Species delimitation 4.0: integrative taxonomy meets artificial intelligence

Although species are central units for biological research, recent findings in genomics are raising awareness that what we call species can be ill-founded entities due to solely morphology-based, regional species descriptions. This particularly applies to groups characterized by intricate evolutionary processes such as hybridization, polyploidy, or asexuality. Here, challenges of current integrative taxonomy (genetics/genomics + morphology + ecology, etc.) become apparent: different favored species concepts, lack of universal characters/markers, missing appropriate analytical tools for intricate evolutionary processes, and highly subjective ranking and fusion of datasets. Now, integrative taxonomy combined with artificial intelligence under a unified species concept can enable automated feature learning and data integration, and thus reduce subjectivity in species delimitation. This approach will likely accelerate revising and unraveling eukaryotic biodiversity.K.K., L.K., P.M., and J.W.’s research is supported by the German Federal Ministry of Education and Research (BMBF) grant 01IS20062, M.H. by the German Ministry of Agriculture and Food (BMEL-BLE) grant 2819NA106, and L.H. by the German Federal Ministry for the Environment, Nature Conservation, Nuclear Safety, and Consumer Protection grant 67KI2086. K.K. also received funding from the German Research Foundation (DFG) Priority Program Taxon-Omics SPP1991 funding line Seed Money for Post-Doc Grants. E.H., S.T., and N.W. are supported by the DFG priority program Taxon-Omics SPP1991 grants Ho4395/10-2, To1400/1-1, and Wa3684/2-1, respectively. J.d.V. is supported by the DFG priority program SPP 2237 MAdLand grant VR 132/4-1. J.d.V. further thanks the European Research Council for funding under the EU Horizon 2020 Research and Innovation Program (Grant Agreement No. 852725; ERC-StG ‘TerreStriAL’). H.L. is supported by the National Natural Science Foundation of China grant 32171813.Peer reviewe

Earthworm invasion into previously earthworm-free temperate and boreal forests

Earthworms are keystone detritivores that can influence primary producers by changing seedbed conditions, soil characteristics, flow of water, nutrients and carbon, and plant–herbivore interactions. The invasion of European earthworms into previously earthworm-free temperate and boreal forests of North America dominated by Acer, Quercus, Betula, Pinus and Populus has provided ample opportunity to observe how earthworms engineer ecosystems. Impacts vary with soil parent material, land use history, and assemblage of invading earthworm species. Earthworms reduce the thickness of organic layers, increase the bulk density of soils and incorporate litter and humus materials into deeper horizons of the soil profile, thereby affecting the whole soil food web and the above ground plant community. Mixing of organic and mineral materials turns mor into mull humus which significantly changes the distribution and community composition of the soil microflora and seedbed conditions for vascular plants. In some forests earthworm invasion leads to reduced availability and increased leaching of N and P in soil horizons where most fine roots are concentrated. Earthworms can contribute to a forest decline syndrome, and forest herbs in the genera Aralia, Botrychium, Osmorhiza, Trillium, Uvularia, and Viola are reduced in abundance during earthworm invasion. The degree of plant recovery after invasion varies greatly among sites and depends on complex interactions with soil processes and herbivores. These changes are likely to alter competitive relationships among plant species, possibly facilitating invasion of exotic plant species such as Rhamnus cathartica into North American forests, leading to as yet unknown changes in successional trajectory

Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy ▿ †

DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C4-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by fluorescence resonance energy transfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {fD = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer

FRAP experiments of <i>E. coli</i> cells expressing (A) DcuS-YFP, (B) CitA-YFP, both showing fluorescence recovery at the cell pole, and (C) aggregated, non-functional DcuR-YFP showing no fluorescence recovery.

<p>DcuS-YFP (pMW407) was expressed in strain IMW262, CitA-YFP (pMW442) in strain IMW279, and DcuR-YFP (pMW1082) in strain IMW238, all in the presence of 133 µM arabinose. (D) The diagram depicts the relative fluorescence intensity of the fluorescent area at the cell pole before and after bleaching over time, normalized against gradual bleaching of the images, each from four independent experiments (standard deviations shown); square, DcuS-YFP; triangle, CitA-YFP; circle, aggregated DcuR-YFP. The mean half-time recovery of DcuS-YFP is 62 s, that of CitA-YFP is 80 s. Four independent experiments each were performed. The pictures illustrate representative examples of the microscopic acquisitions, with the dashed circle indicating the bleached area. Scale bars, 1 µm.</p

Strains of <i>Escherichia coli</i> and plasmids used in this study.

<p>Strains of <i>Escherichia coli</i> and plasmids used in this study.</p

<i>E. coli</i> cells expressing DcuS-YFP in <i>dcuR, dctA</i> and <i>dauA</i> deficient background.

<p>DcuS-YFP (pMW407) fluorescence was monitored in <i>E. coli</i> IMW238 deficient of <i>dcuR</i> (A) or MDO800 deficient of <i>dctA</i> (B); scale bars, 1 µm. (C) DcuS-mYFP (pMW1891) fluorescence was monitored in <i>E. coli</i> EK1 deficient of <i>dauA</i>.</p

Localization of chromosomally expressed or low-level-induced DcuS-YFP in <i>E. coli</i>.

<p>Panels A and D confocal microscopy (overlays of bright field and fluorescence), panels B, C and E epifluorescence (A–C, and E exponentially growing cells). A–B) DcuS-YFP expressed from the chromosome (strain IMW612), C) DcuS-YFP expressed from a plasmid (W3110pMW407), D) stationary phase cells expressing DcuS-YFP at low level (W3110pMW407), E) cells expressing CitA-YFP (W3110pMW422). White triangles indicate polar localized protein clusters. Scale bars, 2 µm.</p

Polar Localization of a Tripartite Complex of the Two-Component System DcuS/DcuR and the Transporter DctA in <i>Escherichia coli</i> Depends on the Sensor Kinase DcuS

<div><p>The C₄-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of <i>E. coli</i> is membrane-bound and reveals a polar localization. DcuS uses the C₄-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PAS_C and the kinase domains of DcuS.</p></div

Co-localization of the related sensor kinases CitA and DcuS in <i>E. coli</i>.

<p>CitA-YFP (pMW442) and DcuS-CFP (pMW408) were coexpressed in IMW262 and fluorescence of YFP (depicted in red) and CFP (depicted in green) were detected separately and merged (overlay image). About 50 cells monitored, with approx. 90% polar localization of the fluorescent proteins. Scale bar, 1 µm.</p