Alx1 is a pivotal transcription factor in a gene regulatory network that controls skeletogenesis throughout the echinoderm phylum. We performed a structure-function analysis of sea urchin Alx1 using a rescue assay and identified a novel, conserved motif (Domain 2) essential for skeletogenic function. The paralogue of Alx1, Alx4, was not functionally interchangeable with Alx1, but insertion of Domain 2 conferred robust skeletogenic function on Alx4. We used cross-species expression experiments to show that Alx1 proteins from distantly related echinoderms are not interchangeable, although the sequence and function of Domain 2 are highly conserved. We also found that Domain 2 is subject to alternative splicing and provide evidence that this domain was originally gained through exonization. Our findings show that a gene duplication event permitted the functional specialization of a transcription factor through changes in exon-intron organization and thereby supported the evolution of a major morphological novelty. The evolution of animal form has occurred through evolutionary modifications to the developmental programs that give rise to anatomy. There is considerable evidence that evolutionary changes in transcriptional networks have played an important role in morphological evolution . FurtherEchinoderms are a valuable experimental model for addressing the evolution of morphological novelty because of their diverse larval and adult body plans. There are five classes of echinoderms: echinoids (sea urchins), holothuroids (sea cucumbers), asteroids (sea stars), ophiuroids (brittle stars) and crinoids (sea lilies). The echinoids consist of two taxa, the euechinoids (thin-spined sea urchins) and the cidaroids (pencil urchins) . Most ecIn sea urchins, the embryonic skeleton is formed by a specialized population of cells known as primary mesenchyme cells (PMCs). These cells are the sole descendants of the large micromeres, four cells that form near the vegetal pole of the cleavage-stage embryo. The specification of the large micromere-PMC lineage occurs during early embryogenesis through a cascade of regulatory (i.e. transcription factor-encoding) genes that are organized into a functional unit termed a gene regulatory network (GRN) . The skealx1 is restricted to the large micromere-PMC lineage. Transcription of the alx1 gene can be detected as early as the 56 cell stage, during the first cell cycle after the large micromeres are born. The level of alx1 transcripts peaks at two developmental stages, at the pre-hatching blastula stage and later at the mesenchyme blastula stage, with expression persisting throughout later embryogenesis  blocks PMC specification while misexpression of alx1 results in the ectopic activation of the skeletogenic program in non-PMC lineages  at the tip of the archenteron  Alx1 (LvAlx1). We developed a rescue assay using morpholino-resistant alx1 mRNA that allowed us to test the function of motifs in the LvAlx1 protein via mutagenesis. We found that large parts of the protein, including the N-terminal region and most of the C-terminal region, were dispensable for LvAlx1 function in PMC specification, PMC patterning, and skeletogenesis. Further dissection of the C-terminal region, however, revealed that a small, novel domain, which we termed Domain 2, harbored motifs essential for LvAlx1 function. We found that LvAlx4 lacked skeletogenic function by our rescue assay; thus, LvAlx1 and LvAlx4 are not functionally redundant. Remarkably, insertion of LvAlx1 Domain 2 into LvAlx4 conferred robust skeletogenic functions on the LvAlx4 chimeric protein. We found that Domain 2 of Alx1 is contained within a short exon regulated by alternative splicing in sea urchins, pencil urchins and sea stars. Lastly, we used cross-species protein expression experiments to show that while Alx1 proteins from closely related sea urchin species are functionally interchangeable, Alx1 proteins from more distantly related echinoderms are not, due to protein-level differences within the C-terminal region but outside the highly conserved Domain 2. Taken together, our findings support the view that exonization of Domain 2 was an early and critically important event in the functional divergence of Alx1 and Alx4 and a major step in the evolution of echinoderm biomineralization. Other protein-level changes in Alx1 have accumulated more recently and limit the functional interchangeability of Alx1 proteins among echinoderms.In the present study, we performed a detailed structure-function analysis of sea urchin (L. variegatus) Alx1, we generated a MO-resistant, GFP-tagged, wild-type Lv-alx1 mRNA (LvAlx1.WT.GFP) by introducing six silent mutations into the MO target site, which was located at the 5’ end of the coding region  . We confWe next tested the ability of various mutant forms of LvAlx1 to rescue PMC specification and patterning. In initial experiments, we generated point mutations in a putative MAPK phosphorylation site which was previously identified in the N-terminal region of Alx1 . The MAPTo more comprehensively examine the role of the N-terminal region, three additional mutants were generated . DeletioC-terminal deletion mutations in LvAlx1 were also generated . SurprisTo identify possible functional domains in the C-terminal region, the sequence between the homeodomain and OAR domain was divided into four regions, designated Domains 1–4, based on splicing patterns and evolutionary conservation . In initTaken together, our deletion studies revealed that large regions of the Alx1 protein, including the N-terminus, the charged domain, Domains 1, 3 and 4, and the OAR domain, were dispensable for function in the embryo. To define a minimal construct sufficient for skeletogenic function, we generated three mutants with progressively larger truncations of the C-terminal region . We obseA potential concern regarding our rescue assay was that there might be variability in the levels of expression of different mutant Alx1 proteins or in their subcellular localization. We confirmed by fluorescence microscopy that mutant Alx1 proteins were expressed at levels very similar to that of LvAlx1.WT.GFP construct . We alsoWe confirmed that rescue of the morphant phenotype was not limited to early PMC specification and early patterning but was also associated with a full rescue of skeletal morphogenesis. For example, embryos co-injected with LvAlx1 MO and LvAlx1.ΔNΔChargedΔOARΔD34.GFP had normal triradiate spicules at the prism stage and elongated skeletal rods at the pluteus stage , confirmalx1 gene.The paralogous LvAlx1 and LvAlx4 proteins share an almost identical homeodomain and a highly conserved OAR domain while the remaining protein sequences are mostly dissimilar . In partStrongylocentrotus purpuratus (SpAlx1), a sea urchin species relatively closely related to L. variegatus (divergence time ~100 million years), has an amino acid sequence almost identical to that of LvAlx1, while the Alx1 protein from a more distantly related echinoderm, the sea star Patiria miniata (PmAlx1) (divergence time >450 million years) is less well conserved (S. purpuratus Alx1 (SpAlx1) or P. miniata Alx1 (PmAlx1) were co-injected into fertilized eggs. SpAlx1 was interchangeable with LvAlx1 but PmAlx1 was not   . This analx1, either within or outside of the echinoderms, supporting the view that this domain arose de novo within the alx1 gene by the recruitment of non-coding sequences. It has been shown that intrinsically disordered regions are enriched in domains obtained through exonization of intronic sequences  exon. In echinozoan alx1 genes , Domains 2 and 3 are contained on a single, separate exon. We analyzed the pattern of splicing in this region by RT-PCR and found that at all developmental stages tested, the wild-type [i.e. Domain (2 + 3)-containing] Lv-alx1 mRNA was the predominant isoform  exon, indicating that this region was also alternatively spliced in this species (data not shown).Newly born exons are alternatively spliced at a much higher frequency than old exons, and it has been argued that alternative splicing may provide an opportunity for natural selection to act on new protein structures without completely eliminating the parental form . We ther isoform . Two min isoform . In addialx1 genes, in an asterozoan, Domains 2 and 3 of Alx1 are encoded by a continuous open reading frame that also includes a portion of the homeodomain , PmAlx1 splice variant 1 contained an in-frame, alternative exon between Domains 3 and 4. In PmAlx1 splice variant 2, an alternative splice site at the 3’ end of Domain 3 was used to join the alternative exon, resulting in a frameshift and premature stop codon. In PmAlx1 splice variant 3, the alternative exon took the place of Domains 2 and 3, which were spliced out using an internal splice site at the 5’ end of Domain 2. In PmAlx1 splice variant 4, the Domain (2 + 3) exon and alternative exon were skipped. We also noted a change in the splicing pattern across development, such that by the late bipinnaria stage, when adult skeletal elements are being produced, levels of the wild-type PmAlx1 which contained the Domain (2 + 3) exon were elevated. These findings revealed a complex pattern of alternative splicing of the alx1 transcript in P. miniata and showed that the splicing pattern is developmentally regulated, with a shift to a greater abundance of the wild-type mRNA isoform (which encodes Domain 2) at stages when skeletogenesis is underway.In contrast to echinozoan eodomain . We noteL. variegatus) embryos. Thus, the N-terminal region (which contains a consensus MAPK phosphorylation site), the conserved OAR domain, and much of the C-terminal region are largely dispensable for these functions. Studies on vertebrate Alx1 have reported that the OAR domain and N-terminal region have modest, attenuating and enhancing influences, respectively, on transcriptional activity  domain, nor did we find signatures of this domain in other proteins, either within or outside echinoderms. Domain 2 is highly conserved among echinoderms, however, which is consistent with its functional importance. The specific biochemical function of Domain 2 remains to be elucidated. Owning to its proximity to the homeodomain, this domain may alter DNA binding directly or may act indirectly by recruiting protein partners that influence the transcriptional activity of Alx1.S. purpuratus embryos has been reported to result in ectopic skeletal spicule formation and a radialized skeletal phenotype (L. variegatus embryos (data not shown). This result was previously interpreted as evidence of the functional conservation of the PmAlx1 and LvAlx1 proteins, but a radialized skeleton can also result from a disruption of ectodermal patterning , and (2) the hemichordate Saccoglossus kowalevskii has a single alx4-like gene. The publicly available genome sequences of a different hemichordate, Ptychodera flava (http://marinegenomics.oist.jp/) confirms the presence of a single Alx4-like protein that clusters with the S. kowlavskii protein (data not shown).The echinoderm alx4 by alx1  Thecturally . (4) In  of alx1 . We proptal exon  and 11. ence see , while talx1 splicing in P. miniata revealed a developmentally regulated pattern, with increased levels of Domain 2-containing mRNA at stages of overt skeletogenesis. Moreover, our cross-species studies demonstrated that the sea star Domain 2 is functional in sea