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The architecture of root systems is an important driver of plant fitness, competition and ecosystem processes. However, the methodological difficulty of mapping roots hampers the study of these processes. Existing approaches to match individual plants to belowground samples are low throughput and species specific. Here, we developed a scalable sequencing-based method to map the root systems of individual trees across multiple species. We successfully applied it to a tropical dry forest community in the Brazilian Caatinga containing 14 species. We sequenced all 42 individual shrubs and trees in a 14 × 14 m plot using double-digest restriction site-associated sequencing (ddRADseq). We identified species-specific markers and individual-specific haplotypes from the data. We matched these markers to the ddRADseq data from 100 mixed root samples from across the centre (10 × 10 m) of the plot at four different depths using a newly developed R package. We identified individual root samples for all species and all but one individual. There was a strong significant correlation between belowground and aboveground size measurements, and we also detected significant species-level root-depth preference for two species. The method is more scalable and less labour intensive than the current techniques and is broadly applicable to ecology, forestry and agricultural biology., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

Recent adaptive radiations are models for investigating mechanisms contributing to the evolution of biodiversity. An unresolved question is the relative importance of new mutations, ancestral variants, and introgressive hybridization for phenotypic evolution and speciation. Here, we address this issue using Darwin's finches and investigate the genomic architecture underlying their phenotypic diversity. Admixture mapping for beak and body size in the small, medium, and large ground finches revealed 28 loci showing strong genetic differentiation. These loci represent ancestral haplotype blocks with origins predating speciation events during the Darwin's finch radiation. Genes expressed in the developing beak are overrepresented in these genomic regions. Ancestral haplotypes constitute genetic modules for selection and act as key determinants of the unusual phenotypic diversity of Darwin's finches. Such ancestral haplotype blocks can be critical for how species adapt to environmental variability and change.

Heterochrony, defined as a change in the timing of developmental events altering the course of evolution, was first recognized by Ernst Haeckel in 1866. Haeckel's original definition was meant to explain the observed parallels between ontogeny and phylogeny, but the interpretation of his work became a source of controversy over time. Heterochrony took its modern meaning following the now classical work in the 1970-80s by Steven J. Gould, Pere Alberch, and co-workers. Predicted and described heterochronic scenarios emphasize the many ways in which developmental changes can influence evolution. However, while important examples of heterochrony detected with comparative morphological methods have multiplied, the more mechanistic understanding of this phenomenon lagged conspicuously behind. Considering the rapid progress in imaging and molecular tools available now for developmental biologists, this review aims to stress the need to take heterochrony research to the next level. It is time to synchronize the different levels of heterochrony research into a single analysis flow: from studies on organismal-level morphology to cells to molecules and genes, using complementary techniques. To illustrate how to achieve a more comprehensive understanding of phyletic morphological diversification associated with heterochrony, we discuss several recent case studies at various phylogenetic scales that combine morphological, cellular, and molecular analyses. Such a synergistic approach offers to more fully integrate phylogenetic and ontogenetic dimensions of the fascinating evolutionary phenomenon of heterochrony., (© 2021 Wiley Periodicals LLC.)

Carotenoid-based polymorphisms are widespread in populations of birds, fish, and reptiles, 1 but generally little is known about the factors affecting their maintenance in populations. 2 We report a combined field and molecular-genetic investigation of a nestling beak color polymorphism in Darwin's finches. Beaks are pink or yellow, and yellow is recessive. 3 Here we show that the polymorphism arose in the Galápagos half a million years ago through a mutation associated with regulatory change in the BCO2 gene and is shared by 14 descendant species. The polymorphism is probably a balanced polymorphism, maintained by ecological selection associated with survival and diet. In cactus finches, the frequency of the yellow genotype is correlated with cactus fruit abundance and greater hatching success and may be altered by introgressive hybridization. Polymorphisms that are hidden as adults, as here, may be far more common than is currently recognized, and contribute to diversification in ways that are yet to be discovered., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults., Competing Interests: The authors have declared that no competing interests exist.

Shifts in flowering time have the potential to act as strong prezygotic reproductive barriers in plants. We investigate the role of flowering time divergence in two species of mountain rose ( Metrosideros ) endemic to Lord Howe Island, Australia, a minute and isolated island in the Tasman Sea. Metrosideros nervulosa and M. sclerocarpa are sister species and have divergent ecological niches on the island but grow sympatrically for much of their range, and likely speciated in situ on the island. We used flowering time and population genomic analyses of population structure and selection, to investigate their evolution, with a particular focus on the role of flowering time in their speciation. Population structure analyses showed the species are highly differentiated and appear to be in the very late stages of speciation. We found flowering times of the species to be significantly displaced, with M. sclerocarpa flowering 53 days later than M. nervulosa . Furthermore, the analyses of selection showed that flowering time genes are under selection between the species. Thus, prezygotic reproductive isolation is mediated by flowering time shifts in the species, and likely evolved under selection, to drive the completion of speciation within a small geographical area. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The strength and spatiotemporal activity of Nodal signaling is tightly controlled in early implantation mouse embryos, including by autoregulation and feedback loops, and involves secreted and intracellular antagonists. These control mechanisms, which are established at the extra-embryonic/embryonic interfaces, are essential for anterior-posterior patterning of the epiblast and correct positioning of the primitive streak. Formation of an ectopic primitive streak, or streak expansion, has previously been reported in mutants lacking antagonists that target Nodal signaling. Here, we demonstrate that loss-of-function of a major bone morphogenetic protein (BMP) effector, Smad5, results in formation of an ectopic primitive streak-like structure in mutant amnion accompanied by ectopic Nodal expression. This suggests that BMP/Smad5 signaling contributes to negative regulation of Nodal. In cultured cells, we find that BMP-activated Smad5 antagonizes Nodal signaling by interfering with the Nodal-Smad2/4-Foxh1 autoregulatory pathway through the formation of an unusual BMP4-induced Smad complex containing Smad2 and Smad5. Quantitative expression analysis supports that ectopic Nodal expression in the Smad5 mutant amnion is induced by the Nodal autoregulatory loop and a slow positive-feedback loop. The latter involves BMP4 signaling and also induction of ectopic Wnt3. Ectopic activation of these Nodal feedback loops in the Smad5 mutant amnion results in the eventual formation of an ectopic primitive streak-like structure. We conclude that antagonism of Nodal signaling by BMP/Smad5 signaling prevents primitive streak formation in the amnion of normal mouse embryos.

Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I-associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling., Competing Interests: The authors have declared that no competing interests exist.

Tracing the precise developmental origin of amnion and amnion-derived stem cells is still challenging and depends chiefly on analyzing powerful genetic model amniotes like mouse. Profound understanding of the fundamental differences in amnion development in both the disc-shaped primate and human embryo and the cup-shaped mouse embryo is pivotal in particular when sampling amniotic membrane from nonprimate species for isolating candidate amniotic stem cells. The availability of molecular marker genes that are specifically expressed in the amniotic membrane and not in other extraembryonic membranes would be instrumental to validate unequivocally the starting material under investigation. So far such amniotic markers have not been reported. We postulated that bone morphogenetic protein (BMP) target genes are putative amniotic membrane markers mainly because deficiency in one of several components of the BMP signaling cascade in mice has been documented to result in defective development of the early amnion. Comparative gene expression analysis of acknowledged target genes for BMP in different extraembryonic tissues, combined with in situ hybridization, identified Periostin (Postn) mRNA enrichment in amnion throughout gestation. In addition, we identify and propose a combination of markers as transcriptional signature for the different extraembryonic tissues in mouse.

Signaling by the many ligands of the TGFβ family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models., (Copyright © 2011. Published by Elsevier Ltd.)

Background: Despite the detailed knowledge obtained over the last decade on the molecular regulation of gastrulation in amniotes, the process of amnion development has been poorly described and illustrated in mice, and conflicting descriptions exist. Understanding the morphogenesis and development not only of the early mouse embryo, but also of its extraembryonic tissues, is crucial for correctly interpreting fate-mapping data and mouse mutants with gastrulation defects. Moreover, the recent isolation from amnion of cells with stem cell features further argues for a better understanding of the process of amnion formation. Here, we revisit the highly dynamic process of amnion formation in the mouse. Amnion development starts early during gastrulation and is intimately related to the formation of the exocoelom and the expansion of the amniotic fold. The authoritative description involves the fusion of two amniotic folds, a big posterior and a smaller anterior fold. We challenged this 'two amniotic folds' model by performing detailed histomorphological analyses of dissected, staged embryos and 3D reconstructions using historical sections., Results: A posterior fold of extraembryonic ectoderm and associated epiblast is formed early during gastrulation by accumulation of extraembryonic mesoderm posterior to the primitive streak. Previously called the "posterior amniotic fold", we rename it the "amniochorionic fold" (ACF) because it forms both amnion and chorion. Exocoelom formation within the ACF seems not to involve apoptosis within the mesoderm. The ACF and exocoelom expand without disrupting the anterior junction of epiblast, extraembryonic ectoderm and visceral endoderm. No separate anterior fold is formed; its absence was confirmed in 3D reconstructions. Amnion and chorion closure is eccentric, close to the anterior margin of the egg cylinder: we name it the "anterior separation point"., Conclusions: Here, we reconcile previous descriptions of amnion formation and provide new nomenclature, as well as an animation, that clarify and emphasize the arrangement of the tissues that contribute to amnion development and the dynamics of the process. According to our data, the amnion and the chorion are formed by a single amniochorionic fold initiated posteriorly. Finally, we give an overview on mutant mouse models with impaired amnion development.

A common characteristic of mammals is the development of extraembryonic supporting tissues and organs that are required for embryonic implantation, survival and development in utero. The amnion is the innermost extraembryonic membrane that eventually surrounds the fetus of amniotes, and contains the amniotic fluid. Next to its function in in utero development, the amnion has been shown to have an important potential for clinical applications. It is mainly used as a dressing to stimulate healing in skin and ocular wounds. Moreover, cells derived from the amniotic membrane and amniotic fluid have been reported to possess stem cell features, like pluripotent differentiation ability. Little is known about the early development of this membrane in humans. The mouse is a powerful genetic model organism that can be used to address the dynamics and the developmental origin of amnion and amnion-derived stem cells. Here, we discuss some fundamental differences in amnion development in the disc-shaped primate embryo and in the cup-shaped mouse embryo. We emphasize the consequences that this may have on the derivation of amniotic "stem" cells. After revision of the different isolation procedures of amniotic (fluid) derived "stem" cells from rodents, we reveal striking differences in the sources used to derive these cells across studies. The profound differences in the development of the extraembryonic membranes and cavities between primates and rodents may result in comparing cell types of different developmental origins, eventually leading to missinterpretations.