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126 results for “nuclear markers”
Deconvolved STED nanoscopy images of the nuclear phosphatidylinositol 4,5-bisphosphate and nuclear speckle marker SON together with deconvolved confocal images of DAPI stained nuclei in human formalin-fixed paraffin-embedded skin warts sections
<p>The collection and analysis of formalin-fixed paraffin-embedded (FFPE) human skin sections was approved by the local ethics-committee at the Department of Pathology, University of Cologne, Germany. Written informed consentwas obtained from all patients in accordance with the Declaration of Helsinki. For biopsy materials from archival paraffin blocks of human skin, an informed consent was obtained from all the subjects and ethical approval obtained from the Ethics Committee at the University of Cologne. Surgically removed human FFPE skin biopsies were sectioned into 4 µm sections. Sections were dewaxed, and indirectly immunofluorescently labeled against nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) using 5 µg/mL rabbit primary polyclonal antibody (Echelon Biosciences Inc. Z-A045, clone 2C11). The primary antibody against nPI(4,5)P2 was recognized by the goat secondary antibody conjugated with Abberrior Star 635P (Abberior 2-0002-007-5). Sections were indirectly immunofluorescently labeled against nuclear speckle marker SON using 1 µg/mL rabbit primary polyclonal antibody (Abcam ab121759). The primary antibody against SON was recognized by the goat secondary antibody conjugated with Abberrior Star 580 (Abberrior ST580-1002). Sections were co-stained by DAPI 1:1000 in PBS for 5 min.</p> <p>Imaging of nPI(4,5)P2-635P channel was performed on Leica TCS SP8 STED 3x inverted DMi8 microscope with pulsed white light laser 470-640 nm 1.5 mW and 775 nm pulse STED laser >1.5 W controlled by Leica Application Suite X software and equipped with HC PL APO CS2 100x/1.40 OIL objective used with Leica Type F immersion oil n=1.518. Unidirectional xyz scanning speed was 400 Hz, line accumulation 8. Pixel size was 20 nm in X and Y. Channel settings: 7% 633 nm laser; 775 Notch filter; 50% 775 nm STED laser; 30% 3D STED; HyD 639-698 nm, photon-counting mode, gain 100, gating 0.3-10 ns. Imaging of SON-580 channel was performed on Leica TCS SP8 STED 3x inverted DMi8 microscope with pulsed white light laser 470-640 nm 1.5 mW and 775 nm pulse STED laser >1.5 W controlled by Leica Application Suite X software and equipped with HC PL APO CS2 100x/1.40 OIL objective used with Leica Type F immersion oil n=1.518. Unidirectional xyz scanning speed was 400 Hz, line accumulation 8. Pixel size was 20 nm in X and Y. Channel settings: 10% 585 nm laser; 775 Notch filter; 80% 775 nm STED laser, 30% 3D STED; Hybrid detector (HyD) 589-616 nm, photon-counting mode, gain 100, gating 0.4-10 ns.</p> <p>Z-stacks of STED images were deconvolved using Huygens Professional 22.10 software (Scientific Imaging B.V.). Data sets were processed using Workflow Processor. The workflow consisted of selecting images, setting up the microscopy and deconvolution parameters and saving deconvolved images as 8-bit TIFF single files for individual channels (which were later used for the quantitative analyses; see below). Microscopy parameters were optimized and set as follows. Sampling intervals were ≤20 nm in X and Y and ≤20 nm in Z. Numerical aperture was 1.4; refractive indexes of the lens immersion oil was 1.518 and of the embedding media 1.458; objective quality was good, coverslip position was 0 µm and imaging direction was downward. For nPI(4,5)P2-635P STED channel the backprojected pinhole was 216 nm; excitation (ex.) and emission (em.) wavelengths (λ) were 633 and 651 nm, resp., ex. fill factor 2. STED depletion mode was pulsed, saturation factor 25, STED λ = 775, STED immunity factor 10 and STED 3X was 30%. Classic MLE algorithm with stabilization of Z-slices was used and signal-to-noise ratio was 5.1. For SON-580 STED channel the backprojected pinhole was 195 nm; excitation (ex.) and emission (em.) wavelengths (λ) were 585 and 602 nm, resp., ex. fill factor 2. STED depletion mode was pulsed, saturation factor 20, STED λ = 775, STED immunity factor 10 and STED 3X was 30%. Classic MLE algorithm with stabilization of Z-slices was used and signal-to-noise ratio was 4.</p>
New genetic markers for Sapotaceae phylogenomics: more than 600 nuclear genes applicable from family to population levels
