AbstractFothergilla is a small genus of deciduous shrubs native to the southeastern United States that depending on circumscription comprises two to four species. Recent treatments recognized only two species in the genus: F.gardenii (tetraploid) and F.major (hexaploid). Until recently, no diploid taxon of Fothergilla was known. However, recent investigations identified a number of diploid populations in Alabama, Florida, Georgia, and South Carolina. A subsequent phylogenomic analysis showed that the diploids segregated into two, well-supported lineages, corresponding to largely allopatric populations. A re-examination of the morphology of diploid plants, in combination with the genetic evidence, has led us to the recognition of two species of diploids in the genus – a resurrected F.parvifolia and a new species  – bringing the total number of recognized species in Fothergilla to four. A revised taxonomic treatment of the genus is provided. Fothergilla L.  is a small genus of deciduous shrubs native to the southeastern United States that depending on circumscription comprises two to four species . F.major based on “having a spike of flowers, three inches or more in length; [...] later flowering, and [...] leaves [...] very broad, and much more toothed” , leaf base (cordate in F.parvifolia vs. cuneate to rounded in F.gardenii), and leaf margin (toothed from below the middle to the apex in F.parvifolia vs. toothed only near the apex in F.gardenii). This circumscription was followed by F.parvifolia as a synonym of F.gardenii (F.gardenii (incl. F.parvifolia) and F.major (incl. F.monticola) (Kearney in  segregatgardenii . In factnticola) .Fothergillagardenii is found in wet savannas and pocosins in the coastal plains of North Carolina, South Carolina, Georgia, Florida, and Alabama, whereas F.major occurs primarily in woodlands, bluffs, and riverbanks of the upper Piedmont and mountains of North Carolina, South Carolina, Georgia, Alabama, Tennessee, and Arkansas , smaller leaves (<5.2 cm wide vs. > 5.2 cm wide in F.major), leaf dentations , base symmetry (symmetric vs. asymmetric in F.major), hypanthium length (3–4.5 mm vs. 4–9.2 mm in F.major), number of stamens per flower (12–24 vs. 22–32 in F.major), and seed size (4.8–6.3 mm long vs. 6.2–7.8 mm long in F.major)  . Weaver F.major and F.gardenii sensu n = 5x = 60) and named the nothospecies F.×intermedia Ranney & Fantz, a finding that cleared up previous controversy as to whether common cultivars (such as ‘Mount Airy’) represented F.major or F.gardenii. No pentaploids have been identified in nature.Although Fothergilla was known. However, recent sampling and cytometric analysis identified a number of diploid populations in Alabama, Florida, Georgia, and South Carolina (F.gardenii and F.major and their relationship to the diploid populations (F.gardenii and F.major). Furthermore, the diploid OTUs segregated into two, well-supported lineages, corresponding to largely allopatric populations. A re-examination of the morphology of diploid plants, in combination with the genetic evidence, has led us here to the recognition of two species of diploids in the genus: a resurrected F.parvifolia and a new species (F.milleri) as described below. A revised taxonomic treatment of the genus is provided.Until recently, no diploid taxon of Carolina . This woulations . These aFothergilla from throughout the southeastern United States, planted and grown in a common garden at the Mountain Horticultural Crops Research and Extension Center in Mills River, North Carolina (Table PSC) sensu IW), and (2) the length of the midvein interval between the junction of the midvein and lowermost secondary vein and the junction of the midvein and the next-most distal secondary vein on the same side of the leaf . We also searched the SERNEC portal  to identify any additional specimens of the diploid taxa we recognized. This search resulted in six additional specimens, which we added to the list of exsiccatae in the taxonomic treatment below . The combined sets of specimens of known ploidy (Table Specimens studied in the course of preparing this revision included: 1) 34 accessions of  34 accesPopulation Aggregation Analysis revealed four distinct aggregate profiles, each corresponding to one of the major lineages identified by F.parvifolia Kearney , the shape is actually a combination of a short cuneate section adjoining the petiole that broadens out laterally into the more general cordate or rounded shape. We here use the terms V-cordate and V-rounded to describe this type of base (the “V” representing the cuneate section) and consider it structurally distinct from the neatly cordate leaf bases of F.parvifolia , F.major (6x), F.milleri (2x), and F.parvifolia (2x). An updated taxonomic treatment follows.Based on these results, Taxon classificationPlantaeSaxifragalesHamamelidaceaeL., Syst. Veg. ed. 13. 418. 1774F9AB60B8-9179-5DF2-813D-AEFB92A8716DFothergillagardenii L., Syst. Veg. ed. 13. 418. 1774 [as F.Gardeni]  × 0.9–2.7(–4.3) mm; petioles 3.9–10.5 mm long, usually ¾ the length of the IL or longer, brown-yellow pubescent; blades mostly spreading, green, narrowly ovate to ovate, (1.8–)2.8–8.7 × (1.0–)2.5–5.3 cm, pinnately 8–11-veined, bases asymmetrical or symmetrical, usually rounded to cuneate, sometimes shallowly cordate, margins crenate to serrate above middle, teeth 3–10, apices acute to obtuse, both surfaces stellate-pubescent, rarely glabrous, abaxial surface sometimes glaucous, IW:IL < or = 0.49 (x̄ = 0.33). Inflorescences appearing before leaves, spikes terminal, appearing lateral on short lower branches, sessile or on short peduncles. Flowers: stamens 10–24, filaments 3.6–13.8 mm long. Capsules 6.6–9.0 × 5.5–6.6 mm. Seeds white to mottled brown or red-brown, ellipsoid to slightly ovoid, 5.1–5.8 × 2.5–3.5 mm, apices mostly obtuse. Genome size and ploidy 3.33–3.76 pg, tetraploid (2n = 4x = 48).Flowering beginning late Mar; fruiting by late Apr through Jul.Acerrubrum L., Amelanchierobovalis (Michx.) Ashe, Aroniaarbutifolia (L.) Pers., Clethraalnifolia L., Cyrillaracemiflora L., Gaylussaciadumosa (Andrews) Torr. & A. Gray, G.tomentosa (A. Gray) Pursh ex Small, Ilexcoriacea (Pursh) Chapm., I.glabra (L.) A. Gray, I.vomitoria Aiton, Kalmiacuneata Michx., K.latifolia L., Liquidambarstyraciflua L., Lyonia Nutt. spp., Magnoliavirginiana L., Perseapalustris (Raf.) Sarg., Pinusserotina Michx., Pteridiumaquilinum (L.) Kuhn, Quercuslaevis Walter, Q.virginiana Mill., Rhododendronviscosum (L.) Torr., Vacciniumcrassifolium Andrews, V.myrsinites Lam., and Zenobiapulverulenta (W. Bartram ex Willd.) Pollard (fide colectoris).This species can be found along the Atlantic coastal plains of North Carolina, South Carolina, and Georgia Fig. . It occuFothergillagardenii was apparently cultivated in England as early as 1765, grown at Kew Gardens by 1789, and English and French plant nurseries were offering seeds for sale by the early 1800s  (mblebees . The autrphidae) .Fothergillagardenii s.l. is considered Vulnerable, currently ranked by NatureServe as follows: G3G4; Alabama (S1), Florida (S1), Georgia (S2), Mississippi (SNR), North Carolina (S3S4), South Carolina (SNR) . In light of the removal of the diploid taxa F.milleri and F.parvifolia from the broader historical concept of F.gardenii, the conservation status of this species, as well as that of the diploid taxa, warrants re-evaluation; such re-evaluation is likely to result in a more imperiled ranking than the G3G4 current assessment, following removal of a significant number of populations and a narrower distribution following its narrower taxonomic circumscription. Due to its sensitive status, we provide only skeletal collections data below..Lynch 43 (NCSC); 2012-078, 13 Jun 2014 (V), Phillips 49 (NCSCm); 12 Jul 1997 (V), Sorrie 9349 (NCUm). McDuffie: Jun 1911 (V), Bartlett 2636 .Georgia. Effingham: 2012-078, 15 Apr 2014 (FL), Coker s.n. (NCU); 14 May 1966 (FL), Blair 415 (NCSC); 21 Jun 1965 (FR), Sawyer 2475 (NCUm); 5 Nov 1956 (V), Ahles & Leisner 21471 (NCUm); 20 Apr 1957 (FL), Ahles & Ramseur 23593 (NCUm); 31 May 2003 (FR), Horn 4437 (BRITm); 25 Jan 1937 (FL), Melvin 3613 (BRITm); 7 Jul 1994 (V), Nifong 400 (NCUm). Brunswick: 8 Jun 1951 (FR), Boyce & Wells 1656 (NCSC); 13 May 1950 (FL), Godfrey & Wiebe 50341 ; 18 Apr 1999 (FL), Hill 31327 (BRIT); 6 Mar 1974 (V), Kologiski 54 (NCSCm); 7 May 1974 (FL), Kologiski 125 (NCSC); 23 Jul 1974 (V), Kologiski 311 (NCSC); 6 Jun 1975 (FR), Kologiski 423 (NCSC); 27 Apr 1976 (FL), Kologiski 551 (NCSC); 18 Apr 1965 (FL), Mullen s.n. (NCSC). Carteret: 2011-096, 26 Mar 2012 (FL), Lynch 9 (NCSC); 2011-096, 2 Jul 2012 (V), Lynch 76 (NCSCm); 2011-096, 13 Jun 2014 (FR), Phillips 50 (NCSC); 2011-103, 21 Mar 2012 (FL), Lynch 3 (NCSC); 2011-103, 2 Jul 2012 (V), Lynch 75 (NCSCm); 22 May 1976 (V), Wilson 1792 (NCUm); 9 Apr 1977 (FL), Wilson 3067 (NCU). Columbus: 25 Apr 1958 (FL), Bell 11417 (NCU). Craven: 19 Apr 1958 (Fl), Radford 31924 (NCU). Cumberland: 11 Oct 1957 (V), Ahles 36621 (NCUm); 28 Apr 1933 (FL), Tallin & Harbison s.n. (NCU). Duplin: 27 Apr 1957 (V), Ahles & Ramseur 23990 (VDBm); 27 Apr 1957 (FR), Radford & Ramseur 23990 (NCUm). Harnett: 10 Apr 1957 (FL), Laing 843 (NCU); 30 Jun 2005 (FR), Sorrie 11634 (NCUm). Hoke: 2011-097, 21 Mar 2012 (FL), Lynch 4 (NCSC); 2011-097, 2 Jul 2012, (V), Lynch 72 (NCSCm); 2011-097, 13 Jun 2014 (FR), Phillips 71 (NCSC); 16 May 1976 (V), Kral 58099 (VDB m); 26 Jun 1975 (FR), Ahles & Haesloop 29627 (NCUm). Lee: 19 Apr 1958 (FL), Stewart 149 (NCU); 7 Jun 1958 (FR), Stewart 451 (NCUm). Montgomery: 9 Oct 1956 (V), Radford 19633 (NCUm). Moore: 17 Jul 1942 (FR), Wicker s.n. (NCUm); 8 Apr 1973 (FL), Carter 449 (NCU); 24 Apr 1949 (FL), Woods 2256 (NCSC). New Hanover: 6 Jun 1929 (FR), Wells s.n. (NCSCm); 29 Jun 1963 (V), McCrary 607 (NCU m); 30 Mar 1991 (FL), Pyne & Seneca 91-013 (NCSC). Onslow: 11 May 1948 (FR), Boyce & Moreland 647 (NCSC); 28 Apr 1951 (FL), Beaman s.n. (NCSC); 2 June 1948 (FR), Boyce & Moreland 700 (NCSCm); 24 Jun 1965 (FR), Wilbur 8398 (BRITm). Pamlico: 12 Oct 1957 (V), Radford 42285 (NCUm). Pender: 17 May 1925 (FR), A.C.W. s.n. (NCSC); 1 Jun 1945 (V), Wells s.n. (NCSC); 19 Apr 1957 (FL), Ahles & Ramseur 23440 (NCU); 25 Apr 1947 (FL), Fox & Wells 162 (NCSC); 12 May 1951 (FR), Fox 4621 (NCSC); 26 Aug 1983 (V), Leonard 8199 (UWFPm); 14 Apr 1925 (FL), Wells s.n. (NCSC). Richmond: 2011-123, 11 Apr 2012 (FL), Lynch 13 (NCSC); 2011-123, 21 Aug 2012 (V), Lynch 74 (NCSCm); 2011-085, 21 Mar 2012 (FL), Lynch 5 (NCSC); 2011-085, 2 Jul 2012 (V), Lynch 73 (NCSC); 2011-085, 13 Jun 2014 (V), Phillips 55 (NCSCm); 19 May 2007 (FL), Boyle 1 (NCU). Robeson: 18 Apr 1956 (FL), Terrell 3019 (NCU). Sampson: 10 Apr 1938 (FL), Godfrey 3394 (NCSC). Scotland: 20 Apr 1999 (FL), Hill 31345 (BRITm); 20 Jun 1957 (FR), Ahles & Haesloop 28637 (NCU); 20 Jun 1957 (FR), Ahles & Haesloop 28601 (NCUm); 4 Jun 2004 (FR), Sorrie 11264 (NCUm).North Carolina. Beaufort: 27 Mar 1949 (FL), Duncan 5923 (NCSC). Charleston: 2012-075, 15 Apr 2014 (FL), Lynch 42 (NCSC); 2012-075, 13 Jun 2014 , Phillips 48 (NCSC m); 2012-77, 15 Apr 2014 (FL), Lynch 37 (NCSC); 2012-077, 21 Aug 2012 (V), Lynch 30 (NCSCm); 2012-77, 13 Jun 2014 (FR), Phillips 53 (NCSC); 2 Apr 1944 (FL), Duncan 5885 (NCSC). Chesterfield: 25 Apr 1968 (FR), Ewel 666 (NCSC); 5 Jun 1956 (V), Radford 12431 (NCUm); 5 Apr 1968 (FL), Leonard & Radford 1219 (NCU). Clarendon: 20 Apr 1957 (FL), Radford 21097 (NCU). Colleton: 5 Apr 1956 (FL), Bell 1862 (NCU); 17 Apr 1974 (FL), Hardin 13420 . Darlington: 25 Mar 1935 (Fl), Matthews s.n. (NCU); 26 Mar 1935 (FL), Matthews & Smith s.n. (NCU); 10 Apr 1940 (FL), Smith 1378 (NCU). Dillon: 7 Apr 1940 (FL), Radford & Stewart 56 (NCU). Dorchester: 20 Jul 1957 (V), Ahles & Leisner 31966 (NCUm). Georgetown: 18 Apr 1987 (FL), Taggart 78 (NCU); Near Georgetown, 28 Jul 1928 (FR), Ashe s.n. (NCUm). Horry: 2012-076, 15 Apr 2014 (FL), Lynch 41 (NCSC); 2012-076, 13 Jun 2014 , Phillips 51 (NCSC m). Lee: 26 Jul 1957 (V), Radford 27396 (NCUm). Marlboro: 4 May 1968 (FL), Leonard & Radford 1218 (NCU).South Carolina. Berkeley: 9 Apr 1944 (FR), Taxon classificationPlantaeSaxifragalesHamamelidaceae2.Lodd., Bot. Cab. 16: Pl. 1520. 