Revised eutherian gene collections.

OBJECTIVES: The most recent research projects in scientific field of eutherian comparative genomics included intentions to sequence every extant eutherian species genome in foreseeable future, so that future revisions and updates of eutherian gene data sets were expected. DATA DESCRIPTION: Using 35 public eutherian reference genomic sequence assemblies and free available software, the eutherian comparative genomic analysis protocol RRID:SCR_014401 was published as guidance against potential genomic sequence errors. The protocol curated 14 eutherian third-party data gene data sets, including, in aggregate, 2615 complete coding sequences that were deposited in European Nucleotide Archive. The published eutherian gene collections were used in revisions and updates of eutherian gene data set classifications and nomenclatures that included gene annotations, phylogenetic analyses and protein molecular evolution analyses.

A new mammal from the Lower Cretaceous Jehol Biota and implications for eutherian evolution.

Here we report on a new Early Cretaceous eutherian represented by a partial skeleton from the Jiufotang Formation at Sihedang site, Lingyuan City, Liaoning Province that fills a crucial gap between the earliest eutherians from the Yixian Formation and later Cretaceous eutherians. The new specimen reveals, to our knowledge for the first time in eutherians, that the Meckelian cartilage was ossified but reduced in size, confirming a complete detachment of the middle ear from the lower jaw. Seven hyoid elements, including paired stylohyals, epihyals and thyrohyals and the single basihyal are preserved. For the inner ear the ossified primary lamina, base of the secondary lamina, ossified cochlear ganglion and secondary crus commune are present and the cochlear canal is coiled through 360°. In addition, plesiomorphic features of the dentition include weak conules, lack of pre- and post-cingula and less expanded protocones on the upper molars and height differential between the trigonid and talonid, a large protoconid and a small paraconid on the lower molars. The new taxon displays an alternating pattern of tooth replacement with P3 being the last upper premolar to erupt similar to the basal eutherian Juramaia. Parsimony analysis places the new taxon with Montanalestes, Sinodelphys and Ambolestes as a sister group to other eutherians. This article is part of the theme issue 'The impact of Chinese palaeontology on evolutionary research'.

Evolutionary transcriptomics implicates new genes and pathways in human pregnancy and adverse pregnancy outcomes.

Evolutionary changes in the anatomy and physiology of the female reproductive system underlie the origins and diversification of pregnancy in Eutherian ('placental') mammals. This developmental and evolutionary history constrains normal physiological functions and biases the ways in which dysfunction contributes to reproductive trait diseases and adverse pregnancy outcomes. Here, we show that gene expression changes in the human endometrium during pregnancy are associated with the evolution of human-specific traits and pathologies of pregnancy. We found that hundreds of genes gained or lost endometrial expression in the human lineage. Among these are genes that may contribute to human-specific maternal-fetal communication (HTR2B) and maternal-fetal immunotolerance (PDCD1LG2) systems, as well as vascular remodeling and deep placental invasion (CORIN). These data suggest that explicit evolutionary studies of anatomical systems complement traditional methods for characterizing the genetic architecture of disease. We also anticipate our results will advance the emerging synthesis of evolution and medicine ('evolutionary medicine') and be a starting point for more sophisticated studies of the maternal-fetal interface. Furthermore, the gene expression changes we identified may contribute to the development of diagnostics and interventions for adverse pregnancy outcomes.

