Phrynocephalus to assess how past environmental changes shaped the evolutionary and biogeographic history of these lizards and especially the impact of paleogeography and climatic factors. Phrynocephalus is one of the most diverse and taxonomically confusing lizard genera. As a key element of Palearctic deserts, it serves as a promising model for studies of historical biogeography and formation of arid habitats in Eurasia. We used 51 samples representing 33 of 40 recognized species of Phrynocephalus covering all major areas of the genus. Molecular data included four mtDNA  and four nuDNA protein-coding genes . AU-tests were implemented to test for significant differences between mtDNA- and nuDNA-based topologies. A time-calibrated phylogeny was estimated using a Bayesian relaxed molecular clock with nine fossil calibrations. We reconstructed the ancestral area of origin, biogeographic scenarios, body size, and the evolution of habitat preference. Phylogenetic analyses of nuDNA genes recovered a well-resolved and supported topology. Analyses detected significant discordance with the less-supported mtDNA genealogy. The position of Phrynocephalus mystaceus conflicted greatly between the two datasets. MtDNA introgression due to ancient hybridization best explained this result. Monophyletic Phrynocephalus contained three main clades: (I) oviparous species from south-western and Middle Asia; (II) viviparous species of Qinghai–Tibetan Plateau (QTP); and (III) oviparous species of the Caspian Basin, Middle and Central Asia. Phrynocephalus originated in late Oligocene (26.9 Ma) and modern species diversified during the middle Miocene (14.8–13.5 Ma). The reconstruction of ancestral areas indicated that Phrynocephalus originated in Middle East–southern Middle Asia. Body size miniaturization likely occurred early in the history of Phrynocephalus. The common ancestor of Phrynocephalus probably preferred sandy substrates with the inclusion of clay or gravel. The time of Agaminae radiation and origin of Phrynocephalus in the late Oligocene significantly precedes the landbridge between Afro-Arabia and Eurasia in the Early Miocene. Diversification of Phrynocephalus coincides well with the mid-Miocene climatic transition when a rapid cooling of climate drove progressing aridification and the Paratethys salinity crisis. These factors likely triggered the spreading of desert habitats in Central Eurasia, which Phrynocephalus occupied. The origin of the viviparous Tibetan clade has been associated traditionally with uplifting of the QTP; however, further studies are needed to confirm this. Progressing late Miocene aridification, the decrease of the Paratethys Basin, orogenesis, and Plio–Pleistocene climate oscillations likely promoted further diversification within Phrynocephalus. We discuss Phrynocephalus taxonomy in scope of the new analyses.We hypothesize the phylogenetic relationships of the agamid genus  Historical biogeography aims to understand the drivers of speciation including the roles played by plate tectonics and climatic change . The easCentral Eurasian deserts cradle a rich herpetofauna . In the Phrynocephalus Kaup, 1825, or toad-headed lizards, is one of the most speciose genera in its family. It contains from 28 to over 42 species and spans arid regions from northwestern China to the western side of the Caspian Sea, across the QTP, and Southwest Asia to the Arabian Peninsula Phrynocepeninsula . They exeninsula . Oviparoeninsula . The invonfusion , especiaPhrynocephalus that cover the entire range of the genus. Specifically, we pursue three main objectives: (1) test the hypothesis that the nuDNA and mtDNA trees give compatible estimations of historical relationships; (2) evaluate hypotheses concerning potential climatic and tectonic drivers of speciation by using time-tree ages of each lineage calibrated based on molecular dating and fossils; and (3) reconstruct the ancestral distributions to differentiate among competing scenarios of historical biogeography. Our work offers the most complete taxon sampling to date by including up to 70% of the diversity of the genus. It helps to resolve longstanding phylogenetic and biogeographic issues of Central Eurasian biogeography and provides insights into the biogeographic consequences of Cenozoic aridization.Herein, we explore a number of unresolved questions by using both mitochondrial and nuclear DNA markers based for 36 species of Phrynocephalus from the collection of Zoological Museum of Moscow University (ZMMU). The primary outgroup included six other Agaminae species from the genera Agama Daudin, 1802, Paralaudakia Baig, Wagner, Ananjeva & Böhme, 2012, Stellagama Baig, Wagner, Ananjeva & Böhme, 2012, and Trapelus Cuvier, 1817 , 1,053 bp of ND2 (NADH-dehydrogenase subunit II), 705 bp of ND4 (NADH-dehydrogenase subunit IV), and 297 bp of Cytb (cytochrome b). The total length of the concatenated mtDNA genes was 2,703 bp (RAG-1 (recombination activating gene), 675 bp of BDNF (brain derived neurotrophic factor), 1,182 bp of AKAP9 (A-kinase anchor protein 9), and 876 bp of NKTR . The total length of these data was 4,188 bp , 0.2 mM dNTPs, and 1 unit of Тaq-polymerase . Protocols for PCR amplification were provided in the Primer pairs for PCR were taken from the literature mtDNA: , 2011 orPhrynocephalus available in GenBank to the final alignments  as well as more distant Southeast Asian agamids  and Australian taxa . The most distant outgroup taxa also included representatives of Chamaeleonidae, Phrynosauridae, Dactyloidae, Iguanidae, Corytophanidae, Tropiduridae, Polychrotidae, Leiocephalidae, Lacertidae, Opluridae, Crotaphytidae, including representative taxa of the following agamid genera: Stellagama, Trapelus, Paralaudakia, and Agama for both nuclear and mtDNA dataset, and additionally representative taxa of the genera Xenagama Boulenger, 1895, Laudakia, BufonicepsPseudotrapelus Fitzinger, 1843 and Calotes for the mtDNA dataset. Details on taxonomy, GenBank accession numbers and associated references were summarized in We added six sequences of ignments . Thirty-Sequences were first aligned using the Clustal W algorithm  in BioEdPhrynocephalus rossikowi Nikolsky, 1898. To test whether the inclusion of distant outgroups can introduce any bias into results of tree inference, the nuclear and mitochondrial concatenations were put through an additional set of reconstructions omitting all non-agamid and non-agamine taxa, respectively. The optimum partitioning schemes for nuclear and mitochondrial alignments were identified with PartitionFinder (Phylogenetic tree reconstructions were performed with the following data sets: (1) each nuclear gene separately; (2) all nuclear genes concatenated; (3) all nuclear genes combined in a species-tree estimation; (4) a concatenation of four mitochondrial genes as in onFinder  using grPhylogenetic trees were reconstructed under the maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) criteria. The ML trees were generated in Treefinder v.March 2011 . For eacBDNF and uncorrelated lognormal relaxed clock models for the other three loci. No calibration information was utilized; the clock rate for BDNF was set to one. We used the same models and partitioning scheme as in the ML analysis. A Yule prior for the species-tree shape and the piecewise constant population size model were assumed. Default priors were used for all other parameters. Two runs of 500 million generations were conducted in BEAST v1.8.0 . We a priori regarded tree nodes with BSP values 75% or greater and BPP values over 0.95 as sufficiently resolved  along tree branches were modeled as a function of time, thus enabling ML estimation of the ancestral states at cladogenic events. Lagrange found the most likely ancestral areas at a node, the split of the areas in the two descendant lineages, and calculated the probabilities of these most likely areas at each node .Middle Asian sun-watchers encompassing Phrynocephalus raddei . P. rossikowi was omitted in the earlier mtDNA study of P. raddei species group.Southern Middle Asian (Turan) s raddei , PhrynocPhrynocephalus axillaris Blanford, 1875 . The versicolor-group had two sublineages: Phrynocephalus hispidus Bedriaga, 1909 from Mongolian Dzungaria and Phrynocephalus sp. 1 from Gansu; and Phrynocephalus przewalskii Strauch, 1876 + Phrynocephalus frontalis Strauch, 1876 + P. versicolor from central China and Mongolia joined with Phrynocephalus kulagini Bedriaga, 1909 from Tuva Republic (Russia).Phrynocephalus guttatus species complex, widespread in plains of Kazakhstan and northern Caspian region (guttatus-group). Within the guttatus-group, P. guttatus, Phrynocephalus alpherakii Bedriaga, 1905 and Phrynocephalus moltschanovi Nikolsky, 1913 clustered together.analyses . SeveralPhrynocephalus and the levels of nodal support .Maximum likelihood, MP, and BI analyses of the concatenated nuclear DNA dataset resulted in highly congruent trees . Exclusi support  coincide support  and werePhrynocephalus received high support as did several species-groups: Microphrynocephalus  from the matrilineal genealogy. The nuDNA topology depicted three main clades ; (2) Oreosaura ; and (3) all other Phrynocephalus . Most notably, the placements of Megalochilus and Oreosaura differed in important ways. Matrilineally, Oreosaura and Megalochilus aligned with Middle and Central Asian “core Phrynocephalus” with strong support (99/88/1.0). In contrast, the nuDNA biparental phylogeny united Megalochilus with Arabian and Iranian species (75/90/0.99), including the P. arabicus-group, P. scutellatus and Microphrynocephalus with Oreosaura forming a sister-group to clades A–D. Other notable conflicts also occurred. The nuclear phylogeny did not depict a shared heritage for P. scutellatus and the Arabian species-group (P. scutellatus (clade B) was the sister-lineage of Microphrynocephalus  Mices-group , as did cephalus . The phyor-group ; 95/91/1or-group .P. mystaceus, P. strauchi, P. scutellatus, Oreosaura, the basal position of Arabian species and Arabian species + Microphrynocephalus. The matrilineal genealogy was forced to the nuclear dataset and vice versa. AU indexes for the basal position of Arabian species or Arabian species + P. scutellatus in the matrilineal genealogy were not statistically rejected by nuclear markers . The alternative nuclear hypotheses for the clades A–E and D–H were not rejected by mitochondrial data. The matrilineal position of P. mystaceus within the lineage (Helioscopus + P. axillaris + P. mystaceus) was rejected by nuclear markers (p = 0.000), and vice versa mitochondrial markers rejected the nuDNA resolution of P. mystaceus + Arabian species + Microphrynocephalus + P. scutellatus (p = 0.000). The AU test for P. strauchi occurring within the raddei-lineage was not rejected statistically by nuclear data (p = 0.752) and the mitochondrial data did not reject the nuDNA resolution of P. strauchi + helioscopus-group (p = 0.857). Existence of the matriline Oreosaura + “guttatus” + “versicolor” was rejected according to nuclear data (p = 0.000) and vice versa the mitochondrial dataset rejected position of Oreosaura within A–E (p = 0.000). Finally, we tested topologies of trees based on each nuclear marker against final topology. The BDNF dataset rejected the topology. In contrast, original RAG-1 topology was rejected. p-Values of the AU-tests of alternative topologies were summarized in We performed additional AU tree-selection test to test for significant differences between matrilineal genealogy and the nuclear phylogeny, including whether one or both datasets rejected alternative placements of particular clades. The test evaluated the conflicting positions of Phrynocephalus originated between the end of Oligocene and beginning of the Miocene (mtDNA: 33.2 Ma (26.4–39.7); nuDNA: 26.9 Ma (22.6–31.7)) and the basal radiation dated to the middle Miocene (mtDNA: 19.3 Ma (14.9–23.5); nuDNA 14.8 Ma (12.0–17.5)).Chronograms from mtDNA and nuDNA data were presented in Lineage-through-time (LTT) plots gave a graphical representation of lineage-accumulation . The mtDPhrynocephalus, thus supporting the hypothesis of a southern origin. Paleogeography of central Eurasia in Miocene–Pliocene was shown in The ancestral areas and biogeographic processes  reconstructed from nuDNA data were shown in Phrynocephalus were given in