18298DC644F4-B01B-567E-B9EB-DF10E90AE717F.monticolaW.W. Ashe 1509  Ashe, Bot. Gaz. 24: 374. 1897. Type. North Carolina, mountains, C. Loddiges Illustration Pl. 1520. Figs Shrub, erect, robust, frequently >1 m tall (to 8 m); stems in clumps of 3 or more, branching. Leaves: stipules ovate to lanceolate, 3.5–11.8 × 1.9–4.0 mm; petioles 8.3–17.1(–18.9) mm, ½ as long or longer than the IL, brown-yellow pubescent; blades mostly spreading, green, broadly ovate or elliptic to suborbiculate, rarely obovate, 3.6–13.7 × 3.2–13.0 cm, most within the upper end of those ranges, pinnately 7–12-veined, bases oblique, occasionally symmetrical, usually V-cordate, rarely nearly rounded, margins crenate or serrate to nearly entire, toothed from at or below middle to the apex, teeth 11–24, apices acute to obtuse, often pubescent, both surfaces glabrous to sparsely stellate-pubescent, abaxial surface sometimes glaucous, IW:IL > or = 0.51 (x̄ = 0.96). Inflorescences usually appearing with leaves, spikes sessile or on short peduncles. Flowers: stamens 14–32, filaments 7.3–16.2 mm long. Capsules 6.8–12.7 × 6.5–8.6 mm. Seeds nearly white to completely brown or red-brown, usually ellipsoid, 5.3–7.4 × 2.7–3.6 mm, apices rounded to obtuse. Genome size and ploidy 5.21–5.25 pg, hexaploid (2n = 6x = 72).Flowering beginning late Mar; fruiting late May through mid-Jul.F.major can be found growing in seepage community with Coastal Plain-affinity species. In this habitat, where burning is frequent, this species may be <1 m in height. However, height variance is apparently not simply a factor of fire frequency. Fothergillamajor varies considerably in habit, ranging from colonial shrubs less than 2 m tall, typically found growing on sandy dampish sites in the southwestern part of the range and lower elevations  to larger tree forms growing to 6 m, with offsets but no rhizomes, typically found in more upland mountain dry forest sites with igneous geology found to the North and East . Commonly associated upland species include Acerrubrum, Aesculussylvatica W. Bartram, Alnusserrulata, Aroniaarbutifolia (Aiton) Willd., Calycanthusfloridus L., Carpinuscaroliniana Walter, Cornusflorida L., Gaylussaciafrondosa (L.) Torr. & A. Gray, Hamamalisvirginiana L., Hepaticaamericana (DC.) Ker Gawl, Hypericumnudiflorum Michx. ex Willd., Ilexopaca Aiton, Kalmialatifolia, Liquidambarstyraciflua, Liriodendrontulipifera L., Magnoliaacuminata (L.) L., Polystichumacrostichoides (Michx.) Schott, Quercusalba L., Q.rubra L., Rhododendroncatawbiense Michx., and Stewartiaovata (Cav.) Weath. (fide colectoris).This species can be found in the mountains and Piedmont of Alabama, Arkansas, Georgia, North Carolina, South Carolina, and Tennessee Fig. . HabitatFothergillamajor is considered Vulnerable rangewide, ranked by NatureServe as follows: G3; Alabama (S2), Arkansas (S1), Georgia (S1), North Carolina (S3), South Carolina (S2), Tennessee (S2); http://explorer.natureserve.org/, accessed 11 Dec 2019). Due to its sensitive status, we provide only skeletal collections data below..Lynch 91 (NCSCm). Cherokee: 12 Apr 1969 (FL), Kral 34269 ; 10 May 1970 (FL), Kral 39052 (BRITm); 14 Jul 1966 (FR), Clark & Landers 5122 . Cullman: 17 Apr 1931 (FL), Ashe s.n. (NCU); 16 Apr 1924 (FR), Wolf s.n. (AUAm); 4 Jul 1911 (FR), Wolf 813 (AUAm); 28 May 1931 (V), Wolf s.n. (VDB). DeKalb: 2008-009, 21 Mar 2012 (FL), Lynch 7 (NCSC); 2008-009, 2 Jul 2012 (V), Lynch 81 (NCSCm); 14 Jul 1966 (FR), Clark & Landers 5023 ; 22 May 1972 (FL), Whetstone 1935 (NCUm). Marshall: 2011-092, s.d. (FL), Lynch 79 (NCSC); 2011-092, 2 Jul 2012 (V), Lynch 17 (NCSCm); 2011-147, 21 Aug 2012 (V), Lynch 24 (NCSCm); 15 Apr 1973 (FL), Kral 49636 . St. Clair: 31 May 1947 , McVaugh 8594 (BRIT m): Unspecified: n.d. (FL), Buckley s.n. (FLAS).Alabama. Blount: 2011-093, 2 Jul 2012 (V), Lynch 10 (NCSC); 2011-082, 2 Jul 2012 (FR), Lynch 80 (NCSCm).Arkansas. Searcy: 2011-082, 26 Mar 2012 (FL), Duncan & McDowell 12200 (NCU); 30 Mar 1951 (FL), Duncan & McDowell 12200 ; 6 May 1951 (FL), Duncan & Venard 12339 (BRIT m). Fulton: 2011-169, 21 Aug 2012 (V), Lynch 28 (NCSC). Lumpkin: 2011-164, 21 Aug 2012 (V), Lynch 27 (NCSCm). Walker: 2011-146, 21 Aug 2012 (V), Lynch 25 (NCSC); 15 Apr 1986 (FL), Coile 4547 (NCSC).Georgia. Bartow: 30 Mar 1951 (FL), Gavalak 3306V90 (BRIT m).Illinois. DuPage: Cultivated at Morton Arboretum, 9 May 1990 (FL), Dewolf & Bruns 2235 (BRIT).Massachusetts. Suffolk: Cultivated at Arnold Arboretum, Harvard University, 13 May 1968 (FL), Gillis 15041 (BRIT m).Michigan. Ingham: Cultivated at Michigan State University, 9 May 1979 (FL), Lynch 81 (NCSCm); 7 Oct 1966 (V), Downs 408 (NCSC); 2 Aug 1977 (FR), Kral 60712 (VDBm); 25 Aug 1989 (FR), Lance & Wood s.n. (NCUm); 1 Sep 1952 (FR), Radford 6676 (NCUm); 9 Sep 1976 (V), Smith 182 (NCSC); 5 Jul 1940 (FR), Stewart 1554 (NCU); 27 May 1964 (FR), Wilbur 7012 (VDBm). Chatham: 25 Apr 1988 (FL), Swab 75 (NCSC); 4 May 1988 (FL), Swab 97 (NCSC); 28 May 1988 (FR), Swab 236 (NCSC). Gaston: 19 May 1919 (FR), Coker s.n. (NCUm). Harnett: 18 Apr 2006 (V), Sorrie & Hart 11770 (NCUm). Montgomery: 2011-122, 21 Aug 2012 (V), Lynch 23 (NCSCm); 11 Oct 2002 (FR), Diamond 1606 (NCUm); 14 Jul 2004 (V), Schwartzman 30 (NCUm); 20 Jul 2004 (V), Weakley s.n. (NCUm); 15 Oct 2004 (V), Weakley s.n. (NCU). Orange: 2011-124, 19 Apr 2012 (FL), Lynch 14 (NCSC); 2011-124, 2 Jul 2012, Lynch 78 (NCSCm); 2011-124, 24 Jun 2014 (FR), Phillips 62 (NCSC); Apr 1899 (FL), Ashe s.n. (NCU); Jun 1899 (FR), Ashe s.n. (NCU); 31 Apr 200 (FL), Wally & Wichmann 125 (NCUm); 4 May 1910 (FR), Clerces s.n. (NCU). Person: 7 Jul 2005 (FR), LeGrand s.n. (NCUm). Polk: 30 May 1930 (FL), Ashe s.n. (NCSC); 20 Apr 1897 (FL), Biltmore 6565 (BRITm); 20 Apr 1897 (FL), Biltmore 708 (NCU). Randolph: 22 Apr 1958 (FL), Melvin s.n. (NCU). Rutherford: 2011-163, 21 Aug 2012 (V), Lynch 26 (NCSCm); 2011-163, 15 Apr 2013 (FL), Lynch 39 (NCSC); 2011-163, 24 Jun 2014 (FR), Phillips 64 (NCSC); 21 Apr 1956 (FL), Bell 2118 (NCU). Stokes: 23 Apr 1950 (FL), Fox et al. 3565 (NCSC); s.d. (FR), Harbison s.n. (NCU); 5 May 1936 (FL), Hunt 13474 ; 7 Jul 1969 (FR), Leonard & Russ 2553 (NCUm); 21 Apr 1974 (FL), Massey 3900 (VDB); 26 Apr 1958 (FL), Matthews 54 (BRIT); 15 Jun 1967 (FR), Radford 45392 ; 4 Jun 1958 (FR), Radford 34675 (VDB m); 9 Apr 1933 (FL), Schallert 3613 (BRIT). Surry: 19 May 1935 (V), Harbison s.n. (NCU). Transylvania: 2011-112, 2 Jul 2012 (V), Lynch 16 (NCSC); 2011-112, 24 Apr 2014 (FL), Lynch 47 (NCSC); 2011-112, 24 Jun 2014 (FR), Phillips 63 (NCSC); 2011-131, 21 Aug 2012 (FR), Lynch 22 (NCSCm); 19 Jun 1965 (FR), Eggers 1262 (VDB m); 26 Jul 1962 (FR), Rodgers 62064a (NCUm); 24 May 2006 (FR), Schwartzman 29 (NCUm). Wake: 28 Oct 2005 (V), Bruhn s.n. (NCU).North Carolina. Burke: 2011-105, 2 Jul 2012 (V), Fogg 1 (UWFP).Pennsylvania. Marion: Cultivated at the Arboretum of the Barnes Foundation, 27 Apr 1968 (FL), Kral 58130 (VDB m); 15 Oct 1988 (V), Hill 20066 (BRIT m); 23 May 1996 (V), Townsend 874 (VDB m). Oconee: 2011-091, 28 Mar 2012 (FL), Lynch 12 (NCSC); 2011-091, 2 Jul 2012 (V), Lynch 77 (NCSCm); 26 Apr 1965 (FL), Radford 44707 (NCU); 20 Apr 1973 (FL), Rogers & Green 73078 (FLAS-2). Pickens: 15 June 1992 (FT), Hill 23387 (BRIT m);South Carolina. Greenville: 17 May 1976 (FR), Sharp 556 ; 30 Apr 1931 (FL), Jennison & Sharp s.n. (NCU); May 3 1936 (Fl), Sharp & Underwood 4205 . Greene: 17 May 1970 (FL), Sharp et al. 45204 . Scott: 2012-065, 21 Aug 2012 (V), Lynch 29 (NCSC m); 2012-065, 15 Apr 2014 (FL), Lynch 44 (NCSC); 23 Apr 1972 (FL), Clebsch s.n. (VDB); 19 Apr 1979 (FL), Whitten & Noss s.n. (FLAS-2); 23 Jun 1978 (FR), Wofford et al. 78-112 (VDB m). Sevier: 1 May 1960 (Fl), Sharp 26818 ; 25 Apr 1970 (FL), Williams 82 (AUA m); 25 Apr 1970 (FL), Pippin 83 (VDB); 2 Apr 1938 (FL), Jennison 53 (VDB m); 26 Apr 1957 (FL), Sharp 21575 (BRIT m).Tennessee. Grainger: 20 Apr 1931 (FL), Meyer 37144 (FLAS).Washington D.C. U.S. National Arboretum, 16 Oct 1990 (V), Taxon classificationPlantaeSaxifragalesHamamelidaceae3.W.D.Phillips & J.E.Haynessp. nov.DC5E3CB6-8D0A-57AA-96CC-7D6838D9DC9Furn:lsid:ipni.org:names:77208304-1Nathan Lynch 70 . Figs Florida. Walton Co.: Voucher specimen from containerized plant at Mountain Horticultural Crops Research and Extension Center, Mills River, NC (leaves and stem collected 19/7/2012), , 2011-088, 19 Jul 2012 (V), Fothergillamilleri is morphologically most similar to F.gardenii, but differs from the latter by leaves held erect (vs. spreading in F.gardenii), blades blue-green or gray-green (vs. green in F.gardenii), petioles 1/3–1/2 the length of the IL (vs. ¾ the length of the IL or longer in F.gardenii), and seed apices acute to acuminate (vs. rounded or obtuse in F.gardenii).Shrub, rhizomatous, erect, to 1.5 m tall, clump-forming, multi-stemmed, branching. Leaves: stipules ovate to lanceolate, 2.5–7.8 × 1.0–3.0 mm; petioles 2.6–8.0 mm long, usually 1/3–1/2 the length of the IL, brown-yellow pubescent; blades erect, blue-green to gray-green, obovate, 3.2–8.0 × 3.0–4.8 cm, pinnately 8–10-veined, bases rounded to truncate, margins crenate to serrate above middle and mostly near the apex, sometimes appearing crenate to entire, apices mostly obtuse to acute, both surfaces conspicuously stellate-pubescent, sometimes sparsely so, abaxial surface not glaucous, IW:IL < or = 0.48 (x̄ = 0.29). Inflorescences appearing before leaves, spikes on short peduncles or sessile. Flowers: stamens 15–22, filaments 4.3–10 mm long. Capsules 7.0–9.0 × 5.0 –7.0 cm. Seeds usually completely brown to red-brown, ovoid, 4.5–6.2 × 2.4–3.2 mm, apices acuminate, often recurved. Genome size and ploidy: 1.70–1.78 pg, diploid (2n = 2x = 24).Flowering beginning late Mar; fruiting late May through the end of Jul.Cyrillaracemiflora or Taxodiumascendens swamp forests. Plant associates include Acerrubrum, Arundinariatecta  Muhl., Clethraalnifolia, Hibiscusaculeatus Walter, Hypericumcistifolium Lam., Juncustrigonocarpus Steud., Dichantheliumscoparium (Lam.) Gould., Osmundastrumcinnamomeum (L.) C. Presl, Rhexiavirginica L., Rhynchosporachalarocephala Fernald & Gale, and Xyrisfimbriata Elliott (fide colectoris).This species can be found in the panhandle of Florida, in Alabama in the Gulf coastal plain, and is also known from one county in Georgia Fig. . This spThe species is named in honor of Dr Ron Miller, Pensacola, Florida, who championed this project, originally suggested that diploid cytotypes