Evolutionary Tinkering Research Articles

Evolutionary developmental biology: Use it or lose it.

A comparison of the development of adapted limbs in mammals uncovers multiple avenues to digit loss and highlights that early patterning events are not untouchable by evolutionary tinkering. See Articles p.41 & p.46 The basic five-digit limb of tetrapods has been altered many times and in many ways during evolution, usually by the progressive loss of digits. Two papers published in this issue of Nature examine the developmental changes underlying digit reduction in mammals. Javier Lopez-Rios et al. look at cattle, where digits three and four are modified to form hooves; digits two and five are vestigial, and the first digit is lost. The first limb bud is shown to be progressively lost as it develops. The Ptch1 gene, which encodes a receptor for the limb-development morphogen Sonic hedgehog (SHH), is upregulated due to evolutionary alteration of a Ptch1 cis-regulatory module that no longer responds to graded SHH signalling during bovine handplate development. Kimberly Cooper et al. show, using a wide range of mammals, that mechanisms of digit loss vary in different lineages. In creatures as varied as the jerboa and the camel, cell death sculpts the tissue in the emerging limb to leave the remaining toes. In other creatures, such as the pig, digit loss is orchestrated by earlier limb patterning events with no increase in cell death. Taken together these findings demonstrate remarkable plasticity in the mechanisms of limb evolution in hooved mammals and rodents, yet reveal a degree of evolutionary convergence.

Brain mechanisms of acoustic communication in humans and nonhuman primates: an evolutionary perspective.

Any account of "what is special about the human brain" (Passingham 2008) must specify the neural basis of our unique ability to produce speech and delineate how these remarkable motor capabilities could have emerged in our hominin ancestors. Clinical data suggest that the basal ganglia provide a platform for the integration of primate-general mechanisms of acoustic communication with the faculty of articulate speech in humans. Furthermore, neurobiological and paleoanthropological data point at a two-stage model of the phylogenetic evolution of this crucial prerequisite of spoken language: (i) monosynaptic refinement of the projections of motor cortex to the brainstem nuclei that steer laryngeal muscles, presumably, as part of a "phylogenetic trend" associated with increasing brain size during hominin evolution; (ii) subsequent vocal-laryngeal elaboration of cortico-basal ganglia circuitries, driven by human-specific FOXP2 mutations.;>This concept implies vocal continuity of spoken language evolution at the motor level, elucidating the deep entrenchment of articulate speech into a "nonverbal matrix" (Ingold 1994), which is not accounted for by gestural-origin theories. Moreover, it provides a solution to the question for the adaptive value of the "first word" (Bickerton 2009) since even the earliest and most simple verbal utterances must have increased the versatility of vocal displays afforded by the preceding elaboration of monosynaptic corticobulbar tracts, giving rise to enhanced social cooperation and prestige. At the ontogenetic level, the proposed model assumes age-dependent interactions between the basal ganglia and their cortical targets, similar to vocal learning in some songbirds. In this view, the emergence of articulate speech builds on the "renaissance" of an ancient organizational principle and, hence, may represent an example of "evolutionary tinkering" (Jacob 1977).

Evolutionary tinkering of the expression of PDF1s suggests their joint effect on zinc tolerance and the response to pathogen attack

