are needed. In general, the estimated prevalence of CCs should be interpreted with caution in small datasets, in isolates from a limited geographical area or datasets with epidemiological duplicates. In the present study, the FR and SI subsets were de-duplicated.In the present study and in the previously published European study , CC7 andL. monocytogenes CCs has already been reported [Interestingly, 6/55 animal clinical isolates from Slovenia belonged to singleton ST184 (lineage II). This CC was not present among the environmental SI isolates and the remaining isolates from the analyzed datasets and was absent from the Institut Pasteur MLST isolates database at the time of writing. Furthermore, a single ST184 human clinical isolate from USA (NCBI BioSample accession number SAMN03277635) was identified in the NCBI Pathogen Detection system. These findings suggest ST184 is a rare clone with a higher prevalence in Slovenia when compared with other countries, which may be adapted to animal hosts and may have a propensity to cause disease; however, further large-scale studies are needed to confirm this finding. Such regional heterogeneity in the prevalence of certain reported –44.L. monocytogenes CCs across different origins of isolation  and across different clinical forms of listeriosis . Due to the high genetic diversity and ubiquitous nature of L. monocytogenes, a large number of isolates is needed to accurately describe its diversity in different environments. To the best of our knowledge, this is the largest study on the prevalence of L. monocytogenes CCs in different clinical forms of listeriosis in animals and in the natural environment in Europe. Furthermore, such large-scale prevalence studies represent a basis for the future studies addressing the virulence and tropism of different L. monocytogenes CCs, i.e. studies on the discovery of genetic traits that are involved in adaptation to a specific ecological niche  and tissue tropism .In conclusion, we show that CC1 and CC4, previously described as the hypervirulent CCs, were the most prevalent CCs among the isolates of both analyzed origins . CC1 was rhombencephalitis-associated, whereas CC6 and CC37 were abortion-associated. CC9, CC14 and CC29 were associated with the natural environment. Our results suggest that animals are mostly exposed to hypervirulent CCs (CC1 and CC4) and that the CC distribution in the natural environment differs from the distribution observed in food. We confirmed the non-homogeneous distribution of L. monocytogenes isolates of different origin  were constructed. Clinical isolates were defined here as isolates originating from confirmed cases of animal listeriosis. The definitions of clinical forms of listeriosis were harmonized with those previously described [L. monocytogenes from the placenta and/or the fetus. Gastroenteritis cases were defined as cases with diarrhea and neutrophilic gastroenteritis, in which L. monocytogenes was isolated from the gastrointestinal content. Mastitis cases were defined as isolation of L. monocytogenes from the udder quarter. Ocular listeriosis cases were defined as isolation of L. monocytogenes from the eye and the presence of ocular inflammation. Rhombencephalitis cases were defined as isolation of L. monocytogenes from the brainstem of animals with neurological signs and presence of pathognomonic changes in the brain tissue, confirmed by histopathology. Septicemia cases were defined as isolation of L. monocytogenes from visceral organs. The natural environment dataset included isolates that originated from the natural environment , excluding isolates from food, food-associated units or confirmed animal listeriosis cases. Both datasets included isolates that had not been pre-selected according to their typing data and neither of the panels included epidemiological duplicates; these were defined as isolates sharing indistinguishable PFGE profiles, originating from the same animal outbreak or farm environment from the same sampling. To increase the number of isolates in both datasets, a total of 205 French (FR) and Slovenian (SI) isolates originating from the period 2008–2018 were supplemented with additional isolates and corresponding metadata originating from scientific publications and public web databases – Listeria Pasteur MLST [In the present study, two large datasets of escribed , 45, 46.eur MLST  and NCBIeur MLST . To ensueur MLST . The comn = 21), bovine (n = 19), caprine (n = 14) and cervine (n = 1) cases. The FR subset of animal clinical isolates consisted of 110 isolates obtained from a prophylactic study including the systematic investigation of causes of abortion in ruminants from 107 farms. This study was conducted by private veterinary laboratories during the 2010–2015 period in the French department of Ille-et-Vilaine (35) within the framework of the OSCAR program [n = 95), caprine (n = 5), ovine (n = 4), cervine (n = 1) and leporine (n = 1) cases. This dataset was supplemented with 185 isolates and their associated metadata  from Switzerland and Great Britain [In total, the animal clinical dataset consisted of 350 isolates originating from cases with a well-defined clinical form of listeriosis, defined above. The isolates were classified according to their associated clinical form: abortion, gastroenteritis, ocular listeriosis, rhombencephalitis or septicemia. The SI subset comprised 55 clinical isolates that were