<p>Some tropical plant families, such as the Sapotaceae, have a complex taxonomy, which can be resolved using Next Generation Sequencing (NGS). For most groups however, methodological protocols are still missing. Here we identified 531 monocopy genes and 227 Short tandem repeats (STR) markers and tested them on Sapotaceae using target capture and NGS. The probes were designed using two genome skimming samples from<em>Capurodendron delphinense</em> and <em>Bemangidia lowryi</em>, both from the Tseboneae tribe, as well as the published <em>Manilkara zapota</em> transcriptome from the Sapotoideae tribe. We combined our probes with 261 additional ones previously published and designed for the entire angiosperm group. On a total of 792 low-copy genes, 638 showed no signs of paralogy and were used to build a phylogeny of the family with 231 individuals from all main lineages. A highly supported topology was obtained at high taxonomic ranks but also at the species level. This phylogeny revealed the existence of more than 20 putative new species. Single nucleotide polymorphisms (SNPs) extracted from the 638 genes were able to distinguish lineages within a species complex and to highlight geographical structuration. STR were recovered efficiently for the species used as reference (<em>C. delphinense</em>) but the recovery rate decreased dramatically with the phylogenetic distance to the focal species. All together, the new loci will help reaching a sound taxonomic understanding of the family Sapotaceae for which many circumscriptions and relationships are still debated, at the species, genus and tribe levels.</p>
Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(4,5)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in control cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were control treated with 1:000 DMSO in the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S5 (Abcam ab5131) 3 µg/mL and mouse ascites IgM anti-PI(4,5)P2 2C11 (Z-A045; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with goat anti-mouse IgM (µ-chain) AF555 (Jackson ImmunoRes. A24126) 10 µg/mL; goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription elongation marker P-S2 and nuclear PI(3,4)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in THZ1 treated cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 3h with 1 µM THZ1 (MedChem HY80013) added to the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S2 (Abcam ab5095) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription elongation marker P-S2 and nuclear PI(3,4)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in control cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were control treated with 1:000 DMSO in the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S2 (Abcam ab5095) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S2 and nuclear PI(3,4)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in DRB treated cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 2h with 100 µM DRB (Sigma D1916) added to the cell culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S2 (Abcam ab5095) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(4,5)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in THZ1 treated cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 3h with 1 µM THZ1 (MedChem HY80013) added to the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S5 (Abcam ab5131) 3 µg/mL and mouse ascites IgM anti-PI(4,5)P2 2C11 (Z-A045; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with goat anti-mouse IgM (µ-chain) AF555 (Jackson ImmunoRes. A24126) 10 µg/mL; goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(3,4)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in THZ1 treated cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 3h with 1 µM THZ1 (MedChem HY80013) added to the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S5 (Abcam ab5131) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(3,4)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in DRB treated cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 2h with 100 µM DRB (Sigma D1916) added to the cell culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S5 (Abcam ab5131) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription elongation marker P-S2 and nuclear PI(4,5)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in control cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were control treated with 1:000 DMSO in the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S2 (Abcam ab5095) 3 µg/mL and mouse ascites IgM anti-PI(4,5)P2 2C11 (Z-A045; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with goat anti-mouse IgM (µ-chain) AF555 (Jackson ImmunoRes. A24126) 10 µg/mL; goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription elongation marker P-S2 and nuclear PI(4,5)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in DRB treated cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 2h with 100 µM DRB (Sigma D1916) added to the cell culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S2 (Abcam ab5095) 3 µg/mL and mouse ascites IgM anti-PI(4,5)P2 2C11 (Z-A045; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with goat anti-mouse IgM (µ-chain) AF555 (Jackson ImmunoRes. A24126) 10 µg/mL; goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(3,4)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in control cells.