Multigenic families of Plant Defensin type 1 (PDF1) have been described in several species, including the model plant Arabidopsis thaliana as well as zinc tolerant and hyperaccumulator A. halleri. In A. thaliana, PDF1 transcripts (AtPDF1) accumulate in response to pathogen attack following synergic activation of ethylene/jasmonate pathways. However, in A. halleri, PDF1 transcripts (AhPDF1) are constitutively highly accumulated. Through an evolutionary approach, we investigated the possibility of A. halleri or A. thaliana species specialization in different PDF1s in conveying zinc tolerance and/or the response to pathogen attack via activation of the jasmonate (JA) signaling pathway. The accumulation of each PDF1 from both A. halleri and A. thaliana was thus compared in response to zinc excess and MeJA application. In both species, PDF1 paralogues were barely or not at all responsive to zinc. However, regarding the PDF1 response to JA signaling activation, A. thaliana had a higher number of PDF1s responding to JA signaling activation. Remarkably, in A. thaliana, a slight but significant increase in zinc tolerance was correlated with activation of the JA signaling pathway. In addition, A. halleri was found to be more tolerant to the necrotrophic pathogen Botrytis cinerea than A. thaliana. Since PDF1s are known to be promiscuous antifungal proteins able to convey zinc tolerance, we propose, on the basis of the findings of this study, that high constitutive PDF1 transcript accumulation in A. halleri is a potential way to skip the JA signaling activation step required to increase the PDF1 transcript level in the A. thaliana model species. This could ultimately represent an adaptive evolutionary process that would promote a PDF1 joint effect on both zinc tolerance and the response to pathogens in the A. halleri extremophile species.

ANRIL/CDKN2B-AS shows two-stage clade-specific evolution and becomes conserved after transposon insertions in simians

BackgroundMany long non-coding RNA (lncRNA) genes identified in mammals have multiple exons and functional domains, allowing them to bind to polycomb proteins, DNA methyltransferases, and specific DNA sequences to regulate genome methylation. Little is known about the origin and evolution of lncRNAs. ANRIL/CDKN2B-AS consists of 19 exons on human chromosome 9p21 and regulates the expression of three cyclin-dependent kinase inhibitors (CDKN2A/ARF/CDKN2B).ResultsANRIL/CDKN2B-AS originated in placental mammals, obtained additional exons during mammalian evolution but gradually lost them during rodent evolution, and reached 19 exons only in simians. ANRIL lacks splicing signals in mammals. In simians, multiple transposons were inserted and transformed into exons of the ANRIL gene, after which ANRIL became highly conserved. A further survey reveals that multiple transposons exist in many lncRNAs.ConclusionsANRIL shows a two-stage, clade-specific evolutionary process and is fully developed only in simians. The domestication of multiple transposons indicates an impressive pattern of “evolutionary tinkering” and is likely to be important for ANRIL’s structure and function. The evolution of lncRNAs and that of transposons may be highly co-opted in primates. Many lncRNAs may be functional only in simians.

Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues

The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or ‘light organ’, which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ∼1/4 into embryogenesis (Naef stage 18) and the light organ ∼3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light-sensing photophore.

Evolutionary rewiring: a modified prokaryotic gene-regulatory pathway in chloroplasts

Photosynthetic electron transport regulates chloroplast gene transcription through the action of a bacterial-type sensor kinase known as chloroplast sensor kinase (CSK). CSK represses photosystem I (PS I) gene transcription in PS I light and thus initiates photosystem stoichiometry adjustment. In cyanobacteria and in non-green algae, CSK homologues co-exist with their response regulator partners in canonical bacterial two-component systems. In green algae and plants, however, no response regulator partner of CSK is found. Yeast two-hybrid analysis has revealed interaction of CSK with sigma factor 1 (SIG1) of chloroplast RNA polymerase. Here we present further evidence for the interaction between CSK and SIG1. We also show that CSK interacts with quinone. Arabidopsis SIG1 becomes phosphorylated in PS I light, which then specifically represses transcription of PS I genes. In view of the identical signalling properties of CSK and SIG1 and of their interactions, we suggest that CSK is a SIG1 kinase. We propose that the selective repression of PS I genes arises from the operation of a gene-regulatory phosphoswitch in SIG1. The CSK-SIG1 system represents a novel, rewired chloroplast-signalling pathway created by evolutionary tinkering. This regulatory system supports a proposal for the selection pressure behind the evolutionary stasis of chloroplast genes.