urchin embryos. It was shown previously that in sea stars, levels of alx1 mRNA are extremely low in the embryo but increase at larval stages, when alx1 is expressed in the skeletogenic centers of the adult rudiment  Alx1 protein during development. Finally, we note that while a role for Domain 2 in adult skeletogenesis seems at odds with our finding that this domain is absent from the single Alx1 protein sequence from crinoids, very limited cDNA sequence data (and no genomic data) are available from this group, and additional splice isoforms may well exist.The presence of a well-conserved Domain 2 in the Alx1 protein of sea stars, which do not form a larval skeleton, suggests that this domain may have both embryonic and adult functions. Indeed, our analysis of rudiment . Our finalx4-like gene lacks Domain 2. Adult hemichordates form small, calcareous skeletal elements termed ossicles, and orthologues of several echinoid biomineralization genes are transcribed during development  would be extremely informative. In addition, analysis of the biochemical function of Domain 2 will provide important insights concerning the mechanisms by which protein domain gain has driven the acquisition of novel structures.In hemichordates, the closest outgroup to echinoderms, the single elopment . This raLytechinus variegatus were acquired from the Duke University Marine Laboratory  and from Pelagic Corp. . Adult Strongylocentrotus purpuratus and Patiria miniata were acquired by Patrick Leahy . Gamete release in sea urchins was induced by intracoelomic injection of 0.5 M KCl and embryos were cultured in artificial seawater (ASW) at 19–24°C (L. variegatus) or at 15° (S. purpuratus) in temperature-controlled incubators.Adult L. variegatus alx1  was amplified by PCR and cloned into the BamHI and ClaI restriction sites of the pCS2 +GFP vector (LvAlx1.WT.GFP). Similar steps were taken to clone S. purpuratus and P. miniata alx1. The accession numbers or IDs of previously identified alx1 and alx4 genes can be found in . Overlap extension PCR was used to generate chimeric constructs. Individual DNA fragments were first amplified from plasmids using primers with overhangs that corresponded to the subsequent piece of DNA. Parental templates were then digested with DpnI. A second round of PCR was then carried out to assemble a full-length chimeric construct, which was subsequently cloned into the pCS2+GFP vector.Total RNA was isolated from mesenchyme blastula stage embryos using the NucleoSpin RNA isolation kit  and cDNA was synthesized using the SuperScript IV First-Strand Synthesis System . Full-length found in . E. tribase.org/ . P. flavoist.jp/ . DeletioMicroinjection of morpholinos (MO)  into fertilized eggs was performed as described . The traPMCs were visualized by immunostaining with monoclonal antibody (mAb) 6a9, which recognizes PMC-specific cell surface proteins of the MSP130 family . InjecteLiving embryos expressing GFP-tagged LvAlx1 (LvAlx1.GFP) were examined in their injection dishes using a compound epifluorescence microscope equipped with a 20X water-immersion objective. For observation of late stage larval skeletogenesis, living embryos were held between coverslips and microscope slides with strips of double sided tape as spacers and photographed using differential interference contrast (DIC) optics. Fixed embryos were examined using 20X and 40X dry objectives. Images were processed using ImageJ (NIH) and Adobe Photoshop CC 2015. The integrated density values of GFP fluorescence were obtained using ImageJ and subsequently normalized with the integrated density values of Texas Red dextran to account for differences in total embryo area and injection volume.Lvalx1 and Pmalx1 splice variants were designed to amplify products 250–650 bp in length. To sequence the splice forms, the PCR products were run on 1% agarose, extracted from the gel, and cloned into the pCS2+ vector.Total RNA was isolated from embryos of different developmental stages and cDNA was synthesized. Specific primer sets for  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Diethard Tautz as the Reviewing and Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Hiroshi Wada (Reviewer #2).Thank you for submitting your article "Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The paper reports analysis of the transcription factor Alx1 in which the authors have experimentally analyzed a protein domain that is critical to initiating skeletogenesis in urchin embryos. By producing chimeric versions of Alx, in which the critical domain of orthologues and paralogues are substituted the authors establish the necessity and sufficiency of the domain. An analysis of Alx gene organization and splice patterns in several related species suggests that a gene duplication event was followed by a recruitment of a new exon from intronic sequences. They conclude that the gain in protein domains provides an evolutionary mechanism for novel morphology to arise.Both reviewers find the experiments, the writing and the conclusions compelling. There are only a few points that need clarification.Essential revisions:1) There is a confusing aspect of the naming of the critical domain of the Alx1 protein. The authors refer to it as a C2 domain and note that their searches indicate it is not a previously characterized protein domain. There is a C2 protein domain that is well characterized and involved in calcium dependent membrane binding of proteins such as PTEN and PKC to specific membranes. As the authors do not refer to this literature, I assume the name they have chosen for the Alx1 protein domain is redundant and may confuse readers. It is therefore necessary to use another nomenclature for the domains to avoid such a confusion.2) It is not clear how the authors established the domain boundaries. It is unclear whether they are based upon protein structure predictions or the position of the splice boundaries. Given that the minimal construct is the homeodomain+C1+C2, is it not possible that there are regions within C1 that are critical to Alx1 function? The Alx4 with the Alx1 C2 domain construct rescues, but the C1 domains of Alx1 and Alx4 are very similar . As well, the authors conclude  that sequence differences in the PmAlx1 outside the C2 domain are critical to its ability to rescue. It seems the C2 domain is necessary, but C1+C2 is sufficient for rescue. The concern here is that the model proposed focuses on the acquisition of the C2 domain, which appears to be defined by splice boundaries. The argument that the domain is acquired by a change in the splice location becomes somewhat circular when the domain is defined only by splice locations.3) In the second paragraph of subsection “The C2 domain is subject to alternative splicing and likely arose by exonization”, the authors describe an analysis that suggests that C3 is intrinsically disordered, but it is not clear how they relate this to the evolution of the C2 domain. It would be necessary to show this in a specific figure and put it into the context of de novo evolution of genes .4) Experiments are reported that indicate that loss of MAPK target sites has no effect on Alx1 function. Previous studies have established that loss of MAPK pathway components inhibits skeletogenesis. It will be necessary to have a discussion of their interpretation of these data. Essential revisions:1) There is a confusing aspect of the naming of the critical domain of the Alx1 protein. The authors refer to it as a C2 domain and note that their searches indicate it is not a previously characterized protein domain. There is a C2 protein domain that is well characterized and involved in calcium dependent membrane binding of proteins such as PTEN and PKC to specific membranes. As the authors do not refer to this literature, I assume the name they have chosen for the Alx1 protein domain is redundant and may confuse readers. It is therefore necessary to use another nomenclature for the domains to avoid such a confusion.We thank the reviewer for pointing out that “C2” was an inappropriate choice for a domain name, as that name is already associated with a well-characterized (and completely different) protein domain. Instead of calling the four regions within the C-terminal region of Alx1 “Domains C1, C2, C3, and C4,” we now assign them the very generic names of “Domains 1, 2, 3 and 4.”2) It is not clear how the authors established the domain boundaries. It is unclear whether they are based upon protein structure predictions or the position of the splice boundaries.The boundaries were based partly on splice junctions and partly on evolutionary conservation, not on protein structure prediction. We have clarified this in the text . As well, the authors conclude  that sequence differences in the PmAlx1 outside the C2 domain are critical to its ability to rescue. It seems the C2 domain is necessary, but C1+C2 is sufficient for rescue. The concern here is that the model proposed focuses on the acquisition of the C2 domain, which appears to be defined by splice boundaries. The argument that the domain is acquired by a change in the splice location becomes somewhat circular when the domain is defined only by splice locations.The relevant experiment here is the one in which we deleted the C1 (now Domain 1) region and showed this has no effect on Alx1 function see . Based o3) In the second paragraph of subsection “The C2 domain is subject to alternative splicing and likely arose by exonization”, the authors describe an analysis that suggests that C3 is intrinsically disordered, but it is not clear how they relate this to the evolution of the C2 domain. It would be necessary to show this in a specific figure and put it into the context of de novo evolution of genes .We thank the reviewer for calling our attention to the recent Wilson et al., (2017) paper, which we now cite. Essentially our argument is that the high degree of intrinsic disorder of the C3 domain supports the hypothesis that the C2+C3 exon arose relatively recently from non-coding sequences. This is consistent with several studies (including the Wilson paper) which show that young proteins (or protein domains) show elevated levels of intrinsic disorder, perhaps because a disordered structure makes new protein sequences less likely to be harmful to cells. We hypothesize that the highly conserved C2 domain has become less disordered over time as it has acquired an important function, while the disorder of C3 has persisted. Our feeling is that this argument is probably not complex enough to warrant a separate figure (as it is just a hypothesis), but we have expanded upon it in the Discussion section.4) Experiments are reported that indicate that loss of MAPK target sites has no effect on Alx1 function. Previous studies have established that loss of MAPK pathway components inhibits skeletogenesis. It will be necessary to have a discussion of their interpretation of these data.Previous studies have shown that MAPK-mediated phosphorylation of a different PMC transcription factor, Ets1, is required for PMC specification. We wondered whether MAPK might also phosphorylate Alx1 and that this might be part of the role of MAPK, but this does not seem to be the case as we can delete the phosphorylation site without affecting Alx1 function. Thus, Ets1 might completely account for the MAPK requirement. We have added statements to this effect in the text .


Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin β1 and β2 chains. We report the crystal structure at 1.85 Å resolution of the laminin β2 LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin β1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4–LE5 junction. These results advance our understanding of laminin structure. •The crystal structure of the laminin β2 LE5-LF-L6 region has been determined.•The LF domain is a β-sandwich with distant homology to the L4 domains in laminin α and γ chains.•A unique α-helical region in the LF domain interacts extensively with LE5.•LE5 and LE6 are arranged in a manner typical of tandem LE domains, despite the insertion of the LF domain. Heterotrimeric (αβγ) laminins are a major constituent of all basement membranes The three short arms of the laminin heterotrimer are composed of long repeats of laminin-type epidermal growth factor-like (LE) domains, capped by laminin N-terminal (LN) domains that mediate the self-interaction of laminins. The LE repeats are interrupted once (β and γ chains) or twice (α chains) by globular domains of unknown function . The insThe LE fold was first described by Stetefeld et al. A recent crystal structure of the second L4 domain of the laminin α2 chain (α2 L4b) revealed an irregular β-sandwich similar to bacterial carbohydrate binding modules, ephrin-binding modules and MAM domains free = 0.210 at 1.85 Å resolution , Glu575 (bidentate) and Asp719 (monodentate), as well as the carbonyl oxygen atoms of residues 598, 601 and 719; the resulting coordination geometry is a pentagonal bipyramid –β10–β5–β8 and β4–β9–β6–β7. The long central strand β10 interacts with β1 and with β3. A disulphide bond, Cys657–Cys685, links the β6–β7 and β8–β9 loops. Strong spherical electron density indicated that a metal ion is bound to the β1–β2 and β2–β3 loops. This ion was assigned as Caipyramid B. Sequen domains C.2 of solvent-accessible surface.The α-helical region of the LF domain consists of three helices held together by a disulphide bond, Cys752–Cys768, and a substantial hydrophobic core  that is conserved in other LF domains C. Helix Clostridium thermocellum2 + site is conserved in the two domains. The loops connecting the strands of the β-sandwich are quite different, however, and the carbohydrate binding site of CBM35 is not conserved in the LF domain. Comparison of the laminin β2 LF domain with the laminin α2 L4b domain A search for related structures using PDBeFold An unexpected feature of the laminin β2 LE5-LF-LE6 structure is the relative arrangement of LE5 and LE6, which closely resembles the canonical arrangement of LE domains that are not separated by an inserted domain. A superposition of the β2 LE5-LF-LE6 structure with domains LE8–LE9 of the laminin γ1 chain ab initio reconstruction resembles a boomerang with bulbous tips. The β1 LN-LE4 crystal structure 2 value of 5.6  was amplified from a cDNA kindly provided by Jeffrey H. Miner. The PCR product was cloned into a modified pCEP-Pu vector P15800.The coding sequence for the LE5-LF-LE6 region of rat laminin β2 (UniProt P02469) was amplified from a cDNA kindly provided by Peter D. Yurchenco. The PCR product was cloned into a modified pCEP-Pu vector The coding sequence for the LN-LE6 region of mouse laminin β1  using a w/w ratio of DNA:PEI of 1:3. The conditioned medium containing the secreted protein was collected 72 h after transfection. The filtered cell culture supernatant was adjusted to a final concentration of 20 mM Na-HEPES (pH 7.5) and loaded onto a 5-ml HisTrap Excel column  using an Äkta Purifier . The column was washed with 20 mM Na-HEPES, 150 mM NaCl (pH 7.5) and the protein was eluted with the same buffer containing 200 mM imidazole. Fractions containing protein were concentrated using a Vivaspin centrifugal device (Sartorius) to a final concentration of 1 mg/ml. The protein solution was dialysed and digested with His-tagged TEV protease (made in Escherichia coli using an expression vector kindly provided by Stephen Curry) in a enzyme:substrate ratio of 1:10 for 18 h at 4 °C. The reaction mixture was loaded onto a 1-ml HisTrap FF column  and the flow through was collected. Fractions containing protein were concentrated using a Vivaspin centrifugal device and further purified on a Superdex 200 10/300 column  using 20 mM Tris–HCl, 150 mM NaCl (pH 7.5) as the running buffer.The laminin β2 LE5-LF-LE6 protein was produced using the FreeStyle™ 293 Expression System (ThermoFisher Scientific) following the manufacturer's procedures. Briefly, 293-F cells were grown in a shaking incubator at 37 °C with 8% COThe laminin β1 LN-LE6 protein was produced in human embryonic kidney 293 c18 cells and purified as described for other laminin short arm fragments 2(NO3)2 or 0.5 mM p-chloromercuribenzoic acid (PCMB), or for 30 s in 500 mM NaI. The crystals were flash-frozen in liquid nitrogen directly from the drops.Screening was done at 20 °C by the sitting-drop vapour diffusion method using 96-well plates (Greiner) and a range of commercial screens. A Mosquito nanolitre robot (TTP Labtech) was used to set up 200 nl drops. The initial crystals of laminin β2 LE5-LF-LE6 were obtained using a protein concentration of 10 mg/ml and condition H4 of the Morpheus screen (Molecular Dimensions), which contains a mixture of amino acids and polyethylene glycols buffered at pH 6.5. Larger crystals were grown in 2 μl hanging drops using the same precipitant solution. The crystals first appeared as heavily intergrown plates, which over the course of a week matured into large single crystals. Heavy-atom derivatives were prepared by soaking the crystals for 18 h in 0.2 mM UOC2 with two copies of laminin β2 LE5-LF-LE6 in the asymmetric unit. CC1/2 was used to determine the resolution limits www.pymol.org).Diffraction data were collected at 100 K at beamlines I04-1 (native data) and I03 (derivative data) of the Diamond Light Source . The data were processed using XDS 2 (pH 7.5) and analysed by small angle X-ray scattering with in-line size exclusion chromatography (SEC-SAXS) at the SWING beamline of the SOLEIL synchrotron . The chromatographic separation of the sample was achieved using an Agilent HPLC with a BioSEC-3 column (300 Å pore size). SAXS data were recorded over a momentum transfer range of 0.0063–0.6162 Å− 1. The data were integrated, buffer-subtracted, and merged using the FOXTROT software G values via Guinier analysis. GNOM max values and generate a P(r) distribution for ab initio envelope determination, which was done using DAMMIF/DAMINN www.cgl.ucsf.edu/chimera).The laminin β1 LN-LE6 protein was concentrated to 9 mg/ml in 20 mM Na-HEPES, 150 mM NaCl, 2 mM CaClThe coordinates of the laminin β2 LE5-LF-LE6 structure have been deposited in the Protein Data Bank under code 5LF2.