<p>U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO<sub>2</sub>. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were control treated with 1:000 DMSO in the culture media.</p> <p>U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S5 (Abcam ab5131) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging.</p> <p>Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels.</p> <p>SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).</p>
Appendix 2 in A new subfamily classification of the Citrus family (Rutaceae) based on six nuclear and plastid markers
<p><b>Appendix 2.</b> Details about the character states of all included taxa for the 13 morphological and karyological characters.</p><table><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th colspan="2">Characters</th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><th>Taxon</th><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td colspan="2">6 7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td><td>13</td></tr><tr><th><i>Acmadenia</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Acradenia</i></th><td>1</td><td>1</td><td>2, 3</td><td>1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Acronychia</i></th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Adenandra</i></th><td>0</td><td>0, 1</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>2, 3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Adiscanthus</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Aegle</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>2</td><td>0</td><td>4</td><td>0</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Aeglopsis</i></th><td>0, 1</td><td>0</td><td>1, 2, 3</td><td>1, 2</td><td>0</td><td>4</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Afraegle</i></th><td>1</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>4</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Agathosma</i> A</th><td>0</td><td>0, 1, 2</td><td>2, 3</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Agathosma</i> B</th><td>0</td><td>0, 1, 2</td><td>2, 3</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Agathosma</i> C</th><td>0</td><td>0, 1, 2</td><td>2, 3</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Amyris</i></th><td>0, 1</td><td>0, 1</td><td>1, 2, 3</td><td>1</td><td>3</td><td>1</td><td>1</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Andreadoxa</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>0</td><td>0</td></tr><tr><th><i>Angostura</i></th><td>0, 1</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0, 1</td><td>1</td><td>0</td></tr><tr><th><i>Asterolasia</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>0, 1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Atalantia</i></th><td>0</td><td>0</td><td>1, 2, 3</td><td>1</td><td>0</td><td>0, 1</td><td>5</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Balfourodendron</i></th><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>4</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Balsamocitrus</i></th><td>0, 1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>4</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Bergera</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>?</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Boenninghausenia</i></th><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>2, 3</td><td>2</td><td>2</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Boronia</i> A</th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>0, 1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Boronia</i> B</th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>0, 1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Boronia</i> C</th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>0, 1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Boronia</i> D (<i>Boronella</i>)</th><td>0</td><td>1, 2</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Bosistoa</i></th><td>0, 1</td><td>0</td><td>2</td><td>1</td><td>1</td><td>2, 3</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Bottegoa</i></th><td>1</td><td>0</td><td>1, 2</td><td>0</td><td>0</td><td>0</td><td>4</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Bouchardatia</i></th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>3</td><td>2</td><td>0, 1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Brombya</i></th><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Burkillanthus</i></th><td>0, 1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>4</td><td>5</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Calodendrum</i></th><td>0</td><td>1, 2</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Casimiroa</i></th><td>0, 1</td><td>0</td><td>1, 2, 3</td><td>0</td><td>0</td><td>1, 2, 3</td><td>0, 1</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Cedrelopsis</i></th><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1, 