Colliding Dynamical Complex Network Models: Biological Attractors versus Attractors from Material Physics

Colliding Dynamical Complex Network Models: Biological Attractors versus Attractors from Material Physics

Stress, adaptation, and speciation in the evolution of the blind mole rat, Spalax, in Israel

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00–2.35Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n=54→S. galili, 2n=52→S. carmeli, 2n=58→S. judaei, 2n=60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.

Evolutionary tinkering with visual photoreception

Eyes have evolved many times, and arthropods and vertebrates share transcription factors for early development. Moreover, the photochemistry of vision in all eyes employs an opsin and the isomerization of a retinoid from the 11-cis to the all-trans configuration. The opsins, however, have associated with several different G proteins, initiating hyperpolarizing and depolarizing conductance changes at the photoreceptor membrane. Beyond these obvious instances of homology, much of the evolutionary story is one of tinkering, producing a great variety of morphological forms and variation within functional themes. This outcome poses a central issue in the convergence of evolutionary and developmental biology: what are the heritable features in the later stages of development that give natural selection traction in altering phenotypic outcomes? This paper discusses some results of evolutionary tinkering where this question arises and, in some cases, where the reasons for particular outcomes and the role of adaptation may not be understood. Phenotypic features include: the exploitation of microvilli in rhabdomeric photoreceptors for detecting the plane of polarized light; different instances of retinoid in the visual pigment; examples of the many uses of accessory pigments in tuning the spectral sensitivity of photoreceptors; selection of opsins in tuning sensitivity in aquatic environments; employing either reflection or refraction in the optics of compound eyes; the multiple ways of constructing images in compound eyes; and the various ways of regenerating 11-cis retinals to maintain visual sensitivity. Evolution is an irreversible process, but tinkering may recover some lost functions, albeit by new mutational routes. There is both elegance and intellectual coherence to the natural processes that produce such variety and functional complexity. But marginalizing the teaching of evolution in public education is a continuing social and political problem that contributes to the reckless capacity of humans to alter the planet without trying to understand how nature works.

A single origin of large colony size in allodapine bees suggests a threshold event among 50 million years of evolutionary tinkering

Evolutionary origins of highly eusocial organization involving morphological castes have been very rare, yet these origins have often led to enormous diversification and ecological success. This suggests that once an apparently severe selective barrier to highly eusocial behaviour is overcome, major new adaptive landscapes open up. One would therefore expect a discontinuity in patterns of evolutionary change across this barrier. However, we do not know if highly eusocial organization has evolved incrementally from less complex societies, or if it has involved some kind of evolutionary leap. Our study examines this issue using colony size data from 33 allodapine bee species, with a crown age of ca. 47 Mya. Our species cover all major allodapine clades, and include Exoneurella tridentata, the only known allodapine with morphologically discrete castes. Phylogenetic analyses indicate a strong effect of phylogeny on the evolution of maximum brood size, but the effect of phylogeny on maximum colony size (number of adults) depends on whether E. tridentata is excluded or included in analyses. We found no evidence of punctuational change in maximum colony or brood sizes over the phylogeny as a whole, but colony and brood sizes in E. tridentata fall well beyond variation among the other allodapines. Colony size in E. tridentata therefore represents an evolutionary outcome that does not fit within the kinds of incremental changes found in other allodapines. We propose that E. tridentata indicates the crossing of an important threshold, and this has entailed some very unusual ecological circumstances.

Gene duplication plays a major role in gene co-option: Studies into the evolution of the motilin/ghrelin family and their receptors

