Comparative genomic analysis of chemosensory-related gene families in gastropods.

Chemoreception is critical for the survival and reproduction of animals. Except for a reduced group of insects and chelicerates, the molecular identity of chemosensory proteins is poorly understood in invertebrates. Gastropoda is the extant mollusk class with the greatest species richness, including marine, freshwater, and terrestrial lineages, and likely, highly diverse chemoreception systems. Here, we performed a comprehensive comparative genome analysis taking advantage of the chromosome-level information of two Gastropoda species, one of which belongs to a lineage that underwent a whole genome duplication event. We identified thousands of previously uncharacterized chemosensory-related genes, the majority of them encoding G protein-coupled receptors (GPCR), mostly organized into clusters distributed across all chromosomes. We also detected gene families encoding degenerin epithelial sodium channels (DEG-ENaC), ionotropic receptors (IR), sensory neuron membrane proteins (SNMP), Niemann-Pick type C2 (NPC2) proteins, and lipocalins, although with a lower number of members. Our phylogenetic analysis of the GPCR gene family across protostomes revealed: (i) remarkable gene family expansions in Gastropoda; (ii) clades including members from all protostomes; and (iii) species-specific clades with a substantial number of receptors. For the first time, we provide new and valuable knowledge into the evolution of the chemosensory gene families in invertebrates other than arthropods.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds.

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species.

Integrating Sequence Capture and Restriction Site-Associated DNA Sequencing to Resolve Recent Radiations of Pelagic Seabirds.

The diversification of modern birds has been shaped by a number of radiations. Rapid diversification events make reconstructing the evolutionary relationships among taxa challenging due to the convoluted effects of incomplete lineage sorting (ILS) and introgression. Phylogenomic data sets have the potential to detect patterns of phylogenetic incongruence, and to address their causes. However, the footprints of ILS and introgression on sequence data can vary between different phylogenomic markers at different phylogenetic scales depending on factors such as their evolutionary rates or their selection pressures. We show that combining phylogenomic markers that evolve at different rates, such as paired-end double-digest restriction site-associated DNA (PE-ddRAD) and ultraconserved elements (UCEs), allows a comprehensive exploration of the causes of phylogenetic discordance associated with short internodes at different timescales. We used thousands of UCE and PE-ddRAD markers to produce the first well-resolved phylogeny of shearwaters, a group of medium-sized pelagic seabirds that are among the most phylogenetically controversial and endangered bird groups. We found that phylogenomic conflict was mainly derived from high levels of ILS due to rapid speciation events. We also documented a case of introgression, despite the high philopatry of shearwaters to their breeding sites, which typically limits gene flow. We integrated state-of-the-art concatenated and coalescent-based approaches to expand on previous comparisons of UCE and RAD-Seq data sets for phylogenetics, divergence time estimation, and inference of introgression, and we propose a strategy to optimize RAD-Seq data for phylogenetic analyses. Our results highlight the usefulness of combining phylogenomic markers evolving at different rates to understand the causes of phylogenetic discordance at different timescales. [Aves; incomplete lineage sorting; introgression; PE-ddRAD-Seq; phylogenomics; radiations; shearwaters; UCEs.].

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

Evolutionary History of Major Chemosensory Gene Families across Panarthropoda.

Chemosensory perception is a fundamental biological process of particular relevance in basic and applied arthropod research. However, apart from insects, there is little knowledge of specific molecules involved in this system, which is restricted to a few taxa with uneven phylogenetic sampling across lineages. From an evolutionary perspective, onychophorans (velvet worms) and tardigrades (water bears) are of special interest since they represent the closest living relatives of arthropods, altogether comprising the Panarthropoda. To get insights into the evolutionary origin and diversification of the chemosensory gene repertoire in panarthropods, we sequenced the antenna- and head-specific transcriptomes of the velvet worm Euperipatoides rowelli and analyzed members of all major chemosensory families in representative genomes of onychophorans, tardigrades, and arthropods. Our results suggest that the NPC2 gene family was the only family encoding soluble proteins in the panarthropod ancestor and that onychophorans might have lost many arthropod-like chemoreceptors, including the highly conserved IR25a receptor of protostomes. On the other hand, the eutardigrade genomes lack genes encoding the DEG-ENaC and CD36-sensory neuron membrane proteins, the chemosensory members of which have been retained in arthropods; these losses might be related to lineage-specific adaptive strategies of tardigrades to survive extreme environmental conditions. Although the results of this study need to be further substantiated by an increased taxon sampling, our findings shed light on the diversification of chemosensory gene families in Panarthropoda and contribute to a better understanding of the evolution of animal chemical senses.