Pregnancy is a complicated process. It has three phases: the body recognizes the embryo, it maintains the pregnancy, and finally, it induces labor. These stages happen in all mammals, but the details are different in humans. Human pregnancy and labor last longer. We menstruate. Our placentas invade deeper into the uterus, and the cues that signal pregnancy is done and induce labor are different than in most other mammals. We are also more likely to have pregnancy complications, including infertility, a dangerous rise in blood pressure called preeclampsia, and premature birth. The reasons for these differences are unknown. Human pregnancy relies on close communication between the placenta and the uterus. The immune system must allow the placenta to grow large enough to support the developing embryo, and blood vessels need to adapt to supply gases and nutrients and to remove waste. Understanding how the genes used by the human uterus are different to those used in other species could help explain why human pregnancies are so unusual. Mika, Marinic et al. compared the genes used by the pregnant human uterus to those used in 32 other species, including monkeys, marsupials and other mammals, birds, and reptiles. The analysis revealed that the humans use almost a thousand genes that other animals do not. These genes have roles in the invasion of the placenta, the growth of blood vessels, and control of the immune system. Several have links to the hormone serotonin, which had not been connected with the uterus before. Mika, Marinic et al. suggest that it might control the length of pregnancy, the timing of labor, and communication between parent and baby. The genes identified here provide a starting point for further investigation of human pregnancy. In the future, this may help to prevent or treat infertility, preeclampsia, or premature birth. A possible next step is to examine our closest living relatives, the great apes. Performing similar experiments using tissues or cells from chimpanzees, gorillas, and orangutans could reveal more about the genes unique to human pregnancy.

Regional effect on the molecular clock rate of protein evolution in Eutherian and Metatherian genomes.

BACKGROUND: Different types of proteins diverge at vastly different rates. Moreover, the same type of protein has been observed to evolve with different rates in different phylogenetic lineages. In the present study we measured the rates of protein evolution in Eutheria (placental mammals) and Metatheria (marsupials) on a genome-wide basis and we propose that the gene position in the genome landscape has an important influence on the rate of protein divergence. RESULTS: We analyzed a protein-encoding gene set (n = 15,727) common to 16 mammals (12 Eutheria and 4 Metatheria). Using sliding windows that averaged regional effects of protein divergence we constructed landscapes in which strong and lineage-specific regional effects were seen on the molecular clock rate of protein divergence. Within each lineage, the relatively high rates were preferentially found in subtelomeric chromosomal regions. Such regions were observed to contain important and well-studied loci for fetal growth, uterine function and the generation of diversity in the adaptive repertoire of immunoglobulins. CONCLUSIONS: A genome landscape approach visualizes lineage-specific regional differences between Eutherian and Metatherian rates of protein evolution. This phenomenon of chromosomal position is a new element that explains at least part of the lineage-specific effects and differences between proteins on the molecular clock rates.

Molecular analysis suggests that Namibian cheetahs (Acinonyx jubatus) are definitive hosts of a so far undescribed Besnoitia species.

BACKGROUND: Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the internal transcribed spacer-1 (ITS1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide that are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Also related, but not as closely, is B. besnoiti, the cause of bovine besnoitiosis. METHODS: Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing. RESULTS: One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites, including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti and Neospora caninum, were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but also showed clear divergences. CONCLUSIONS: Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while prey species of the cheetah may act as intermediate hosts.

When the past informs our future.

Comparing the genes expressed at the maternal-fetal interface in different species helps to pinpoint those that contribute to a healthy pregnancy by regulating the activity of the immune system.

Evolutionary transcriptomics implicates HAND2 in the origins of implantation and regulation of gestation length.

The developmental origins and evolutionary histories of cell types, tissues, and organs contribute to the ways in which their dysfunction produces disease. In mammals, the nature, development and evolution of maternal-fetal interactions likely influence diseases of pregnancy. Here we show genes that evolved expression at the maternal-fetal interface in Eutherian mammals play essential roles in the evolution of pregnancy and are associated with immunological disorders and preterm birth. Among these genes is HAND2, a transcription factor that suppresses estrogen signaling, a Eutherian innovation allowing blastocyst implantation. We found dynamic HAND2 expression in the decidua throughout the menstrual cycle and pregnancy, gradually decreasing to a low at term. HAND2 regulates a distinct set of genes in endometrial stromal fibroblasts including IL15, a cytokine also exhibiting dynamic expression throughout the menstrual cycle and gestation, promoting migration of natural killer cells and extravillous cytotrophoblasts. We demonstrate that HAND2 promoter loops to an enhancer containing SNPs implicated in birth weight and gestation length regulation. Collectively, these data connect HAND2 expression at the maternal-fetal interface with evolution of implantation and gestational regulation, and preterm birth.