We have studied experimentally the interaction of isolated needle-like domains created in an array via local switching using a biased scanning probe microscope (SPM) tip and visualized via piezoelectric force microscopy (PFM) at the non-polar cuts of MgO-doped lithium niobate (MgOLN) crystals. It has been found that the domain interaction leads to the intermittent quasiperiodic and chaotic behavior of the domain length in the array in a manner similar to that of polar cuts, but with greater spacing between the points of bias application and voltage amplitudes. It has also been found that the polarization reversal at the non-polar cuts and domain interaction significantly depend on humidity. The spatial distribution of the surface potential measured by Kelvin probe force microscopy in the vicinity of the charged domain walls revealed the decrease of the domain length as a result of the partial backswitching after pulse termination. The phase diagram of switching behavior as a function of tip voltage and spacing between the points of bias application has been plotted. The obtained results provide new insight into the problem of the domain interaction during forward growth and can provide a basis for useful application in nanodomain engineering and development of non-linear optical frequency converters, data storage, and computing devices. Local polarization switching via the biased conductive tip of a scanning probe microscope (SPM) has been studied since the first experimental realization of piezoresponse force microscopy (PFM) ,2. FurthA recent study of the local switching at the polar cut of lithium niobate crystals allowed to reveal various regimes of domain interaction depending on the switching parameters: temperature, humidity, applied bias, and the spacing between the points of bias application . It was It must be stressed that local switching by a biased SPM tip at thick polar cut LN plates creates needle-like domains of a sub-micron diameter with the charged domain walls . The knoThe studied domain interaction has also been described in the correlated nucleation effect, which led to discrete domain switching ,19 and tuniform, intermittent quasiperiodic, and chaotic—and plotted the phase diagram of the switching behavior as a function of tip voltage and the spacing between the points of bias application. The obtained results provide new insight into the problem of domain interaction during forward growth and can provide a basis for the useful application of nanodomain engineering and development of non-linear optical frequency converters, data storage, and computing devices.Here, we have experimentally studied the length change of interacting needle-like domains created by a biased SPM tip and visualized by PFM at the non-polar cut of MgO-doped lithium niobate (MgOLN) crystals. It has been revealed that the domain interaction leads to effects that are similar to those of polar cuts, but with the greater spacing between the points of bias application and voltage amplitudes. The decrease in domain length as a result of partial backswitching after pulse termination has been measured by Kelvin probe force microscopy (KPFM). We separated three main regimes of domain interaction—The choice of the studied material (congruent lithium niobate doped by 5 mol % MgO) is due to the low switching threshold fields and speedup of the bulk screening process as a result of doping. Moreover, the essential increase of the bulk conductivity leads to a larger optical damage threshold, thus stimulating the wide application of MgOLN in various nonlinear optical devices . Investigated samples (15 mm × 10 mm) of MgOLN were cut from 1-mm-thick wafers with a surface roughness of about 1 nm . The bottom surface was glued to the conductive substrate by silver paste.The arrays of isolated needle-like domains were created by the application of a series of rectangle voltage pulses using a conductive SPM tip  with a constant distance between switching points. We used a pulse duration of 500 ms and amplitudes ranging from 60 to 140 V. The pulses were generated using an NI-6251 multifunction Data Acquisition Board  and high-voltage amplifier Trek-677B . The domain structure was visualized using Probe NanoLaboratory NTEGRA Aura  in PFM mode. Commercial probes NSC18 with a titanium–platinum conductive coating  with a radius of curvature R = 35 nm, resonance frequency f = 70 kHz, and spring constant k = 3.5 N/m were used. The piezoresponse measurements were realized by the application of 1–2 V AC voltage with a frequency close to the contact resonance. The spatial distribution of the surface potential was measured by a closed loop two-pass KPFM with the 0.1 V AC voltage applied to the tip and a 100 nm tip–surface distance at the second pass. The variable relative humidity was controlled using a homemade air humidifier. In order to study domain–domain interactions at the non-polar cut, we wrote the arrays of isolated needle-like domains in line with various periods. Decrease in the spacing between the points of bias application allowed us to find that, for short enough periods, the length of the domains in the arrays essentially changed  . According to the results of studying the local polarization switching at the polar cut, we expected the domain sizes to decrease as the relative humidity increased, due to the formation of the water meniscus on the SPM tip . We showWe found that increasing the relative humidity up to 40% results in complete domain backswitching (disappearance of the switched domains) after pulse termination . Analysis of the electric field distribution by KPFM demonstrated the existence of the electric field produced by charged domain walls of the needle-like domains. The lower value of the surface potential in the area close to the domain end b has beeWe did not observe any significant differences between sizes of domains produced in dry air and with a relative humidity of about 20%. Moreover, we found that such humidity essentially decreased a general “background” of the surface potential caused by the spread of the injected charge. For this reason, all further experiments were conducted with a relative humidity of about 20%.uniform  uniform a, with adoubling b; and (3(chaotic c. The smn+1 − xn = q(xn), where xn+1 and xn are the lengths of the previous and subsequent domains, respectively, and q is the empirical function, whose behavior depends on the domain interaction. For the characterization of the different regimes, we plotted the normalized difference of the neighboring domains length versus the normalized length of one of them: xuniform regime realized for the large spacing between the points of bias application and low voltage, the domain interaction function q(xn) is almost constant, which leads to the dense position of the experimental points at the plot  and spacing between the points of bias application (domain spacing) allowed for the phase diagram to be obtained . The phauniform, intermittentquasiperiodic, and chaotic—and built a phase diagram. The obtained results provide new insight into the problem of domain interaction during forward growth and can provide a basis for the useful nanodomain engineering applications: optical and non-linear optical frequency converters, data storage, and computing devices.In conclusion, we experimentally studied the interaction of isolated domains at the non-polar cuts of the doped lithium niobate. It was found that the interaction was organized in a manner similar to that of polar cuts, but with different spacing and voltage amplitude characteristics. The polarization switching at the non-polar cuts of the lithium niobate and domain interaction were found to be significantly modified by the humidity, so we limited our studies to a relative humidity range of up to 20%. We separated three main regimes of interaction—


Protein domain prediction is one of the most powerful approaches for sequence-based function prediction. Although domain instances are typically predicted independently of each other, newer approaches have demonstrated improved performance by rewarding domain pairs that frequently co-occur within sequences. However, most of these approaches have ignored the order in which domains preferentially co-occur and have also not modeled domain co-occurrence probabilistically.We introduce a probabilistic approach for domain prediction that models ‘directional’ domain context. Our method is the first to score all domain pairs within a sequence while taking their order into account, even for non-sequential domains. We show that our approach extends a previous Markov model-based approach to additionally score all pairwise terms, and that it can be interpreted within the context of Markov random fields. We formulate our underlying combinatorial optimization problem as an integer linear program, and demonstrate that it can be solved quickly in practice. Finally, we perform extensive evaluation of domain context methods and demonstrate that incorporating context increases the number of domain predictions by ∼15%, with our approach dPUC2 (Domain Prediction Using Context) outperforming all competing approaches.http://github.com/alexviiia/dpuc2.dPUC2 is available at Bioinformatics online. Plasmodium falciparum proteome . Let iF be the domain family of the domain instance iD; note that domain instances are individual predictions, while domain families correspond to Pfam HMMs. Our goal is to identify Given a set of candidate domains, we develop an approach that identifies the most likely subset of domains under a probabilistic domain co-occurrence model. Let iD asiX is the subsequence of the predicted domain, iX given the HMM of iF , and iX under the null model R of independent and identically distributed amino acids  model repeating domains. Pair probabilities, corresponding to the proportion of all domain pairs that are of a given family pair, are estimated using a symmetric Dirichlet prior with parameter α, yieldingn is the number of domain families  and iF preceding an instance of family jF is estimated asWe use a directed ‘context network’ of family pair counts α = 1.Counts We find optimal domain architectures with two strategies: ‘positive elimination’ (PE), followed by ILP. PE discards domains with poor HMMER3 and context scores, returning trivial solutions (see below) or passing a smaller set to the ILP solver. The combinatorial ILP problem is solved using the lp_solve C library  in the ILP ensures that, out of a set of domains C that all conflict with each other, only one is chosen –(5) correspond to the ‘AND’ operation of polytope . In contF for which All tests in the main body of this article use Pfam 25  across a range of noise levels, we used ‘-E 1e-2 --domE 1e-2’ to set a permissive P-value threshold. For measurements of runtime, we used ‘--cpu 0’ to use a single thread , and ‘-E 1e-4 --domE 1e-4’ to set final P-value thresholds, as this is how our tool dPUC2 would be used in practice.Domains are predicted with hmmscan from HMMER 3.0. We used: ‘--F1 1e-1 --F2 1e-1 --F3 1e-2’ to set ‘stage 1/2/3’ i in the test, let i with domain predictions  (RevSeq (Reverse Sequence) pools domain predictions from real and reversed UniRef50 sequences . Different methods select domains from this set, treating all domains as belonging to one sequence . Selectehttp://github.com/alexviiia/RandProt). Real and random sequence domains are merged, and then MarkovR FDRs are computed similarly to the RevSeq FDR test (MarkovR (Markov Random) generates random sequences from a second-order Markov model of UniRef50 . Further, each ortholog group is pruned so that no two sequences share >90% identity. This results in a total of 79 386 ortholog families that consist of 525 258 sequences. We note that the two other previously introduced tests  labels domains in a sequence as TP or FP if domains of the same clan are present in its orthologs or not, respectively , as descE-value thresholds are varied. Both Extended Standard Pfam and E-values remove conflicting domains  ranking by P-value.We first compare how well our approach identifies domains as compared with Pfam when using several different approaches for setting thresholds. The ‘Standard Pfam’ predicts domains using the Pfam domain and sequence thresholds, but removing domain instances of the same clan that overlap strictly (one or more amino acids), keeping only the most significant prediction per conflict . Lastly, we also test our previous dPUC1 approach, which does not take into account directional context. In this case, due to runtime limitations, it is not feasible to run dPUC1 directly in large-scale testing; instead, the dPUC1 objective is solved using the dPUC2 ILP outlined here. We did not test the methods of We also compare the performance of our method to three previous methods that use domain context. First, DAMA  are simply varied, across the entire range of estimated FDRs , dPUC2 identifies more domains than DAMA but also results in higher estimated FDRs; this is due in part to dPUC2 keeping all Standard Pfam predictions in its input (see below). Thus, while overall dPUC2 performs better than DAMA, when using highly confident domain predictions, dPUC2 and DAMA trade off coverage and noise in the MarkovR test. Finally, in every FDR test, dPUC2 also improves upon dPUC1, illustrating the benefit of incorporating directional context. Directional context reduces the FDR of predictions produced for the same inputs; for example, when using candidate domains with P < 1e-4, dPUC2 results in a lower FDR than dPUC1 . Using a 3.2 GHz processor with 16 GB RAM, we find that the HMMER3 step that predicts candidate domains averages 0.455 second per protein (s/protein) in wallclock runtime when run in a single thread; this is comparable to a previous reported average of ∼1 s/protein  predicting binding sites for sets of transcription factors.In the future, it is likely that context-based methods could be further improved by using q-values  instead Supplementary DataClick here for additional data file.