2</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Chloroxylon</i></th><td>1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>3</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Choisya</i></th><td>1</td><td>1</td><td>1, 2</td><td>1</td><td>1</td><td>1</td><td>2</td><td>2</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Chorilaena</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Citropsis</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>0</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Citrus</i> A</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Citrus</i> B</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>Taxon</th><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td colspan="2">Characters 6 7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td><td>13</td></tr><tr><th><i>Citrus</i> C</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Clausena</i></th><td>1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Clymenia</i> (<i>Citrus</i> s.l.)</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Cneoridium</i></th><td>0</td><td>1</td><td>1</td><td>1</td><td>3</td><td>1, 2</td><td>0</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Cneorum</i></th><td>0</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Coatesia</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Coleonema</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Comptonella</i></th><td>0, 1</td><td>1</td><td>1</td><td>0, 1</td><td>0, 1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Conchocarpus</i> A</th><td>0, 1</td><td>0, 1</td><td>1, 2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0, 1</td><td>1</td><td>0</td></tr><tr><th><i>Conchocarpus</i> B (<i>Almeidea</i>)</th><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Correa</i></th><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td></tr><tr><th><i>Crossosperma</i></th><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0, 1</td><td>1</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Crowea</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Cyanothamnus</i></th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Desmotes</i></th><td>0</td><td>1</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Dictamnus</i></th><td>1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>2</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>0</td><td>0</td></tr><tr><th><i>Dictyoloma</i></th><td>1</td><td>0</td><td>2</td><td>0</td><td>1</td><td>2</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Dinosperma</i></th><td>0, 1</td><td>0, 1, 2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>0, 1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Diosma</i></th><td>0</td><td>0, 1</td><td>2</td><td>0, 1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Diplolaena</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Drummondita</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Dryades</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>?</td><td>?</td><td>0</td><td>0, 1</td><td>1</td><td>0</td></tr><tr><th><i>Dutaillyea</i></th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0, 1</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Empleurum</i></th><td>0</td><td>0</td><td>1</td><td>0</td><td>1, 3</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Eremocitrus</i> (<i>Citrus</i> s.l.)</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Eriostemon</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Ertela</i></th><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>1, 2</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Erythrochiton</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0, 1</td><td>1</td><td>0</td></tr><tr><th><i>Esenbeckia</i></th><td>0, 1</td><td>0, 1</td><td>2, 3</td><td>0</td><td>0, 1</td><td>1</td><td>2</td><td>0, 1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Euchaetis</i></th><td>0</td><td>0, 1</td><td>2</td><td>0, 1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Euodia</i></th><td>0, 1</td><td>1</td><td>1</td><td>0, 1</td><td>1</td><td>1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Fagaropsis</i></th><td>1</td><td>1</td><td>1, 2, 3</td><td>0, 1</td><td>0</td><td>0</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Feroniella</i></th><td>1</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Flindersia</i></th><td>0, 1</td><td>0, 1</td><td>2</td><td>1</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 3</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Fortunella</i> (<i>Citrus</i> s.l.)