might still exist, and ultimately found them. Dr Miller’s extensive effort and field work (with colleagues) provided the foundation for this research and establishment of ex situ living collections, including accessions in the U.S. National Plant Germplasm System.F.major. Consequently, only skeletal collections data are provided below.The conservation status of this species must be reassessed. It is presently known from only seven counties, and would appear to have an imperilment status at least as severe as that of .Lynch 1 (NCSC); 2011-087, 2 Jul 2012 (V), Lynch 68 (NCSCm); 2011-087, 13 Jun 2014 (FR), Phillips 58 (NCSC); 16 Jun 1984 (FR), Hedges 156 (UWFPm); 19 Apr 2001 (FR), Schotz 1830 (UNA [online!]). Covington: 25 Jun 1906 (FR), Harper 108 (NY [online!]). Escambia: 11 Apr 1964 (FL), Kral 19698 (AUAm). Geneva: 25 Jul 1968 (V), Kral 32029 (VDBm); 26 Apr 1969 (FL), Kral 34524 (VDBm); 7 Sep 1994 (V), Simmers s.n. (HTTU [online!]). Monroe: 15 Jun 1959 (V), Grelen s.n. (FLASm).Alabama. Baldwin: 2011-087, 21 Mar 2012 (FL), Lynch 2 (NCSC); 2011-083, 2 Jul 2012 (FL), Lynch 67 (NCSC); 2011-083, 13 Jun 2014 (FR), Phillips 57 (NCSCm); 20 Apr 1991 (FL), Burkhalter 12638 ; 5 Aug 1990 (V), Burkhalter 12211 (UWFPm); 13 Jul 1996 (V), Burkhalter 15064 (UWFP); 18 May 1993 (FR), Naczi 3028 (KNK [online!]). Walton: 8 Apr 1899 (FL), Biltmore Herbarium 7609b (FLASm); 2011-088, 28 Mar 2012 (FL), Lynch 11 (NCSU); 2012-060, 19 Mar 2013 (FL), Lynch 34 (NCSC); 2012-060, 2 Jul 2012 (V), Lynch 15 (NCSC); 2012-060, 13 Jun 2014 (FR) Phillips 59 (NCSC); 18 Aug 1990 (V), Orzell and Bridges 14757 .Florida. Okaloosa: 2011-083, 21 Mar 2012 (FL), Lynch 8 (NCSC); 2011-178, 2 Jul 2012 (V), Lynch 69 (NCSCm).Georgia. Taylor: 2011-178, 26 Mar 2012 (FL), Taxon classificationPlantaeSaxifragalesHamamelidaceae4.Kearney in Small, Fl. S.E. U.S. 509, 1331. 1903988E10E7-FD07-532B-8B40-80EC41C51904Kearney s.n. . Figs Georgia. Wayne Co., Jesup, dry soil, 4 June 1893 (FR), Shrub, rhizomatous, erect, to 1 m tall, clump-forming, multi-stemmed, branching. Leaves: stipules ovate to lanceolate, 3.5–6.0 × 8.0–13.0 mm, curved downward; petioles 4.4–17.8 mm long, usually nearly as long to longer than the IL, brown-yellow pubescent; blades drooping, green, mostly ovate, 3.2–12.1 × 3.0–6.2 cm, pinnately 8–10-veined, bases cordate, margins crenate to serrate from middle, apices acute, adaxial and abaxial surfaces not glaucous, stellate-pubescent, sometimes sparsely so, IW:IL > or = 0.62 (x̄ = 0.96). Inflorescences appearing before leaves, spikes on short peduncles or sessile. Flowers: stamens 15–20 per flower, filaments 6.6–9.3 mm long. Capsules 7.5–10.0 × 5.0 –7.6 mm. Seeds usually completely brown to red-brown, ovoid, apex obtuse, 4.5–6.2 × 2.4–3.2 mm, apices obtuse to acute. Genome size and ploidy 1.73–1.82 pg, diploid (2n = 2x = 24).Flowering Mar–May; fruiting May–Sep.This species range is restricted to Georgia, South Carolina, and Alabama Fig. . BecauseF.major. Consequently, only skeletal collections data are provided below.The conservation status of this species needs to be assessed. It is presently known from only eight counties, and would appear to have an imperilment status at least as severe as that of .Harbison 1033 (NCUm).Alabama. Montgomery: 12 Sep 1899 (FR), Cuthbert s.n. (FLAS). Brantley: 23 Aug 1947 (V), Thorne & Norris 6284 (GEO [online!]). Emanuel: 2011-170, 2 Jul 2012 (V), Lynch 19 (NCSCm); 2011-170, 15 Mar 2013 (FL), Lynch 33 (NCSC); 2011-170, 14 Jun 2014 (FR), Phillips 60 (NCSC). Long: 2011-171, 2 Jul 2012 (V), Lynch 20 (NCSCm); 2011-171, 14 Mar 2013 (FL), Lynch 32 (NCSC); 2011-171, 13 Jun 2014 (FR), Phillips 61 (NCSC); Tattnall: 2011-168, 19 Mar 2013 (FL), Lynch 35 (NCSC); 2011-168, 2 Jul 2012 (V), Lynch 18 (NCSCm). Wayne: 31 Aug 1904 (V), Biltmore Herbarium 14940 (NY [online!]).Georgia. Augusta-Richmond: 2 Apr 1898 (FL), Lynch 40 (NCSC); 2012-084, 13 Jun 2014 (V), Phillips 56 (NCSCm). Lexington: 13 Sept 1988 (V), Pittman 9139817 (BRITm); 27 May 1957 (FR), Radford 23378 (NCUm).South Carolina. Aiken: 2012-084, 15 Apr 2014 (FL),


Numerous individual studies have investigated the diagnostic value of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG detection for nasopharyngeal carcinoma (NPC), but the conclusions remain controversial. This meta-analysis aimed to determine the value of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG detection in the diagnosis of NPC.PROSPERO registration number: CRD42019145532. PubMed, EMBASE, Cochrane Library, and Chinese data libraries  were searched up to January 2019. The pooled sensitivity, specificity, and positive likelihood, negative likelihood, and diagnostic odds ratios were conducted in this meta-analysis. Summary receiver operating characteristic curves evaluated the test-performance global summary. Publication bias was examined by Deek’s funnel plot asymmetry test.EBV-DNA in diagnosis of NPC were: 0.76 (95% CI 0.73–0.77), 0.96 (95% CI 0.95–0.97), 14.66 (95% CI 9.97–21.55), 0.19 (95% CI 0.13–0.28), 84 (95% CI 50.45–139.88), 0.96 (SE: 0.001), and 0.55 (95% CI 0.54–0.57), 0.96 (95% CI 0.96–0.97), 12.91 (95% CI 9.55–17.45), 0.35 (95% CI 0.29–0.43), 39.57 (95% CI 26.44–59.23), 0.94 (SE: 0.002) for the EA-IgA, and 0.85 (95% CI 0.84–0.85), 0.89 (95% CI 0.88–0.89), 6.73 (95% CI5.38–8.43), 0.17 (95% CI 0.12–0.23), 43.03 (95% CI 31.51–58.76), 0.93 (SE: 0.007) for the VCA-IgA, and 0.86 (95% CI 0.85–0.88), 0.87 (95% CI 0.88–0.90), 7.55 (95% CI 5.79–9.87), 0.16 (95% CI 0.13–0.19), 50.95 (95% CI 34.35–75.57), 0.94 (SE: 0.008) for the EBNA1-IgA, and 0.70 (95% CI 0.69–0.71), 0.94 (95% CI 0.94–0.95), 9.84 (95% CI 8.40–11.54), 0.25 (95% CI 0.21–0.31), 40.59 (95% CI 32.09–51.35), 0.95 (SE: 0.005) for the Rta-IgG. The EBV-DNA had larger AUC compared with other EBV-based antibodies (P < 0.05), while the difference between EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG was not statistically significant (P > 0.05).Forty-seven studies with 8382 NPC patients (NPC group) and 15,089 individuals without NPC (Control group) were included in this meta-analysis. The sensitivity, specificity, positive likelihood (+ LR), negative likelihood (-LR), DOR and AUC of EBV-DNA, VCA-IgA, EBNA1-IgA and Rta-IgG detection have high accuracy in early diagnosis NPC. In addition, EBV-DNA detection has the higher diagnosis accuracy in NPC. On the other hand, EA-IgA is suitable for the diagnosis but not NPC screening.The online version contains supplementary material available at 10.1186/s12935-021-01862-7. Nasopharyngeal carcinoma (NPC) is the most common malignant tumor in head and neck surgery and it is highly prevalent in southern China and Southeast Asia . UnfortuPROSPERO registration number: CRD42019145532.Literature review was separately conducted by two investigators that queried online databases, including PubMed, EMBASE, Cochrane Library, and Chinese data libraries , and the search concluded in January 2019, using the following keywords: nasopharyngeal carcinoma, Epstein-Barr virus, capsid antigen-IgA, early antigen antibody, nuclear antigen antibody, BRLF1 transcription activator IgG, EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG.Studies that assessed the performance of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG detection for untreated NPC identification;All patients included in the study were diagnosed using a reference test ;Studies that used a pre-specified threshold;Studies that clearly stated the number of true positive, false positive, false negative, and true negative results in the diagnosis of NPC or these values could be calculated from the data;Studies that provided a clear definition of the control sources ;In cases of multiple reports describing the same population, the most recent or most complete report was selected.Reported results were insufficient for construction of the 2 × 2 table;Studies that failed to clearly define the control types;The NPC group contained other tumors;Basic research, review articles, comments, letters, case reports, abstracts in conference, responding letters and experimental animal studies.Study quality assessment was conducted using the diagnostic accuracy (QUADAS) II checklist . StudiesQ test and inconsistency index (I2) were used to estimate the heterogeneity within studies [P < 0.05 or I2 > 50%. If statistically significant heterogeneity existed, meta-analysis was performed using the random effects model, otherwise, a fixed effect model was used.Standard methods recommended for meta-analyses of diagnostic test evaluations were used to perform this meta-analysis . Review  studies . HeterogThe accuracy indexes of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG was pooled by meta-analysis, such as sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and AUC. The likelihood ratios (PLR and NLR) are clinically meaningful for the measurement of diagnostic accuracy; PLR > 10 and NLR < 0.1 are considered high . The DORP values were two sides and P < 0.05 was regarded as statistically significant.Sensitivity analyses were performed to explore the sources of heterogeneity of the included studies by removing each included study consecutively. The heterogeneity was investigated by meta-regression according to different covariates, including publication year (Year ≥ 2011 or < 2011), NPC size (NPC ≥ 100 or NPC < 100), control sources , detection method, and article language (English or Chinese). Publication bias was examined by Deek’s funnel plot asymmetry test. All In this meta-analysis, 47 publications on the role of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG concentrations in the diagnosis of NPC that met the criteria for inclusion were included in the analysis –58. FiguI2 = 77.5%, P < 0.001; EA-IgA:77.3%, P < 0.001; VCA-IgA: 87.0%, P < 0.001; EBNA1-IgA:78.9%, P < 0.001; Rta-IgG: 60.5%, P < 0.001) indicated significant heterogeneity among the studies. The result showed that there was no threshold effect in the pooled analysis of EBV-DNA (P < 0.001), EA-IgA (P < 0.001), VCA-IgA (P < 0.001), EBNA1-IgA (P < 0.001) and Rta-IgG (P < 0.001).The inconsistency index (EBV-DNA: P < 0.05), while EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG were no statistically different from each other (P > 0.05) in Table The pooled sensitivity, specificity, PLR, NLR, DOR and AUC for the value of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG in the diagnosis of NPC are displayed in Table P < 0.0095).The sensitivity analysis showed that the results were not affected by the exclusion of any individual trial. As meta-regression result indicated that publication year, NPC or control size, control sources, detection method, cutoff value, and article language are not the DOR heterogeneity of EBV-DNA, VCA-IgA, EBNA1-IgA and Rta-IgG, whereas detection method was possible DOR heterogeneity sources for the EA-IgA (P = 0.14), EA-IgA (P = 0.26), EBNA1-IgA (P = 0.56) and Rta-IgG (P = 0.16), other than VCA-IgA (P = 0.03)  and 0.89 (95% CI 0.88–0.90) [CI 0.90–0.92) and specificity 0.92 (95% CI 0.92–0.93), with SROC 0.98. But the Li’s study existed language bias and publication bias. For the Rta-IgG, Cui et al.pooled 17 studies involving 2658 NPC patients, and the results pointed out that the sensitivity of Rta-IgG for detecting NPC was 90.83 (95% CI 0.78–0.87), the