RH and 27 ± 2°C. Sprout was removed, dried in hot air oven at 50 ± 2°C for 24 hr and milled to pass through 40 mesh sieve. For fermented sorghum flour, 13.3 mg% diastase and 2 mg % pepsin (on the basis of whole sorghum flour weight) was added to cooked (88 ± 2°C) sorghum flour and left for 1 hr. Lactobacillus plantarum (107 cfu/g) was inoculated and incubated at temperature 30 ± 2°C for 48 hr. The fermented slurry was dried at 50 ± 2°C in hot air oven for 24 hr and milled to pass through 40 mesh sieve. The lower yield of sorghum flour was obtained compared to whole and malted sorghum flour. Germination of sorghum reduced phytate, tannin, and oxalate by 40%, 16.12% and 49.1%, respectively, whereas fermentation of sorghum flour reduced above by 77%, 96.7% and 67.85%, respectively. There was no significant change in hydrogen cyanide in malted sorghum flour compared to whole sorghum flour, but fermentation of sorghum flour reduced hydrogen cyanide by 52.3%. Bulk density and viscosity was significantly reduced by the malting and fermentation, whereas water absorption capacity and oil absorption capacity was markedly increased by the malting and fermentation. Fermented flour was good due to reduced ANF and improved functional property despite of lower yield.The research was aimed to observe the effect of malting and fermentation on antinutritional component and functional characteristics of sorghum flour. For whole sorghum flour, cleaned sorghum grain was milled to pass through 40 mesh sieve. For malting, cleaned sorghum grain was steeped in 0.2% calcium hydroxide solution for 24 hr and then germinated for 48 hr at 90%  The pH dropped from 6.2 to 4.3. Fermented slurry was dried at 50 ± 2°C for 24 hr. Dried fermented sorghum cake was milled in grinder  and passed through 40 mesh size screen (HT/Standard sieves)  and passed through 40 mesh size screen (HT/Standard sieves). For malted sorghum flour, cleaned sorghum was steeped in 0.2% calcium hydroxide solution for 24 hr. The ratio of the solution and sorghum was 2:1. Water was drained and washed with distilled water and germinated in the RH chamber at 90% RH and 27 ± 2°C for 48 hr. Sprouts were manually removed and dried at 50 ± 2°C for 24 hr. Dried malted sorghum was milled in grinder  and passed through 40 mesh size screen (HT/Standard sieves)  was used to perform analysis of variance (one way) to determine significant differences using the least significant difference (LSD) at 5% significance level. Triplicate data were used for computation.33.1The yield of the flour followed by the different processes applied is shown in Figure The low yield of flour may be due to removal of the bran and germ after sieving  of the Whole sorghum flour was found to be 126.72%. There is a significant increase in the water holding capacity of germinated and fermented sorghum flour. Water absorption of the flour is mainly depends upon protein, carbohydrate, their interaction and nature (Echendu, Onimawo, & Somtochi,4Malting and fermentation significantly reduced antinutritional components of sorghum flour compared to malting and unprocessed. However, malting did not produce any significant changes in the hydrogen cyanide content of sorghum flour. Malting and fermentation reduce bulk density and viscosity but increased oil absorption capacity. Fermented sorghum flour was better due to reduce antinutritional factor and improved functional properties.None declared.


Much of the fossil record for dogs consists of mandibles. However, can fossil canid mandibles be reliably identified as dogs or wolves? 3D geometric morphometric analysis correctly classifies 99.5% of the modern dog and wolf mandibles. However, only 4 of 26 Ust’-Polui fossil mandibles, a Russian Arctic site occupied from 250BCE to 150CE, were identified as dogs and none of the 20 Ivolgin mandibles, an Iron Age site in southern Russia, were identified as dogs. Three of the Ust’-Polui mandibles and 8 of the Ivolgin mandibles were identified as wolves. In contrast, all 12 Ivolgin skulls and 5 Ust’-Polui skulls were clearly identified as dogs. Only the classification of the UP6571 skull as a dog (Dog Posterior Probability = 1.0) was not supported by the typical probability. Other evidence indicates these canids were domesticated: they were located within human dwellings, remains at both sites have butchery marks indicating that they were consumed, and isotope analysis of canid and human remains from Ust’-Polui demonstrate that both were consuming freshwater protein; indicating that the humans were feeding the canids. Our results demonstrate that the mandible may not evolve as rapidly as the cranium and the mandible is not reliable for identifying early dog fossils. Previous research on canid fossil mandibles and skulls has employed Euclidean distances for identification7. We conducted a three-dimensional geometric morphometric analysis of fossil mandibles and skulls from Ust’-Polui, Ivolgin, and Alaska to determine whether these are dog or wolf fossils19. The Ust’-Polui and Ivolgin sites date to the late Holocene, post-dating the advent of dog domestication by millennia19. The Ust’-Polui archaeological site is in Salekhard, Russia, in the Arctic . Ust’-Polui has produced thousands of artifacts and faunal remains, including disarticulated skeletal elements from over 100 canids, nearly all of which were originally identified as dogs based on their relative small sizes compared to Arctic wolves19. At least two of the canid skeletons found at this site were fully articulated burials . Radiocarbon dating and dendrochronology indicate this fortified site was occupied from ~250BCE to 150CE by foragers19. The Ivolgin site is in the steppe region of southern Russia near Ulan-Ude . Ivolgin consisted of a series of earth ramparts and a wood stockade that surrounded over 50 wooden dwellings17. This town was probably occupied by the Xiongnu, a confederation of Iron Age pastoral groups, from ~300BCE to 200CE17. Scattered faunal remains were found throughout Ivolgin, with over 90% identified as domestic animals . At least 59 specimens from Ivolgin were previously identified as dogs based on size comparisons with southern Siberian wolves16. The only wild canid identified was fox16. At both Ust’-Polui and Ivolgin, some of the canid remains display butchery marks, indicating they were consumed by people. Additionally, we analyzed carbon-dated late Pleistocene canid fossils from Alaskan permafrost deposits that were all genetically classified as wolves20. Finally, Alaskan canids carbon-dated near 1600CE and genetically classified as dogs also were analyzed21. These two sets of Alaskan canid fossils were included to determine whether genetic identifications correspond with those based on morphology.The date and location of dog domestication is a contentious issue whether the evidence being considered is genetic or morphological7. As demonstrated in our previous analysis of the canid skull6, distance measurements are problematic for multiple reasons including: autocorrelation, isometry, overlap between dogs and wolves (making identification impossible), spurious correlations in multivariate analyses, and non-normal distributions22. Capturing 3D coordinates from mandibles and skulls provides a more accurate representation of their inherent 3D shapes than one-dimensional distances and ratios  are widely recognized as powerful and sophisticated diagnostic tools for investigating biological shape15. Procrustes superimposition of the coordinate configurations removes information related to size by scaling all configurations to the same centroid size while translating and rotating the landmark configurations using a least-squares fit of homologous landmarks25. Thus, Procrustes coordinates only contain information pertaining to shape, having removed information related to size, position, and rotation25. Here we re-analyze the canid skulls and mandibles from the Ust’-Polui, Ivolgin, and Alaska sites using 3D GMM to assess whether they can be accurately identified as dogs or wolves. We compared the fossil mandibles and skulls to a large dataset of modern mesaticephalic (wolf-like) dogs and a global assemblage of both modern and fossil wolves. We hypothesize that the fossil skulls will be accurately identified as wolves or dogs; however, it remains to be seen if these fossil mandibles can be reliably categorized.Traditional morphometric analyses of fossil canid mandibles and skulls aimed at identifying dogs depend mostly on one-dimensional measures of length and widthPC1 = 0.99, P < 0.001) from the large, robust jaws of wolves and dog breeds such as German Shepherds to the smaller mandibles of breeds like the Fox Terrier . The curvature of the mandible distinguishes dogs from wolves along PC2  revealed that modern dog and wolf mandibles are separated within the first three principal components, which account for 92.3% of the total mandible form-space variance Fig. . PC1 capier Fig. . Wolves ier Fig. . Dogs diPC2 Fig. .Figure 199.5% of modern canid mandibles were correctly identified as either dogs or wolves with 100% accuracy using a resampling procedure involving one-thousand iterations of a cross-validation Quadratic Discriminant Analysis (QDA) to ensure equal dog and wolf sample sizes, with posterior probability greater than 0.90 . Furthermore, the classifications were supported by the typical probabilities.20. Our results also verified that 2 of the Alaskan mandibles dated near 1600CE (AMNH30436 and AMNH70932) are dogs  dogs, ancient dogs, and the global assemblage of both modern and fossil wolves from our previous studynce Fig. . The Ustnce Fig. 6. The rens Table . Only thns Table . The lat21, our analysis only classified 2 of these mandibles as dogs  and elevated δ15N values   and elk  at the site have far more positive δ13C values , and much lower δ15N values . Bone collagen δ15N values show enrichment of 3–5‰ along the food chain, providing an indication of trophic level27. The canids and humans at Ust’-Polui, all with δ15N values above 12‰, were regularly consuming food items with higher δ15N values than those of these large-bodied terrestrial herbivores. In the Arctic, such elevated δ15N values appear more consistent with dietary reliance on freshwater or marine fauna. Neither freshwater fish or marine mammals are well represented in our isotope data, but other studies show that Arctic freshwater fish have far more negative δ13C values than marine mammals and fish, ranging from around −30‰ to −20‰, with the two groups of marine fauna often having more positive values than terrestrial ungulates35. Offsets in δ13C between prey collagen and predator collagen are around 1‰36. The very negative δ13C values for the canids and humans at Ust’-Polui suggest both were regularly consuming freshwater fish; remains of such fish are highly abundant at the site19. Such dietary patterns are inconsistent with those of wolves living in the Arctic, where water bodies are frozen for much of the year38, but are consistent with people partially provisioning dogs with their own food items, a historically well-documented practice in many areas39.Surprisingly, only 15% of the mandible specimens from the Ust’-Polui site were classified as dogs despite the very high accuracy of this procedure in correctly assigning known specimens to their group. In addition, 69% of the mandible specimens from Ust’-Polui and 60% of the mandible specimens from Ivolgin were unclassified as either dogs or wolves. The unclassified mandibles are found outside the wolf mandible morphospace because they are smaller than the wolves and outside the dog mandible morphospace because of their wolf-like shape. However, all but one of the skull specimens from both sites were identified as dogs. Interestingly, we saw a similar pattern in the fossil canids from Alaska. Although 4 of 1600CE Alaskan fossil mandibles had been genetically identified as dogs21.The lack of