Phrynocephalus suggested that the most likely substrate type for the ancestor consisted of soft substrate, i.e., loose sands with non-differentiated proluvial sediments, such as clay or gravel.Results of habitat evolution modelling for Phrynocephalus was likely significantly smaller (size category 87–97 mm) than its sister taxa Laudakia, Stellagama, and Paralaudakia . Most species of Phrynocephalus have been found to be smaller than their common ancestor (size categories 47–77 mm); however, several lineages of Phrynocephalus have subsequently increased their body size , while further miniaturization was suggested for the Phrynocephalus interscapularis-group (37–47 mm).Results of ancestral state reconstructions of maximum SVL evolution for each taxon were shown in Phrynocephalus.PhrynocephalusPhylogenetic placement of . Our results are in accordance with previous studies on the higher phylogenetic relationships within the subfamily Agaminae  is poorly resolved; the old age of this radiation, which we estimate as early to mid-Oligocene  is unambiguous in all analyses.Agaminae  , subgenus Oreosaura (lineage E), P. guttatus- and P. versicolor-groups , P. mystaceus , P. helioscopus (lineage F), and P. arabicus–P. maculatus (lineage C). The P. guttatus- and P. versicolor-matrilines are sister-groups, although concatenated analyses of nuDNA data nest the former within the latter , P. arabicus–P. maculatus-group, P. scutellatus and P. mystaceus . The phylogenetic relationships between these four groups are essentially unresolved. This groups includes oviparous species inhabiting Arabia, Near to Middle East, and Middle Asia. Many of them associate with sand dunes. Monophyly of the P. arabicus–maculatus group and Microphrynocephalus  has strong support.Clade I  containsOreosaura). Phylogenetic relationships within this group remain unresolved with the exception of a sister-species relationship between Phrynocephalus theobaldi Blyth, 1863 and Phrynocephalus forsythii Anderson, 1872. In the concatenated analysis of nuDNA exons  of Oreosaura appear to be deeply nested within the core clade of Phrynocephalus and nests with the P. helioscopus-group, while P. mystaceus, P. arabicus, Phrynocephalus longicaudatusP. maculatus) and Microphrynocephalus occupy the basal position in the Phrynocephalus radiation. The potential explanation of these differences may be connected with habitat preferences; adaptation to types of substrate may drive convergent changes and morphology. Both Tibetan viviparous Oreosaura and P. helioscopus-group members occur mostly on solid (rocky or clay) substrates and are superficially similar in external morphology, sharing robust body habitus, shortened tail, and other features. At the same time, sand-dwelling Microphrynocephalus, P. mystaceus and P. arabicus and Bufoniceps share such characters as presence of enlarged scale fringes on toes, enlarged or keeled scales on throat, eyelids or jaws, flattened body and tail, which are considered to be adaptive for life on large wind-blown sand dunes. Trapelus and Bufoniceps. However, convergent similarity between send-dwelling Phrynocephalus and Bufoniceps potentially could lead to the basal position of P. mystaceus.Our phylogeny has better resolution and wider taxon sampling than previously published mtDNA-based genealogies  and the Mito-nuclear discordance due to ancient hybridization. The main difference between mt- and nuDNA trees is the positions of P. mystaceus: the nuDNA-based topology strongly suggests that P. mystaceus as a sister-group with respect to the Middle-Eastern P. interscapularis-group , P. scutellatus and the Arabian P. arabicus–P. maculatus-group , whereas the basal differentiation of Phrynocephalus is estimated as the middle Miocene , but Phrynocephalus and reassigned it to monotypic Bufoniceps, which he treated as a sister-clade of Phrynocephalus, Subsequent molecular analyses supported this arrangement , and morphological —within the clade remains unclear. Finally, P. mystaceus, to which the name MegalochilusThe recognition of subgenera within clade II  unites v clade I  joins a ov, 2014  are miniov, 2014  and geogMegalochilus, or splitting it into a number of smaller taxa, including Megalochilus, Phrynosaurus, Microphrynocepahlus, and an unnamed taxon for the P. arabicus–P. maculatus species group. Our analyses lack samples from a number of Middle Eastern species, which likely fall into clade I, including P. ornatus ornatus, Phrynocephalus clarkorum Anderson et Leviton, 1967, Phrynocephalus lutensisPhrynocephalus luteoguttatus Boulenger, 1887 and, most importantly, large-sized and psammophilous P. euptilopus. Accordingly, we suggest that further taxon sampling and additional nuDNA-markers be evaluated before making subgeneric changes in the interest of maintaining taxonomy stability.Two alternative taxonomic decisions are possible: recognizing the whole of clade I as Taxonomic differentiation within species complexes. Our results indicate that in many cases the currently adopted taxonomy is incomplete and does not reflect the actual biodiversity within Phrynocephalus. We briefly review these cases and provide corresponding taxonomic recommendations.MicrophrynocephalusLineage A. . This group of miniaturized species inhabit wind-blown sands of southern Middle Asia (Turan) and Middle East. Our analyses include P. interscapularis, Phrynocephalus sogdianusP. ornatus vindumi Golubev, 1998 only. Although unsampled, P. ornatus ornatus, P. clarkorum, P. luteoguttatus, P. lutensis most likely belong to this group based on morphological characters . Later, P. sogdianus, Kurjalakum Sands, occurs approximately 50 km from the type locality and the Vakhsh River Valley separates it from the type locality. Our substantial divergence in mtDNA sequences (uncorrected genetic p-distance = 3.9–4.3% for COI) coincides with morphological and distributional differences and favors the recognition of P. sogdianus as a full species.aracters . P. sogdPhrynocephalus scutellatusLineage B. . The phylogenetic position of this small-sized species, which inhabits clay or gravel deserts on the Iranian Plateau, remains unclear within clade I. The recent molecular study of P. scutellatus might consist of a species complex that requires reconsideration.Lineage C. Arabian group. Our analyses include psammophilous P. arabicus s.l., which inhabits aeolian sand dunes from the Arabian Peninsula to westernmost Iran, and P. maculatus, which occurs on hard substrates and has two presently recognized subspecies: P. m. maculatus (Anderson) from the Iranian Plateau and P. m. longicaudatus from the Arabian Peninsula. Our sampling lacks Phrynocephalus golubewii Shenbrot et Semenov, 1990 from Turkmenistan and Phrynocephalus sakoi Melnikov, Melnikova, Nazarov, Al-Johany & Ananjeva, 2015 from Oman. P. arabicus complex, considered Phrynocephalus nejdensisPhrynocephalus macropeltisP. arabicus s.l. from western Iran as the new species Phrynocephalus ahvazicus Melnikov, Melnikova, Nazarov, Rajabizadeh, Al-Johany, Amr & Ananjeva, 2014. However, this taxonomic action was based primarily on small to moderate genetic distances between these forms (p-distance 2.7–6.0%) and differences observed in coloration of living animals. Because P. ahvazicus of P. arabicus s.l. pending a further re-assessment of the taxonomy of the P. arabicus species complex. Previously, P. maculatus s.l. occurred with respect to P. arabicus. Our multilocus nuDNA-based phylogeny agrees with the matrilineal genealogy and indicates that P. longicaudatus from the Arabian Peninsula is a sister-taxon of P. arabicus (p-distance = 8.0% for COI). This differs from P. maculatus from Iran being reconstructed as a sister-taxon to the clade joining P. arabicus and P. longicaudatus. Based on the principle of monophyly, as well as genetic and distributional differences, we raise P. longicaudatus to full species status as P. longicaudatus . Nuclear markers also reveal deep divergence between these two lineages, one of which is restricted to East Khorasan Province, Iran (P. mystaceus 2) and another occupies the rest of species range in Middle Asia (P. mystaceus 1). Thus, the taxonomy of this complex requires further study.OreosauraLineage E. . This clade consists of viviparous highland species inhabiting deserts of the QTP and our analyses evaluate Phrynocephalus erythrurus, P. forsythii, P. putjatai, P. vlangalii, and P. theobaldi within the complex. The phylogenetic relationships among these species remain essentially unresolved. P. helioscopus–Phrynocephalus persicus-group.Lineage F.  This group, which was until recently considered to be a widespread polytypic species P. helioscopus s.l., occurs in the montane deserts from western and northern Iran, Transcaucasia, the Turan Region, and Middle Asia to the Caspian Basin in the west and westernmost China and Mongolia in the east. P. helioscopus has a robust, tuberculate morphology and inhabits mostly hard substrates in clay or clay/gravel deserts. Previous phylogenetic analyses of mtDNA (COI) and nuDNA (interSINE-PCR) by P. helioscopus complex (Middle Asia and adjacent territories) and P. persicus De Filippi, 1863 complex (Iran and Transcaucasia), both of which contained a number of highly divergent lineages. Subsequent analysis of morphological characters resulted in recognizing seven subspecies within P. helioscopus and three within P. persicus . P. saidalievi based on differences in karyotype and our analyses strongly support this arrangement. Based on the principle of monophyly, along with the molecular and morphological analyses of Phrynocephalus meridionalis Dunayev, Solovyeva, Poyarkov in Solovyeva, 2012 comb. et stat. nov. This species is the sister-taxon of P. saidalievi and was originally described as a subspecies of P. helioscopus from the Surkhandarya Region of southern Tajikistan. The species differs markedly in its mtDNA sequences (p = 10.0–10.6% in COI), nuclear markers and morphology   and Phrynocephalus reticulatusP. re. reticulatusP. re. bannikovi Darevsky, Rustamov et Shammakov, 1976 from Turkmenistan and P. re. strauchi from Ferghana Valley in Uzbekistan and Tajikistan; P. boettgeri a junior synonym of P. raddei. Agama ocellata Lichtenstein in Eversmann, 1823 and demonstrated that it was the senior synonym of P. reticulatusP. ocellatus  was widely accepted for over 25 years  . Until r25 years . Despite25 years : 56 proppriority . Herein,P. raddei boettgeri–P. ocellatus group and suggest that P. strauchi is their sister-group. Their relationships within Phrynocephalus remain unresolved (P. strauchi is unresolved and P. raddei boettgeri–P. ocellatus form a well-supported sister-group relationships with P. helioscopus–P. persicus (lineage F). This clearly supports giving full-species status of P. strauchi as suggested by P. bannikovi and P. raddei raddei from Turkmenistan. Further studies are required to clarify phylogenetic relationships within this group.Our mtDNA analyses suggest monophyly of the resolved . Concateresolved  and specresolved  analysesP. axillarisLineage H. . Oviparous P. axillaris from sand deserts of Taklimakan and adjacent parts of western China is a highly divergent lineage according to our concatenated (P. guttatus–P. versicolor-group). These lizards have a slender habitus, share habitat preferences and have bright red to blue axillary spots used in intraspecific communication  inhabits various types of deserts ranging from sand dunes to gravel deserts. They occur in the Middle Asia from Caspian Basin to the westernmost China. P. guttatus, P. moltschanovi, and P. alpheraki from the Caspian and Aral basins and Ili Depression in eastern Kazakhstan; (2) Phrynocephalus kuschakewitschi Bedriaga, 1905 and P. incertus Bedriaga, 1905 from the Balkhash Lake Basin in eastern Kazakhstan; and (3) Phrynocephalus melanurus from the Zaysan Depression in northeastern Kazakhstan and Junggar Depression of northwestern China. Significant differentiation in mtDNA sequences (p=4.0–7.9% for COI), morphology and distribution argue for recognizing these forms of P. guttatus as full species. However, further research including more variable nuclear DNA-markers is necessary before doing so, especially in the Balkash Lake Basin in Eastern Kazakhstan, which cradles the highest species diversity of this group and where gene flow between species might take