specificity was 0.92 (95% CI 0.90–0.93) [Previous meta-analyses have been published on the value of some EBV-based markers in the detection of NPC. For the EBV-DNA, Han et al.conducted a meta-analysis based on 18 studies involving 1492 NPC cases and 2461 health controls in Asians, in which the pooled sensitivity and specificity of EBV-DNA detection for NPC were 0.73 (95% 88–0.90) . Further88–0.90) . The Han88–0.90) , which wP < 0.05). Additionally, the pooled DOR of EBV-DNA (84.00) that differed 33.05–44.43 was higher than other EBV-based antibodies. These results indicated that EBV-DNA detection had higher accuracy in diagnosis of NPC. In addition, a meta-analysis included 8128 NPC cases showed that pre-EBV-DNA levels can also be a prognostic indictor for patients with NPC [To our knowledge, this is the first meta-analysis to determine the usefulness of EA-IgA and EBNA1-IgA and compare the accuracy EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgA in diagnosis of NPC. In this study, the highest sensitivity was EBNA1-IgA (0.86), and the specificity of EBV-DNA (0.96) and EA-IgA (0.96) were highest. Besides, the sensitivity of EA-IgA (0.55) was lowest, and screening for NPC using only EA-IgA may lead to misdiagnosis, but the specificity was high, which indicated that EA-IgA was suitable for the diagnosis but not screening of NPC. EA-IgA or EBV-DNA detection combined with other indicators may also improve the sensitivity and specificity for the serological diagnosis of NPC , 51. Thewith NPC . A recenwith NPC , seventywith NPC . In addiwith NPC , 71. In with NPC , 72, it Several strengths of present meta-analysis should be highlighted. This study compered five EBV-related diagnostic markers for NPC with comprehensive calculations of their diagnostic performance, shedding light on the value of these tests in clinical settings. In the context of current availability of studies on EBV-DNA and immunoglobin antibody tests in the literature, this meta-analysis covers a large sample size pooled from rigorously included studies, and the results were stable. However, this meta-analysis should be interpreted with caution due to certain limitations. First, there was large heterogeneity among the included studies with differences in characteristics of the study and participants. The inability to obtain raw data on patient age and gender may have led to the heterogeneity and hindered a more detailed analysis. Second, most of the included populations were Chinese, which could lead to population selection bias and should not allow for generalization to other ethnicity groups, rendering further research needed. Third, technical methods for testing of EBV-related markers vary across different studies, including the inconsistent cut-off values and different antigen sets used despite of a same generic name. Use of enzyme-linked immunosorbent assay or immunofluorescence assay may have contributed to the different results . FinallyNotwithstanding heterogeneity of currently available data, the studies included in our analysis are of a large sample size and high-quality, thus providing a considerable power. EBV-DNA, VCA-IgA, EBNA1-IgA and Rta-IgG detection have high accuracy in early diagnosis NPC and can improve the effectiveness of screening. In addition, EBV-DNA detection has the higher diagnosis accuracy in NPC. On the other hand, EA-IgA is suitable for the diagnosis but not NPC screening. Further well-designed clinical trials need to be carried out in order to improve early diagnosis rate.Additional file 1: Forest plots of sensitivity, specificity, PLR, NLR, DOR for acoustic analysis of EBV-DNA, EA-IgA, VCA-IgA, EBNA1-IgA and Rta-IgG, and Funnel plots for publication bias test.


AbstractPhyllodiaptomus (Phyllodiaptomus) roietensissp. nov. was collected from temporary water bodies in Roi Et and Nakhon Ratchasima provinces in northeastern Thailand and Kampong Thom Province in central Cambodia. The new species is closely related to Phyllodiaptomus (P.) surinensis Sanoamuang & Yindee, 2001 in that it shares common morphological characters in the males: urosomites 2–3, P5 intercoxal sclerite, right P5 Exp-2, and left P5 Exp. Minor differences on the right antennule, right caudal ramus, P5 basis and Enp exist. The females differ in their Pdg 5, genital double-somite, and P5. An updated key to the species of the genus Phyllodiaptomus Kiefer, 1936 is provided. Phyllodiaptomus Kiefer, 1936, is among the most common freshwater copepods in Southeast Asia (Phyllodiaptomus (Phyllodiaptomus) blanci  from Uzbekistan; P. (Ctenodiaptomus) annae  from Sri Lanka; P. (P.) tunguidus Shen & Tai, 1964 from China; P. (P.) longipes Kiefer, 1965 from Indonesia; P. (C.) sasikumari Ranga Reddy & Venkateswarlu, 1989 and P. (C.) wellekensae Dumont & Ranga Reddy, 1993 from India; P. (C.) praedictus Dumont & Ranga Reddy, 1994, P. (P.) christineae Dumont, Ranga Reddy & Sanoamuang, 1996, P. (P.) surinensis Sanoamuang & Yindee, 2001, and P. (P.) thailandicus Sanoamuang & Teeramaethee, 2006 from Thailand; and P. (P.) irakiensis Khalaf, 2008 from Iraq. In addition, P. (C.) praedictussulawesensis as a subspecies of P. (C) praedictus from Indonesia; this subspecies was later found in the Philippines  roietensis sp. nov. from two localities in Roi Et and Nakhon Ratchasima provinces, northeast Thailand, and two localities in Kampong Thom Province in central Cambodia . After 10 min the animals were transferred to pure glycerol. The animals were dissected and prepared in a glycerin-mounted slide under a stereomicroscope at 40–100-x magnifications. The dissected specimens were mounted in pure glycerin on a glass slide and sealed under a cover glass with transparent nail varnish. All un-dissected specimens were stored in 70% ethanol in 1.5 mL microtubes.All appendages and body ornamentation were examined at 1000-x magnification under an Olympus CX31 compound microscope. The drawings were made using an Olympus U-DA drawing tube mounted on a compound microscope. The final versions of the drawings were made using the CORELDRAW 12.0 graphic program.SEM) were dehydrated in an ethanol series  for 15 min at each concentration. Specimens were dried in a critical-point dryer and were mounted on stubs using adhesive tape under a stereomicroscope. Dried specimens were coated with gold in a sputter-coater. The SEM photographs were taken using a scanning electron microscope (FEI Helios NanoLab G3 CX).Specimens for scanning electron microscopy  and at the Applied Taxonomic Research Center, Khon Kaen University (Thailand) (KKU).Specimens were deposited at the Abbreviations used in this paper are as follows:ae aesthetasc;Enp endopod;Exp exopod;Exp/Enp-n exopodal segment n/endopodal segment n;Pdg pediger;Pdg 1–5 pedigers 1–5;P1–P5 legs 1–5;sp spine.The descriptive terminology follows Diaptominae Kiefer, 1932Sub-family Phyllodiaptomus Kiefer, 1936Genus Phyllodiaptomus Dumont, Ranga Reddy & Sanoamuang, 1996Subgenus Taxon classificationAnimaliaCalanoidaDiaptomidae0B9F97DA-0E9E-5345-88D4-29E2AF212FBEhttp://zoobank.org/59131C6D-A0DE-4BE0-9383-20C0DA8A709D-1.A pool in the rice field at Ban Nakae, Khilek Subdistrict, Pathum Rat District, Roi Et Province, northeastern Thailand; pH of water 8.6, water conductivity 126 µS cmHolotype: one adult male completely dissected , Ban Nakae , Khilek Subdistrict, Pathum Rat District, Roi Et Province, northeastern Thailand; collected on 12 June 1999 by L. Sanoamuang. Allotype: one adult female completely dissected ; same data as for holotype. Paratypes: two adult females and three adult males undissected preserved in 70% ethanol (NHMUK 2019.9–13), one adult female completely dissected (KKU-COP-2019-S-01); one adult female with eggs and three adult males undissected preserved in 70% ethanol (KKU-COP-2019-T-01); same data as for holotype.15°10'55"N, 102°23'46"E), Than Lalot Subdistrict, Phimai District, Nakhon Ratchasima Province, northeastern Thailand; collected on 17 October 2017 by N. Plangklang; (2) a roadside canal, Tropeang Chouk village , Baray District, Kampong Thom Province, central Cambodia; collected on 14 June 2007 by R. Chaicharoen; (3) a temporary pond, Kropeu village , Steung Sen District, Kampong Thom Province, central Cambodia; collected on 14 June 2007 by R. Chaicharoen.(1) a temporary pond, Ban Non Lakki , 3+ae (II), 1+ae (III), 1(IV), 1+ae (V), 1 (VI), 1+ae (VII), 1+sp (VIII), 2+ae (IX), 1 (X), 1 (XI), 1+ae+sp (XII), 1 (XIII), 1+ae (XIV), 1 (XV), 1+ae (XVI), 1 (XVII), 1 (XVIII), 1+ae (XIX), 1 (XX), 1 (XXI), 2 (XXII), 2 (XXIII), 2 (XXIV), 4+ae (XXV).Antennule Fig.  symmetriExp-1–7 with 1, 3, 1, 1, 1, 1, and 1 inner seta, respectively; Exp-7 with three additional apical setae. Enp-1 with two inner median setae. Enp-2 with eight inner and seven apical setae.Antenna Fig.  biramousEnp-1 with four inner distal setae; Enp-2 with nine apical setae plus tiny spinules along posterior surface. Exp-1–4 with 1, 1, 1, 3 setae, respectively.Mandible Fig.  with sixEnp with seven apical setae. Exp with six setae.Maxillule Fig.  with 13 Enp-1 and 2 with three setae each.Maxilla Fig.  with twoEnp-1–6 with 2, 3, 2, 2, 2, 4 setae, respectively.Maxilliped Fig.  with fouExp longer than Enp, Exp and Enp three-segmented except P1 Enp bi-segmented. Armature formula of P1–P4 as follows :P1–P4 Fig.  with rouExp-1. Exp-1 sub-rectangular, more than twice as long as wide, longer than Enp. Exp-2 triangular, right side stout and shorter than left one; with row of strong spinules along margins and proximolateral spine at basal Exp-3; with two longitudinal grooves on anterior view , 3+ae (II), 1+ae (III), 1 (IV), 1+ae (V), 1 (VI), 1+ae (VII), 1+sp (VIII), 2+ae (IX), 1+sp (X), 1+sp (XI), 1+ae+sp (XII), 1+ae+sp (XIII), 2+ae+sp (XIV), 2+ae+sp (XV), 2+ae+sp (XVI), 1+sp (XVII), 1+sp (XVIII), 2+ae+sp (XIX), 3+sp (XX), 2 (XXI), 4+ae (XXII); geniculated between segments 18 and 19; segment 20 (antepenultimate) with serrated process distally (3–4 teeth), and with longitudinal hyaline membrane along outer margin.Antennule Figs , 7G, H aLeft antennule, antenna, mouthparts, and P1–P4 as in female.Exp-1. Enp  roietensis sp. nov. with the male P5 Exp-2 displays an affinity to the subgenus Phyllodiaptomus sensu Exp-2, medially concave in posterior view, principal lateral spine inserted on distal to mid-outer margin and three accessary spines arranged from proximal, middle, and distal, respectively; the left Exp-2 with patch of strong spinules along medial margin.Exp-1 (4) Exp-2 oval and concave, with strong, flat, curved principal spine and three accessary spines, and (5) bi-lobed Enp. The left P5 with long and narrow hyaline lamella along inner margin, Exp-2 with patch of strong spinules along medial margin, and bi-segmented Enp.The male of the new species has serrated outgrowth on the antepenultimate segment of the right antennule. The right caudal ramus with small chitinous spine near distal margin on ventral side and triangular prominence along proximal one-third length of outer margin. The P5 intercoxal sclerite produced, with convex distal margin. The right P5 with (1) short, strong spine on posterior lobe of coxa, (2) triangular hyaline lamella on proximal inner margin and large chitinous outgrowth on posterior surface of basis, (3) acute distal outer corner of Pdg 5 wings, left wing