consistent identification of the mandibles as either dogs or wolves could indicate several things. The mandibles seem unlikely to be from small wolves or hybrids because neither wolf nor hybrid crania were found at either site; all of the skulls from both sites were clearly identified as dogs. It also seems improbable that the mandibles are from some other type of canid, as one would expect their crania also to be present at these sites, both of which have been extensively excavated. Perhaps most telling, even some of the Late Holocene mandibles from Alaska were not morphologically identified, despite the identification of the skulls as dogs as well as their confirmation as dogs from genetic informationContextual and dietary information from both Ivolgin and Ust’-Polui provide supporting evidence for the presence of dogs at these two sites. The mandibles from Ivolgin were found with remains other domestic animals within a fortified town. Analysis of the canid skulls at both Ust’-Polui and Ivolgin confirm that numerous dogs are present at both locations but failed to show the presence of wolves. Further, there are butchery marks on the canid remains at both of these sites, and the isotope analysis indicates that the canids at Ust’-Polui had similarly structured diets as the humans buried there, and these diets included freshwater fish.Overall, these results indicate that the rate of evolutionary modification of the dog mandible may not keep pace with cranial shape change, and that the variation in mandible shape that differentiates modern dogs and wolves mostly emerged relatively late in the domestication process, perhaps with the advent of modern intensive breeding. Fossil mandibles from even Late Holocene dogs may be mistakenly classified as small wild canids and should not be relied on as the only evidence for specimen identification. Future studies comparing shape variation in canid skulls and mandibles may elucidate the lack of coevolution observed in our dataset.40 all other specimens were digitized by AGD with a Microscribe digitizer. Fossils include 26 mandibles from Ust’-Polui19, 20 from Ivolgin17, 8 late Pleistocene canids from Alaska20 , 4 canids from near 1600CE Alaska21 , and another 7 canids that are likely from around 1600 CE Alaska (but have not been carbon-dated) . 37 three-dimensional coordinates were captured from the mandibles of 121 North American wolves, 85 Eurasian wolves, and 240 adult dogs  19, 6 fossil skulls from Ivolgin 17, 4 late Pleistocene skulls from Alaska20 , and 4 skulls from near 1600CE Alaska21 . 36 three-dimensional coordinates were recorded from skulls of 258 North American wolves, 57 European wolves, and 91 adult dogs . The following fossils were included in the wolf sample: Eliseevichi MAE 447/5298, Goyet, and Trou Balleux. The following fossils were included in the dog sample: Shamanka II, Ust’-Belaia, three Egyptian mummified dogs from the Saite–Ptolemaic period, and four Neolithic and one Gallo-Roman dog from France. For more details on the specimens used in the cranial analysis please see Drake et al.6.Ct-scans of fossil skulls from Ivolgin and Ust’-Polui, as well as the following fossil specimens: Eliseevichi MAE 447/5298 , Goyet , Trou Balleux , Shamanka II , and Ust’-Belaia  were converted into Polygon files and digitized in IDAV Landmark softwareThe majority of dog specimens are housed in the Albert Heim Collection at the Natural History Museum in Berne, Switzerland. Most of the wolf specimens are from the University of Alaska Museum in Fairbanks, Alaska. Other specimens are from the Smithsonian Institution’s National Museum of Natural History in Washington, DC, the Museum of Vertebrate Zoology at the University of California in Berkeley, California, the Natural History Museum in Berne, Switzerland, and the Zoology Department at the Natural History Museum, London.41 was conducted with the R programming language. Landmark software was used to warp a 3D Ct-scan of a wolf mandible and a wolf skull to the average shape of the known dog and wolf specimens and then warped along the PC axes40. Many of the methods used in this analysis are similar to those used in Drake et al.6. Here we detail any differences in methodology.Geometric morphometric analysiset al.6. For the analysis of the skulls we followed the same procedure, using PCs 1–6.We used a resampling procedure to balance the sample sizes of the wolf and dog groups. A test developed by Anderson determined that eigenvalues from PC 9 onwards were nearly equal and therefore not useful for our analysis. We ran 1,000 iterations of the resampling procedure and in each round we used the Anderson test to determine whether the first 9 PCs were useful. If they were not, we eliminated that round and ran the resampling procedure again. For each successful round of resampling we used the 9 PCs to conduct a quantitative discriminant function analysis (QDFA) of dogs and wolves. The posterior probability and typical probabilities were calculated in the same manner as in Drake The datasets analysed during the current study are available from the corresponding author on reasonable request.Supplementary Video S2Supplementary Video S3Supplemental Information


The key to interpreting the contribution of a disease-associated mutation in the development and progression of cancer is an understanding of the consequences of that mutation both on the function of the affected protein and on the pathways in which that protein is involved. Protein domains encapsulate function and position-specific domain based analysis of mutations have been shown to help elucidate their phenotypes.In this paper we examine the domain biases in oncogenes and tumour suppressors, and find that their domain compositions substantially differ. Using data from over 30 different cancers from whole-exome sequencing cancer genomic projects we mapped over one million mutations to their respective Pfam domains to identify which domains are enriched in any of three different classes of mutation; missense, indels or truncations. Next, we identified the mutational hotspots within domain families by mapping small mutations to equivalent positions in multiple sequence alignments of protein domains We find that gain of function mutations from oncogenes and loss of function mutations from tumour suppressors are normally found in different domain families and when observed in the same domain families, hotspot mutations are located at different positions within the multiple sequence alignment of the domain.http://strubiol.icr.ac.uk/extra/MOKCa).By considering hotspots in tumour suppressors and oncogenes independently, we find that there are different specific positions within domain families that are particularly suited to accommodate either a loss or a gain of function mutation. The position is also dependent on the class of mutation. We find rare mutations co-located with well-known functional mutation hotspots, in members of homologous domain superfamilies, and we detect novel mutation hotspots in domain families previously unconnected with cancer. The results of this analysis can be accessed through the MOKCa database ( All cancers depend on mutations in critical genes that confer a selective advantage to the tumour cell. Knowledge of these mutations is fundamental to understanding the biology of cancer initiation and progression, and to the development of targeted therapeutic strategies. The genes that harbour the driver mutations that contribute to the disease process are traditionally classified as either as ‘tumour suppressors’ or as oncogenes, dependent on their role in cancer development.When mutations (or epigenetic silencing) of the protein products of tumour suppressors result in their loss of function (LOF), cancer progression occurs. Driver alterations in these genes are typically molecularly recessive in nature, with both copies of the gene requiring a LOF defect. In oncogenes, an increase in activity, or a change of function is required for tumorigenesis. These genes tend to exhibit a molecularly dominant mode of action, and usually only one faulty copy of the gene is required to provide an oncogenic phenotype .When mutations from cohorts of patients are sequenced and the alterations mapped to a single genome, the mutational spectra in tumour suppressors and oncogenes tend to differ. In tumour suppressors small mutations are often liberally dispersed along the length of the gene. This is because the protein products can be disrupted with damaging mutations at a multitude of positions , 3. DrivSeveral groups have used the differences in these mutational patterns to automatically distinguish between tumour suppressor and oncogenes . For insAs well as discriminating between tumour suppressors and oncogenes, there are several approaches to detect which genes are likely to be drivers, irrespective of their biological function: Statistical methods have been successfully applied to identify recurrently mutated genes within large cohorts of sequenced tumours eg , 8). How. How8]).An alternative approach to identifying drivers uses sequence and structural data to predict whether a missense mutation, or small insertion/deletion (indel) could contribute to disease by impacting on the function of the encoded protein , 12. SeqWhile most analysis of cancer mutations has been gene-centric, considering encoded proteins as a whole, a few studies have focused on the individual protein domains affected –22. LargProteome-wide analyses have previously been performed to identify domains enriched in missense mutations , 26, 27 http://strubiol.icr.ac.uk/extra/MOKCa).Here we examine the domain biases in oncogenes and tumour suppressors, and have also compared them with genes not assigned to these roles and find that their domain compositions substantially differ. We have mapped over 1 million mutations from whole-exome sequencing cancer genomic projects including data from over 30 different types of cancer and identified which domains are recurrently mutated in tumour suppressors, oncogenes and throughout the genome. We have divided the mutations into three different classes; missense, truncations or indels. Finally we identified the mutational hotspots within domain families by mapping small mutations to equivalent positions in multiple sequence alignments of protein domains. Examining the differences in the distribution of the positions of domain hotspots, between tumour suppressors and oncogenes, has enabled us to identify key positions of activating mutations in a variety of domain types. This has enabled us to identify putative gain of function mutations in proteins previously unassociated with cancer that may be actionable with current therapies. The results of this analysis can be accessed through the MOKCa database  and oncogenes of more transcription factors (P = 0.0023).Although generally segregating onto different pathways, the functions of the large majority of the proteins in oncogenes and tumour suppressors were somewhat similar see , with thNext we analysed the domain compositions within tumour suppressors and oncogenes. In total 5523 Pfam domain families were identified within the 17537 proteins analysed. Tumour suppressor proteins contained 197 different types of Pfam domains with the most frequently observed domains including Helicase_C (7), DEAD (4), SET (4), HMG-box (3), F-box-like (3), ARID (3), and PHD finger  domains and the C-terminal domain from DNA mismatch repair proteins . Of the 310 Pfam domain types found in our set of oncogenes the most frequently observed were Pkinase_Tyr (26), Homeobox (16), HLH (14), Ets (9), and SH2 (9) domains.We only found 44 domain types common to tumour suppressor and oncogenes. The majority of these were either protein binding modules  or modules evolved to bind to nucleic acids . Using a support vector machine classifier and a 10-fold cross validation protocol, we achieved a ROC AUC sore of 0.72 (see Supplementary Methods) suggesting that the classifier has some predictive value.We ran the classifier on 37 genes labelled as both oncogene and tumour suppressor in the CGC. We found that 17 of the genes were predicted to be tumour suppressors with probabilities greater than 0.78, including DDB2, TP53 and DAXX. Nine genes were classified as oncogenes with probabilities greater than 0.83, including ERBB4, BCL10 and BTK. We could not resolve the classification of 11 genes using this approach see .Although this classification approach may give a guide to the gene's predominant cancer role within the cell, there is increasing evidence in the literature that depending on cell type and cancer type, many genes can function as both a tumour suppressor and as an oncogene dependent on the alteration in question.To define the mutational ‘load’ that the different domain types are subjected to in cancers, we mapped mutations from Cosmic v71 (WGS) whole genome sequencing cancer studies onto the Pfam domains identified above. Mutations were grouped into three subsets; missense, truncating (nonsense or frameshift), and indels (inframe insertion and deletions). In total, 727,525 missense, 69,414 truncation and 2,958 indel mutations from over 30 different types of cancer were mapped to Pfam domains within the human genome.The most frequently reported mutational event that changes the protein product of tumour suppressors (62%) is the missense substitution. However, only 15 domain families were significantly enriched in missense mutations  and P53 (TP53) each from a single protein . We detected 37 domains from our set of oncogenes that were significantly enriched in missense mutations  modules. These are small beta-sandwiches and function in proteins that are part of the innate immune system. Several sushi containing proteins have been implicated in the development of tumour cells and their loss correlates with poor prognosis , 39.Similarly, in the 225 domains showing enrichment in truncations, 196 were not present in the current cancer gene set documented in the Cancer Gene Census see . Sushi dOf the 38 domains significantly enriched in indels, 31 were not present in our cancer gene lists .As well as identifying which domain families were enriched in mutations, we also wanted to identify the key positions within a domain, that when mutated, were particularly suited to causing a loss or change in function of the protein the domain occurs in. To achieve this we created multiple sequence alignments for each domain family and counted the mutations at each position in the alignment  and MAP2K4 (G252R) have mutations co-located with the CDK12 R882L/Q mutations. The CDK12 R882L mutation has been shown to impair kinase activity, possibly by breaking critical interactions in the active conformation of the kinase between phosphorylated threonine 893 and the activation loop [Co-located mutations in the WD40 tumour suppressors FBXW7 (T385K) and TBL1XR1 (Y395H) are also likely to be loss of function. The WD40 domain is especially sensitive to position specific disruption by missense mutations because the way in which its fold is stabilized. WD40 domains consist of a β-propeller structure containing between six to eight propeller ‘blades’. These blades are each formed by a four-stranded antiparallel β-sheet, which are joined by β-hairpins. The blades are arranged symmetrically about a central axis, and the inside edge of each propellers comprise side chains that form a network of hydrogen bonds with each other, and internal water molecules that maintain the domain's stability  and the Helicase_C domains .The sparsity of both truncation and indel data meant that almost all the tumour suppressor hotspots were derived from single proteins. Truncation hotspots were observed in VHL and P53, in the RhoGAP domain in PIK3R1, and the RB_A domain in retinoblastoma associated protein. Several protein kinase domains had truncating mutations at position 14 in the domain multiple sequence alignment which would result in complete loss of function of the kinase in BUB1B (E813*), MAP3K1 (Q1247fs*26) and STK11 (D53fs*11).TP53 exhibited the most indel hotspots with hotpots observed in DNA binding domains (P53) and the P53_tetramer tetramerisation motif. In several cases multiple variants were observed at the same hotspot. This included the P53 domain where there was a deletion of residue 113 F or several residues FLH, and at position 155 there was an insertion of DSTPPPGT and a deletion of residues TR recorded.Within oncogenes we identified 85 missense hotspot in 46 domain families, 10 indel hotspots within 9 domains and 42 truncation hotspots in 30 domain families see , 12, 13.We observed the well known, high frequency mutations in the Ras , isocitrate dehydrogenase  and tyrosine protein kinase domains (BRAF V600E etc). These highly recurrent mutations have been extensively analysed and are thought to cause a gain/change of function of the protein by changing the canonical conformation of the protein.The small GTPases  are molecular switches cycling between the GTP-bound active and GDP-bound inactive conformations. They have co-located hotspots that are implicated in a large variety of cancers. When mutated at position 12, the bulky side chain of the mutants are thought to lower the GTPase activity through a steric interference of the catalytic process . This leIDH1 and IDH2 catalyse the oxidative carboxylation of isocitrate to α-ketoglutarate. Mutational hotspots at R132H in IDH1, and R140Q and R172K in IDH2 alter the progression of this reaction. Recent structural work suggests that the R132H IDH1 mutation hampers the conformational change from the initial isocitrate binding state to the pre-transition state, thus causing an impairment of enzyme function . This alOther less will documented co-located missense hotspot mutations were found in the guanine nucleotide binding protein domains . GNAS R201H somatic mutation is an activating mutation resulting in constitutively activated G-alpha protein and the downstream cAMP cascade, independent of TSH signalling . This reIn the rhodopsin seven transmembrane helix domain family the (7tm_1) the thyrotropin receptor (TSHR) A623V activating mutations  are co-lFew of the truncation hotspots in oncogenes were observed in more than one protein, suggesting that truncating mutations, if they result in a consequence, may be specific to the context of the domain within the larger protein, rather than to the domain itself.Although the indel data was sparse there was still some evidence that co-located indel hotspot mutations in oncogenes are activating. Co-located deletions E746_A750delELREA and E746_T751delELREAT both cause activation of EGFR [In total we identified 341 mutational hotspots within 66 domains in our cancer gene set. The hotspots in tumour suppressors and oncogenes occurred in different domain types except in 6 domains  and when they were observed in the same domain type, they were found with in different locations in the domain  can be thought of being in equilibrium between the open and closed conformations. Usually, other protein kinases phosphorylate the activating residues (S/T/Y) - moving the conformational equilibrium towards the open, active conformation, whereas protein phosphatases remove the phosphate groups shifting the conformational equilibrium back to the closed, inactive conformation. These processes leads to highly regulated control of the conformation and activation of kinase domains.Dependant on their location within the kinase domain, missense mutations will often be better tolerated in one or other conformation of the protein kinase resulting in an alteration of the conformational equilibrium and constitutive activation (or in some cases deactivation) of the protein kinase. This is reflected in that the positions of the hotspots are generally different in the oncogenes and tumour suppressor flavours of this domain. This may not be the case in position 117 of the Pkinase_Tyr domain. Ten oncogene kinases have a mutation in this position, including the documented activating mutations FGFR2 N549S/K/H, the FGFR1 N546K and EGFR R776H mutations. However, the tumour suppressor MAP3K13 has an A218T mutation of unknown consequence at this position, which suggests that it may be possible to have a driver mutation that deactivates the protein at this position alternatively A218T may be an activating mutation.The final part of our analysis was to assess how many of the genome–wide hotspots we could putatively assign as activating/gain of function, or as loss of function. In total there were 954 missense hotspots in 423 domain families, 113 indels in 93 domain families and 506 truncations in 382 domain families of which ∼11% were co-located with an oncogene or tumour suppressor hotspot.We were able to identify mutations in genes not previously related to cancer that aligned with well-established caner hotspots. These included 14 tyrosine protein kinase domains that had missense mutations co-located with activating BRAF V600E mutation including kinase suppressor of ras 2 (KSR2) p.R724W (117) , mixed lOf the remaining 89% of hotspots, 94% are located in ∼700 domain families not yet associated with well-documented oncogenes and tumours suppressors. This included a significant hotspot mutation in the AAA+ domain (PF00004), a large diverse protein family belonging to the AAA superfamily of P-loop NTP hydrolases, that utilise ATP to create conformational changes that are transduced into mechanical forces on macromolecule substrates. There is a mutation located at position 110 in the MSA of the domain. This includes mutations in WRN1P1 a DNA damage sensor (R306Q), the 26S protease regulatory subunit 6 (PSMC2) (R258H), and in paraplegin (SPG7) R391W. Structural analysis by SAAPdat  and mCSMProtein sequences from COSMIC v71  were mapSomatic mutation data was extracted from the “Whole Genome Sequencing” (WGS) version of the COSMIC database V71 and processed using the MOKCa update protocol. 2,399,998 mutations from 15051 patient samples in 30 cancer types were mapped to the UniProt protein sequences. In total, 1,077,825 (45%) mutations could be mapped to conserved Pfam domains .The mutations were classified into three subsets. Missense mutations, where usually a single base substitution changes the protein product by a single amino acid. Truncating mutations, which incorporate nonsense mutations and frameshift insertions and deletions. Truncations may just disrupt a single domain or result in complete destruction of the protein for example by nonsense mediated decay. Finally, inframe insertions and deletions (indels) were grouped together as they are relative infrequent, and both have the possibility of causing more severe disruptions to the protein product than a missense mutations. In total there were 727,525 missense, 69414 truncations and 2,958 indels mapped to 17,536 protein domains.The panther functional classification website was used to define the function of the proteins assigned as tumour suppressors and oncogenes. The DAVID website  was usedTo find the domains enriched in mutations in tumour suppressors and oncogenes we compared the mutational frequency for each domain to the mutational frequency of a dataset of 450 “random” domains not related to cancer using a chi-square association test . A BonfeFor the genome-wide study, the mutational burden in each single domain type was compared to that in all other domain types using a chi-square association test. Data was normalized by domain frequency, number of samples and domain length.A suite of Perl programs was used to generate and analyse hotspot domain positions. A multiple sequence alignment (MSA) was generated for all human domain fasta sequences, for each Pfam family using the MUSCLE (v3.8.31) alignment program . Each muA binomial test was used to identify which positions had a significant number of mutations. If