SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120.

IMPORTANCE: Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

Fusion of Aza- and Oxadiborepins with Furans in a Reversible Ring-Opening Process Furnishes Versatile Building Blocks for Extended π-Conjugated Materials.

A modular synthesis of both difurooxa- and difuroazadiborepins from a common precursor is demonstrated. Starting from 2,2'-bifuran, after protection of the positions 5 and 5' with bulky silyl groups, formation of the novel polycycles proceeds through opening of the furan rings to a dialkyne and subsequent re-cyclization in the borylation step. The resulting bifuran-fused diborepins show pronounced stability, highly planar tricyclic structures, and intense blue light emission. Deprotection and transformation into dibrominated building blocks that can be incorporated into π-extended materials can be performed in one step. Detailed DFT calculations provide information about the aromaticity of the constituent rings of this polycycle.

High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity.

Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1 ) with high affinity (app. KD  = 0.76 µm± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1 . Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD  = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1 .

Dithiophene-Fused Oxadiborepins and Azadiborepins: A New Class of Highly Fluorescent Heteroaromatics.

Access to dithiophene-fused oxadiborepins and the first azadiborepins attained via a modular synthesis route are presented. The new compounds emit intense blue light, some of which demonstrate fluorescence quantum yields close to unity. Cyclic voltammetry (CV) revealed electrochemically reversible one-electron reduction processes. The weak aromatic character of the novel 1,2,7-azadiborepin ring is demonstrated with in-depth theoretical investigations using nucleus-independent chemical shift (NICS) scans and anisotropy of the induced current density (ACID) calculations.

Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame-encoded peptides in top-down proteomics.

Short open reading frame-encoded peptides (SEP) represent a widely undiscovered part of the proteome. The detailed analysis of SEP has, despite inherent limitations such as incomplete sequence coverage, challenges encountered with protein inference, the identification of posttranslational modifications and the assignment of potential N- and C-terminal truncations, predominantly been assessed using bottom-up proteomic workflows. The use of top-down based proteomic workflows is capable of providing an unparalleled level of characterization information, which is of increased importance in the case of alternatively encoded protein products. However, top-down based analysis is not without its own limitations, for which efficient separation prior to MS analysis is a major issue. We established a sample preparation approach for the combined bottom-up and top-down proteomic analysis of SEP. Key improvements were made by the application of solid phase extraction (SPE), which supported enrichment of proteins below ca. 20 kDa, followed by 2D-LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterization of 36 proteoforms mapping to 12 SEP from the archaeon Methanosarcina mazei strain Gö1, with the concurrent detection and identification of several posttranslational modifications in SEP. BIOLOGICAL SIGNIFICANCE: Small or short open reading frames (sORF) have been widely neglected in genome research in the past. With their increasing discovery, the question about the presence and molecular function of their translation products, the short open reading frame-encoded peptides (SEP), arises. As these small proteins are usually below the 10 kDa range, the number of peptides identifiable by bottom-up proteomics is limited which hampers both the identification and the recognition of potential posttranslational modifications. The presented top-down approach allowed for the detection of full length SEP, as well as of terminally truncated proteoforms, and further enabled the identification of disulfide bonds in these small proteins. This demonstrates, that this yet widely undiscovered part of the proteome undergoes the same modifications as classical proteins which is an essential step for future understanding of the biological functions of these molecules.

Two novel heteropolymer-forming proteins maintain the multicellular shape of the cyanobacterium Anabaena sp. PCC 7120.

Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - for example, MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, for example, keratin and α/ß tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament-forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide independently in vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice.

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation.

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.

Exopeptidase Assisted N- and C-Terminal Proteome Sequencing.