more elongated in posterio-lateral direction; posterior and dorsal spines short and strong. Genital double-somite with posterolateral directed process on right side. One pair of genital spines on lateral side slightly symmetrical and strong. P5 Exp-2 with conveyor canal on anterior surface. P5 with bi-segmented Enp.Female with asymmetrical roietensis is taken after the type locality, Roi Et Province. The name with the Latin suffix “-ensis” is the adjective for a location.The specific name Dentodiaptomusjavanus , Eodiaptomussanoamuangae Ranga Reddy & Dumont, 1998, Mongolodiaptomuscalcarus , M.malaindosinensis , Neodiaptomuslaii Kiefer, 1974, and Phyllodiaptomus (Phyllodiaptomus) christineae Dumont, Ranga Reddy & Sanoamuang, 1996.Known only from four temporary water bodies from Roi Et and Nakhon Ratchasima provinces, Thailand and Kampong Thom Province, Cambodia Fig. . PresencPhyllodiaptomus has been recorded in Asia, including south China, Turkey, Israel, Uzbekistan, Iran, Iraq, India, Sri Lanka, Nepal, Indonesia, Thailand, Laos, Philippines and Cambodia (P. (C.) annae, P. (C.) wellekensae, and P. (C.) sasikumari) are endemic to India; two species (P. (P.) thailandicus and P. (P.) surinensis) are endemic to Thailand; P. (P.) tunguidus, P. (P.) irakiensis, and P. (P.) longipes are endemic to China, Iraq, and Indonesia, respectively. Only P. (P.) blanci is widely distributed, extending across many countries. Five species have been recorded in Thailand, namely P. (C.) praedictus, P. (P.) christineae, P. (P.) thailandicus, P. (P.) surinensis, and P. (P.) roietensis sp. nov. (P. (P.) surinensis and P. (P.) roietensis sp. nov. are rare. In 3,000 samples collected within Thailand, each has been recorded in only two localities in the northeast. This is in contrast to another endemic Thai species, P. (P.) thailandicus, which is widely distributed in both temporary and permanent water bodies in the east and south of Thailand  roietensis sp. nov., Mongolodiaptomusloeiensis Watiroyram & Sanoamuang, 2017, and Mongolodiaptomusmekongensis Sanoamuang & Watiroyram, 2018 differ from P. (P.) surinensis and other diaptomids by having additional spines on segments 17–19  thailandicus. However, the characteristic that differentiates the new diaptomid from P. (P.) thailandicus is the presence of a single process on each side of the genital double-somite; P. (P.) thailandicus has two processes only on the right side  surinensis have unique females which can be easily differentiated. The female P5 Exp-2 of the new species is obviously asymmetrical compared with that of P. (P.) surinensis which has a slightly asymmetrical P5 Exp-2. Exp-2 in females is species-specific and unique to the genus Phyllodiaptomus: the new species has two longitudinal ridges on the anterior surface versus multi-longitudinal ridges in P. (P.) surinensis  surinensis. The new species has substantial left genital double-somite proximal bulging versus only slight asymmetry in P. (P.) surinensis. The genital double-somite of P. (P.) surinensis has a bi-lobed hyaline outgrowth ventrally, which is absent in the new species. The genital spines in the female of the new species are oriented to a posterolateral direction in dorsal view, whereas they are pointed to the lateral direction in P. (P.) surinensis. The new species has tiny spinules on Pdg 4–5 laterally; these are present dorsally in P. (P.) surinensis  roietensis sp. nov. has a number of morphological differences from other members of the blanci-species group as follows:The male of P. (P.) longipes.a) Antepenultimate segment with a serrated process versus smooth in P. (P.) thailandicus, P. (P.) christineae, and P. (P.) blanci.b) Urosomite(s) with a long hair or hair-like setae versus bare in P. (P.) surinensis and P. (P.) tunguidus. However, a ventral prominence is also present on the left ramus of P. (P.) tunguidus (but is absent in the left ramus of the new species) and there are only two prominences in the new species but five in P. (P.) surinensis.c) Right caudal ramus with ventral prominences as in P. (P.) irakiensis and P. (P.) thailandicus. The new species has a round or semi-circular distal margin versus triangular in P. (P.) blanci, P. (P.) christineae, P. (P.) longipes, and P. (P.) tunguidus.d) Intercoxal sclerite is modified distally into single lobe versus two lobes in P. (P.) thailandicus and slender in P. (P.) christineae.e) Right P5 coxal spine is strong and acute versus rectangular in P. (P.) irakiensis and P. (P.) blanci. In addition, three species, P. (P.) longipes, P. (P.) christineae, and P. (P.) tunguidus, have a longitudinal ridge on the posterior surface, which is absent in the new species (the first one has two minute prominences on the ridge). The right P5 basis has a triangular hyaline lamella at inner margin versus elongated in P. (P.) christineae, P. (P.) longipes, and P. (P.) tunguidus, and round in P. (P.) blanci. The left P5 basis has inner lamella versus bare in P. (P.) irakiensis and P. (P.) longipes, digitiform in P. (P.) tunguidus, and small in P. (P.) blanci. The new species lacks any ornamentation on the anterior surface but P. (P.) surinensis has two minute lateral spines (see f) Right P5 basis with a three-lobed chitinous prominence on posterior surface versus bare in ines see : fig. 54Exp-2 with three accessary lateral spines versus bare in P. (P.) tunguidus and P. (P.) blanci, and one in P. (P.) irakiensis, P. (P.) christineae, and P. (P.) longipes.g) Right P5 Enp with a bi-lobed shape versus conical in the rest of the species except P. (P.) surinensis.h) Right P5 Enp versus one-segmented in P. (P.) thailandicus, P. (P.) surinensis, P. (P.) blanci, P. (P.) christineae, and P. (P.) longipes.i) Left P5 with bi-segmented P. (P.) surinensis. However, there are three major differences among the males, i.e. the right caudal ramus, left P5 basis, and left P5 Enp as described above. The fine detail on its inner hyaline lamella on the right P5 basis is also different: triangular in the new species versus oval bi-lobed in P. (P.) surinensis.With regard to the comparative morphology above, the male of the new species is most similar to those of Phyllodiaptomus (P. (P.) tunguidus, P. (P.) blanci, P. (P.) longipes, P. (C.) annae, P. (C.) wellekensae, and P. (C.) sasikumari); he also gave morphological descriptions of these six species. In this study, the key is updated as follows:Males:Females:


Adiantum philippense revealed bioactive Nigrospora sphaerica from the leaf segment. Chemical and biological profiling through TLC–bioautography and hyphenated spectroscopic techniques confirmed the presence of phomalactone as an antimicrobial metabolite.Endophyte bestows beneficial aspects to its inhabiting host, along with a contribution to diverse structural attributes with biological potential. In this regard, antimicrobial profiling of fungal endophytes from medicinal plant Escherichia coli by disc diffusion assay. The MIC was found to be significant against both Escherichia coli and Xanthomonas campestris in the case of bacteria and dermatophyte Candida albicans at 150 μg/ml, respectively.The chemical investigation of the broth extract by bioassay-guided fractionation confirmed phomalactone as a bioactive antimicrobial secondary metabolite. The antimicrobial activity of phomalactone was found to be highest against Nigrospora sphaerica exhibiting a broad spectrum of antimicrobial activity against human and phytopathogenic bacteria and fungi. This work is the first report regarding the antibacterial activity of phomalactone.Overall, the results highlighted the antimicrobial potential of phomalactone from the endophyte  Taxomyces andreanae inhabiting Taxus brevifolia -5, 6-dihydroxy-2 h-pyran-2-one].HPLC: The purified compound eluted at RT-4.89 with 86% purity  : 2.05 , 2.80 , 4.53 , 4.76  5.67–5.69 , 6.03–6.04  : 17.6 (C-9), 63.8 (C-5), 122.3 (C-3), 132.7 (C-8), 144.8 (C-4), 162.1 , 83.3 (C-6), 123.5 (C-7). MS : 154.06, (100%), 155.07 (M + 1) , 1750–1720 (ether), 1680–1600 , 1448 (CH3-bond), 1050–1150 (> C=O (1160) stretch), 1380 (–CH-bond) . The results obtained indicated that Gram-negative bacteria were found to be more susceptible when compared to Gram-positive.The MIC was found to be lowest against Nigrospora sphaerica has also been reported from a vast range of host viz., Ginkgo biloba [Moringa oleifera [Phoenix dactylifera [Saccharum arundinaceum [N. sphaerica, extracted with ethyl acetate showed a significant antimicrobial activity against bacteria and fungus viz., S. aureus, B. subtilis, S. epidermidis, E. coli, K. pneumonia, P. aeruginosa, S. typhi, S. flexneri, X. campestris, V. parahaemolyticus, and C. albicans, respectively. Several bioactive compounds have been reported from N. sphaerica which suggests the potential for discovery of pharmaceutically relevant substances. Previously, it was reported N. sphaerica to produce new antimicrobial bioactive secondary metabolites, such as nigrosporolides [o biloba , Moringaoleifera , Phoenixtylifera  and Saccdinaceum . In thisorolides , nigrosporolides , lactoneorolides , epoxydoorolides , and nigorolides .Nigrospora [Xylaria [Aspergillus [Verticillium [Drechslera [Hirsutella (entomopathogenic) [Paecilomyces (entomopathogenic) [Phoma [Aspergillus fumigatus [Phomalactone is quite insecticidal  and has grospora , 33, Xyl[Xylaria , Aspergiergillus , Verticiicillium , Drechslechslera , Hirsutehogenic) , Paecilohogenic) , and Pho) [Phoma . Phomalaumigatus , nematocumigatus , immunomumigatus , insectiumigatus , and phyumigatus .Xylaria sp. exhibited to possess weak activity at 13 μg/ml against protozoan Plasmodium falciparum was reported by Carlos et al. 2008. A study conducted by Kumudini et al. 2015 reported phomalactone from N. sphaerica had larvicidal, adulticide activity against Aedes aegypti and Anopheles quadrimaculatus mosquitoes with LD50 value 0.64 μg/org and 0.20 μg/org, respectively. The investigation of biopotential endophytes inhabiting Zoysia japonica Steud by Kim et al. 2001 revealed phomalactone isolated from N. sphaerica exhibited potent fungitoxic activity against Phytophthora infestans and Phytophthora capsici. Its ecological role(s) in each of these species has not been rigorously studied, but its phytotoxic, fungitoxic, and insecticidal activities may be important to the various fungi that produce phomalactone.Phomalactone from endophyte N. sphaerica from medicinal plant A. philippense. Chemical investigation through bioautography N. sphaerica displayed a broad spectrum of antimicrobial activity, which indicated that endophytic N. sphaerica as a potent producer of bioactive phomalactone derivative with great potential as a natural product drug molecule.In conclusion, this is the first report on the isolation of Additional file 1: Supplementary Figure 1. HPLC analysis of purified bioactive compound phomalactone.Additional file 2: Supplementary Figure 2. LCMS spectra of purified bioactive compound phomalactone.Additional file 3: Supplementary Figure 3. Proton (1H) NMR Spectra of purified bioactive compound phomalactone.Additional file 4: Supplementary Figure 4.13C NMR Spectra of purified bioactive compound phomalactone.Additional file 5: Supplementary Figure 5. FT-IR Spectra of purified bioactive compound phomalactone.