place .th mtDNA  and nuDNth mtDNA  and 4 daP. versicolor-group that inhabit lowland deserts of northern Central Asia. Whereas the nuDNA analyses do not resolve relationships within this group, mtDNA results suggest the presence of two matrilines, one containing populations from the Mongolian part of the Junggar Depression (Mongolian Jungaria), previously assigned to P. versicolor hispida . Our analyses are in concordance the previous results by P. przewalskii and P. frontalis. Despite the presence of two matrilines, the nuDNA markers do not differ between the two and P. przewalskii Strauch, 1876 appears to be the senior synonym for this taxon.Lineage I comprises the species of  hispida . ConcatePhrynocephalus versicolor is a wide-ranging species that inhabits the Mongolian and Chinese Gobi Desert as far northwards as the Tuva Republic in southern Siberia (Russia). Traditionally, two subspecies were recognized: P. v. kulagini from Tuva and northern Mongolia and P. v. versicolor from the rest of species’ range. Previous studies , nuDNA genes, and morphology. These results are supported by morphological differentiation  skin covering the tympanum, (2) lateral fringes of elongate scales on digits and eyelids, (3) countersunk jaws, (4) flattened body and tail, and (5) a characteristic burial behavior by lateral oscillations of the body . Two species of clade I (P. scutellatus and P. maculatus) demark independent shifts to firmer ground . Clade II demonstrates adaptation to high elevation, stony or gravel deserts; a reversal to sand habitats occurs for P. forsythii, which inhabits the sandy Taklimakan Desert at lower elevations. Clade III likely had a sand-dwelling ancestor  or gravel . All members of the P. raddei–P. helioscopus group are specialized to life on hard substrates  and show no reversal to sand habitats, with the exception of P. helioscopus varius, which occurs in sandy areas of pine forests in the northernmost limit of its distribution in the Altai Region of Russia  to 123 mm (P. mystaceus)  found in the aeolian sand dunes of the Middle East and Turan are the most miniaturized group of Phrynocephalus (SVLmax 47 mm). An example of an overt reversal in body size occurs in P. mystaceus, which is the largest member of the genus and is also specialized to large floating sand dunes. Similar change in body size possibly took place in psammophilous P. euptilopus, which appears to be closely related to P. interscapularis–P. luteoguttatus, but is much larger (SVLmax 63 mm) (P. maculatus (SVLmax 91 mm) and P. putjatai (SVLmax 84 mm).staceus) . The weiAgaminae . Accordix 63 mm) . Some spPhrynocephalusTime-tree and origin of . Estimates of divergence and diversification times for Phrynocephalus vary among authors. Phrynocepalus represented an ancient radiation and diverged about 35 Ma. According to immunological data, Phrynocephalus from other Agaminae took place around 11 Ma, while their allozyme data provided an estimate of 6 Ma. Using relaxed clock dating, Phrynocephalus including P. interscapularis diverged 28.9 Ma (21.1–36.2 Ma). Excluding P. interscapularis, the estimated origin dated to 22.4 Ma (16.5–30.6 Ma) for mtDNA, and 15.8 Ma (11.8–23.0 Ma) for nuDNA.Phrynocephalus diverged from other Agaminae in early Oligocene around 33.2 Ma (19.92–45.69 Ma), or based on nuDNA data in late Oligocene around 26.9 Ma (22.44–31.27 Ma). The basal differentiation within the genus took place in early Miocene  or mid-Miocene  based on mtDNA and nuDNA data, respectively . Some researchers . However, despite recent progress based on molecular phylogenetics   . This scPhrynocephalus into clades I–III coincides perfectly with onset of the MMCT (14.8 Ma). The increasing aridification provided a diversity of desert habitats for Phrynocephalus to occupy as they started to disperse out of the ancestral area in Middle East and Turan , which diversified in response to ongoing aridification of the Indian subcontinent in the late Miocene , which occurred between 14.8 and 14.1 Ma. . At thatnd Turan . A simil Miocene .Oreosaura (clade II) supposedly colonized the QTP 13.5–10.0 Ma and diversification within this clade began around 3.8 Ma ago  by Phrynocephalus may represent a promising model group for such studies.The ancestors of viviparous 8 Ma ago  and 6. OPhrynocephalus (Middle East and Turan)   and 6. Cand 5 Ma . The vic5 Ma ago .Phrynocephalus in Southwest Asia best explains their apparent lag in radiation.Oviparous clade III encompasses the highest number of species and has the largest distribution in ranging from the Middle East and Asia Minor to Turan and Middle and Central Asia. Cladogenesis started in the late Miocene (ca. 8.9–7.1 Ma) and was likely influenced by progressive aridification of central Eurasia, orogenesis and changes in level of the Paratethys Sea. The age of the Central Asian deserts is questionable. The 22 Ma estimation of the onset of aridification in northwestern China  correspoP. raddei–P. helioscopus group adapted to life on gravel or clay deserts. The highest diversity within this group occurs on alluvial plains of Middle Asia and Turan. The P. helioscopus–P. persicus species complex has largest distribution penetrating westwards to Transcaucasia and eastern Asia Minor, and northwards to the Caspian Basin, lowland deserts of Middle and Central Asia as far as western Mongolia   , while oPhrynocephalus and infer its biogeographic history. The ancestral area of the Agaminae and factors that influenced its diversification remains uncertain. The origin of Phrynocephalus dates to the late Oligocene (26.9 Ma) and this precedes the formation of the mid-Miocene landbridge that connected Africa and Asia. The common ancestor of Phrynocephalus appears to have been a ground-dwelling, miniaturized agamine adapted to sand habitats. The basal divergence of Phrynocephalus into three major clades appears to have occurred in the Middle East or southern Middle Asia (Turan) around 14.8 Ma. This corresponds well with the mid-Miocene climatic transition—climate cooling that coincided with aridification and spreading of xerophytic plants across Mediterranean and Paratethys Basins. Subsequent drying up of the Paratethys Sea formed vast desert habitats that Phrynocephalus appears to have occupied. Two oviparous clades dispersed independently to lowland deserts of the Arabian Peninsula, Middle and Central Asia. Orogenetic processes and Paratethys Basin dynamics appear to have driven further cladogenesis, which Pliocene–Pleistocene climate oscillations built upon. Substantial variation in body size and morphology occurs in the oviparous lizards. Viviparous Oreosaura occupied the QTP around 13.5–10 Ma. Cladogenesis in this group dates between the late Oligocene and mid-Pliocene depending on the dataset . This estimate coincides well with the divergence time of another viviparous group of lizards inhabiting Central Asia—the racerunner subgenus Pareremias (Lacertidae)  , which wertidae) .Climatic changes during the Cenozoic, including the ongoing aridification of central Eurasia, shaped the biodiversity of the region . Most re10.7717/peerj.4543/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4543/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.4543/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.4543/supp-4Supplemental Information 4See Click here for additional data file.10.7717/peerj.4543/supp-5Supplemental Information 5ML BSP/BI BPP values are given for resolved nodes only.Click here for additional data file.10.7717/peerj.4543/supp-6Supplemental Information 6ML BSP/BI BPP values are given for resolved nodes only.Click here for additional data file.10.7717/peerj.4543/supp-7Supplemental Information 7Color marking of species groups corresponds to Click here for additional data file.10.7717/peerj.4543/supp-8Supplemental Information 8ML BSP/BI BPP values are given for resolved nodes only.Click here for additional data file.10.7717/peerj.4543/supp-9Supplemental Information 9ML BSP/MP BSP values are given for strongly supported nodes.Click here for additional data file.10.7717/peerj.4543/supp-10Supplemental Information 10ML BSP/MP BSP values are given for strongly supported nodes.Click here for additional data file.10.7717/peerj.4543/supp-11Supplemental Information 11ML BSP/MP BSP values are given for strongly supported nodes.Click here for additional data file.10.7717/peerj.4543/supp-12Supplemental Information 12ML BSP/MP BSP values are given for strongly supported nodes.Click here for additional data file.10.7717/peerj.4543/supp-13Supplemental Information 13Node values correspond to estimated divergence times (in Ma). Grey-bars correspond to 95%-confidence intervals. Color marking of species groups corresponds to Click here for additional data file.10.7717/peerj.4543/supp-14Supplemental Information 14Node values correspond to estimated divergence times (in Ma). Grey-bar corresponds to 95%-credibility interval. Color marking of species groups corresponds to Click here for additional data file.10.7717/peerj.4543/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.4543/supp-16Supplemental Information 16ID/source–museum voucher/isolate ID; Locality–geographic locality of origin. No exact locality information is available for specimens obtained via pet trade.Click here for additional data file.10.7717/peerj.4543/supp-17Supplemental Information 17Specimen ID/source: Museum ID or GB–sequences downloaded from GenBank.Click here for additional data file.10.7717/peerj.4543/supp-18Supplemental Information 18Total length (in b.p.), number of conservative (Cons.), variable (Var.) and parsimony-informative (Pars.-Inf.) sites are given (data presented only for the ingroup).Click here for additional data file.10.7717/peerj.4543/supp-19Supplemental Information 19“F,” “L”–forward primer, “R,” “H”–reverse primer.Click here for additional data file.10.7717/peerj.4543/supp-20Supplemental Information 20Node–tree node used for calibration, for node names see Click here for additional data file.10.7717/peerj.4543/supp-21Supplemental Information 21Geographic regions: AR–Near East and Arabia, MI–Asia Minor and Transcaucasia, KZ–Kazakhstan, northern Caspian and Ciscaucasian deserts, CA–Central Asia, TU–Turan, TI–Tibet, ME–Middle East. Maximum SVL (maxSVL) values are given in mm. Substrate states (Substrate) are classified as: (1) loose sand dunes; (2) sands with non-differentiated proluvial sediments, e.g. gravel or clay; (3) gravel and stone deserts; (4) clay soils and salines; (5) clay soils mixed with gravel; (6) large rocks and cliffs.Click here for additional data file.10.7717/peerj.4543/supp-22Supplemental Information 22Numbers encode: (1) loose sand dunes; (2) sands with non-differentiated proluvial sediments, e.g. gravel or clay; (3) gravel and stone deserts; (4) clay soils and salines; (5) clay soils mixed with gravel; (6) large rocks and cliffs; see Click here for additional data file.10.7717/peerj.4543/supp-23Supplemental Information 230–test hypothesis , H1–original analysis ; AU–AU-test value.HClick here for additional data file.10.7717/peerj.4543/supp-24Supplemental Information 240–test hypothesis, H1–original topology; nu_vs_mt–testing nuclear topology on mitochondrial dataset, mt_vs_nu–testing mitochondrial topology on nuclear dataset; AU–AU-test value.HClick here for additional data file.10.7717/peerj.4543/supp-25Supplemental Information 25For node names see Supplementary Figure S1. Estimated age is given in (Ma) for nuDNA and mtDNA datasets; results of ML biogeographic area reconstruction in Lagrange  is given for nuDNA topology only. For biogeographic areas definition see Click here for additional data file.10.7717/peerj.4543/supp-26Supplemental Information 26Click here for additional data file.10.7717/peerj.4543/supp-27Supplemental Information 27Click here for additional data file.10.7717/peerj.4543/supp-28Supplemental Information 28Click here for additional data file.10.7717/peerj.4543/supp-29Supplemental Information 29Click here for additional data file.10.7717/peerj.4543/supp-30Supplemental Information 30Click here for additional data file.10.7717/peerj.4543/supp-31Supplemental Information 31Click here for additional data file.10.7717/peerj.4543/supp-32Supplemental Information 32Click here for additional data file.10.7717/peerj.4543/supp-33Supplemental Information 33Click here for additional data file.