Comparative genomic analysis of eutherian interferon genes.

The eutherian interferons were implicated as paradigmatic effector proteins in innate and acquired immunity. Yet, familiar interferon classification and nomenclature disagreed with functional genomics, phylogenetic and evolutionary analyses. Using eutherian comparative genomic analysis protocol and 35 public eutherian reference genomic sequence data sets, the present analysis attempted to resolve major disagreements in descriptions of eutherian interferons. Among 836 eutherian interferon potential coding sequences, the tests of reliability of eutherian public genomic sequences annotated, in aggregate, 495 complete coding sequences that were partitioned into supercluster IF1 and supercluster IF2 gene data sets. There were 29 human class 2 cytokine genes described, including initially described human IF1IC2 gene. There was convergence between nonhomologous eutherian supercluster IF1 and supercluster IF2 protein primary structures including common cysteine amino acid residues implicated in disulfide bonding. The integrated gene annotations, phylogenetic analysis and protein molecular analysis proposed revised and updated classification and nomenclature of eutherian interferons.

Comparative genomic analysis of eutherian fibroblast growth factor genes.

BACKGROUND: The eutherian fibroblast growth factors were implicated as key regulators in developmental processes. However, there were major disagreements in descriptions of comprehensive eutherian fibroblast growth factors gene data sets including either 18 or 22 homologues. The present analysis attempted to revise and update comprehensive eutherian fibroblast growth factor gene data sets, and address and resolve major discrepancies in their descriptions using eutherian comparative genomic analysis protocol and 35 public eutherian reference genomic sequence data sets. RESULTS: Among 577 potential coding sequences, the tests of reliability of eutherian public genomic sequences annotated most comprehensive curated eutherian third-party data gene data set of fibroblast growth factor genes including 267 complete coding sequences. The present study first described 8 superclusters including 22 eutherian fibroblast growth factor major gene clusters, proposing their updated classification and nomenclature. CONCLUSIONS: The integrated gene annotations, phylogenetic analysis and protein molecular evolution analysis argued that comprehensive eutherian fibroblast growth factor gene data set classifications included 22 rather than 18 homologues.

Eutherian-Specific Gene TRIML2 Attenuates Inflammation in the Evolution of Placentation.

Evolution of highly invasive placentation in the stem lineage of eutherians and subsequent extension of pregnancy set eutherians apart from other mammals, that is, marsupials with short-lived placentas, and oviparous monotremes. Recent studies suggest that eutherian implantation evolved from marsupial attachment reaction, an inflammatory process induced by the direct contact of fetal placenta with maternal endometrium after the breakdown of the shell coat, and shortly before the onset of parturition. Unique to eutherians, a dramatic downregulation of inflammation after implantation prevents the onset of premature parturition, and is critical for the maintenance of gestation. This downregulation likely involved evolutionary changes on maternal as well as fetal/placental side. Tripartite-motif family-like2 (TRIML2) only exists in eutherian genomes and shows preferential expression in preimplantation embryos, and trophoblast-derived structures, such as chorion and placental disc. Comparative genomic evidence supports that TRIML2 originated from a gene duplication event in the stem lineage of Eutheria that also gave rise to eutherian TRIML1. Compared with TRIML1, TRIML2 lost the catalytic RING domain of E3 ligase. However, only TRIML2 is induced in human choriocarcinoma cell line JEG3 with poly(I:C) treatment to simulate inflammation during viral infection. Its knockdown increases the production of proinflammatory cytokines and reduces trophoblast survival during poly(I:C) stimulation, while its overexpression reduces proinflammatory cytokine production, supporting TRIML2's role as a regulatory inhibitor of the inflammatory pathways in trophoblasts. TRIML2's potential virus-interacting PRY/SPRY domain shows significant signature of selection, suggesting its contribution to the evolution of eutherian-specific inflammation regulation during placentation.