Escherichia coli is the causative agent of diarrhea in infants and animals worldwide. Many isolated strains recovered from pigs with postweaning diarrhea are multidrug resistance (MDR), and hybrids of E. coli are potentially more virulent, as enterotoxigenic E. coli (ETEC)/Shiga-toxigenic E. coli (STEC) hybrids. Here, we used whole-genome sequencing to analyze clinical isolates of the five colistin-resistant E. coli. The E. coli CAU15104, CAU15134, and CAU16060 belonged to ETEC/STEC hybrids, displaying the same serotype O3:H45 and sequence type ST4214. The E. coli CAU16175 and CAU16177 belonged to atypical enteropathogenic E. coli (aEPEC), display O4:H11 and O103:H2, ST29, and ST20, respectively. The E. coli CAU16175 carries six plasmids. An IncHI2-type plasmid, pCAU16175_1, harbors an IS26-enriched MDR region, which includes 16 antimicrobial-resistant genes. An IncFII-type plasmid, pCAU16175_3, harbors mcr-1.1, tet(M), and blaTEM−1B, whereas mcr-1.1 is located within a Tn2 derivative. Our findings indicate that the ETEC/STEC strains of the O3:H45 serotype as well as the aEPEC strains of the O4:H11 and O103:H2 serotypes are associated with postweaning diarrhea in swine and that some of diarrheagenic E. coli contains IS26-enriched MDR region and the mcr-1 gene located within a Tn2 derivative on IncFII plasmid.Diarrheagenic  Escherichia coli (DEC) is a leading cause of infectious diarrhea in humans and animals around the world (E. coli has six well-described pathotypes: enteropathogenic E. coli (EPEC), which is subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli, enteroaggregative E. coli, diffusely adherent E. coli, and enterohemorrhagic E. coli, which is subgroup of Shiga-toxigenic E. coli (STEC)  encoded by ltc, and heat-stable enterotoxin (ST) encoded by st (stx2e (E. coli strains are defined as forming the attaching and effacing (A/E) lesions mediated by genes located on the locus of enterocyte effacement (LEE) pathogenicity island (including eae) in the intestinal epithelium but not produce Shiga-like toxin (Stx)  are present during PWD  , 4. All i (STEC) . Enteroted by st . In pigst (stx2e . Enteropin (Stx) . Moreoveand stx− . Three pring PWD , 10. Thering PWD . Atypicaring PWD .E. coli isolates, 94.15% of the strains were MDR, with antimicrobial resistance rates ranging from 2.34% for meropenem to 90.05% for nalidixic acid  Gram-negative Enterobacteriaceae has been increasing worldwide between humans and animals . In the xic acid . Among tmcr-1), which has received widespread attention in different species of Enterobacteriaceae found in animals and humans around the world since it was first reported (mcr-1 (mcr-2 to mcr-10) have been detected (mcr-1 (mcr-1.1 to mcr-1.22), mcr-2 (mcr-2.1 to mcr-2.3), mcr-3 (mcr-3.1 to mcr-3.30), mcr-4 (mcr-4.1 to mcr-4.6), mcr-5 (mcr-5.1 to mcr-5.4), and mcr-8 (mcr-8.1 and mcr-8.2) (E. coli isolates (mcr-1 were higher than the rates of mcr-1–positive E. coli (64.6 vs. 49.2%)  .26 and Tn2, which is a member of the IS6 family and Tn3 family, respectively, play a key role in the dissemination of antimicrobial-resistant genes in Gram-negative bacteria  and transposons (Tns), arguably most numerous autonomous transposable elements, are crucial to shape their host genomes, particularly important in the bacterial antimicrobial resistance . IS26 anbacteria , 26. IS2 of IS26 . Tn2 is E. coli. . ISSwi1-with Tn2 . ISApl1,of mcr-1 , 30.E. coli isolates recovered from pigs with PWD and to characterize the complete sequence of a Tn2 derivative carrying mcr-1.1 on IncFII plasmid, pCAU16175_3, from a swine aEPEC isolate.In the present study, we used whole-genome sequencing (WGS) to analyze virulence and resistant genes of the five clinical colistin-resistant E. coli strains were obtained from feces samples or small intestinal content from pigs with diarrhea in China between 2014 and 2016 and identified with polymerase chain reaction (PCR) amplification of the uspA gene  based on the multilocus sequence typing and caused severe damage to IPEC-J2 cells , and two MDR aEPEC isolates (E. coli CAU16175 and CAU16177) were randomly chosen for WGS.A total of 455 Escherichia coli ATCC 25922 was used as the quality control. According to the MIC determined for each antimicrobial, the isolates were defined as “susceptible,” “intermediate,” or “resistant.”The susceptibility of the five isolates to 18 antimicrobials were tested by determining the minimum inhibitory concentration (MIC) using the US Clinical and Laboratory Standards Institute (CLSI) broth micro method . The resE. coli isolates and further used WGS by the Oxford Nanopore Technologies MinION platform to get complete genome of the E. coli CAU16175.Bacterial isolates were recultured from stock; DNA was extracted using a TIANamp Bacteria DNA kit . We used WGS by the Illumina Hiseq platform to get draft genome of the five https://cge.cbs.dtu.dk). Insertion sequence typing was carried out using the ISFinder database (https://www-is.biotoul.fr/). The complete genome sequences were initially annotated with Rapid Annotation using the Subsystem Technology server (http://rast.nmpdr.org) and curated manually using the BLAST server (https://blast.ncbi.nlm.nih.gov/Blast). The obtained plasmid sequences were aligned with homologous plasmid sequences from NCBI using the BRIG tool . The serotype, multilocus sequence type, plasmid type, antimicrobial-resistant gene, and virulence gene detection were performed using the Center for Genomic Epidemiology server (RIG tool .E. coli CAU16175 as the donor and the E. coli J53 (resistant to sodium azide) as the recipient. The transconjugant was screened on BHI agar plates containing sodium azide (150 mg/L) and colistin (4 mg/L). The presence of mcr-1 in the transconjugant was confirmed by PCR, and the MICs of antimicrobial agents for the transconjugant were determined using the agar dilution method. The conjugation experiment was repeated three times, and the conjugation frequencies were calculated as the number of transconjugants per recipient. The transconjugant strains were distinguished from donor occurring natural mutants of sodium azide–resistant E. coli by verifying eae, which is marker gene for E. coli CAU16175 donor.The conjugation experiment was carried out using the E. coliCAU15104, CAU15134, CAU16060, and CAU16177 isolates have been deposited at GenBank under SRA accession no. SRR10828049, SRR10828048, SRR10828047, and SRR10828046, respectively. The complete genome sequences of the E. coli CAU16175 have been deposited at GenBank under SRA accession no. SRR10813965.The draft genome sequences of the Antimicrobial susceptibility testing of the five isolates showed that they were MDR exhibiting resistant to at least three different classes of antimicrobials . All theE. coli CAU15104, CAU15134, and CAU16060 belonged to ETEC/STEC  hybrid strains, display the same serotype O3:H45 and sequence type ST4214, which are identical sequence type with the strains swine19 (LVMV00000000), swine22 (LVMY00000000), swine54 (LVMV00000000), and swine67 (LVOR00000000) from the NCBI database, all carrying fedA, fedF, iha, stb, ltcA, sepA, stx2A, and stx2B virulence genes and antimicrobial-resistant genes conferring resistant to KAN (aph(3′)-Ia), STR , TE [tet(A) and tet(M)], SXT  and PB (mcr-1.1)  and aac(6′)Ib-cr (associated to quinolone and aminoglycoside antibiotic). The E. coli CAU15134 and CAU16060 carry dfrA1 (associated to SXT), mcr-3.1 (associated to PB), oqxA, and oqxB, which are efflux pump conferring antibiotic resistance, and exhibit resistant to AMP associated to blaTEM−1B.The clinical isolates of the mcr-1.1) , 2. Besitibiotic . The E. E. coli CAU16175 and CAU16177, belonged to aEPEC , display different serotype and sequence type, O4:H11 and O103:H2, ST29 and ST20, respectively. They both carry cif, eae, espA, espB, espJ, gad, iss, nleB, and tir virulence genes and antimicrobial-resistant genes conferring resistance to beta-lactam antibiotic (blaOXA−1), KAN (aph(3′)-Ia), GEN (aac(3)-IVa), STR (aadA1 or aadA2 or strA or strB), TE [tet(A) and/or tet(M)], SXT , and PB (mcr-1.1 and/or mcr-3.1) (E. coli CAU16175 and CAU16177 both carry aac(6′)Ib-cr (associated to quinolone and aminoglycoside antibiotic), aph(4)-Ia (associated to aminoglycoside antibiotic), ARR-3 (associated to rifamycin antibiotic), catB3, cmlA1, and floR (associated to phenicol antibiotic) .The clinical isolates of the mcr-3.1) , 2. In aibiotic) . InteresE. coli CAU16175 has a total GC content of 50.47% with a single chromosome and six plasmids. The 5.62 Mb chromosome has a GC content of 50.66%. The six plasmids sequences of E. coli CAU16175 range in length from 6.99 to 190.22 kb with a GC content from 47.10 to 51.35% (mdf (A) antimicrobial-resistant gene. The pCAU16175_1 (IncHI2 type) harbors an IS26-enriched MDR region, which includes blaOXA−1 and 15 additional antimicrobial-resistant genes. The pCAU16175_2 (IncFIB type) harbors katP, sepA, and perA virulence genes. The pCAU16175_3 (IncFII type) harbors mcr-1.1, tet(M), and blaTEM−1B antimicrobial-resistant genes. The pCAU16175_5 harbors celb virulence gene (The 6.07-Mb complete genome of o 51.35% . The chrnce gene .mcr-1) increased to 8 and 16 mg/L, respectively  database. An overall nucleotide sequence identity (99.66–99.86%) with query coverages of 90–99% to pSH16G4498 (MH522423), pSH16G2457 (MH522421), pHNYJC8 (KY019259), pHNLDF400 (KY019258), pHXY0908 (KM877269), and other 25 IncHI2-type plasmids were observed (6 