Mycobacterium abscessus accounts for a large proportion of lung disease cases caused by rapidly growing mycobacteria. The association between clarithromycin sensitivity and treatment outcome is clear. However, M. abscessus culture and antibiotic susceptibility testing are time-consuming. Clarithromycin susceptibility genotyping offers an alternate, rapid approach to predicting the efficacy of clarithromycin-based antibiotic therapy. M. abscessus lung disease patients were divided into two groups based upon the clarithromycin susceptibility genotype of the organism isolated. A retrospective analysis was conducted to compare the clinical features, microbiological characteristics, and treatment outcomes of the two groups. Several other potential predictors of the response to treatment were also assessed. Sixty-nine patients were enrolled in the clarithromycin-resistant genotype group, which included 5 infected with rrl 2058-2059 mutants and 64 infected with erm(41)T28-type M. abscessus; 31 were in the clarithromycin-sensitive group, i.e., 6 and 25 patients infected with genotypes erm(41)C28 and erm(41) M type, respectively. The results showed that lung disease patients infected with clarithromycin-sensitive and -resistant M. abscessus genotypes differed significantly in clarithromycin-based combination treatment outcomes. Patients infected with the clarithromycin-sensitive genotype exhibited higher initial and final sputum-negative conversion and radiological improvement rates and better therapeutic outcomes. Multivariate analysis demonstrated that genotyping was a reliable and, more importantly, rapid means of predicting the efficacy of clarithromycin-based antibiotic treatment for M. abscessus lung disease. Mycobacterium abscessus infections is the most challenging  has increased significantly in recent years –3. Of al–llenging , 5. M. abrosis 6–.M. abscessus is among the most antibiotic-resistant pathogens known (M. abscessus lung disease (M. abscessus infections (ns known . Althoug disease . Currentfections .M. abscessus to CLA. Two genotypes confer CLA resistance: a point mutation (A to C or A to G) in the 2058-2059 locus of the 23S rRNA (rrl) gene confers acquired resistance (erm(41) gene, which exhibits a T/C polymorphism at the 28th nucleotide, confers inducible resistance when the 28th nucleotide is thymidine [erm(41)T28] (erm(41), i.e., genotype erm(41)C28 (erm(41) nucleotides 64 and 65, or deletion of nucleotides 159 to 432, also results in the loss of erm(41) gene function (M type) and a gain in CLA sensitivity (Genotypic variations influence the sensitivity of sistance . An inta(41)T28] , 15. Altsitivity , 16.M. abscessus can be divided into M. abscessus subsp. abscessus and M. abscessus subsp. massiliense based upon the integrity or absence of the erm(41) gene. Korean and Japanese researchers first reported that M. abscessus subsp. abscessus and M. abscessus subsp. massiliense exhibited disparate clinical and microbiological characteristics (erm(41)C28] genotype had a prognostic advantage  sequevar. Sixty-nine (69%) patients were enrolled in the CLA-resistant genotype group, which included 5 (7.2%) rrl 2058-2059 mutant- and 64 (92.8%) erm(41)T28-type-infected patients; 31 (31%) belonged to the CLA-sensitive genotype group, which included 6 (19.4%) erm(41)C28- and 25 (80.6%) erm(41) M-type-infected patients. No significant differences were found in the ages and genders of the two groups (P = 0.045). Furthermore, a significantly greater incidence of cavity-like manifestations occurred in computed tomography (CT) scans of patients infected with isolates with the CLA-resistant genotype than in patients infected with isolates with the CLA-sensitive genotype . CT scans of the CLA-sensitive-group patients, on the other hand, displayed a higher incidence of a tree-in-bud pattern .One hundred o groups . The proM. abscessus isolates manifest two distinct colony morphotypes: smooth and rough. The colony morphology of the isolates associated with both CLA susceptibility groups did not differ significantly (M. abscessus characterized by a rough-type colony exhibited a higher incidence of cavities in CT images (P = 0.043) (ficantly . Patient= 0.043) . The colM. abscessus isolates to 10 antibiotics tested is shown in rrl 2058-2059 mutant isolates exhibited acquired resistance to CLA, i.e., they were resistant on day 3 of exposure and prior to induction. Twenty-seven of the 64 erm(41)T28 isolates also exhibited acquired resistance; 36 isolates were induced by 14 days exposure to CLA; and one isolate showed abnormal CLA sensitivity despite expressing an erm(41)T28 gene, albeit with a wild-type rrl gene. In sharp contrast, no CLA resistance was observed within the CLA-sensitive genotype group. Notably, although most isolates in the CLA-resistant genotype group were insensitive to CLA, only one isolate was insensitive to amikacin treatment. A considerable number of isolates in both the CLA-sensitive and CLA-resistant genotype groups were sensitive to linezolid. A large number of isolates in both groups were resistant to moxifloxacin, doxycycline, imipenem, and tobramycin; no significant difference in resistance to these antibiotics was found between groups.The sensitivity of all the P = 0.011). Times to initial sputum conversion also differed significantly between the CLA-sensitive and CLA-resistant genotype groups (P = 004). Sputum relapse after initial conversion to negative occurred in both groups and did not differ significantly. The proportion of patients whose sputa converted and remained culture-negative during the follow-up period was significantly greater in the CLA-sensitive than in the CLA-resistant genotype group . Radiographic improvement rates were significantly higher in patients infected with the CLA-sensitive genotype group isolates than in patients infected with the CLA-resistant genotype group (P = 0.006). The effective treatment response evaluated by radiology and microbiology was also significantly greater for the CLA-sensitive genotype group than for the CLA-resistant genotype group (P < 0.001).All patients enrolled in the study were treated with a standard combination of antibiotics based upon CLA. Patients infected with the CLA-sensitive genotype group isolates were significantly more likely to demonstrate initial sputum conversion    . All othM. abscessus lung disease patients with the CLA susceptibility genotype of clinical isolates. We found that the treatment results for patients infected with isolates with the CLA-sensitive M. abscessus genotype were far superior to the results for patients infected with isolates with the CLA-resistant genotype evaluated in terms of sputum conversion rate, duration of initial sputum conversion, radiological improvement, and efficacy. Treatment outcome, however, was independent of all other factors examined, which included BMI, colony morphology, and radiological images.The study reported here was the first undertaken to explore and correlate the differences in treatment outcomes of M. abscessus complex was first divided into M. abscessus subsp. abscessus and M. abscessus subsp. massiliense based upon differences in the rpoB gene (erm(41) genotype influenced the sensitivity of these subtypes to CLA in vitro T28, erm(41)C28, and erm(41) M type. In the study described here, clinical isolates were grouped into these genotypes rather than M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and the responses of patients to standard, CLA-based treatment were assessed and compared. The response of the CLA-sensitive genotype group was significantly superior to that of the CLA-resistant genotype group judged in terms of the sputum conversion rate, radiological improvement, duration of initial sputum conversion results, and treatment efficacy. While lung disease patients infected with M. abscessus subsp. abscessus [erm(41)C28 genotype] isolates may exhibit a better response to combination CLA treatment, the response of patients infected with M. abscessus subsp. massiliense isolates expressing the 2058-2059 rrl mutation was often much worse. As such, CLA susceptibility genotyping is more accurate than subtyping as an approach to predicting the treatment outcomes of patients with M. abscessus lung disease .In 2006, the poB gene . In 2011in vitro . The cli studies –20. PatiM. abscessus lung disease treatment C28 genotypes; consequently, their characteristics may not be representative. Solidifying their characteristics will require the enrollment of more patients infected with isolates exhibiting the rrl mutant and erm(41)C28 genotypes in future studies. Second, a minority of patients relapsed following initially successful treatment . Conceivably, these relapses were due to subsequent infection by a different M. abscessus strain or genotype. In the absence of dynamic follow-up, our study failed to determine whether recurrence occurred due to reinfection by a different M. abscessus strain.The study described here has several limitations. First, only a relative small number of isolates exhibited the M. abscessus genotype isolates. The CLA-sensitive genotype group was significantly superior in sputum conversion rate, initial sputum conversion time, radiological improvement, and treatment efficacy. Accurate genotyping is an important factor in predicting the efficacy of combination therapy with CLA-based antibiotics. Rapid genotyping should help clinicians optimize therapeutic strategies, especially in cases of critically ill patients who cannot wait weeks for culture and susceptibility testing. Genotyping would also be effective as a diagnostic approach in areas where facilities for mycobacterial culture and susceptibility testing are unavailable.In conclusion, there was a significant difference in treatment outcomes for patients infected with CLA-resistant and -sensitive M. abscessus lung disease was conducted between January 2012 and December 2015 at the Shanghai Pulmonary Hospital. Patient inclusion criteria were as follows: (i) age, >16 years; (ii) underwent initial diagnosis and treatment at the Shanghai Pulmonary Hospital in accordance with the 2007 American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) guidelines; (iii) received oral CLA-based combination treatment; (iv) follow-up period lasted more than 6 months. The exclusion criteria were as follows: (i) age, <16 years; (ii) history of NTM lung disease; (iii) lack of critical visit data , failure to follow up, or death from non-M. abscessus lung disease-related causes; (iv) treatment did not include oral CLA; (v) history of long-term macrolide drug treatment; (vi) diagnosed with active tuberculosis or received antituberculosis treatment within 3 months prior to study enrollment; (vii) coinfected with another nontuberculosis mycobacterium; (viii) refused to sign informed consent form; (ix) AIDS. In addition, patients with cystic fibrosis were not included in the study; notably, cystic fibrosis is extremely rare among Asian patients. A detailed, patient enrollment flow chart is shown in A retrospective review of the medical records of all patients with M. abscessus isolates used in this study were preserved in the Clinical Microbiology Laboratory of Shanghai Pulmonary Hospital. Shanghai Pulmonary Hospital is one of the designated treatment centers for tuberculosis and NTM disease in China, attracting NTM disease cases nationwide. M. abscessus isolates were obtained from sputum and bronchoalveolar lavage fluid. Samples were transferred to Lowenstein-Jensen (L-J) agar plates after treatment with 4% NaOH. Smears prepared from the bacterial colonies that grew were stained and examined microscopically to identify the acid-fast organisms. To select further NTM, positive colonies were inoculated and cultured in L-J medium containing 0.5 mg/ml P-nitrobenzoic acid and 5 mg/ml 2-thiophenecarboxylic acid hydrazide for 1 to 2 weeks at 