Due to mechanisms such as proteolytic processing or alternative translation starts, in vivo proteoforms do not necessarily correspond directly to those encoded in the genome. Therefore, the knowledge of protein termini is an indispensable prerequisite to understand protein functions. So far, sequencing of protein N- and C-termini has been limited to single purified protein species, while the proteome-wide identification of N- and C-termini relies on the generation of single, terminal proteotypic peptides followed by chemical enrichment or depletion strategies to facilitate their detection via mass spectrometry (MS). To overcome the numerous limitations in such approaches, we present an alternative concept that readily enables unbiased ladder sequencing of protein N- and C-termini. The approach combines exopeptidase digestions of the proteome with two-dimensional chromatographic separation and tandem-MS. We demonstrate the potential of the methodology by analyzing the N- and C-terminome of S. cerevisiae, identifying 2190 N-termini and 1562 C-termini. In conclusion, the presented method largely expands the proteomics toolbox enabling N- and C-terminal sequential characterization of entire proteomes.

Identification and characterization of novel filament-forming proteins in cyanobacteria.

Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

A novel septal protein of multicellular heterocystous cyanobacteria is associated with the divisome.

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.

Degradome of soluble ADAM10 and ADAM17 metalloproteases.

Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.

Combination of SCX Fractionation and Charge-Reversal Derivatization Facilitates the Identification of Nontryptic Peptides in C-Terminomics.

The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC-MS/MS analysis. Charge-reversal improved both MS and MS/MS spectra quality of peptides carrying nonbasic C-terminal residues, allowing the identification of a high number of noncanonical C-termini not identified in nonderivatized samples. Further, we could show that C-terminal 18O labeling introduced during proteolytic processing of the samples is not suitable to distinguish internal from C-terminal peptides. The presented workflow enables the simultaneous identification of proteins by internal peptides and additionally provides data for the C- and N-terminome. Applying the developed workflow for the analysis of a Saccharomyces cerevisiae proteome allowed the identification of 734 protein C-termini in three independent biological replicates, and additional 789 candidate C-termini identified in two or one of three biological replicates, respectively. The developed analytical workflow allowed us to chart the nature of the yeast C-terminome in unprecedented depth and provides an alternative methodology to assess C-terminal proteolytic protein processing.

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis.

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.

Ylide-Functionalized Phosphines: Strong Donor Ligands for Homogeneous Catalysis.

Phosphines are important ligands in homogenous catalysis and have been crucial for many advances, such as in cross-coupling, hydrofunctionalization, or hydrogenation reactions. Herein we report the synthesis and application of a novel class of phosphines bearing ylide substituents. These phosphines are easily accessible via different synthetic routes from commercially available starting materials. Owing to the extra donation from the ylide group to the phosphorus center the ligands are unusually electron-rich and can thus function as strong electron donors. The donor capacity surpasses that of commonly used phosphines and carbenes and can easily be tuned by changing the substitution pattern at the ylidic carbon atom. The huge potential of ylide-functionalized phosphines in catalysis is demonstrated by their use in gold catalysis. Excellent performance at low catalyst loadings under mild reaction conditions is thus seen in different types of transformations.

SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets.

While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific 'network wiring'. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib-a tyrosine kinase inhibitor (TKI)-in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI.

Novel NEDD1 phosphorylation sites regulate γ-tubulin binding and mitotic spindle assembly.

During cell division, microtubules organize a bipolar spindle to drive accurate chromosome segregation to daughter cells. Microtubules are nucleated by the γ-TuRC, a γ-tubulin complex that acts as a template for microtubules with 13 protofilaments. Cells lacking γ-TuRC core components do nucleate microtubules; however, these polymers fail to form bipolar spindles. NEDD1 is a γ-TuRC-interacting protein whose depletion, although not affecting γ-TuRC stability, causes spindle defects similar to the inhibition of its core subunits, including γ-tubulin. Several residues of NEDD1 are phosphorylated in mitosis. However, previously identified phosphorylation sites only partially regulate NEDD1 function, as NEDD1 depletion has a much stronger phenotype than mutation of these residues. Using mass spectrometry, we have identified multiple novel phosphorylated sites in the serine (S)557-S574 region of NEDD1, close to its γ-tubulin-binding domain. Serine to alanine mutations in S565-S574 inhibit the binding of NEDD1 to γ-tubulin and perturb NEDD1 mitotic function, yielding microtubule organization defects equivalent to those observed in NEDD1-depleted cells. Interestingly, additional mutations in the S557-T560 region restore the capacity of NEDD1 to bind γ-tubulin and promote bipolar spindle assembly. All together, our data suggest that the NEDD1/γ-tubulin interaction is finely tuned by multiple phosphorylation events in the S557-S574 region and is critical for spindle assembly. We also found that CEP192, a centrosomal protein similarly required for spindle formation, associates with NEDD1 and modulates its mitotic phosphorylation. Thus CEP192 may regulate spindle assembly by modulating NEDD1 function.