</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Galipea</i></th><td>0, 1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Geijera</i></th><td>0</td><td>0</td><td>0, 1, 2</td><td>0</td><td>1</td><td>1</td><td>3</td><td>1</td><td>3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Geleznowia</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Glycosmis</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>0, 1</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Halfordia</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Haplophyllum</i></th><td>0, 1</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1, 2, 3</td><td>2</td><td>2</td><td>0</td><td>1, 2</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Harrisonia</i></th><td>1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Helietta</i></th><td>1</td><td>0, 1</td><td>1, 2</td><td>0</td><td>0</td><td>1</td><td>4</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Hortia</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td><td>0, 1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>Taxon</th><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td colspan="2">Characters 6 7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td><td>13</td></tr><tr><th><i>Ivodea</i></th><td>0</td><td>0, 1, 2</td><td>1, 2</td><td>0, 1</td><td>1</td><td>1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Leionema</i> A</th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0, 1</td><td>0</td></tr><tr><th><i>Leionema</i> B</th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0, 1</td><td>0</td></tr><tr><th><i>Limonia</i></th><td>1</td><td>0</td><td>1, 2, 3</td><td>1</td><td>0</td><td>4</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Lunasia</i></th><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0, 1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Luvunga</i></th><td>0, 1</td><td>0</td><td>0, 1, 2</td><td>1</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Maclurodendron</i></th><td>0</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Macrostylis</i></th><td>0</td><td>0, 1</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Medicosma</i></th><td>0, 1</td><td>0, 1, 2</td><td>1</td><td>0, 1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Melicope</i> A</th><td>0, 1</td><td>1</td><td>1</td><td>0, 1</td><td>1</td><td>1</td><td>3</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Melicope</i> B</th><td>0, 1</td><td>1</td><td>1</td><td>0</td><td>0, 1</td><td>1</td><td>3</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Melicope</i> C</th><td>0, 1</td><td>1, 2</td><td>1</td><td>0, 1</td><td>0, 1</td><td>1</td><td>3</td><td>2</td><td>0, 1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Melicope</i> D</th><td>0, 1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0, 1</td><td>3</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Melicope</i> E (<i>Platydesma</i>)</th><td>0</td><td>0, 1</td><td>1</td><td>1</td><td>0</td><td>2, 3</td><td>3</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Merope</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>0</td><td>1, 2</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Merrillia</i></th><td>1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>3</td><td>0</td><td>0</td><td>?</td><td>0</td><td>1</td><td>0</td><td>0</td></tr><tr><th><i>Metrodorea</i></th><td>0, 1</td><td>1</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>0, 1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Microcitrus</i> (<i>Citrus</i> s.l.)</th><td>0</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Microcybe</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Micromelum</i></th><td>0, 1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Monanthocitrus</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>0</td><td>1, 2, 3</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Muiriantha</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td></tr><tr><th><i>Murraya</i></th><td>1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Myrtopsis</i></th><td>0</td><td>1</td><td>1, 2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>?</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Naringi</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>0, 1</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Nematolepis</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0, 1</td><td>0</td></tr><tr><th><i>Neobyrnesia</i></th><td>0</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Neoraputia</i></th><td>0, 1</td><td>0, 1</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Neoschmidea</i></th><td>0</td><td>0</td><td>1, 2</td><td>1</td><td>1</td><td>1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Orixa</i></th><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Pamburus</i></th><td>0</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Paramignya</i></th><td>0</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Peltostigma</i></th><td>0, 1</td><td>0</td><td>0, 1, 2, 3</td><td>1, 2</td><td>0</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Pentaceras</i></th><td>1</td><td>0</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1, 