37°C. Bacterial isolates that grew rapidly were selected for molecular typing by PCR. The bacteria were digested with 1 mg/ml lysozyme and 1 mg/ml proteinase K, and the DNA was extracted with phenol-chloroform. First, the rpoB gene was amplified by PCR, and the DNA sequences were determined. To confirm the M. abscessus complexes, 754 bp of the DNA segment was subjected to BLAST analysis. Second, the erm(41) gene was amplified, and the DNA sequence was analyzed to identify and differentiate M. abscessus subsp. massiliense, M. abscessus subsp. abscessus, and M. abscessus subsp. bolletii. Finally, the PRA-hsp65 gene was compared to an online reference (http://app.chuv.ch/prasite/index.html) to confirm the M. abscessus subsp. abscessus and M. abscessus subsp. bolletii identifications. M. abscessus subsp. bolletii was excluded from the study because it is essentially absent in China. Identified M. abscessus subsp. abscessus and M. abscessus subsp. massiliense isolates, stored at −80°C, were subsequently recovered for microbiology and molecular biology studies.All the clinical M. abscessus isolates by growth on Middlebrook 7H10 agar plates supplemented with 10% oleic acid-albumin-dextrose-catalase. The colonies were classified macroscopically as smooth or rough. If isolates gave rise to colonies of both morphotypes, a colony of each type was analyzed separately, and the identity was established by whole-genome sequencing.Single colonies were obtained from frozen erm(41) , rll wild type, and rrl 2058-2059 mutant.Genomic information for all isolates was obtained by whole-genome sequencing. Single nucleotide polymorphism (SNP) analysis was performed using the NCBI GenBank database and BLAST algorithm. The following genotypes were of specific interest: http://bioinf.spbau.ru/en/spades) were used to assemble the genome draft (http://quast.bioinf.spbau.ru/).Detailed methods were published previously by us . DNA wasme draft . The assNC_010397.1) M. abscessus strain served as the reference for rrl and erm(41)T28, CR5701 (HQ127366.1) was used as the reference strain for erm(41)C28, and CCUG48898 (AP014547.1) was the reference for M type.The NCBI Nucleotide BLAST program was used for SNP analysis. The standard ATCC 19977 . CLA resistance was assessed at 3 days and 14 days after M. abscessus exposure. Antibiotics' susceptible and resistant breakpoints were interpreted according to Clinical and Laboratory Standards Institute (CLSI) document M24-A2. Staphylococcus aureus  served as the control reference strain.Antibiotic sensitivity was determined by the microdilution method. Sulfonamides, moxifloxacin, cefoxitin, amikacin, doxycycline, tigecycline, CLA, linezolid, imipenem, and tobramycin are among the most common antibiotics used to treat All patients were treated with antibiotics as follows: an initial 4-week course of amikacin  combined with cefoxitin  by intravenous administration. CLA was also administered orally from the beginning of therapy. After 4 weeks, an oral regimen of CLA combined with levofloxacin or moxifloxacin was given. If an adverse reaction to either amikacin or cefoxitin occurred, the regimen was replaced with imipenem (500 mg three times a day), linezolid (600 mg once every 12 h), or tigecycline . CLA was administered continually throughout the course of treatment as recommended in the guidelines.All the patients underwent chest CT examination, as well as sputum smears and culture, regularly. Therapeutic efficacy was determined according to the results of microbiological examination and radiological changes. The clinical characteristics, sputum culture conversion rate and time, radiological improvement rate, and microbiological characteristics of each genotype group were compared. Culture conversion was defined as three consecutive negative cultures from sputum specimens. Effective treatment was defined as sputum culture negative or significant pulmonary lesion resolution without recurrence during the observation period. Ineffective treatment included failure to achieve culture and smear conversion, recurrence after initial culture conversion, and appearance of increased or stable lesions in CT scans.t test or the Mann-Whitney U test for continuous variables and the Pearson χ2 test or Fisher exact test for categorical variables. P values of <0.05 were considered statistically significant in a 2-tailed analysis. Times to initial culture conversion were compared using the Kaplan-Meier method. Potential predictors of the treatment response were assessed by multivariable logistic regression. In the logistic regression models, variables with P values of <0.1 in the univariable analysis were included in the multivariable analysis.All statistical analyses were conducted using SPSS20.0 . The data were compared using Student's M. abscessus isolates sequenced in this study are available at DDBJ/ENA/GenBank under BioProject PRJNA398137.The accession numbers for all the


Asclepias spp.) are currently the focus of restoration programs in the USA aimed at reversing a decline in populations of the milkweed-dependent monarch butterfly (Danaus plexippus); however, little is known of the benefits of these plants to other beneficial insects. Beneficial insects  attracted to two milkweed species  in central Washington State, WA, USA were identified and counted on transparent sticky traps attached to blooms over five seasons. Combining all categories of beneficial insects, means of 128 and 126 insects per trap were recorded for A. speciosa and A. fascicularis, respectively. Predatory and parasitic flies dominated trap catches for A. speciosa while parasitic wasps were the most commonly trapped beneficial insects on A. fascicularis. Bees were trapped commonly on both species, especially A. speciosa with native bees trapped in significantly greater numbers than honey bees. Beneficial insect attraction to A. speciosa and A. fascicularis was substantial. Therefore, these plants are ideal candidates for habitat restoration, intended to enhance conservation biological control, and for pollinator conservation. In central Washington, milkweed restoration programs for enhancement of D. plexippus populations should also provide benefits for pest suppression and pollinator conservation.Native plant and beneficial insect associations are relatively unstudied yet are important in native habitat restoration programs for improving and sustaining conservation biological control of arthropod pests in agricultural crops. Milkweeds ( Eriogonum spp.) and stinging nettles (Urticadioica L.).Restoring native plants and habitats is increasingly seen as a critical part of enhancing and sustaining conservation biological control of insects and mites in agricultural crop pest management ,2,3. NatAsclepias L. is a genus in the Apocynaceae containing at least 76 species of perennial herbaceous plants known as “milkweeds” that occur throughout the United States into southern Canada [Danaus plexippus L.) and two other danaid species in North America [D. plexippus since the late 1990s [Asclepias spp. has been recognized for some time [Asclepias spp. to other beneficial insects like predators and parasitoids has not received the same attention. To date, the tachinid fly parasitoid of stink bugs, Trichopoda pennipes (F.), is the only natural enemy of a herbivore demonstrated to be attracted to a milkweed species (Asclepias curassavica L.) [n Canada . Milkwee America . An appa America ,16 has bte 1990s  as part te 1990s . Organizome time ,22,23,24vica L.) .Asclepias, A. speciosa Torr. and A. fascicularis Decne occurring in an agriculturally intensive area of central Washington. Some horticultural industries in this region, for example wine grapes, have low inputs of pesticides and depend upon conservation biological control for much of their arthropod pest management [Here, we report the results of a field study on the attraction of predators, parasitoids and pollinators to the two endemic species of nagement ,27. Restnagement .Asclepias speciosa Torr.) and narrow-leaved milkweed (Asclepias fascicularis Decne) using transparent sticky traps. Milkweed plants were located growing in riparian or natural areas at six locations , Satus pass , Snow Mountain , Yakima , Prosser , and Horn rapids   in central Washington by counting and identifying beneficial insects attracted to blooming showy milkweed (9.26° W) . AsclepiAsclepias plants. Plants with traps were at least 5 m from other plants/traps at each site and traps were attached to plants using flexible wires and positioned to provide a sticky surface immediately above or adjacent to the flowers. Traps were left in place for 12–14 days before removal and were replaced if blooming continued. In a few instances, follow-up trapping occurred on the same plants (when plant numbers were limited) but usually different plants were chosen. Traps collected from the field were transported to the laboratory and stored at −30 °C until examined under a stereomicroscope.Transparent sticky traps  were used, avoiding trap color as a potential influence on insect attraction. Transparent sticky traps were used in earlier studies in Washington on predatory and parasitic insects attracted to native plants and were successful in trapping a wide diversity of these insects. Traps were attached to plants as soon as blooming commenced. At each site and on each occasion trapping was conducted, three traps were placed on three Erythroneura spp.) and lygus bug (Lygus spp.) pests were also recorded.All insects were identified to family or species and counted. The incidence and abundance of 34 species, genera or groups of winged beneficial insects were recorded . NumbersAnagrus wasps (Mymaridae), other parasitic wasps  and bees (Apoidea). Bees were separated into honey bees (Apis mellifera L.) and native bees. Bumblebees and larger wasps such as yellow jackets and hornets were often able to extricate themselves from the sticky material.Beneficial insects were condensed into 10 categories: lacewings (Chrysopidae), ladybeetles (Coccinellidae), predatory true bugs , predatory thrips (Aeolothripidae), predatory and parasitic flies , ichneumonid and braconid wasps , t-test was used for comparing two groups. Repeated-measures analysis of variance (ANOVA) with means separated using the Holm-Sidak method was used for comparing multiple groups .Trapping data were log (log x) transformed prior to analyses to improve normality of variances and then back-transformed for reporting. Student’s A. speciosa plants during the period May 16 to August 25. Fifteen traps were used on flowering A. fascicularis during June 24–10 and July 2012–2014 , grape leafhoppers (Erythroneura spp.) and western flower thrips ). Traps on A. speciosa caught 0.2 and 0.05 individuals per trap of Lygus bugs and leafhoppers, respectively, during trapping from 2010–2013. Numbers of F. occidentalis were not recorded but were usually less than 10 per trap.Beneficial insects dominated trap catches throughout the study. Very few pest insects were encountered. The only pests recorded were lygus bugs  showed no significant difference in numbers of beneficial insects trapped on A. speciosa (97.6 ± 9.2) and A. fascicularis (105.6 ± 7.6) .Combining all categories of beneficial insects and analyzed over all trapping years for each A. speciosa than any other beneficial insect group (mean: 63.7/trap)  (Significantly greater numbers of predatory and parasitic flies were attracted to < 0.001) .A. fascicularis (mean: 89.3/trap)  (Parasitic wasps (41.4), bees (19.0) and predatory bugs (7.0) were the next most commonly attracted beneficials with relatively small numbers of coccinellids (1.4), ichneumonids/braconids (1.4), mymarids (1.4), predatory thrips (1.1) and lacewings (0.6) trapped . Parasit< 0.001) . Predato< 0.001) .Asclepias spp. were Dolichopodidae (long-legged flies) and Empididae (dagger flies) accounting for 19%–50% of