Understanding the Early Evolutionary Stages of a Tandem Drosophilamelanogaster-Specific Gene Family: A Structural and Functional Population Study.

Gene families underlie genetic innovation and phenotypic diversification. However, our understanding of the early genomic and functional evolution of tandemly arranged gene families remains incomplete as paralog sequence similarity hinders their accurate characterization. The Drosophila melanogaster-specific gene family Sdic is tandemly repeated and impacts sperm competition. We scrutinized Sdic in 20 geographically diverse populations using reference-quality genome assemblies, read-depth methodologies, and qPCR, finding that ∼90% of the individuals harbor 3-7 copies as well as evidence of population differentiation. In strains with reliable gene annotations, copy number variation (CNV) and differential transposable element insertions distinguish one structurally distinct version of the Sdic region per strain. All 31 annotated copies featured protein-coding potential and, based on the protein variant encoded, were categorized into 13 paratypes differing in their 3' ends, with 3-5 paratypes coexisting in any strain examined. Despite widespread gene conversion, the only copy present in all strains has functionally diverged at both coding and regulatory levels under positive selection. Contrary to artificial tandem duplications of the Sdic region that resulted in increased male expression, CNV in cosmopolitan strains did not correlate with expression levels, likely as a result of differential genome modifier composition. Duplicating the region did not enhance sperm competitiveness, suggesting a fitness cost at high expression levels or a plateau effect. Beyond facilitating a minimally optimal expression level, Sdic CNV acts as a catalyst of protein and regulatory diversity, showcasing a possible evolutionary path recently formed tandem multigene families can follow toward long-term consolidation in eukaryotic genomes.

Smelling in the dark: Phylogenomic insights into the chemosensory system of a subterranean beetle.

The chemosensory system has experienced relevant changes in subterranean animals, facilitating the perception of specific chemical signals critical to survival in their particular environment. However, the genomic basis of chemoreception in cave-dwelling fauna has been largely unexplored. We generated de novo transcriptomes for antennae and body samples of the troglobitic beetle Speonomus longicornis (whose characters suggest an extreme adaptation to a deep subterranean environment) in order to investigate the evolutionary origin and diversification of the chemosensory gene repertoire across coleopterans through a phylogenomic approach. Our results suggested a diminished diversity of odourant and gustatory gene repertoires compared to polyphagous beetles that inhabit surface habitats. Moreover, S. longicornis showed a large diversity of odourant-binding proteins, suggesting an important role of these proteins in capturing airborne chemical cues. We identified a gene duplication of the ionotropic coreceptor IR25a, a highly conserved single-copy gene in protostomes involved in thermal and humidity sensing. In addition, no homologous genes to sugar receptors or the ionotropic receptor IR41a were detected. Our findings suggest that the chemosensory gene repertoire of this cave beetle may result from adaptation to the highly specific ecological niche it occupies, and that gene duplication and loss may have played an important role in the evolution of gene families involved in chemoreception. Altogether, our results shed light on the genomic basis of chemoreception in a cave-dwelling invertebrate and pave the road towards understanding the genomic underpinnings of adaptation to the subterranean lifestyle at a deeper level.

Cryptic species delineation in freshwater planarians of the genus Dugesia (Platyhelminthes, Tricladida): Extreme intraindividual genetic diversity, morphological stasis, and karyological variability.