Quantitative analysis of forebrain pallial morphology in monotremes and comparison with that in therians.

We have made quantitative volumetric analyses of cerebral cortical (pallial) structures in the brains of three species of monotreme (Ornithorhynchus anatinus, Tachyglossus aculeatus, Zaglossus bruijni) and compared the findings with similar measurements in a range of therian mammals (6 marsupials and 50 placentals). We have found that although the iso- and periallocortical grey matter volume of the monotremes is about what would be expected for their brain size, the proportion of iso- and periallocortical white matter in monotremes is substantially lower than that in the forebrains of therians. This suggests that the forebrains of the three monotremes have fewer association, commissural and/or projection connections than those of similarly sized forebrains of therian mammals. We also found that the iso- and periallocortex of the platypus is relatively smooth-surfaced compared to similarly sized brains of therian mammals, with a distinct caudal shift in the positioning of cortical white matter in the forebrain, consistent with expansion of the posterior thalamic radiation. Central laminated olfactory structures (anterior olfactory nucleus and piriform cortex) are large in the tachyglossid monotremes (Tachyglossus aculeatus and Zaglossus bruijni) and large in xenarthran placental mammals, suggesting convergence of the forebrain structure of monotreme formivores with that of similarly specialized therians like the xenarthrans Myrmecophaga tridactyla and Dasypus novemcinctus.

Novel and divergent genes in the evolution of placental mammals.

Analysis of genome sequences within a phylogenetic context can give insight into the mode and tempo of gene and protein evolution, including inference of gene ages. This can reveal whether new genes arose on particular evolutionary lineages and were recruited for new functional roles. Here, we apply MCL clustering with all-versus-all reciprocal BLASTP to identify and phylogenetically date 'Homology Groups' among vertebrate proteins. Homology Groups include new genes and highly divergent duplicate genes. Focusing on the origin of the placental mammals within the Eutheria, we identify 357 novel Homology Groups that arose on the stem lineage of Placentalia, 87 of which are deduced to play core roles in mammalian biology as judged by extensive retention in evolution. We find the human homologues of novel eutherian genes are enriched for expression in preimplantation embryo, brain, and testes, and enriched for functions in keratinization, reproductive development, and the immune system.

Investigating Difficult Nodes in the Placental Mammal Tree with Expanded Taxon Sampling and Thousands of Ultraconserved Elements.

The phylogeny of eutherian mammals contains some of the most recalcitrant nodes in the tetrapod tree of life. We combined comprehensive taxon and character sampling to explore three of the most debated interordinal relationships among placental mammals. We performed in silico extraction of ultraconserved element loci from 72 published genomes and invitro enrichment and sequencing of ultraconserved elements from 28 additional mammals, resulting in alignments of 3,787 loci. We analyzed these data using concatenated and multispecies coalescent phylogenetic approaches, topological tests, and exploration of support among individual loci to identify the root of Eutheria and the sister groups of tree shrews (Scandentia) and horses (Perissodactyla). Individual loci provided weak, but often consistent support for topological hypotheses. Although many gene trees lacked accepted species-tree relationships, summary coalescent topologies were largely consistent with inferences from concatenation. At the root of Eutheria, we identified consistent support for a sister relationship between Xenarthra and Afrotheria (i.e., Atlantogenata). At the other nodes of interest, support was less consistent. We suggest Scandentia is the sister of Primatomorpha (Euarchonta), but we failed to reject a sister relationship between Scandentia and Glires. Similarly, we suggest Perissodactyla is sister to Cetartiodactyla (Euungulata), but a sister relationship between Perissodactyla and Chiroptera remains plausible.

Macrosystematics of eutherian mammals combining HTS data to expand taxon coverage.