flies trapped. Tachinids accounted for 3.0%–7.0% and syrphids 0.5%–2.0% ; F = 1.607, df = 4, 12, p = 1.607 (fascicularis)) ) .Apis mellifera L.) and native bees trapped on the two Asclepias spp. was assessed in 2013 (speciosa) and 2012–2013 (fascicularis). A significantly greater numbers of native bees (mean 14.7/trap) than honey bees (5.9/trap) were trapped on A. speciosa . Similarly, greater numbers of native bees (5.9/trap) than honey bees (0.1/trap) were trapped on A. fascicularis  (The relative numbers of honey bees (= 0.001) .A. speciosa  but were comparable in numbers  on A. fascicularis . Minute pirate bugs (Orius spp.) were the dominant predatory bugs trapped on both Asclepias spp., significantly so on A. speciosa   significantly outnumbered acariphagous coccinellids (35) trapped on < 0.001) . CombiniDanaus plexippus) which has suffered a substantial population decline during the past two decades [D. plexippus [D. plexippus and being attractive to pollinators [Milkweeds are currently receiving much attention and interest in North America because of their role as the sole host plant for immature stages of the monarch butterfly  per trap. Of 100 flowering plant species evaluated by our laboratory for beneficial insect attraction in central Washington, A. speciosa and A. fascicularis are ranked in the top 20. Ten species of flowering buckwheats (Eriogonum spp.) attracted means of 48.5–167.7 beneficial insects per trap and were considered to have potential in habitat-restoration strategies for improving biological control in Washington viticulture [Medicago sativa L. , considered highly attractive to beneficial insects [Asclepias speciosa and A. fascicularis therefore appear to be at the higher end of the beneficial insect attraction scale and should have value in habitat-restoration programs aimed at improving crop pest management.Combining data for all categories and all years, iculture . Sticky- insects  in July A. speciosa (>60/trap). Most of these insects were dolichopodids (long-legged flies) and empidids (dagger flies). Empidids both as adults and larvae are predatory on a wide range of arthropods from aphids to mosquito larvae [Trichopoda pennipes, were attracted to tropical milkweed (Asclepias currassavica) in Georgia, GA, USA [Asclepias spp. Tachinid fly attraction to milkweeds may be a factor in the importance of these parasitoids in the regulation of D. plexippus larval populations [Asclepias spp., carnivorous flies were not greatly attracted to Eriogonum spp. or stinging nettles (Urtica dioica L.) [Carnivorous flies were the dominant beneficial insects attracted to o larvae  as are do larvae . Tachini GA, USA  and thisulations . In contoica L.) ,12.A. fascicularis (mean 10.1/trap) with parasitic wasps  dominating the trap catches for this species (mean 89.3/trap). Parasitic wasps were the second most numerous group of beneficial insects attracted to A. speciosa (mean 41.4/trap).Predatory and parasitic flies did not appear to be strongly attracted to A. speciosa (mean 20.6/trap) comparable to Eriogonum niveum Douglas ex Benth., a buckwheat species that attracted most bees in a central Washington study [A. fascicularis attracted few bees (mean 6.0/trap). Native bees accounted for 71% of the bees attracted to A. speciosa and 98% of the bees attracted to A. fascicularis. In contrast, nearly six times as many honeybees as native bees were recorded visiting blooms of Asclepias incarnata L. in Michigan, USA [Asclepias syriaca L. in Illinois [Asclepias species in Indiana and Arizona showed that the relative frequency of visits by honeybees and native bees (primarily bumblebees) varied but overall was balanced [A. speciosa and A. fascicularis. Most of our trapping sites were in agricultural areas with high numbers of honeybees.Bees were strongly attracted to on study . In contgan, USA . HoneybeIllinois . Studiesbalanced ,24. BumbOrius spp. (Anthocoridae), were trapped in good numbers on A. speciosa and A. fascicularis . They are also strongly attracted to flowering and non-flowering stinging nettles in central Washington [Eriogonum [Orius spp. comprised 94% of the predatory bugs we trapped in this study and accounted for >95% of bugs in James et al. [Orius tristicolor (White) was recorded on 64 plant species including A. speciosa in a central Washington study [Predatory bugs, primarily shington  and someriogonum . Orius ss et al. . Orius ton study .Eriogonum spp. [Aphis nerii Boyer de Fonscolombe) but they were absent in this study. When present, they will likely increase attraction of aphidophagous ladybeetles as well as other aphid predators. Milkweeds rarely support mite populations and only 58 mite-feeding ladybeetles were trapped during our five-year study.Other groups of beneficial insects were trapped at levels <2.0/trap. The number of ladybeetles trapped (mean 1.4/trap) was similar to numbers trapped on most num spp.  and stinnum spp. . MilkweeA. speciosa and A. fascicularis, attract a range of predators, parasitoids and pollinators during their blooming period from May−August. This is the first detailed evaluation of Asclepias spp. as pest natural enemy attractants and hopefully will encourage similar investigations on other milkweed species. Provision of a milkweed (Asclepias currassavica) insectary habitat in peanut-cotton farmscapes in Georgia, GA, USA increased tachinid fly parasitism of pest stink bugs, while aiding monarch butterfly and pollinator conservation [A. speciosa and A. fascicularis to provide a similar role enhancing pest management in central Washington agriculture. The beneficial insects most attracted to A. speciosa and A. fascicularis  play a significant role in suppressing a range of pest insects  affecting a variety of crops  in central Washington. Planting A. speciosa and/or A. fascicularis in non-cropped locations  near to crops should provide benefits to biological control and integrated pest-management programs. Benefits will likely vary according to the pests and natural enemies involved, but the use of milkweeds as plants that enhance and sustain biological pest management clearly deserves evaluation in specific locations and crops. Asclepias speciosa also appears to be a significant resource for native bees which are receiving increased attention as important pollinators for some crops [Asclepias speciosa and A. fascicularis are two of the major milkweed species used as larval hosts of monarch butterflies in the western U.S. Significantly, conservation and proliferation of A. speciosa and A. fascicularis are critical to increasing populations of D. plexippus in the west [This study has shown that the two milkweed species occurring in central Washington, ervation . Clearlyme crops . Asclepithe west . The inf


Helicobacter pylori is a gastrointestinal pathogen known to be associated with cardiovascular disease (CVD). However, most analyses about the effect of H. pylori infection have been done in patients with a history of CVD but not in healthy subjects. We evaluated the association between H. pylori infection and subclinical atherosclerosis by using cardiac multidetector computed tomography (MDCT) in healthy subjects without previous CVD. From December 2007 to February 2014, 463 subjects who underwent the rapid urease test (CLO test), pulse-wave velocity (PWV) measurement, and MDCT for a self-referred health check-up were enrolled to this study. Helicobacter pylori infection was defined on the basis of CLO test positivity on endoscopic gastric biopsy. Significant coronary artery stenosis was defined as ≥50% stenosis in any of the major epicardial coronary vessel on MDCT. The CLO-positive subjects had a lower high-density lipoprotein-cholesterol (HDL-cholesterol) level compared to the CLO-negative subjects. The incidence of significant coronary stenosis was higher in the CLO-positive group . Furthermore, the number of subjects with coronary artery calcium score >0 and log{(number of segments with plaque)+1} were also significantly higher in the CLO-positive group. However, there was no statistical difference in the number of subjects with coronary artery calcium score >100, the prevalence of any plaque nor the plaque characteristics . Pulse-wave velocity (PWV) was neither associated with CLO test positivity. The CLO-positive group was 3-fold more likely to have significant coronary artery stenosis even after adjusting for confounding factors . In a healthy population, current H. pylori infection was associated with subclinical but significant coronary artery stenosis. The causal relationship between H. pylori infection and subclinical atherosclerosis in a “healthy” population remains to be investigated in the future. Chlamydia pneumonia, cytomegalovirus, or Helicobacter pylori [Several studies have reported that cardiovascular disease (CVD) may be associated with certain microorganisms such as r pylori . Those rr pylori . Additior pylori .Helicobacter pylori is a gram-negative bacterium that lives in the stomach and a carcinogen leading to stomach cancer [H. pylori is especially high in Asians [H. pylori infection and CVD has received considerable attention especially in Asian countries [h cancer ,5. The in Asians . Becausen Asians ,8, the rountries –12.H. pylori has been suggested as a possible contributor to CVD progression but the results from the previous studies were quite controversial. Some reports showed that the seroprevalence of H. pylori was not correlated with coronary artery disease and they insisted that the infection status of H. pylori did not determine the risk of CVD [H. pylori infection among patients with previous history of CVD and supported a possible connection between H. pylori and the development of CVD [H. pylori infection is significantly associated with subclinical atherosclerosis. The individuals with subclinical atherosclerosis are at a higher risk of developing clinical atherosclerosis including coronary heart disease compared to the individuals without subclinical diseases [H. pylori and subclinical atherosclerosis is significant, earlier diagnosis and possibly eradication of H. pylori might be necessary for preventing atherosclerosis progression, especially in the high-risk population. Additionally, most studies on H. pylori infection and CVD have relied on antibody tests for defining the H. pylori infection status [k of CVD –16. In ct of CVD ,17–21. Idiseases –24. If tn status ,20, the n status ,26.H. pylori infection, diagnosed by using an endoscopic CLO test, was associated with subclinical atherosclerosis using pulse-wave velocity (PWV) and cardiac multidetector computed tomography (MDCT) in a healthy population.In this study, we aimed to evaluate whether current H. pylori infection and subclinical atherosclerosis in healthy population, subjects with previous coronary artery disease were excluded. Subjects with thyroid dysfunction and renal insufficiency were omitted as changes in thyroid or renal function influence the level of serum lipids along with coronary atherosclerosis [This study included 527 consecutive subjects undergoing esophagogastroduodenoscopy with the CLO test, PWV, and MDCT as part of the self-referred health check-up program at Gangnam Severance Hospital Health Promotion Center, Seoul, Korea from December 2007 to February 2014. All participants were divided into two study groups according to the CLO positivity. To avoid confounding bias, we excluded possible confounding medical conditions known to be associated with the health outcome. The exclusion criteria were as follows: (i) history of coronary artery disease, (ii) significant hypothyroidism/hyperthyroidism (thyroid-stimulating hormone >4.5 μIU/mL or free thyroxine >2.0 ng/dL), (iii) significant renal insufficiency (creatinine >1.5 mg/dL), (iv) significant CRP (C-reactive protein) elevation (>10.0 mg/L) and (v) subjects with any GI  medications. Since we intended to investigate the