The keystone of planarian taxonomy traditionally has been the anatomy of the copulatory apparatus. However, many planarian species comprise asexual fissiparous populations, with the fissiparous animals not developing a copulatory apparatus, thus precluding their morphological identification. Incorporation of molecular data into planarian systematics has been of great value, not only in the identification of fissiparous individuals but also as an additional source of information for determining species boundaries. Nevertheless, the discrepancy between morphological and molecular data has highlighted the need for extra sources of taxonomic information. Moreover, a recent study has pointed out that fissiparous reproduction may lead to high levels of intraindividual genetic diversity in planarians, which may mislead molecular analyses. In the present study we aim to test a new up-to-date integrative taxonomic procedure for planarians, including intraindividual genetic data and additional sources of taxonomic information, besides morphology and DNA, using Dugesia subtentaculata sensu lato as a model organism, a species with an intricate taxonomic history. First, we used three different methods for molecular species delimitation on single locus datasets, both with and without intraindividual information, for formulating Primary Species Hypotheses (PSHs). Subsequently, Secondary Species Hypotheses (SSHs) were formulated on the basis of three types of information: (1) a coalescent-based species delimitation method applied to multilocus data, (2) morphology of the copulatory apparatus, and (3) karyological metrics. This resulted in the delimitation of four morphologically cryptic species within the nominal species D. subtentaculata. Our results provide evidence that the analysis of intraindividual genetic data is essential for properly developing PSHs in planarians. Our study reveals also that karyological differentiation, rather than morphological differentiation, may play an important role in speciation processes in planarians, thus suggesting that the currently known diversity of the group could be highly underestimated.

Gene duplications in the E. coli genome: common themes among pathotypes.

BACKGROUND: Gene duplication underlies a significant proportion of gene functional diversity and genome complexity in both eukaryotes and prokaryotes. Although several reports in the literature described the duplication of specific genes in E. coli, a detailed analysis of the extent of gene duplications in this microorganism is needed. RESULTS: The genomes of the E. coli enteroaggregative strain 042 and other pathogenic strains contain duplications of the gene that codes for the global regulator Hha. To determine whether the presence of additional copies of the hha gene correlates with the presence of other genes, we performed a comparative genomic analysis between E. coli strains with and without hha duplications. The results showed that strains harboring additional copies of the hha gene also encode the yeeR irmA (aec69) gene cluster, which, in turn, is also duplicated in strain 042 and several other strains. The identification of these duplications prompted us to obtain a global map of gene duplications, first in strain 042 and later in other E. coli genomes. Duplications in the genomes of the enteroaggregative strain 042, the uropathogenic strain CFT073 and the enterohemorrhagic strain O145:H28 have been identified by a BLASTp protein similarity search. This algorithm was also used to evaluate the distribution of the identified duplicates among the genomes of a set of 28 representative E. coli strains. Despite the high genomic diversity of E. coli strains, we identified several duplicates in the genomes of almost all studied pathogenic strains. Most duplicated genes have no known function. Transcriptomic analysis also showed that most of these duplications are regulated by the H-NS/Hha proteins. CONCLUSIONS: Several duplicated genes are widely distributed among pathogenic E. coli strains. In addition, some duplicated genes are present only in specific pathotypes, and others are strain specific. This gene duplication analysis shows novel relationships between E. coli pathotypes and suggests that newly identified genes that are duplicated in a high percentage of pathogenic E. coli isolates may play a role in virulence. Our study also shows a relationship between the duplication of genes encoding regulators and genes encoding their targets.

Chance and predictability in evolution: The genomic basis of convergent dietary specializations in an adaptive radiation.

The coexistence of multiple eco-phenotypes in independently assembled communities makes island adaptive radiations the ideal framework to test convergence and parallelism in evolution. In the radiation of the spider genus Dysdera in the Canary Islands, species diversification occurs concomitant with repeated events of trophic specialization. These dietary shifts, to feed primarily on woodlice, are accompanied by modifications in morphology (mostly in the mouthparts), behaviour and nutritional physiology. To gain insight into the molecular basis of this adaptive radiation, we performed a comprehensive comparative transcriptome analysis of five Canary Island Dysdera endemics representing two evolutionary and geographically independent events of dietary specialization. After controlling for the potential confounding effects of hemiplasy, our differential gene expression and selective constraint analyses identified a number of genetic changes that could be associated with the repeated adaptations to specialized diet of woodlice, including some related to heavy metal detoxification and homeostasis, the metabolism of some important nutrients and venom toxins. Our results shed light on the genomic basis of an extraordinary case of dietary shift convergence associated with species diversification. We uncovered putative molecular substrates of convergent evolutionary changes at different hierarchical levels, including specific genes, genes with equivalent functions and even particular amino acid positions. This study improves our knowledge of rapid adaptive radiations and provides new insights into the predictability of evolution.

DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets.

We present version 6 of the DNA Sequence Polymorphism (DnaSP) software, a new version of the popular tool for performing exhaustive population genetic analyses on multiple sequence alignments. This major upgrade incorporates novel functionalities to analyze large data sets, such as those generated by high-throughput sequencing technologies. Among other features, DnaSP 6 implements: 1) modules for reading and analyzing data from genomic partitioning methods, such as RADseq or hybrid enrichment approaches, 2) faster methods scalable for high-throughput sequencing data, and 3) summary statistics for the analysis of multi-locus population genetics data. Furthermore, DnaSP 6 includes novel modules to perform single- and multi-locus coalescent simulations under a wide range of demographic scenarios. The DnaSP 6 program, with extensive documentation, is freely available at http://www.ub.edu/dnasp.

Rapid Functional and Sequence Differentiation of a Tandemly Repeated Species-Specific Multigene Family in Drosophila.

Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, our understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated, and tandemly expanded in Drosophila melanogaster, contributing to increased male-male competition. Using various types of massively parallel sequencing data, we studied the organization, sequence evolution, and functional attributes of the different Sdic copies. By leveraging long-read sequencing data, we uncovered both copy number and order differences from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, we also detected signatures of two episodes of diversifying selection, which have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. Our results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection.

The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

DOMINO: development of informative molecular markers for phylogenetic and genome-wide population genetic studies in non-model organisms.

MOTIVATION: The development of molecular markers is one of the most important challenges in phylogenetic and genome wide population genetics studies, especially in studies with non-model organisms. A highly promising approach for obtaining suitable markers is the utilization of genomic partitioning strategies for the simultaneous discovery and genotyping of a large number of markers. Unfortunately, not all markers obtained from these strategies provide enough information for solving multiple evolutionary questions at a reasonable taxonomic resolution. RESULTS: We have developed Development Of Molecular markers In Non-model Organisms (DOMINO), a bioinformatics tool for informative marker development from both next generation sequencing (NGS) data and pre-computed sequence alignments. The application implements popular NGS tools with new utilities in a highly versatile pipeline specifically designed to discover or select personalized markers at different levels of taxonomic resolution. These markers can be directly used to study the taxa surveyed for their design, utilized for further downstream PCR amplification in a broader set taxonomic scope, or exploited as suitable templates to bait design for target DNA enrichment techniques. We conducted an exhaustive evaluation of the performance of DOMINO via computer simulations and illustrate its utility to find informative markers in an empirical dataset. AVAILABILITY AND IMPLEMENTATION: DOMINO is freely available from www.ub.edu/softevol/domino CONTACT: elsanchez@ub.edu or jrozas@ub.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Computational prediction of the phenotypic effects of genetic variants: basic concepts and some application examples in Drosophila nervous system genes.

Predicting the phenotypic impact of mutations is a central challenge in population and functional genetics. The analysis of DNA and amino acid sequence variation in an evolutionary context is a robust approach to infer the fitness effects of genetic variants. In this review, we discuss the most popular methods based on this approach, covering both theoretical and practical aspects, and introduce compelling software for predicting the functional effects of mutations, and to highlight functionally relevant nucleotide or amino acid candidate positions. Furthermore, we provide some examples of their application to Drosophila genes affecting significant aspects of the development, physiology and function of the nervous system.

High Gene Family Turnover Rates and Gene Space Adaptation in the Compact Genome of the Carnivorous Plant Utricularia gibba.