Extant genes can be modified, or ‘tinkered with’, to provide new roles or new characteristics of these genes. At the genetic level, this often involves gene duplication and specialization of the resulting genes into particular functions. We investigate how ligand-receptor partnerships evolve after gene duplication. While significant work has been conducted in this area, the examination of additional models should help us better understand the proposed models and potentially reveal novel evolutionary patterns and dynamics. We use bioinformatics, comparative genomics and phylogenetic analyses to show that preproghrelin and prepromotilin descended from a common ancestor and that a gene duplication generated these two genes shortly after the divergence of amphibians and amniotes. The evolutionary history of the receptor family differs from that of their cognate ligands. GPR39 diverges first, and an ancestral receptor gives rise to receptors classified as fish-specific clade A, GHSR and MLNR by successive gene duplications occurring before the divergence of tetrapods and ray-finned fish. The ghrelin/GHSR system is maintained and functionally conserved from fish to mammals. Motilin-MLNR specificity must have arisen by ligand-receptor coevolution after the MLN hormone gene diverged from the GHRL gene in the amniote lineage. Conserved molecular machinery can give rise to new neuroendocrine response mechanisms by the co-option of duplicated genes. Gene duplication is both parsimonious and creative in producing elements for evolutionary tinkering and plays a major role in gene co-option, thus aiding the evolution of greater biological complexity.

Evo-devo and accounting for Darwin's endless forms

Evo-devo has led to dramatic advances in our understanding of how the processes of development can contribute to explaining patterns of evolutionary diversification that underlie the endless forms of animal life on the Earth. This is increasingly the case not only for the origins of evolutionary novelties that permit new functions and open up new adaptive zones, but also for the processes of evolutionary tinkering that occur within the subsequent radiations of related species. Evo-devo has time and again yielded spectacular examples of Darwin's notions of common ancestry and of descent with modification. It has also shown that the evolution of endless forms is more about the evolution of the regulatory machinery of ancient genes than the origin and elaboration of new genes. Evolvability, especially with respect to the capacity of a developmental system to evolve and to generate the variation in form for natural selection to screen, has become a pivotal focus of evo-devo. As a consequence, a balancing of the concept of endless forms in morphospace with a greater awareness of the potential for developmental constraints and bias is becoming more general. The prospect of parallel horizons opening up for the evolution of behaviour is exciting; in particular, does Sean Carroll's phrase referring to old genes learning new tricks in the evolution of endless forms apply equally as well to patterns of diversity and disparity in behavioural trait-space?

The single active X in human cells: evolutionary tinkering personified

All mammals compensate for sex differences in numbers of X chromosomes by transcribing only a single X chromosome in cells of both sexes; however, they differ from one another in the details of the compensatory mechanisms. These species variations result from chance mutations, species differences in the staging of developmental events, and interactions between events that occur concurrently. Such variations, which have only recently been appreciated, do not interfere with the strategy of establishing a single active X, but they influence how it is carried out. In an overview of X dosage compensation in human cells, I point out the evolutionary variations. I also argue that it is the single active X that is chosen, rather than inactive ones. Further, I suggest that the initial events in the process-those that precede silencing of future inactive X chromosomes-include randomly choosing the future active X, most likely by repressing its XIST locus.

The National Educational Technology Plan Doesn't Live up to its Call for Revolutionary Transformation

The National Educational Technology Plan (NETP) calls for the revolutionary transformation of the American educational system. The plan correctly points to the decisions of the late 1800s that still direct educational policy today, and correctly calls the school change efforts evolutionary tinkering. The author, a pioneer in online education efforts in the United States, expected the plan to offer a blueprint for radical transformation of American education. He states his disappointment in the plan's lack of either that blueprint or recommendation of a process for that transformation. He then presents his outline of a process for the creation of a revolutionary transformational model of education.

Functional and Structural Diversification of the Anguimorpha Lizard Venom System

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this ∼130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding domains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A(2) toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms.