4</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Perryodendron</i></th><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>3</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Phebalium</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Phellodendron</i></th><td>1</td><td>1</td><td>2</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Philotheca</i> A</th><td>0</td><td>0</td><td>1, 2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0, 1</td><td>0</td></tr><tr><th><i>Philotheca</i> B</th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0, 1</td><td>0</td></tr><tr><th><i>Phyllosma</i></th><td>0</td><td>0</td><td>2</td><td>0, 1</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Picrella</i></th><td>0, 1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0, 1</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Pilocarpus</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>0</td><td>0</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Pitavia</i></th><td>0</td><td>1, 2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Pitaviaster</i></th><td>0</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>Taxon</th><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td colspan="2">Characters 6 7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td><td>13</td></tr><tr><th><i>Pleiospermium</i></th><td>0, 1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>1</td><td>5</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Plethadenia</i></th><td>1</td><td>0, 1</td><td>1</td><td>1</td><td>2</td><td>0</td><td>2</td><td>?</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Poncirus</i> (<i>Citrus</i> s.l.)</th><td>1</td><td>0</td><td>1, 2, 3</td><td>2</td><td>0</td><td>1, 2, 3</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Psilopeganum</i></th><td>1</td><td>0</td><td>1, 2</td><td>1</td><td>1</td><td>2, 3</td><td>2</td><td>2</td><td>?</td><td>2</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Ptaeroxylon</i></th><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Ptelea</i></th><td>1</td><td>0</td><td>1, 2, 3</td><td>0</td><td>0</td><td>1</td><td>2, 4</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Rauia</i></th><td>0, 1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0</td><td>1</td><td>0</td></tr><tr><th><i>Ravenia</i></th><td>0, 1</td><td>1</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Rhadinothamnus</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0, 1</td><td>0</td></tr><tr><th><i>Ruta</i></th><td>1</td><td>0</td><td>1, 2</td><td>1</td><td>0</td><td>4</td><td>2</td><td>2</td><td>0, 1, 2</td><td>1, 2</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Sarcomelicope</i></th><td>0</td><td>1, 2</td><td>1</td><td>1</td><td>0, 1</td><td>1</td><td>1</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Severinia</i></th><td>0</td><td>0</td><td>1, 2, 3</td><td>1</td><td>0</td><td>0, 1</td><td>5</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Sheilanthera</i></th><td>0</td><td>0</td><td>2</td><td>0</td><td>1</td><td>0, 1</td><td>2</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Sigmatanthus</i></th><td>1</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Skimmia</i></th><td>0</td><td>0</td><td>1, 2, 3</td><td>0</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Sohnreyia</i></th><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0, 1</td><td>4</td><td>0, 1, 2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Spathelia</i></th><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0, 1</td><td>4</td><td>0, 1, 2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Spiranthera</i></th><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Swinglea</i></th><td>1</td><td>0</td><td>2</td><td>1</td><td>0</td><td>4</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Tetractomia</i></th><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>3</td><td>2</td><td>?</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><th><i>Tetradium</i></th><td>1</td><td>1</td><td>1, 2</td><td>0</td><td>1</td><td>0, 1</td><td>3</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Thamnosma</i></th><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>2, 3</td><td>2</td><td>2</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Toxosiphon</i></th><td>0, 1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>?</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><th><i>Triphasia</i></th><td>0, 1</td><td>0</td><td>0, 2</td><td>1</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0, 1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Vepris</i></th><td>0, 1</td><td>0, 1</td><td>1</td><td>0, 1</td><td>0, 3</td><td>0, 1</td><td>1</td><td>2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Wenzelia</i></th><td>0</td><td>0</td><td>2</td><td>1</td><td>0</td><td>3</td><td>0</td><td>0</td><td>?