Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing.

Understanding macrophage heterogeneity in tissue repair is a major challenge. Here, we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing. We use a modified version of the original Smart-seq2 protocol to increase speed and accuracy.

Convergent and lineage-specific genomic differences in limb regulatory elements in limbless reptile lineages.

Loss of limbs evolved many times in squamate reptiles. Here we investigated the genomic basis of convergent limb loss in reptiles. We sequenced the genomes of a closely related pair of limbless-limbed gymnophthalmid lizards and performed a comparative genomic analysis including five snakes and the limbless glass lizard.

Recapitulating Evolutionary Divergence in a Single Cis-Regulatory Element Is Sufficient to Cause Expression Changes of the Lens Gene Tdrd7.

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved noncoding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis.

Six reference-quality genomes reveal evolution of bat adaptations.

Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus).

Author Correction: A genomics approach reveals insights into the importance of gene losses for mammalian adaptations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly.

Background: Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes.

Results: Here, we report a high-quality genome assembly of the tegu lizard, Salvator merianae, the first lacertoid with a sequenced genome. We combined 74X Illumina short-read, 29.

Phenotype loss is associated with widespread divergence of the gene regulatory landscape in evolution.

Detecting the genomic changes underlying phenotypic changes between species is a main goal of evolutionary biology and genomics. Evolutionary theory predicts that changes in cis-regulatory elements are important for morphological changes. We combined genome sequencing, functional genomics and genome-wide comparative analyses to investigate regulatory elements in lineages that lost morphological traits.

REforge Associates Transcription Factor Binding Site Divergence in Regulatory Elements with Phenotypic Differences between Species.

Elucidating the genomic determinants of morphological differences between species is key to understanding how morphological diversity evolved. While differences in cis-regulatory elements are an important genetic source for morphological evolution, it remains challenging to identify regulatory elements involved in phenotypic differences. Here, we present Regulatory Element forward genomics (REforge), a computational approach that detects associations between transcription factor binding site divergence in putative regulatory elements and phenotypic differences between species.

A genomics approach reveals insights into the importance of gene losses for mammalian adaptations.

Identifying the genomic changes that underlie phenotypic adaptations is a key challenge in evolutionary biology and genomics. Loss of protein-coding genes is one type of genomic change with the potential to affect phenotypic evolution. Here, we develop a genomics approach to accurately detect gene losses and investigate their importance for adaptive evolution in mammals.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL).

Controlling for Phylogenetic Relatedness and Evolutionary Rates Improves the Discovery of Associations Between Species' Phenotypic and Genomic Differences.

The growing number of sequenced genomes allows us now to address a key question in genetics and evolutionary biology: which genomic changes underlie particular phenotypic changes between species? Previously, we developed a computational framework called Forward Genomics that associates phenotypic to genomic differences by focusing on phenotypes that are independently lost in different lineages. However, our previous implementation had three main limitations. Here, we present two new Forward Genomics methods that overcome these limitations by (1) directly controlling for phylogenetic relatedness, (2) controlling for differences in evolutionary rates, and (3) computing a statistical significance.

Iterative error correction of long sequencing reads maximizes accuracy and improves contig assembly.

Next-generation sequencers such as Illumina can now produce reads up to 300 bp with high throughput, which is attractive for genome assembly. A first step in genome assembly is to computationally correct sequencing errors. However, correcting all errors in these longer reads is challenging.

Are hemipenial spines related to limb reduction? A spiny discussion focused on gymnophthalmid lizards (Squamata: Gymnophthalmidae).

Calcified spines in the hemipenial surface occur convergently in several gymnophthalmid lizard species and in advanced snakes. Based on the pronounced degrees of limb reduction in these distantly related lineages, such hemipenial structures were suggested to play a functional role in couple-anchoring during copulation, partly assuming the function of the limbs during mating. Herein, we assessed the hemipenial morphology of virtually all the valid genera of the family Gymnophthalmidae to test for a phylogenetic correlation between limb reduction and the presence of calcified hemipenial spines.

Through the looking glass: the spectacle in gymnophthalmid lizards.