each individual mutation were to affect a random residue across the domain the frequency of mutations at each site would follow a binomial distribution. As such our null model states that there is an equal probability of a mutation occurring at each residue on the given domain.Where n is the total number of mutations in the domain, k is the number of mutations falling at a specific residue and p the probability of any mutation affecting a specific residue we can find the probability of observing k mutations falling at any specific point in the domain by calculating the probability of a minimum of k mutations at that point and comparing it to our null model.Missense, truncations and indels were tested independently and only positions where mutations occurred at least two were analysed. The results were amended by a Bonferroni correction. The overlap of hotspots between different mutational types were visualised with jvenn web application http://strubiol.icr.ac.uk/extra/MOKCa) was developed to structurally and functionally annotate, and where possible predict, the phenotypic consequences of disease-associated mutations in proteins implicated in cancer. The initial database focused on protein kinases, but has now been extended include all the proteins from the human genome that are mutated in cancer.The MOKCa database  have been mapped to their position in UniProt sequences. COSMIC use their own reference sequences (Ensembl transcripts), and although most COSMIC protein sequences (∼17000) match perfectly when mapped to UniProt sequences, for the remaining ∼4000 sequences the relationship is more complicated. Each COSMIC sequence was aligned with their corresponding UniProt sequence and when the sequences are not identical the alignment was stored in the database. This allows us to identify the position of the mutation with regard to the UniProt sequence, which provides the authoritative reference.Each mutation is described its alteration to the protein structure, eg V600E. When this mutation has been reported on more one occasion each mutation is stored as the same aggregate and an aggregate count given. Different genetic changes that result in the same mutation are presented together at the protein level. Each disease type in which this mutation has been recorded is also presented on the protein overview page.Functional annotations for each protein using a variety of databases have incorporated this into the new MOKCa database. These annotations include the identification and position of Pfam domain assignments within the protein sequence, and the positions of residues known or predicted to be affected by post-translational modifications including phosphorylation, glycosylation, and ubiquitination. Gene Ontology (GO) annotations and Prosite patterns  have alsThe amino acid sequence for every Pfam-annotated domain for which COSMIC records a cancer-associated mutation has been scanned against the Protein Data Bank (PDB)  using PSTo identify which mutations mapped onto residues with structural density in the PDB file, PDB sequence to structure alignments from the SIFTS  initiative were utilized.http://strubiol.icr.ac.uk/extra/mokca/ and can be searched by gene name or by UniProt accession. Users can also “browse the data from the gene data. To help identify those proteins we have identified subsets of proteins that are frequently mutated in cancer this includes, protein kinases [The new web-interface for MOKCa database can accessed at kinases , oncogen kinases , protein kinases .In this study we have used recurrence to identify hotspot positions of somatic missense, indel and truncating mutations on over 5000 Pfam domain families. We analysed the data in tumour suppressors and oncogenes separately as we were particularly keen to find hotspots involved in activated proteins, and found that mutational hotspots in tumour suppressors and oncogenes usually occur in different types of domains, when they do occur in the same domain family, they occur at different positions in the domain. Our analysis also suggests that there may only be a small subset of domain types that can easily be activated by single small mutations.Missense hotspots were frequently conserved in multiple members of Pfam domain families, however truncations were conserved far less frequently with many truncational hotspots occurring only in individual proteins. This may be because truncations often obliterate the functioning protein due to processing of the transcript by nonsense-mediated decay, so its position within a domain is far less crucial than for missense mutations. The large number of truncation hotspots observed in the whole genome dataset, suggest that there may be a large number of tumour suppressors not yet documented. Current statistical methods for analysing cohorts of cancer patients are designed to identify statistically significant mutations in single genes. Many of the tumour suppressors are part of large protein complexes where failure of any single component will result in loss of function of the complex as a whole. The mutational burden is thus distributed over all components of the complex, with no individual subunit being affected at a sufficient level to generate a statistically detectable signal.Using the Cosmic v71 (WGS) we identified several indel mutations conserved in multiple member of domain families. As more genome sequencing studies are undertaken and the algorithms used to detect indels improve, it is likely that more indel hotspots will be identified.We have also used our oncogene and tumour suppressor hotspots to identify co-located hotspots in 167 proteins as yet, not associated with cancer. This information enables us to assign putative gain or loss of function mutations in these proteins that may contribute to cancer progression. Using the biological knowledge associated with protein domains, such as structural information and evolutionary conservation, enables the transfer of knowledge from well studied oncogenes to less well studies homologues can lead to testable hypotheses of the effect of rare mutations in large cancer genomics datasets, and may lead to tractable therapeutic intervention points.http://strubiol.icr.ac.uk/extra/MOKCa).The domain hotspots identified within this study are available though the MOKCa database where mutations are annotated by driver types (


Austinogebia edulis, being abundant in the intertidal zone of western Taiwan, constructs deep burrows (>1 m). This study highlights the potential of mud shrimps to modify sediment characteristics of the tidal flat by its burrowing behavior. We studied the structure of the burrow wall, compared the difference in the sediment composition of the burrow and the background sediment, and compared the organic content inside the burrow wall. This study was carried out from September 2015 to November 2016 in three areas of the western coast of Taiwan, namely Shengang, Hanbow, and Wangong. The present study found significant differences between burrow wall and the burrow lumen. The diameter of the burrow wall was double as wide as the inner burrow lumen at the opening and gradually increased to 10 times of the burrow lumen at 30 cm depth. The burrow wall of A. edulis showed low permeability and increased the sheer strength. Statistically, a significant difference was noticed in the comparison between the sediment composition of the burrow wall and the background . An accumulation of 3.63 for fine sand  and 9 for clay  was found in the upper burrow wall of A. edulis. This indicated that they somehow chose finer particles to build burrows. This will gradually change the sediment distribution—vertically and horizontally. The burrow wall consisted of a 24 times higher organic matter content than one individual of mud shrimp. The burrow may provide organic material as a potential food source. The mud shrimp thus transforms the sediment characteristics as an ecological engineer, which is expected to have a significant ecological impact on the ecosystem.The mud shrimp  Mudflats are coastal wetlands that are formed by the sedimentation of mud layers during tidal movements . GeneralAustinogebia edulis . Figure C1 in S1 File. Comparison of the void ratio of the mud shrimp burrow wall and the ambient sediment in 3 sampling areas. Figure C2 in S1 File. Comparison of ash free dry weight of background sediment and mud shrimp burrow wall using one-way analysis of variance, followed by Tukey’s test. Figure C3 in S1 File. Relative weight of ash-free dry weight of one individual of mud shrimp and one whole mud shrimp burrow wall.Figure A1 in S1 File. The volume of the burrow wall and the burrow lumen. Pie chart—the proportion of the burrow wall and the burrow lumen in a mud block. Figure B1 in S1 File. Accumulated percentage and proportion of sediments in burrow wall and surroundings from three sampling areas. Shengang (a), Hanbao (b), and Wangong (c). Table B2 in S1 File.. Results of Student’s t-test comparison for proportion (%) of each size category of sediment between habitat (control) and burrow. n is number of samples. WSC is Wentworth Size Class. [* Significant at the (XLSX)Click here for additional data file.


Establishing an accurate timescale for the history of life is crucial to understand evolutionary processes. For this purpose, relaxed molecular clock models implemented in a Bayesian MCMC framework are generally used. However, these methods are time consuming. RelTime, a non-Bayesian method implementing a fast, ad hoc, algorithm for relative dating, was developed to overcome the computational inefficiencies of Bayesian software. RelTime was recently used to investigate the timing of origin of animals, yielding results consistent with early strict clock studies from the 1980s and 1990s, estimating metazoans to have a Mesoproterozoic origin—over a billion years ago. RelTime results are unexpected and disagree with the largest majority of modern, relaxed, Bayesian molecular clock analyses, which suggest animals originated in the Tonian-Cryogenian (less that 850 million years ago). Here, we demonstrate that RelTime-inferred divergence times for the origin of animals are spurious, a consequence of the inability of RelTime to relax the clock along the internal branches of the animal phylogeny. RelTime-inferred divergence times are comparable to strict-clock estimates because they are essentially inferred under a strict clock. Our results warn us of the danger of using ad hoc algorithms making implicit assumptions about rate changes along a tree. Our study roundly rejects a Mesoproterozoic origin of animals; metazoans emerged in the Tonian-Cryogenian, and diversified in the Ediacaran, in the immediate prelude to the routine fossilization of animals in the Cambrian associated with the emergence of readily preserved skeletons. Timescales are essential to evolutionary biology, calibrating biological processes to human and geological timescales and elucidating the tempo and mode of evolution, from viral strains  to the eRelTime eschews fossil calibrations, estimating relative, rather than absolute, evolutionary timescales. RelTime relative timescales can be transformed into absolute timescales using calibration anchors that are considered reliable a priori, to re-scale branch lengths to absolute time . AdvocatRegression analyses indicate that RelTime inferred relative divergence times are clearly not proportional to the corresponding Bayesian relative divergence times a. CruciaThe fundamental difference between relative divergence times estimated using Phylobayes and RelTime begs the question of which set of relative rates and dates is the more reliable. We investigated how inferred rates of evolution change as the tree is traversed from the tips to the root. We found that RelTime estimates of relative rates do not vary along the entire tree c, differR2=0.99), with the largest SE associated with the root  are summarised in