obtained during the 2008–2018 period as part of national disease surveillance activities, which included brain samples from ruminants with clinical neurological signs, or during routine monitoring of raw milk. The SI isolates originated from ovine  from Austria, the Czech Republic, Finland, Germany, Great Britain, Greece, Italy, Portugal and Switzerland [In total, the natural environment dataset consisted of 253 isolates. It comprised 40 SI isolates originating from the natural environment that were obtained as part of national research projects that investigated the prevalence of tzerland , 25–27.Lm [L. monocytogenes was digested with ApaI and AscI restriction endonucleases and the obtained fragments were separated using the CHEF-DR II system  according to the recommended electrophoretic protocol. PFGE profiles were analyzed using the BioNumerics v7.6.2 software .A total of 95 isolates  from the SI subset and 110 animal clinical isolates from the FR subset were PFGE-typed according to the method recommended by the EURL Lm . BrieflyListeria MLST scheme [Among the 205 isolates from the FR and SI subsets, 78 isolates (26 SI and 52 FR isolates) underwent WGS. Of these, eight isolates were sequenced on an Ion Torrent PGM System , whereas 70 isolates were sequenced on an Illumina NextSeq500 System (USA) due to its higher throughput. Highly concordant results for both technologies have been previously shown with regard to bacterial typing . WGS datT scheme . The curAscI-ApaI profiles) and CC. The method uses the high concordance between PFGE clusters and corresponding CCs . Briefly, a reference panel of 396 isolates typed with PFGE and MLST was used and imported into BioNumerics as a bundle [AscI-ApaI dendrogram of the 396 reference isolates and all the analyzed isolates was constructed using the following parameters: unweighted pair group method with arithmetic average (UPGMA), similarity calculated using the Dice coefficient, tolerance, and optimization set to 1%. The isolates sharing ≥85% similarity with the reference isolates of a known MLST type were considered as belonging to the same CC. When two clusters were associated with the same CC, the clusters were merged. When several isolates from different CCs were part of the same cluster, the CCs were merged (e.g. clone CC4-CC217). When the isolates did not share PFGE clusters with any of the isolates typed by MLST, CC was not assigned [Among the 205 isolates from the FR and SI subset of the constructed datasets, 127 isolates were not typed using WGS. To obtain the CC affiliation of these isolates, their CC was mapped to their PFGE profiles as described previously . The lata bundle , 18. A cp < 0.05. The statistical analysis was confined to the CCs that showed a frequency of n > 10 in any given dataset.Statistical analyses were performed using GraphPad Prism v6.01 . All associations of the CCs or phylogenetic lineages with their corresponding traits  were tested for their significance using Fisher’s exact test. Therefore, association with a given trait did not depend exclusively on CC prevalence per se. A statistical significance was determined at L. monocytogenes STs was constructed using BioNumerics v7.6.2 software . For this purpose, the Institut Pasteur Listeria MLST scheme [https://cran.r-project.org/web/packages/iNEXT/) and ggplot2 v3.0.0 (https://cran.r-project.org/web/packages/ggplot2/) package for R.The minimum spanning tree of T scheme  was impoT scheme  was usedAdditional file 1: Table S1. A complete database of Listeria monocytogenes isolates from the animal clinical cases.Additional file 2: Table S2. A complete database of Listeria monocytogenes isolates from the natural environment.Additional file 3: Figure S1. Percentage of Listeria monocytogenes isolates of each clonal complex (CC) according to the origin of isolation. The percentage of isolates of each CC in the clinical dataset (y axis) was plotted against the percentage of isolates of each CC in the natural environment dataset (x axis). The 11 most common CCs in the combined clinical and environmental dataset are shown, representing 85.3% of all isolates (n = 603); each CC is indicated by a circle whose size reflects the number of isolates. Lineage I was significantly associated with a clinical origin (p < 0.0001) and lineage II with the natural environment (p < 0.0001). CCs that were significantly associated with their origin of isolation are shown in bold: CC1 was significantly (p < 0.0001) associated with a clinical origin, whereas CC9 (p < 0.0001), CC29 (p = 0.0013) and CC14 (p = 0.0185) were significantly associated with the natural environment.Additional file 4: Figure S2. Number of Listeria monocytogenes isolates of each clonal complex (CC) in the animal clinical dataset according to the clinical form of listeriosis. The complete animal clinical dataset (n = 350) consisted of the French (FR) subset (n = 110), the Slovenian (SI) subset (n = 55) and the supplementary European dataset (n = 185). Only the most frequent CCs are shown, except for the SI subset.Additional file 5: Figure S3. Number of Listeria monocytogenes isolates of each clonal complex (CC) in the complete natural environment dataset (n = 253) and the Slovenian (SI) subset of the natural environment dataset (n = 40). CC9 (p < 0.0001), CC29 (p = 0.0013) and CC14 (p = 0.0185) were significantly associated with the natural environment. Only the most frequent CCs are shown.