family insert sequences (IS26/IS15DI/IS15DIV/IS1006/ISEc59) . The structure of the MDR region comprises twelve IS6 family insert sequence  that flank containing different antimicrobial-resistant genes. The MDR region contains floR, sul2, aph(4)-Ia, aac(3)-IVa, aac(6')-Ib-cr, blaOXA−1, catB3, ARR-3, sul1 (two copies), dfrA12, aph(3')-Ia, sul3, aadA1, cmlA1, aadA2b, and tet(A). The MDR region is also interspersed with a number of different mobile elements including ΔTnAs3, ΔISVsa3, ISVsa3, ISAba1, ΔTn5393, ΔTn2, ΔIS15, and ΔTnAs1.The 190.22-kb plasmid observed . We chos/ISEc59) . The pCAin China . In the pCAU16175_3 was blasted against the GenBank Nucleotide collection (nr/nt) database. An overall nucleotide sequence identity (94.46–97.88%) with query coverages of 80–89% to pEC1515-3 (CP021847), pEC974-3 (CP021843), pH1038-142 (KJ484634), pFORC_081_1 (CP029058), plasmid R1 (KY749247), and other 27 IncFII-type plasmids were observed (mcr-1pKP81-BE (KU994859) for comparison analysis , which are close to the mcr-1.1 gene.The 76.63-kb plasmid observed . We chosanalysis . Althoug2 derivative (mcmM (encoded microcin M). A five-nucleotide direct duplication (TATTT) was identified flanking the structure. Unlike the Tn2, this structure harbors mcr-1–pap2-ΔISApl1 and IS1X2-hp-tet(M)-IS15DI, deletes the resolvase (tnpR), and truncates the transposase (tnpA). Besides, the striking features of the insertion sites of ISApl1 were found, which includes high AT content and 2-bp direct repeats (DRs) (AA)  (AA) . From thRs) (AA) . The mcrE. coli isolates recovered from feces samples or small intestinal content from pigs with diarrhea in China between 2014 and 2016 to know the E. coli pathotype and the antimicrobial susceptibility of the isolates -Ia), phenicol-resistant genes (cmlA1 and floR), tetracycline-resistant genes [tet(A) and tet(M)], trimethoprim-resistant gene (dfrA12), sulfonamide-resistant genes , streptomycin-resistant genes (strA and strB), and colistin-resistant genes (mcr-1.1). The ST4214 ETEC/STEC also harbored fimbrial adhesin genes (fedA and fedF), adherence gene (iha), heat-labile enterotoxin (LT) gene (ltcA), heat-stabile enterotoxin (ST) gene (stb), Shigella extracellular protein gene (sepA), and Shiga toxin 2 variant e genes (stx2A and stx2B). We observed the ST4214 strains were recovered from different provinces  and years  in China, which indicated this clone strains have spread in swine and should be brought to our attention.The clinical isolates of the ype ST10 . In the E. coli CAU16175 and CAU16177 belonged to aEPEC, displaying different serotype and sequence type, O4:H11 and O103:H2, ST29 and ST20, respectively. For aEPEC, serotypes O45 and O123 are frequently occurring in diarrheagenic pigs , aminoglycoside-resistant genes (aac(3)-IVa, aac(6′)Ib-cr, aph(3′)-Ia and aph(4)-Ia), phenicol-resistant genes , tetracycline-resistant genes [tet(A)], trimethoprim-resistant gene (dfrA12), sulfonamide-resistant genes , rifampicin-resistant gene (ARR-3), and colistin-resistant genes (mcr-1.1). Interestingly, E. coli CAU16177 also harbored mcr-3.1. Four mcr-1– and mcr-3-positive E. coli have been reported, and three E. coli isolates were recovered from pigs , non–LEE-encoded effector gene (nleB), glutamate decarboxylase gene (gad), increased serum survival gene (iss), a marker gene for EPEC, intimin gene (eae), and translocated intimin receptor gene (tir).In addition, the clinical isolates of the nic pigs , 42. TheE. coli CAU16175 carries IS26-enriched MDR region in pCAU16175_1 (IncHI2 type). The IncHI2-type plasmids have been discovered as genetic elements mediating the transmission of MDR genes . The emergence of the mcr-1 gene can be traced back to the E. coli isolated in the 1980s, and the outbreak of chicken-derived mcr-1–containing E. coli started in 2009 , we noticed the mcr-5 gene and the mcr-3.6 gene could be mobilized by Tn6452 and Tn6518 (belonged to Tn2 family). For the Tn6452, the mcr-5 gene was embedded within a Tn3-family Tn with 38-bp inverted repeats and flanked by 5-bp DRs, which were usually generated during the insertion  can be found below: The animal study was reviewed and approved by the Animal Ethics Committee of the China Agricultural University under the protocol CAU20140616-1.LG and YZ conceived and designed the experiments. LG, JW, SW, JS, and XW performed the experiments. LG analyzed the sequencing data and wrote the manuscript. YZ revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hibiscus schizopetalus (Dyer) Hook.f.  is an ornamental plant. The aim was to investigate its antimicrobial and antioxidant activities. In vitro antiviral, antibacterial, and antioxidant activities of the 70% ethanolic extract (Et-E) of the aerial parts of the plant were determined. The Dichloromethane Fraction (DCM-F) and the n-Butanol Fraction (Bu-F) were assessed using Liquid chromatography–mass spectrometry (LC-MS). The DCM-F showed higher antiviral activities against Coxsackie B4 (CoxB4) viruses (IC50 = 64.13 µg/mL) and adenoviruses (IC50 = 54.88 µg/mL) than acyclovir . The DCM-F showed higher anti-helicobacter pylori activity (MIC = 3.9 µg/mL) than clarithromycin (MIC = 1.95 µg/mL). The DCM-F inhibited Herpes Simplex Virus (HSV) Type I (IC50 = 29.85 µg/mL) and HSV Type II (IC50 = 74.17 µg/mL). The Bu-F showed higher anti-mycobacterial activity (MIC = 7.81 µg/mL) than isoniazid (MIC = 0.24 µg/mL) and higher antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA)(MIC = 7.81 µg/mL) than vancomycin (MIC = 3.9 µg/mL). Antioxidant assays included total antioxidant capacity (TAC), 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), and iron reducing power. The Bu-F showed the highest antioxidant activity. Chemical profiles were analyzed using HPLC-HR–ESI–MS to identify the metabolites responsible for these biological activities. We identified more than 60 metabolites that belong to anthocyanins, flavonoids, phenolics, terpenes, sterols, and fatty acids. In conclusion, Hibiscusschizopetalus is endowed with metabolites that could be used against viruses and antibiotic-resistant bacteria. They can also be potent antioxidants. Antimicrobial resistance to classical antibiotics is a major problem that negatively impacts treatments of common infectious diseases caused by fungi, bacteria, viruses, and parasites. In order to overcome this major problem, natural products offer a viable alternative .Hibiscus is a member of Malvaceae family and is cultivated globally as an ornamental and medicinal plant -2,5-diphenyl tetrazolium bromide (MTT) was performed spectrophotometrically using a Perkin-Elmer ELISA reader (HTS 7000 plus) at 540 nm . The minimum concentration required for the inhibition of bacterial growth (MIC) and the concentration required for inhibition of 90% of the bacterial growth (MIC90) were determined from the corresponding dose-response curve. The MTT assay was done using an automatic BioTek plate reader at 620 nm. Activity against n method . H. pyloM. tuberculosis (M. Tub ATCC 27294) was detected using the Microplate Alamar Blue Assay (MABA) [M. tuberculosis inoculum (105 CFU/mL) was added. Plates were incubated at 37 °C for 4 days, then 20 μL of Alamar Blue solution and 12.5 μL of 20% Tween 80 were added. The plates were re-incubated at 37 °C for 24 h. The color intensity was measured using an ELISA microplate reader at 590 nm. The experiment was done in triplicates.The anti-mycobacterial activity against y (MABA) . Isoniaz(MRSA ATCC 27294) was assessed using the microdilution method in the presence of tetrazolium salts [6 CFU/mL), and 50 μL of the sample dilutions (0.24–125 μg/mL) were added. The plates were incubated at 37 °C for 24 h. To each well, 40 μL of XTT were added and incubated for 1 h in the dark at 37 °C. For each well, the color intensity was measured at 492 nm.The anti-MRSA activity um salts . VancomyA.Total antioxidant capacity assay (TAC).The antioxidant activity of the samples was assessed using the phosphomolybdenum method ,49. AbsoB.2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging assay.Hibiscus extract and its fractions [The ABTS radical scavenging assay was used to assess the free radical scavenging activity of the ractions ,49. MeasC.2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging assay.The anti-oxidant activity was measured as previously described  AbsorbanD.Reducing power assay.The reducing power was assessed by the evaluating the ability of the samples to change potassium ferricyanide to potassium ferrocyanide. Absorbance was measured at 700 nm ,49. The Experiments for antioxidants were done in triplicates and means were calculated.m/z 150–1500. The crude methanolic extract and fractions for mass spectrometry were prepared at 1 mg/mL. Metabolomic analysis of the samples was done using LC-HR-ESI-MS ,51. We uThe data were imported and analyzed using MZmine 2.20 . The masp < 0.05.Data analysis was done using analysis of variance (ANOVA). The significance of differences between means was done using Duncan’s multiple range tests at H. schizopetalus is endowed with a lot of chemical compounds (more than 60 metabolites) that could be clinically used in fighting viruses, such as adenoviruses, HSVI, and CoxB4. They can also be used as effective therapies against antibiotic resistant pathogens such as M. tuberculosis, MRSA, and H. pylori. Finally, they can be potent antioxidants that can protect cells from free radical damage in cancer, heart diseases, and other serious health conditions.