In the last few years high-throughput sequencing technologies have permitted significant advances in mammalian phylogenetic studies from a genomic perspective. However, these studies have been restricted to a sparse number of species with available reference genomes. Thus, several issues inside the eutherian mammals phylogeny remain unresolved. This may be due in part to limited taxon sampling, as taxonomic density is known to affect phylogenetic resolution. In this context, we present a protocol to increase taxon coverage using high-throughput sequencing data (RNA or DNA) generated for other biological studies and available in public databases. Following this procedure we addressed pending or controversial issues concerning the phylogenetic position of Dermoptera, Pholidota and Chiroptera, considering multiple and independent loci. Also for Chiroptera and Arctoidea we evaluated the relationships of the lineages that compose it. Although the maximum number of genes used is moderate (95), in some cases taxon coverage doubles that of previous related studies. Globally, all coalescent-based (STAR, MP-EST and ASTRAL) and concatenated (IQ-TREE and BEAST2) methods used for species tree reconstruction were consistent to each other and most of interrogated nodes received high statistical support.

Extracellular matrix in epitheliochorial, endotheliochorial and haemochorial placentation and its potential application for regenerative medicine.

A placenta is defined as structural approximation of maternal and foetal tissues to perform physiological exchange. Associated processes of differentiation and the establishment of its cells take place within the extracellular matrix (ECM) that provides a rich environment of collagens, fibronectins, cytokines and other components. Placental ECM is promising for tissue regeneration purposes, because it has immune tolerance capacities that may cause only minimal rejections of transplants with immunological differences between donor and recipient. However, specific characteristics of ECM during evolution of the structurally very diverse mammalian placenta are not yet revealed. We here address the major aspects of placental types, that is non-invasive (epitheliochorial), medium (endotheliochorial)-to-high (haemochorial) invasive nature of the interhemal barrier between the foetal and maternal blood system as well as their main components of ECM with special reference to species that are commonly used as animal models for human placentation and in the potential applications for regenerative medicine.

Multivariate Meta-Analysis of Brain-Mass Correlations in Eutherian Mammals.

The general assumption that brain size differences are an adequate proxy for subtler differences in brain organization turned neurobiologists toward the question why some groups of mammals such as primates, elephants, and whales have such remarkably large brains. In this meta-analysis, an extensive sample of eutherian mammals (115 species distributed in 14 orders) provided data about several different biological traits and measures of brain size such as absolute brain mass (AB), relative brain mass (RB; quotient from AB and body mass), and encephalization quotient (EQ). These data were analyzed by established multivariate statistics without taking specific phylogenetic information into account. Species with high AB tend to (1) feed on protein-rich nutrition, (2) have a long lifespan, (3) delayed sexual maturity, and (4) long and rare pregnancies with small litter sizes. Animals with high RB usually have (1) a short life span, (2) reach sexual maturity early, and (3) have short and frequent gestations. Moreover, males of species with high RB also have few potential sexual partners. In contrast, animals with high EQs have (1) a high number of potential sexual partners, (2) delayed sexual maturity, and (3) rare gestations with small litter sizes. Based on these correlations, we conclude that Eutheria with either high AB or high EQ occupy positions at the top of the network of food chains (high trophic levels). Eutheria of low trophic levels can develop a high RB only if they have small body masses.

The Transcriptomic Evolution of Mammalian Pregnancy: Gene Expression Innovations in Endometrial Stromal Fibroblasts.

The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type "neo-ESF" in contrast to "paleo-ESF" which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation.

Anterior commissure versus corpus callosum: A quantitative comparison across mammals.