association between clerosis . We exclclerosis ,31. We fWe reviewed the participants’ questionnaire at the time of the health check-up, which included medical history, concomitant medication use, and other medico-social history information. After an overnight fasting for ≥8 hours, the fasting plasma glucose, total cholesterol, HDL-cholesterol, triglyceride, calcium, and C-reactive protein levels were checked from the peripheral venous blood samples by means of adequate enzymatic methods . The Friedewald formula was used to calculate the low-density lipoprotein-cholesterol (LDL-cholesterol) level.2 within and/or adjacent to the vessel lumen, and classified according to the presence/proportion of intraplaque calcification. The plaques were divided into calcified , mixed  or soft . Log{(number of segments with plaque) + 1} was used to analyze the number of segments with plaque.Coronary artery calcium scores (CACSs), coronary artery stenosis, and the number, presence and the characteristics of intracoronary plaques were evaluated by using an MDCT scanner . A standard prospective electrocardiogram-gating protocol with a step-and-shoot technique  was used. The CACS was measured  and described as Agatston scores. The CACS was interpreted as having either no coronary calcium (CACS 0 vs. >0) or severe coronary calcium (CACS ≤100 vs. >100). Coronary arteries were segmented to 3 major arteries and 12 small branches. Significant coronary artery stenosis was defined as at least ≥50% stenosis in any coronary artery/branch. A plaque was defined as a structure >1 mmBrachial-ankle PWV (ba-PWV) was assessed by using a volume plethysmographic instrument . The ba-PWV was estimated as the value of brachial-ankle distance divided by the blood transit time. An automatic device recorded electrocardiograms, phonocardiograms and the blood pressure (BP) at both the brachial artery and the posterior tibial artery after a stable rest for ≥5 min in supine position. The average value of the right and left sides was defined as the mean PWV.H. pylori infection status with a commercial CLO kit . Each gastric mucosal biopsy specimen was immediately transferred into the CLO kit and analyzed after 30 min of reaction to determine the test positivity according to the color change of the reaction kit.Biopsy of the gastric mucosa was performed to assess the P-value of ≤0.05 was considered statistically significant.Continuous variables with normal distribution are presented as mean ± standard error (SE), and those with a skewed distribution, such as CACS or the number of coronary segments with any plaque, were log transformed for analyses. Categorical variables are presented as absolute numbers and percentages. Intergroup comparisons with age adjustment were performed by using analysis of covariance for continuous variables and the generalized estimating equation for categorical variables. Adjusted odds ratios (ORs) for significant coronary artery stenosis in the CLO-positive group compared with the negative group were estimated with logistic regression analysis models. The SPSS statistical package  was used for all statistical analyses. A P = 0.01). Therefore, comparisons throughout this study were performed after adjustment for age as a confounding variable. The CLO-positive group had a lower mean HDL-cholesterol level  and a higher mean triglyceride level . There were no differences in the prevalence of diabetes and hypertension nor the use of antidiabetic, antihypertensive, and antiplatelet agents between the two groups. Subjects in the CLO-positive group were more likely to take a lipid-lowering agent compared to those in the CLO-negative group .A total of 463 subjects  were analyzed for the current study and divided into two groups according to the CLO test result. There were 224 (48.4%) CLO-positive and 239 (51.6%) CLO-negative subjects, respectively. The clinical and biochemical characteristics of the study subjects are presented in P = 0.05) than in the CLO-negative group. However, there were no statistical differences in the presence of severe coronary artery calcium (CACS >100), the mean value of log(CACS + 1) or the mean PWV between the CLO-positive and CLO-negative subjects. There was also no difference between the two groups not only in the prevalence of any plaque but also in the characteristics of plaque . However, the number of segments with plaque was higher in the CLO-positive group . More importantly, the incidence of significant coronary artery stenosis (≥50% stenosis of any major vessel) was significantly higher in the CLO-positive subjects than in the CLO-negative subjects   was significantly higher in the CLO-positive group  .H. pylori infection is an independent risk factor for subclinical atherosclerosis, we evaluated the adjusted risk for significant coronary artery stenosis in the CLO-positive subjects compared with the CLO-negative subjects by analyzing odds ratio (P = 0.01) and also after adjusting for several additional factors that may influence coronary artery stenosis, such as systolic blood pressure (BP), fasting glucose, HDL-cholesterol, anti-hypertension/diabetic medications, lipid-lowering agents, and antiplatelet agents .To investigate whether current ds ratio . The CLOH. pylori infection is significantly associated with significant coronary artery stenosis in a healthy population. The elevated risk of coronary artery stenosis in H. pylori-infected subjects was associated with a lower HDL-cholesterol level. This is the first report demonstrating that active/current H. pylori infection can be a risk for subclinical atherosclerosis in healthy subjects without previous CVD.In this study, we demonstrated that current H. pylori can be divided into invasive and noninvasive methods. The serologic H. pylori IgG test, urea breath test, and stool antigen assay are noninvasive, whereas the CLO test can only be performed with stomach tissue obtained from invasive endoscopic biopsy [H. pylori IgG test is fast, cheap, and noninvasive, most of the studies on H. pylori infection and CVD were performed using this test [H. pylori seropositivity and CVD [H. pylori infection itself [H. pylori infection and CVD [H. pylori infection is associated with subclinical atherosclerosis, it can be inferred that the eradication of H. pylori infection might help prevent the progression of atherosclerosis. In this context, this study provides a theoretical basis for studying the effect of H. pylori eradication on preventing the future progression of atherosclerosis.The diagnostic tests for c biopsy . As the his test ,20,32–35 and CVD ,18,20,32 and CVD –35. One n itself . Most imn itself  Owing to and CVD . In cont and CVD . If currH. pylori in many cases, the CLO test may fail to detect the presence of H. pylori because of a decreased bacterial burden in atrophic gastritis, particularly in the presence of intestinal metaplasia [H. pylori infection and CVD compared with previous studies with the serologic test.However, the CLO test is not without limitations. For example, although atrophic gastritis is explicitly associated with taplasia . Neverthtaplasia . The spetaplasia . Moreovetaplasia . Hence, H. pylori infection and overt coronary artery disease [H. pylori infection and subclinical coronary atherosclerosis. This study is distinct from previous studies because we did not observe CVD events but the incidence of subclinical coronary atherosclerosis in a relatively large number of healthy subjects without previous CVD. Atherosclerosis is a pathologic process narrowing the coronary, cerebral, and peripheral arteries due to the formation of atheromatous plaques [H. pylori infection and subclinical atherosclerosis allows investigators to confirm that earlier vessel-wall changes can be induced by H. pylori infection. Some reports have shown the association between H. pylori infection and the degree of carotid atherosclerosis, but not with that of coronary atherosclerosis [H. pylori infection, and linked this test with direct visualization of the coronary artery with cardiac MDCT in a relatively large number of healthy subjects. The significant correlation between H. pylori infection and subclinical coronary atherosclerosis in this study might be able to support H. pylori eradication as a potential cardiovascular prevention strategy.Several studies have reported the correlation between the prevalence of  disease ,17–21. H plaques . Studyinclerosis –41. To oH. pylori infection can induce atherosclerosis has not been clearly established yet [H. pylori infection [Helicobacter pylori carries lipopolysaccharide and can upregulate certain cytokines in the host, such as tumor necrosis factor-α (TNF-α) [H. pylori infection may also directly induce atherosclerosis. Endothelial dysfunction induced by vacuolating cytotoxin A secreted from H. pylori [H. pylori are all potential mechanisms of atherosclerosis that have been reported to be directly induced by H. pylori infection. The possibility of H. pylori induced-atherosclerosis independent of HDL-cholesterol levels is also supported by our data showing that H. pylori itself is associated with a higher incidence of significant coronary artery stenosis despite adjusting for HDL-cholesterol level. This significant association between H. pylori and significant coronary artery stenosis, irrespective of disturbed HDL-cholesterol levels, suggests that H. pylori infection may aggravate coronary atherosclerosis through both lipid metabolism-dependent and independent pathways.How shed yet ,29. In tnfection ,42–45. H (TNF-α) ,35. Inhi (TNF-α) , leading (TNF-α) . Besides. pylori ,49, mole. pylori , enhance. pylori , oxidati. pylori , and pla. pylori  by H. pyH. pylori seropositivity and coronary atherosclerosis. In a previous study, the degree of coronary atherosclerosis assessed by CACS was significantly associated with H. pylori seropositivity [H. pylori infection needs to be further investigated in a larger study population.CACS has also been used for suggesting the association between sitivity . In our sitivity ,55, the H. pylori infection and increased arterial PWV has been documented previously [H. pylori infection is thought to induce systemic inflammation through cytokines such as interleukin-6 [H. pylori infection [H. pylori was identified in coronary atherosclerotic plaque [H. pylori might be able to aggravate coronary atherosclerosis by direct invasion into the coronary vessels preferentially compared to other vessels regardless of systemic inflammation.The link between eviously ,57. As cleukin-6 ,51, not leukin-6 . Howevernfection ,57. In ac plaque ,59,60, HH. pylori in atherosclerosis [H. pylori eradication treatment previously because this was not included in the questionnaire of the health check-up. Nevertheless, our study is powerful and unique in demonstrating the association between subclinical atherosclerosis and H. pylori infection compared to other studies because our dataset is composed of extensive data encompassing MDCT, PWV, and CLO-test, the results of which are difficult to gather comprehensively.Our study has several limitations. First, this study lacks the measurement of cytotoxin-associated gene-A (CagA), which has been known to determine the pathogenicity of clerosis –63. It wclerosis ,65. LastH. pylori infection is associated with subclinical but significant coronary artery stenosis in a healthy population. This result suggests the possibility that H. pylori eradication might be worthwhile in preventing coronary artery disease. Further studies are warranted to investigate whether CLO test positivity could be a new marker for the assessment of future coronary artery disease risk and the underlying mechanism of how H. pylori infection may lead to coronary atherosclerosis.In conclusion, current


Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human and animal granulocytic anaplasmosis and tick-borne fever of ruminants. This obligate intracellular bacterium evolved to use common strategies to establish infection in both vertebrate hosts and tick vectors. Herein, we discuss the different strategies used by the pathogen to modulate cell apoptosis and establish infection in host cells. In vertebrate neutrophils and human promyelocytic cells HL-60, both pro-apoptotic and anti-apoptotic factors have been reported. Tissue-specific differences in tick response to infection and differential regulation of apoptosis pathways have been observed in adult female midguts and salivary glands in response to infection with A. phagocytophilum. In tick midguts, pathogen inhibits apoptosis through the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, while in salivary glands, the intrinsic apoptosis pathways is inhibited but tick cells respond with the activation of the extrinsic apoptosis pathway. In Ixodes scapularis ISE6 cells, bacterial infection down-regulates mitochondrial porin and manipulates protein processing in the endoplasmic reticulum and cell glucose metabolism to inhibit apoptosis and facilitate infection, whereas in IRE/CTVM20 tick cells, inhibition of apoptosis appears to be regulated by lower caspase levels. These results suggest that A. phagocytophilum uses different mechanisms to inhibit apoptosis for infection of both vertebrate and invertebrate hosts. Anaplasma phagocytophilum is an obligate intracellular bacterium transmitted primarily by Ixodes spp. ticks with a great impact on both human health and animal production worldwide [A. phagocytophilum is a cocoid, gram negative bacterium that infects host immune cells, mainly neutrophils, and endothelial cells of vertebrate hosts. Inside the cell, it forms dense intracellular microcolonies called morulae [A. phagocytophilum has a wide host range that coincides with the widespread distribution of the tick vectors [A. phagocytophilum have been reported which vary in host preferences, host responses, and tick vectors, with differences between ruminant and human/dog strains [A. phagocytophilum include human granulocytic anaplasmosis (HGA) and equine and canine granulocytic anaplasmosis [orldwide ,2. A. ph morulae . A. phag vectors ,5,6. Dif strains ,7,8,9. T strains . Diseaselasmosis ,2. Tick lasmosis ,3. The pA. phagocytophilum has evolved mechanisms to subvert host response to facilitate infection, multiplication, and transmission [Intracellular bacteria infecting host immune cells have evolved to avoid destruction within the cells. The mechanisms employed by these intracellular pathogens include diverse molecular pathways to invade cells and manipulate host defence mechanisms ,12. Apopsmission . These msmission ,19,20,21A. phagocytophilum employs common strategies to infect vertebrate host and tick cells, including the inhibition of cell apoptosis, but the infection also affects mechanisms involving other genes and proteins that have been described only in vertebrate host cells [I. scapularis ticks except for the presence of the perforin ortholog [st cells . Signallst cells —both of st cells . There iortholog . Additioortholog ,25,26,27A. phagocytophilum infection on the inhibition of cell apoptosis, which appears to be a key adaptation mechanism to facilitate infection and survival of A. phagocytophilum in both vertebrate hosts and ticks.In this review, we focused on the effect of A. phagocytophilum is emerging as a human pathogen in the U.S., Europe and Asia [A. phagocytophilum in humans has been detected in neutrophils and bone marrow progenitor cells, and to a lesser extent in monocytes and macrophages [A. phagocytophilum proteins [A. phagocytophilum infection delays neutrophil apoptosis in vivo [A. phagocytophilum has the ability to inhibit both extrinsic and intrinsic apoptotic pathways in neutrophils at multiple levels. A. phagocytophilum inhibits the intrinsic pathway of the spontaneous neutrophil apoptosis by protecting the mitochondrial membrane integrity [A. phagocytophilum Type IV secretion system (T4SS) [bcl-2 gene and degradation of the X-linked inhibitor of apoptosis protein , alongside with inhibition of mitochondria-mediated apoptotic caspase-3 processing and cleavage of pro-caspase-8, caspase-8, and caspase-9 have been reported [A. phagocytophilum also activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and kinases such as the phosphoinositide kinase-3 (PI3K)/protein kinase B (Akt) and p38 MAP kinase signalling [A. phagocytophilum regulates the expression of the anti-apoptotic protein myeloid leukemia cell differentiation Mcl-1, thus inhibiting apoptosis and stimulating IL-8 autocrine secretion, leading to the recruitment of neutrophils [A. phagocytophilum infection up-regulates the expression of anti-apoptotic members of the Bcl-2 family, blocks anti-CD95-induced programmed cell death in human neutrophils, and blocks clustering of CD95 at the cell surface during spontaneous neutrophil apoptosis, as determined by several microarray analyses [A. phagocytophilum to survive in a host cell-derived vacuole and replicate to develop into morulae within infected human cells [and Asia , where Hand Asia . Infectirophages . Neutropproteins ,27,29,30 in vivo  and infe in vivo . A. phagntegrity . Bacterim (T4SS) ,27. Ats-m (T4SS) ,27. The m (T4SS) . Activatreported ,33. A. pgnalling ,35. A. ptrophils ,33. A. panalyses  and is uA. phagocytophilum-induced apoptosis delay appears to be a neutrophil-specific process and not a global consequence of infection [A. phagocytophilum infection are quite complex and operate at different levels depending on the host cell type [The rickettsia can be propagated in the laboratory in undifferentiated human promyelocytic cells HL-60 and in HL-60 cells differentiated into neutrophil-like cells, potential precursors of the myelomonocytic lineage . In contnfection . Neverthnfection . For insnfection , thus suell type   gene was found down-regulated in experimentally-infected sheep, which had not been reported before [Apoptosis is inhibited in ovine cells as confirmed by an ex vivo study on ovine neutrophils infected in vivo with a sheep isolate of tophilum . Gene exnt hosts . For insd before ,45. Thesd before .A. phagocytophilum can control bacterial infection through the activation of innate immune responses and cytoskeleton rearrangement to promote phagocytosis and autophagy [A. phagocytophilum-induced apoptosis inhibition in human neutrophils [A. phagocytophilum infection in pigs may result in low infection levels or infection clearance, thus contributing to the low percentage of infection prevalence detected in this species in most regions and suggests that they may have a low impact as reservoir hosts for A. phagocytophilum [A. phagocytophilum evolved mechanisms to subvert innate immune responses and inhibit apoptosis in vertebrate hosts, some species may still activate innate immune protective mechanisms to control infection, therefore highlighting species-specific differences in host response to infection [In fact, pigs naturally and experimentally infected with utophagy . Gene extrophils ,35,46. Ctophilum . These rnfection .A. phagocytophilum is transmitted by several tick species [Ixodes ricinus complex, I. scapularis, and I. pacificus in the USA [I. ricinus in Europe [A. phagocytophilum infection of ticks and tick cells, but tissue-specific differences in tick response to infection and differential regulation of apoptosis pathways have been observed [A. phagocytophilum infection inhibits cell apoptosis through the activation of the JAK/STAT pathway and inhibits the mitochondrial intrinsic apoptosis pathway to establish infection in I. scapularis tick midguts and salivary glands. Reactive oxygen species (ROS)-mediated damage to midgut epithelial cells results in activation of the JAK/STAT pathway, which in turn inhibits apoptosis that facilitates infection of tick salivary glands [A. phagocytophilum infection of I. scapularis nymphs [A. phagocytophilum inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating porin (voltage-dependent anion-selective channel) expression, resulting in the inhibition of cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection [A. phagocytophilum infection in ticks. The α-fodrin (spectrin α-chain) is also involved in A. phagocytophilum infection/multiplication and the tick cell response to infection in I. scapularis [ species , ticks o the USA  and I. rn Europe  being thn Europe ,49. Tickobserved ,24,50. Aobserved  recentlyy glands . The JAKnfection . Porin infection . Thus, bnfection ,54 appeaapularis . The patA. phagocytophilum can be maintained in the tick cell lines—IDE8 and ISE6—that were originally derived from I. scapularis embryos [I. ricinus embryos [Anaplasma interactions, differences exist between cultured tick cells and in comparison with tick tissues in the apoptotic response to pathogen infection [I. scapularis ISE6 cells resembles that of tick hemocytes, while the response in I. ricinus IRE/CTVM20 cells appears to be more closely related to that reported previously in infected tick midguts [A. phagocytophilum infection inhibits ISE6 tick cell apoptosis through down-regulation of porin expression, causing lower cytochrome c protein levels to inhibit the intrinsic apoptosis pathway and facilitate infection [neogenin in ISE6 tick cells [I. scapularis nymphs or adult female midguts and salivary glands in response to A. phagocytophilum infection [ embryos ,56,57, a embryos ,59. Althnfection ,51,59,60ck cells . Howevernfection   and up-regulation of Janus kinase (JAK) involved in activation of the JAK/STAT pathway, up-regulation of FAS implicated in the extrinsic apoptosis pathway, and up-regulation of cytochrome c and bcl-2 interacting protein of the intrinsic apoptosis pathway [FAS suggests a possible effect of A. phagocytophilum infection on the inhibition of the extrinsic apoptosis pathway [I. scapularis tick salivary glands and ISE6 cells, but not in I. ricinus IRE/CTVM20 cells [I. ricinus IRE/CTVM20 cells appear to be more similar to I. scapularis midguts after infection with A. phagocytophilum [I. ricinus IRE/CTVM20 infected cells  and glucose metabolic pathways by lowering protein levels of phosphoenolpyruvate carboxykinase (PEPCK), mitogen-activated protein kinase (MKK), and apoptosis signal-regulating kinase 1 (ASK1), resulting in the inhibition of tick cell apoptosis in order to increase pathogen infection [A. phagocytophilum induces protein misfolding to counteract the tick cell response and facilitate infection, but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells [A. phagocytophilum may benefit from the tick cell ability to limit rickettsial infection through PEPCK inhibition, leading to decreased glucose metabolism and the availability of essential metabolites for bacterial growth, which also results in the inhibition of cell apoptosis that increases infection of tick cells [In nfection . A. phaged cells . These red cells . Additiock cells  (Figure A. phagocytophilum infection also manipulates I. scapularis tick cell epigenetics, as demonstrated by Cabezas-Cruz, et al. [A. phagocytophilum manipulates tick HMEs to regulate transcription and apoptosis in a tissue-specific manner to facilitate infection, but preserving tick fitness to guarantee survival of both pathogens and ticks [, et al. . Their rnd ticks .A. phagocytophilum has evolved common molecular mechanisms—including the inhibition of cell death—to establish infection in tick vectors and vertebrate hosts as a result of co-evolution and adaptation to a large number of tick and reservoir host species [A. phagocytophilum during host-pathogen and tick-pathogen interactions will provide insights into new strategies for the prevention and control of HGA and TBF.Intracellular bacteria elicit a diverse range of host protective responses. Amongst them, host cell death is critical to influence disease outcome.  species ,15,62. U