The diversity of protein turnover and abundance under nitrogen-limited steady-state conditions in Saccharomyces cerevisiae.

To establish more advanced models of molecular dynamics within cells, protein characteristics such as turnover rate and absolute instead of relative abundance have to be analyzed. We applied a proteomics strategy to analyze protein degradation and abundance in Saccharomyces cerevisiae. We used steady-state chemostat cultures to ascertain well-defined growth conditions and nitrogen limited media, which allowed us to rapidly switch from (14)N to (15)N-isotope containing media and to monitor the decay of the (14)N mono-isotope signals in time. We acquired both protein abundance information and degradation rates of 641 proteins. Half-lives of individual proteins were very diverse under nitrogen-limited steady-state conditions, ranging from less than 30 min to over 20 h. Proteins that act as single physical complexes do not always show alike half-lives. For example the chaperonin-containing TCP-1 complex showed similar intermediate half-lives ranging from 7 to 20 h. In contrast, the ribosome exhibited a wide diversity of half-lives ranging from 2.5 to over 20 h, although their cellular abundances were rather similar. The stabilities of proteins involved in the central sugar metabolism were found to be intermediary, except for the glycolytic enzymes Hxk1p and Fba1p and the TCA-cycle proteins Lsc2p and Kgd1p, which showed half-lives of over 20 h. These data stress the need for inclusion of quantitative data of protein turn-over rates in yeast systems biology.

Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels.

BACKGROUND: The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast nat3Δ strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in nat3Δ. RESULTS: Comparing by proteomics WT and nat3Δ strains, using metabolic 15N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation. CONCLUSIONS: Protein expression levels change only marginally in between WT and nat3Δ. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in nat3Δ revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.

Introgressive hybridization and the evolutionary history of the herring gull complex revealed by mitochondrial and nuclear DNA.

BACKGROUND: Based on extensive mitochondrial DNA (mtDNA) sequence data, we previously showed that the model of speciation among species of herring gull (Larus argentatus) complex was not that of a ring species, but most likely due more complex speciation scenario's. We also found that two species, herring gull and glaucous gull (L. hyperboreus) displayed an unexpected biphyletic distribution of their mtDNA haplotypes. It was evident that mtDNA sequence data alone were far from sufficient to obtain a more accurate and detailed insight into the demographic processes that underlie speciation of this complex, and that extensive autosomal genetic analysis was warranted. RESULTS: For this reason, the present study focuses on the reconstruction of the phylogeographic history of a limited number of gull species by means of a combined approach of mtDNA sequence data and 230 autosomal amplified fragment length polymorphism (AFLP) loci. At the species level, the mtDNA and AFLP genetic data were largely congruent. Not only for argentatus and hyperboreus, but also among a third species, great black-backed gull (L. marinus) we observed two distinct groups of mtDNA sequence haplotypes. Based on the AFLP data we were also able to detect distinct genetic subgroups among the various argentatus, hyperboreus, and marinus populations, supporting our initial hypothesis that complex demographic scenario's underlie speciation in the herring gull complex. CONCLUSIONS: We present evidence that for each of these three biphyletic gull species, extensive mtDNA introgression could have taken place among the various geographically distinct subpopulations, or even among current species. Moreover, based on a large number of autosomal AFLP loci, we found evidence for distinct and complex demographic scenario's for each of the three species we studied. A more refined insight into the exact phylogeographic history within the herring gull complex is still impossible, and requires detailed autosomal sequence information, a topic of our future studies.