The anatomy and development of the eyelids in squamate reptiles are still relatively unknown, considering its variation within the group. The neotropical Gymnophthalmini are traditionally characterized by having lost the eyelids, but their structure is not well described. In this study, the embryonic development and the adult morphology of the gymnophthalmid eye, with special attention to the eyelids, the nictitating membrane, and the spectacle are described.

Digit evolution in gymnophthalmid lizards.

The tetrapod limb is a highly diverse structure, and reduction or loss of this structure accounts for many of the limb phenotypes observed within species. Squamate reptiles are one of the many tetrapod lineages in which the limbs have been greatly modified from the pentadactyl generalized pattern, including different degrees of reduction in the number of limb elements to complete limblessness. Even though limb reduction is widespread, the evolutionary and developmental mechanisms involved in the formation of reduced limb morphologies remains unclear.

A comparative analysis of the post-cranial skeleton of fossorial and non-fossorial gymnophthalmid lizards.

Squamates are found in a wide range of habitats and show a corresponding diversity of morphologies that can often be correlated with locomotor mode. The evolution of a snake-like body form, frequently associated with fossoriality, from a typical lacertiform morphology involves changes in the morphology of vertebrae, girdles, and limbs; the changes are mainly manifested by the reduction or loss of limbs and body elongation. In this study, we describe the axial and appendicular skeletons of six closely related gymnophthalmid species.

Rediscovery of the earless microteiid lizard Anotosaura collaris Amaral, 1933 (Squamata: Gymnophthalmidae): a redescription complemented by osteological, hemipenial, molecular, karyological, physiological and ecological data.

More than a century after its discovery by Ernest Garbe, and almost 80 years after its original description, we obtained a series of specimens of the earless gymnophthalmid Anotosaura collaris, the type species of the genus, up to now known only by a single specimen. On the basis of the material obtained at and close to the type locality we redescribe the species, adding information about the external and hemipenial morphology, osteology and karytoype. Molecular data confirm its sister relationship with Anotosaura vanzolinia as well as the close relationship of Anotosaura with the Ecpleopodini Colobosauroides and Dryadosaura.

Skeletal development in the fossorial gymnophthalmids Calyptommatus sinebrachiatus and Nothobachia ablephara.

The development of the cartilaginous and bony elements that form the skull and axial and appendicular skeleton is described in detail for the post-ovipositional embryonic development of the fossorial gymnophthalmid species Calyptommatus sinebrachiatus and Nothobachia ablephara. Both species have a snake-like morphology, showing an elongated body and reduced or absent limbs, as well as modifications in skull bones for burrowing, such as complex articulation surfaces and development of bony extensions that enclose and protect the brain. Similar morphological changes have originated independently in several squamate groups, including the one that led to the snake radiation.

Embryonic development of the fossorial gymnophthalmid lizards Nothobachia ablephara and Calyptommatus sinebrachiatus.

The evolutionary history of the lizard family Gymnophthalmidae is characterized by several independent events of morphological modifications to a snake-like body plan, such as limb reduction, body elongation, loss of external ear openings, and modifications in skull bones, as adaptive responses to a burrowing and fossorial lifestyle. The origins of such morphological modifications from an ancestral lizard-like condition can be traced back to evolutionary changes in the developmental processes that coordinate the building of the organism. Thus, the characterization of the embryonic development of gymnophthalmid lizards is an essential step because it lays the foundation for future studies aiming to understand the exact nature of these changes and the developmental mechanisms that could have been responsible for the evolution of a serpentiform (snake-like) from a lacertiform (lizard-like) body form.

Comparative cranial osteology of fossorial lizards from the tribe Gymnophthalmini (Squamata, Gymnophthalmidae).

Squamates (lizards, snakes and amphisbaenians) are represented by a large number of species distributed among a wide variety of habitats. Changes in body plan related to a fossorial habit are a frequent trend within the group and many morphological adaptations to this particular lifestyle evolved convergently in nonrelated species, reflecting adaptations to a similar habitat. The fossorial lifestyle requires an optimal morphological organization for an effective use of the available resources.