</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Zanthoxylum</i> A</th><td>0, 1</td><td>0</td><td>1, 2</td><td>0</td><td>1</td><td>1</td><td>3</td><td>1, 2</td><td>1, 2, 3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Zanthoxylum</i> B</th><td>0, 1</td><td>0</td><td>1, 2</td><td>0</td><td>1</td><td>1</td><td>3</td><td>1, 2</td><td>1, 2, 3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Zanthoxylum</i> C</th><td>0, 1</td><td>0</td><td>0, 1, 2, 3</td><td>0</td><td>1</td><td>1</td><td>3</td><td>1, 2</td><td>1, 2, 3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Zanthoxylum</i> D (<i>Toddalia</i>)</th><td>0, 1</td><td>0</td><td>1, 2, 3</td><td>0</td><td>0</td><td>1</td><td>1</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th><i>Zieria</i></th><td>0, 1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0, 1</td><td>2</td><td>2</td><td>1, 2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table><p><b>1.</b> Leaf type: (0) simple/unifoliolate; (1) compound. <b>2.</b> Phyllotaxis: (0) alternate; (1) opposite; (2) whorled. <b>3.</b> Flower merosity: (0) 3-merous; (1) 4-merous; (2) 5-merous; (3) polymerous. <b>4.</b> Stamen whorls: (0) haplostemonous; (1) diplostemonous; (2) more than two whorls. <b>5.</b> Carpel connation: (0) syncarpous; (1) syncarpous (style only); (2) apocarpous (no joined style); (3) one carpel. <b>6.</b> Ovules per locule: (0) 1; (1) 2; (2) 3–5; (3) 6–10; (4)>10. <b>7.</b> Fruit type: (0) berry; (1) drupe; (2) dehiscent with seeds detached; (3) dehiscent with seeds attached; (4) samara; (5) hesperidium. <b>8.</b> Endosperm: (0) lacking; (1) scanty; (2) copious. <b>9.</b> Chromosomes: (0) 9–10; (1) 14–18; (2) 28–36; (3) ≥64. <b>10.</b> Growth form: (0) shrubs or trees; (1) subshrubs; (2) perennial herbs. <b>11.</b> Flower symmetry: (0) actinomorphic; (1) zygomorphic. <b>12.</b> Tubular corolla: (0) petals free and not forming a tube; (1) petals coherent or connate and forming a corolla tube. <b>13.</b> Seeds: (0) not winged; (1) winged</p><p>(Continues)</p><p>(Continues)</p>
Standardized nuclear markers improve and homogenize species delimitation in Metazoa
<p><span>Species are the fundamental units of life and evolution. Their recognition is essential for science and society. Molecular methods have been increasingly employed for the identification of animal species, despite several challenges. </span></p> <p><span>Here, we explore with genomic data from nine animal lineages a set of nuclear</span><span> </span><span>markers, namely metazoan-level universal single-copy orthologs (metazoan USCOs), for their use in species delimitation. Our data sets include arthropods and vertebrates. We use various data assembly strategies and employ coalescent-based species inference as well as population admixture analyses and phenetic methods.</span></p> <p><span>We demonstrate that metazoan USCOs well distinguish closely related morphospecies and consistently outperform classical mitochondrial DNA barcoding in discriminating closely related species in different animal taxa. USCOs overcome the general shortcomings of mitochondrial DNA barcodes, and due to standardization across Metazoa, also those of other approaches. They accurately assign samples not only to lower but also to higher taxonomic levels. </span></p> <p><span>Metazoan USCOs provide a powerful and unifying framework for DNA-based species delimitation and taxonomy in animals and their employment could result in a more efficient use of research data and resources.</span></p>
Рис. 3. Снижение чисΛенности гусениц при возΑействии разΛичных географических штаммов ВЯП. СтоΛбцы: коΛичество погибших во время эксперимента гусениц поΑ опреΑеΛенной инфекционной нагрузкой. ПоказатеΛи (в процентах) привеΑены в Λевой части табΛицы. СтреΛки: минимаΛьное и максимаΛьное коΛичество погибших во время очереΑного учета гусениц и крестообразные маркеры: среΑнее коΛичество погибших во время очереΑного учета гусениц. ПоказатеΛи (в абсоΛютных значениях) в правой части табΛицы. По горизонтаΛи: номер титра, от боΛьшего к меньшему Fig. 3. Decrease in the number of caterpillars under the influence of various geographical strains of NPV. Columns: number of caterpillars killed during the experiment under a certain infectious load. Indicators (in percent) are shown on the left side of the table. Arrows: the minimum and maximum number of deaths during the next track count and cross markers: the average number of deaths during the next track count. Indicators (in absolute values) on the right side of the table. Horizontal: titre, from highest to lowest in Sensitivity of caterpillars of the gypsy moth (Lymantria dispar, Erebidae) from the South of Khabarovsk Territory to various strains of nuclear polyhedrosis virus