Utricularia gibba is an aquatic carnivorous plant with highly specialized morphology, featuring fibrous floating networks of branches and leaf-like organs, no recognizable roots, and bladder traps that capture and digest prey. We recently described the compressed genome of U. gibba as sufficient to control the development and reproduction of a complex organism. We hypothesized intense deletion pressure as a mechanism whereby most noncoding DNA was deleted, despite evidence for three independent whole-genome duplications (WGDs). Here, we explore the impact of intense genome fractionation in the evolutionary dynamics of U. gibba's functional gene space. We analyze U. gibba gene family turnover by modeling gene gain/death rates under a maximum-likelihood statistical framework. In accord with our deletion pressure hypothesis, we show that the U. gibba gene death rate is significantly higher than those of four other eudicot species. Interestingly, the gene gain rate is also significantly higher, likely reflecting the occurrence of multiple WGDs and possibly also small-scale genome duplications. Gene ontology enrichment analyses of U. gibba-specific two-gene orthogroups, multigene orthogroups, and singletons highlight functions that may represent adaptations in an aquatic carnivorous plant. We further discuss two homeodomain transcription factor gene families (WOX and HDG/HDZIP-IV) showing conspicuous differential expansions and contractions in U. gibba. Our results 1) reconcile the compactness of the U. gibba genome with its accommodation of a typical number of genes for a plant genome, and 2) highlight the role of high gene family turnover in the evolutionary diversification of U. gibba's functional gene space and adaptations to its unique lifestyle and highly specialized body plan.

Streptococcus gallolyticus subsp. gallolyticus from human and animal origins: genetic diversity, antimicrobial susceptibility, and characterization of a vancomycin-resistant calf isolate carrying a vanA-Tn1546-like element.

The aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41 Streptococcus gallolyticus subsp. gallolyticus isolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely related S. gallolyticus subsp. gallolyticus genomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of the vanY-vanZ region and partial duplication of the vanH gene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion, S. gallolyticus subsp. gallolyticus isolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.

Functional evidence that a recently evolved Drosophila sperm-specific gene boosts sperm competition.

In many species, both morphological and molecular traits related to sex and reproduction evolve faster in males than in females. Ultimately, rapid male evolution relies on the acquisition of genetic variation associated with differential reproductive success. Many newly evolved genes are associated with novel functions that might enhance male fitness. However, functional evidence of the adaptive role of recently originated genes in males is still lacking. The Sperm dynein intermediate chain multigene family, which encodes a Sperm dynein intermediate chain presumably involved in sperm motility, originated from complex genetic rearrangements in the lineage that leads to Drosophila melanogaster within the last 5.4 million years since its split from Drosophila simulans. We deleted all the members of this multigene family resident on the X chromosome of D. melanogaster by chromosome engineering and found that, although the deletion does not result in a reduction of progeny number, it impairs the competence of the sperm in the presence of sperm from wild-type males. Therefore, the Sperm dynein intermediate chain multigene family contributes to the differential reproductive success among males and illustrates precisely how quickly a new gene function can be incorporated into the genetic network of a species.

Molecular population genetics of the insulin/TOR signal transduction pathway: a network-level analysis in Drosophila melanogaster.

The IT-insulin/target of rapamycin (TOR)-signal transduction pathway is a relatively well-characterized pathway that plays a central role in fundamental biological processes. Network-level analyses of DNA divergence in Drosophila and vertebrates have revealed a clear gradient in the levels of purifying selection along this pathway, with the downstream genes being the most constrained. Remarkably, this feature does not result from factors known to affect selective constraint such as gene expression, codon bias, protein length, and connectivity. The present work aims to establish whether the selective constraint gradient detected along the IT pathway at the between-species level can also be observed at a shorter time scale. With this purpose, we have surveyed DNA polymorphism in Drosophila melanogaster and divergence from D. simulans along the IT pathway. Our network-level analysis shows that DNA polymorphism exhibits the same polarity in the strength of purifying selection as previously detected at the divergence level. This equivalent feature detected both within species and between closely and distantly related species points to the action of a general mechanism, whose action is neither organism specific nor evolutionary time dependent. The detected polarity would be, therefore, intrinsic to the IT pathway architecture and function.