Mammals rely on two major pathways to transfer information between the two hemispheres of the brain: the anterior commissure and the corpus callosum. Metatheria and monotremes rely exclusively on the anterior commissure for interhemispheric transfer between the isocortices and olfactory allocortices of each side, whereas Eutheria use a combination of the anterior commissure and an additional pathway exclusive to Eutheria, the corpus callosum. Midline cross-sectional area of the anterior commissure and corpus callosum were measured in a range of mammals from all three infraclasses and plotted against brain volume to determine how midline anterior commissure area and its size relative to the corpus callosum vary with brain size and taxon. In Metatheria, the square root of anterior commissure area rises in almost direct proportion with the cube root of brain volume (i.e. the ratio of the two is relatively constant), whereas among Eutheria the ratio of the square root of anterior commissure area to the cube root of brain volume declines slightly with increasing brain size. The total of isocortical and olfactory allocortical commissure area rises more rapidly with increasing brain volume among Eutheria than among Metatheria. This means that the midline isocortical and olfactory allocortical commissural area of metatherians with large brains (about 70 ml) is only about 50% of that among eutherians with similarly sized brains. On the other hand, isocortical and olfactory allocortical commissural area is similar in Metatheria and Eutheria at brain volumes around 1 ml. Among the Eutheria, some groups make less use of the anterior commissure pathway than do others: soricomorphs, rodents and cetaceans have smaller anterior commissures for their brain size than do afrosoricids, erinaceomorphs and proboscideans. The findings suggest that use of the anterior commissural route for isocortical commissural connections may have placed limitations on interhemispheric transfer of information among the metatherians, but only when brain size reaches 50 ml or more.

What was the ancestral function of decidual stromal cells? A model for the evolution of eutherian pregnancy.

In human and mouse, decidual stromal cells (DSC) are necessary for the establishment (implantation) and the maintenance of pregnancy by preventing inflammation and the immune rejection of the semi-allograft conceptus. DSC originated along the stem lineage of eutherian mammals, coincidental with the origin of invasive placentation. Surprisingly, in many eutherian lineages decidual cells are lost after the implantation phase of pregnancy, making it unlikely that DSC are necessary for the maintenance of pregnancy in these animals. In order to understand this variation, we review the literature on the fetal-maternal interface in all major eutherian clades Euarchontoglires, Laurasiatheria, Xenarthra and Afrotheria, as well as the literature about the ancestral eutherian species. We conclude that maintaining pregnancy may not be a shared derived function of DSC among all eutherian mammals. Rather, we propose that DSC originated to manage the inflammatory reaction associated with invasive implantation. We envision that this happened in a stem eutherian that had invasive placenta but still a short gestation. We further propose that extended gestation evolved independently in the major eutherian clades explaining why the major lineages of eutherian mammals differ with respect to the mechanisms maintaining pregnancy.

Connectivity and neurochemistry of the commissura anterior of the pigeon (Columba livia).

The anterior commissure (AC) and the much smaller hippocampal commissure constitute the only interhemispheric pathways at the telencephalic level in birds. Since the degeneration study from Zeier and Karten (), no detailed description of the topographic organization of the AC has been performed. This information is not only necessary for a better understanding of interhemispheric transfer in birds, but also for a comparative analysis of the evolution of commissural systems in the vertebrate classes. We therefore examined the fiber connections of the AC by using choleratoxin subunit B (CTB) and biotinylated dextran amine (BDA). Injections into subareas of the arcopallium and posterior amygdala (PoA) demonstrated contralateral projection fields within the anterior arcopallium (AA), intermediate arcopallium (AI), PoA, lateral, caudolateral and central nidopallium, dorsal and ventral mesopallium, and medial striatum (MSt). Interestingly, only arcopallial and amygdaloid projections were reciprocally organized, and all AC projections originated within a rather small area of the arcopallium and the PoA. The commissural neurons were not GABA-positive, and thus possibly not of an inhibitory nature. In sum, our neuroanatomical study demonstrates that a small group of arcopallial and amygdaloid neurons constitute a wide range of contralateral projections to sensorimotor and limbic structures. Different from mammals, in birds the neurons that project via the AC constitute mostly heterotopically organized and unidirectional connections. In addition, the great majority of pallial areas do not participate by themselves in interhemispheric exchange in birds. Instead, commissural exchange rests on a rather small arcopallial and amygdaloid cluster of neurons.