Рис. 3. Снижение чисΛенности гусениц при возΑействии разΛичных географических штаммов ВЯП. СтоΛбцы: коΛичество погибших во время эксперимента гусениц поΑ опреΑеΛенной инфекционной нагрузкой. ПоказатеΛи (в процентах) привеΑены в Λевой части табΛицы. СтреΛки: минимаΛьное и максимаΛьное коΛичество погибших во время очереΑного учета гусениц и крестообразные маркеры: среΑнее коΛичество погибших во время очереΑного учета гусениц. ПоказатеΛи (в абсоΛютных значениях) в правой части табΛицы. По горизонтаΛи: номер титра, от боΛьшего к меньшему Fig. 3. Decrease in the number of caterpillars under the influence of various geographical strains of NPV. Columns: number of caterpillars killed during the experiment under a certain infectious load. Indicators (in percent) are shown on the left side of the table. Arrows: the minimum and maximum number of deaths during the next track count and cross markers: the average number of deaths during the next track count. Indicators (in absolute values) on the right side of the table. Horizontal: titre, from highest to lowest
Fig. 11 in Introgression at the emerging secondary contact zone of magpie Pica pica subspecies (Aves: Corvidae): integrating data on nuclear and mitochondrial markers, vocalizations, and field observations
Fig. 11 Rates of chatter calls in different magpie populations and individuals. a Each mark represents average chattering rate for a single bird from five populations indicated by colours. Figures are numbers for the outliers: 1, 2—jankowskii from the mixed population of Argun'; 3, 4, 5—hybrid birds from the hybridogeneous population of Kerulen. b Each mark represents average chattering rate for a series of chatterings of one selected individual representing jankowskii, leucoptera, and hybrid birds, respectively. Green mark—pair #6 jankowskii from Vladivostok; gray—pair #43 leucoptera from Tsasuchei, Transbaikalia; blue—pair #24 hybrids from Kerulen, eastern Mongolia. X-axis—number of elements per second in a total series of chattering; Y-axis— number of elements per second in a series of 5 elements of chattering
Fig. 12 in Introgression at the emerging secondary contact zone of magpie Pica pica subspecies (Aves: Corvidae): integrating data on nuclear and mitochondrial markers, vocalizations, and field observations
Fig. 12 Violin plot diagram of the chatter call speed (elements per second) of Eurasian magpie populations across regions. X-axis presents a set of populations; Y-axis—elements per second. Box outlines the interquantile range (25%, 75%), whiskers represent range without outliers, central bar is the median, red dot is the mean, and figure shape is the probability density. The brackets on the top denote statistically significant pairwise differences (GamesHowell test, p<0.05)
Fig. 9 in Introgression at the emerging secondary contact zone of magpie Pica pica subspecies (Aves: Corvidae): integrating data on nuclear and mitochondrial markers, vocalizations, and field observations
Fig. 9 Population genetic structure based on unlinked SNP markers. Scatter plots of principal component analysis (PCA) show individual variation in components one and two (a) and three and four (b). The amount of variance explained by each PC is shown in parentheses. I—leucoptera,
Fig. 7 in Introgression at the emerging secondary contact zone of magpie Pica pica subspecies (Aves: Corvidae): integrating data on nuclear and mitochondrial markers, vocalizations, and field observations
Fig. 7 Bayesian skyline plots (BSPs) for effective female population sizes for haplogroups, subspecies, and populations of Pica pica. a Comparison of 6 haplogroups, depicted in the network Fig. 4. b Comparison of 6 subspecies. c Comparison of 4 populations of P. p. jankowskii. d Comparison of 3 populations of P. p. leucoptera.
Fig. 6 in Introgression at the emerging secondary contact zone of magpie Pica pica subspecies (Aves: Corvidae): integrating data on nuclear and mitochondrial markers, vocalizations, and field observations
Fig. 6 Mismatch distribution of nucleotide differences in populations representing different haplogroups as at Figs. 4 and 5. X-axis— number of nucleotide differences; Y-axis—proportion (frequency). Solid lines—expected distributions (under expectation of population growth); dashed lines—observed distributions. a Haplogroup 1:
ScienceDex guides
Understand access before you commit
These curated guides explain access requirements, typical timelines, costs, and reuse considerations for widely used research datasets.
Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
ABODe is a University of Edinburgh DataShare dataset for behavior classification in group-housed mice using home-cage video, identities, bounding boxes, ground-plate positions, and annotator labels.
DANDI Archive for NWB datasets
DANDI is a BRAIN Initiative archive for publishing and sharing neurophysiology data, including electrophysiology, optophysiology, and behavioral data packaged as NWB and related standards.
International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.