Tumor metastasis accounts for the majority of cancer-related deaths; it is therefore important to develop preclinical models that faithfully recapitulate disease progression. Here, we generated paired organoids derived from primary tumors and matched liver metastases in the same colorectal cancer (CRC) patients. Despite the fact that paired organoids exhibit comparable gene expression and cell morphology, organoids from metastatic lesions demonstrate more aggressive phenotypes, tumorigenesis, and metastatic capacity than those from primary lesions. Transcriptional analyses of the paired organoids reveal signature genes and pathways altered during the progression of CRC, including SOX2. Further study shows that inducible knockdown of SOX2 attenuated invasion, proliferation, and liver metastasis outgrowth. Taken together, we use patient-derived paired primary and metastatic cancer organoids to model CRC metastasis and illustrate that SOX2 is associated with CRC progression and may serve as a potential prognostic biomarker and therapeutic target of CRC. To the Editor,Tumor heterogeneity plays a key role in cancer progression and therapy resistance . HoweverInvasion is a fundamental step in tumor progression toward metastasis. To study collective invasion, we cultured paired organoids in a 3D invasion matrix Fig. a. AlthouP < 0.05; fold change > 2.5) that were significantly upregulated in metastatic organoids  inducible expression of shRNA targeting SOX2 was established in metastatic organoids Fig. SC and D. nes Fig. E-G. Furtnts Fig. H. We thents Fig. . Taken tIn summary, the present study highlights the potential of patient-derived paired primary and metastatic cancer organoids as an experimental model for investigating CRC progression. We identified a significantly dysregulated gene between paired organoids, SOX2, which could be a prognostic biomarker, and perhaps a potent therapeutic target in the treatment of CRC.Additional file 1: Supplementary Table S1. Summary of patient-derived CRC organoid lines and corresponding clinical data.Additional file 2: Supplementary Figures. Figure S1. Paired organoids derived from primary and metastatic CRC recapitulate the histopathological structure of parental tumor. A and B, Organoids architecture resembles parental tumor epithelium. Representative bright-field images of organoids together with H&E staining of parental tumors and patient-derived organoids. The scale bar represents 100 μm. C and D, Representative IHC sections for the intestinal epithelial marker CDX2. The scale bar represents 100 μm. Figure S2. Organoids derived from liver metastatic lesions exhibited the most aggressive phenotypes with significant high tumorigenic and metastatic potential. A, Representative micrographs of organoids in 3D invasion assay. Tumor organoids showed the smooth and protrusive leading fronts, respectively. B, Micrographs of the paired organoids stained with phalloidin–F-actin (right). The scale bar represents 100 μm. C, qRT-PCR analysis of MMP-2 in paired organoid lines. Error bars indicate SEMs. *P=0.0207, **P =0.0083, ***P=0.0008 , ***P=0.0006 . D, Western blot analysis of the MMP-2 protein expression in paired organoid lines. α-tubulin was used as a loading control. E, Representative IHC sections for Ki67 in human colorectal tumor tissues and paired organoid lines. The scale bar represents 100 μm. F, Representative IHC sections for Ki67 in organoid xenografts and organoids derived from xenografts. The scale bar represents 100 μm. G, Representative gross image, histopathology and Ki67 staining of whole liver from primary organoid xenografts. The scale bar of the whole liver represents 1cm. The black scale bar represents 100 μm. Figure S3. A, Representation of the up-regulated genes in 13L organoids. Error bars indicate SEMs. *P = 0.0283, ***P = 0.0004, ****P < 0.0001 (one-way ANOVA). B, qRT-PCR analysis of the up-regulated genes in 13L organoids. Values were normalized to mean levels in 13a organoids. Error bars indicate SEMs. *P=0.02, ***P=0.0008 ****P < 0.0001 (one-way ANOVA). C, RNA sequencing analysis of SOX2 in human colorectal cancer tissues and normal colon tissues. D, qRT-PCR analysis of SOX2 in tumor tissues and paired normal colon tissues. Values were normalized to mean levels in normal colon tissues. Error bars indicate SEMs. **P = 0.0018, ****P < 0.0001 (one-way ANOVA). E, Western blot analysis of the SOX2 protein expression in tumor tissues and paired normal colon tissues. α-tubulin was used as a loading control. F, Representative IHC sections for SOX2 in human colorectal tumor tissues and paired normal colon tissues. The scale bar represents 100 μm. Figure S4. Silencing SOX2 in metastatic organoids attenuated cell invasion and proliferation and suppressed liver metastasis in vivo. A, qRT-PCR analysis of SOX2 in paired organoid lines (left). Values were normalized to mean levels in 21a organoids. Error bars indicate SEMs. ****P < 0.0001(Unpaired t test). Western blot analysis of the SOX2 protein expression in paired organoid lines. α-tubulin was used as a loading control (right). B, Representative IHC sections for SOX2 in human colorectal tumor tissues and organoids. The scale bar represents 100 μm. C, qRT-PCR analysis of SOX2 in the 13L-shRNA-1/2 (left) and 21L-shRNA-1/2 (right) organoids after 3 days of Dox-induction. The level of SOX2 was compared to that in the untreated sample. Error bars indicate SEMs. ***P=0.0001, ****P < 0.0001 (two-way ANOVA). D, Western blot analysis of the expression of SOX2 in the 13L-shRNA-1/2 and 21L-shRNA-1/2 organoids after 3 days of Dox-induction. α-tubulin was used as a loading control. E, Representative micrographs of Dox-transduced the 21L-shRNA-1/2 organoids after 5 days and stained with phalloidin–F-actin. The scale bar represents 100 μm. F, Proliferation of the 21L-shRNA-1/2 organoids were examined by CTG cell viability assays following 3- or 5-days growth in the presence or absence of Dox. Error bars indicate SEMs. **p=0.0019, ****P < 0.0001 (two-way ANOVA). G, Representative micrographs of colonies arising from the 21L-shRNA-1/2 organoids (top), with magnified insets showing colonies (bottom). The scale bar represents 200 μm. Colony forming efficiency in G was calculated and compared (right). Error bars indicate SEMs. **P=0.0015 (two-way ANOVA). H, Representative Ki67 immunostaining of liver metastases of the 13L-shRNA-1/2 organoids (left) and ratio of Ki67-positive tumor cells in liver metastases (right). Scale bars, 200 μm (top) and 100 μm (bottom). Each dot indicates individual mice. Error bars indicate SEMs. *p=0.0197, **P=0.0044 (two-way ANOVA). Figure S5. SOX2 is overexpressed in primary organoids. A, qRT-PCR analysis of SOX2 in primary organoid lines. Values were normalized to mean levels in control organoids. Error bars indicate SEMs. ****P < 0.0001 (one-way ANOVA). B, Western blot analysis of the SOX2 protein expression in primary organoid lines. α-tubulin was used as a loading control. C, Representative micrographs of colonies arising from the control, LV-Vector and LV-SOX2 primary organoid lines. The scale bar represents 200 μm. D-F, Colony forming efficiency in C was calculated and compared. Error bars indicate SEMs. ***P=0.0008, ****P < 0.0001 (one-way ANOVA). G, Proliferation of the control, LV-Vector and LV-SOX2 primary organoid lines were examined by CTG cell viability assays. Error bars indicate SEMs. ***P=0.0008, ****P < 0.0001 (one-way ANOVA). H, Representative micrographs of organoids in 3D invasion assay. Arrowheads, protrusive leading fronts. Micrographs of the paired organoids stained with phalloidin–F-actin. The scale bar represents 100 μm.Additional file 3. Supplementary Materials and Methods.


Correction to: Mol Neurodegeneration 15, 34 (2020)https://doi.org/10.1186/s13024-020-00383-7The original article [incorrectly displays 6th author, Björn Oskarsson’s name with middle initial. The correct presentation can be viewed in this Correction article. article  incorrec


In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained from a teaching hospital in China. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX−M−55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX−M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes  were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened.The emergence and spread of carbapenemase genes, colistin resistance genes  Escherichia coli as the leading pathogen  bacteria are rapidly increasing, which poses a serious threat to clinical therapy and public health WHO, . In 2019Escherichia coli is one of the predominant carriers of blaNDM; some lineages, such as ST167, ST410, and ST617, and popular plasmids  are associated with blaNDM global dissemination in E. coli  in E. coli from pigs in China can reduce the clinical efficacy of these agents   gene has recently been increasingly reported in E. coli from different sources , mainly in China  in E. coli , and carbapenemases in human or animal gut microbiomes , and carbapenemases is worrisome, as options for therapy are limited. Therefore, there is an urgent need for evaluating and monitoring the prevalence of pathogenic bacteria carrying mcr, tet(X), and carbapenemases. In this study, we investigated the prevalence and characterization of mobilized resistance genes tet(X4), mcr-1, and blaNDM in E. coli strains isolated from a teaching hospital in Jiangsu Province, China.A previous study reported the discovery of E. coli strains were isolated from patients in a teaching hospital in Jiangsu Province, China. The presence of tet(X), mcr-1, and carbapenemase genes  was detected by PCR and sequencing according to previously described protocols -carrying E. coli is described in From November 2019 to November 2021, 298 non-duplicate blaNDM/mcr-1/tet(X)-carrying E. coli isolates were tested for susceptibility to 14 antimicrobial agents, namely, ampicillin, cefotaxime, meropenem, gentamycin, amikacin, streptomycin, tetracycline, tigecycline, chloramphenicol, nalidixic acid, ciprofloxacin, colistin, fosfomycin, and sulfamethoxazole/trimethoprim, by using the agar dilution method or broth microdilution method (limited to colistin and tigecycline). E. coli ATCC 25922 was used as the quality control strain. The results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) M100, 30th edition .The on CLSI, . StreptoblaNDM/mcr-1/tet(X)-carrying E. coli isolates as donor bacteria and E. coli C600 (high-level streptomycin resistance) as the recipient strain following a previously described protocol , colistin (for mcr-1), or tigecycline [for tet(X)]. They were further confirmed by detecting the presence of blaNDM, mcr-1, and tet(X) using PCR. Transfer frequencies were calculated as the number of transconjugants per recipient. Conjugation experiments were performed at least in triplicate.Conjugation experiments were performed using blaNDM/mcr-1/tet(X)-carrying E. coli isolates using a TIAN amp Bacteria DNA kit  and sequenced by the Illumina Hiseq platform. The 150 bp pair-end raw reads were trimmed with the NGSQC toolkit v.2.3.3 and assembled into contigs using SPAdes v.3.8.2 , XYEH3783 (blaNDM − 5), and XYEH3934 [tet(X4)], were sequenced using Nanopore MinION and assembled with Unicycler version 0.4.9. The remaining blaNDM/mcr-1-carrying plasmid contigs were assembled into a complete plasmid sequence using PCR and Sanger sequencing (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The genome sequences were analyzed for multi-locus sequence typing (MLST) and resistance genes using MLST and ResFinder, respectively (https://cge.food.dtu.dk//services). Analysis and annotation of all blaNDM/mcr-1/tet(X4)-carrying plasmids were performed using the RAST server , ResFinder, PlasmidFinder (https://cge.food.dtu.dk/services/PlasmidFinder/), BLAST, and the Gene Construction Kit 4.5 . Blast Ring Image Generator (BRIG) version 0.95 was used to visualize the blaNDM-carrying plasmid comparisons -positive E. coli strains and blaNDM-positive E. coli ST167 in this study and the NCBI database was constructed based on core genome single nucleotide polymorphisms (SNPs) using ParSNP v. 1.2 (https://github.com/marbl/parsnp), respectively. ChiPlot (http://chiplot.online/#) was used to visualize the phylogenetic tree.The phylogenetic tree of all blaNDM/mcr-1/tet(X4)-carrying plasmids have been deposited in GenBank under accession number PRJNA901528.The genome sequences and blaNDM gene has been globally distributed as the third most common carbapenemase-encoding gene  or blaNDM − 6 (n = 1) (E. coli were resistant to meropenem (MIC ≥ 8 mg/L), ampicillin, cefotaxime, gentamicin, tetracycline, chloramphenicol, nalidixic acid, ciprofloxacin, and sulfamethoxazole/trimethoprim , ST167 (n = 2), ST361 (n = 1), and ST1193 (n = 1), respectively  . None ofethoprim . Resistaectively . E. coliecipient .blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids  were obtained from four ST156 NDM-5-producing E. coli strains. These IncX3 plasmids were identical, except that two copies of IS1294 were inserted into the accessory replication gene bis and DNA relaxase gene taxC, respectively, in pYUXEEH1271-NDM and pYUXEEH1278-NDM  of animal or human origin  and F2:A1:B1 plasmid pYUXYEH3783-NDM . These IncFII plasmids showed high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae  from food-producing animals or humans  have been identified in Enterobacteriaceae from animals, humans, and the environment , ampicillin, cefotaxime, nalidixic acid, ciprofloxacin, and some other agents , tet(A) (n = 3), fosA3 (n = 3), and rmtB (n = 1) (All r agents . In addi (n = 1) . The pre (n = 1) .mcr-1-positive E. coli strains could transfer mcr-1 to E. coli C600 by conjugation with frequencies of 10−6 to 10−4 transconjugants/recipient  from E. coli and pSCS23  from Salmonella  , was inserted near dnaJ to generate 5-bp direct repeats (5′-GAAAA-3′)  . The blaAAAA-3′) . The co-tet(X4) in E. coli from pigs in China in 2019  has been rarely reported in E. coli from patients. It was detected in 4.5%  fecal samples of hospital patients in Guangdong province, and only 0.07%  E. coli isolates from patients in China were identified to carry tet(X4) -positive E. coli isolate, XYEH3934. This strain exhibited high-level tigecycline resistance (MIC = 32 mg/L) and was also resistant to ampicillin, cefotaxime, streptomycin, tetracycline, chloramphenicol, nalidixic acid, fosfomycin, and sulfamethoxazole/trimethoprim (tet(X4) to E. coli C600 by conjugation.Since the discovery of the plasmid-mediated tigecycline resistance gene ethoprim . Howevertet(X) and another four resistance genes  were co-located on an IncX1 plasmid pYUXYEH3934-2 with a size of 31,816 bp. It was highly similar to other tet(X4)-carrying IncX1 plasmids from porcine E. coli in China, such as plasmids pYUYZMP62 (MW439254) and pDW28-tet(X4) (ON390803), as well as tet(X4)-positive plasmids from Escherichia and Klebsiella (tet(X4) was also associated with ISCR2 in pYUXYEH3934-2 (tet(X4) is mainly mediated by the horizontal transfer of insertion sequences  and plasmids such as IncX1 in the present study -positive E. coli strains has been previously described, such as E. coli ST877, ST10, and ST48 between animals and humans  and four plasmids . Among tebsiella . Howeverebsiella , which mebsiella , tet(X4)EH3934-2 . The rap Aminov, . HoweverblaNDM/mcr-1/tet(X4)-positive E. coli isolates in the present study. The results showed that the 15 E. coli strains were divided into four clades  and Italy (n = 1) were clustered in one clade with a close relationship  was observed in E. coli isolates from patients in one hospital in this study, their dissemination in E. coli associated with some successful pandemic plasmids  is of great concern. Conversely, E. coli strains from food or food-producing animals in China show a higher prevalence of blaNDM, mcr-1, and tet(X)  can be found below: LS and JW conceived the study. LS, G-ZS, YJ, C-YM, H-YW, and G-MK carried out the experiments. LS, YJ, Z-YW, and JW analyzed the data. JW wrote the manuscript. XJ revised the manuscript. All authors have read and approved the final version of the manuscript.