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Author SummaryMany protein families are so diverse that it is hard to determine their ancestral functions and to understand how their derived functions evolved. The existence of so many different functions within protein families often creates the impression that complex, novel functions must have evolved repeatedly and independently. Nuclear receptors (NRs) are a large family of related proteins that regulate key biological processes in animals by binding to specific DNA sequences and triggering expression of nearby target genes. Many NRs are activated by a specific hormone or other small molecule, but some do not require a ligand, and still others are incapable of activating gene expression and so act primarily as repressors of transcription. To understand how the functional diversity of NRs evolved, we reconstructed the structural and functional characteristics of the ancient protein from which the entire family evolved, using genomic, biochemical, functional, and structural analyses in a phylogenetic framework. We show, contrary to current belief, that the ancestral NR was a ligand-activated transcriptional activator that existed in the earliest period of animal evolution. Our analysis reveals how the extraordinary functional diversity of modern receptors was generated by subtle tinkering with this ancestral template—slightly reshaping the ligand cavity, stabilizing the protein's active conformation so it no longer required a ligand, or disabling the protein's capacity to activate transcription without affecting its other properties. We predict that, when sufficient data are gathered to allow detailed evolutionary reconstructions in other protein families, it will become apparent that most protein functional diversity evolved by tinkering with ancient functions; invoking the evolution of wholesale “novelty” will seldom be necessary.

Syntax: Its Evolution and Its Representation in the Brain

Poeppel (2008) observes that there is no clear correspondence between units of analysis in linguistics (especially the abstract and arbitrary-looking principles of syntax) and biological units of neuroscience, concluding that current neurolinguistic research presents a case of cross-sterilization, rather than cross-fertilization. Here the proposal is developed that decomposing syntax into intermediate evolutionary layers, into its evolutionary primitives, not only makes syntax compatible with gradualist accounts, but it also renders it more tangible and less abstract. In this approach, at least some complexities (and oddities) of syntax, such as Subjacency effects and the small clause core, can be seen as side-effects/by-products of evolutionary tinkering. It is conceivable that such evolutionary considerations are a necessary missing ingredient in any attempt to establish links between the postulates of syntax and the units of neuroscience. This article considers concrete linguistic data and suggestions as to where and how to look for neurobiological correlates of syntax.

Evolutionary Tinkering with Conserved Components of a Transcriptional Regulatory Network

Author SummaryConserved metabolic machineries direct energy production and investment in most life forms. However, variation in the transcriptional regulation of the genes that encode this machinery has been observed and shown to contribute to phenotypic differences between species. Here, we show that the regulatory circuits governing the expression of central metabolic components (in this case the ribosomes) in different yeast species have an unexpected level of evolutionary plasticity. Most transcription factors involved in the regulation of expression of ribosomal genes have in fact been reused in new ways during the evolutionary time separating S. cerevisiae and C. albicans to generate global changes in transcriptional network structures and new ribosomal regulatory complexes.

Phylogenomics of the oxidative phosphorylation in fungi reveals extensive gene duplication followed by functional divergence

BackgroundOxidative phosphorylation is central to the energy metabolism of the cell. Due to adaptation to different life-styles and environments, fungal species have shaped their respiratory pathways in the course of evolution. To identify the main mechanisms behind the evolution of respiratory pathways, we conducted a phylogenomics survey of oxidative phosphorylation components in the genomes of sixty fungal species.ResultsBesides clarifying orthology and paralogy relationships among respiratory proteins, our results reveal three parallel losses of the entire complex I, two of which are coupled to duplications in alternative dehydrogenases. Duplications in respiratory proteins have been common, affecting 76% of the protein families surveyed. We detect several instances of paralogs of genes coding for subunits of respiratory complexes that have been recruited to other multi-protein complexes inside and outside the mitochondrion, emphasizing the role of evolutionary tinkering.ConclusionsProcesses of gene loss and gene duplication followed by functional divergence have been rampant in the evolution of fungal respiration. Overall, the core proteins of the respiratory pathways are conserved in most lineages, with major changes affecting the lineages of microsporidia, Schizosaccaromyces and Saccharomyces/Kluyveromyces due to adaptation to anaerobic life-styles. We did not observe specific adaptations of the respiratory metabolism common to all pathogenic species.

Origin and Evolution of the Unique Tetra-Domain Hemoglobin from the Hydrothermal Vent Scale Worm Branchipolynoe

Hemoglobin is the most common respiratory pigment in annelids. It can be intra or extracellular, and this latter type can form large multimeric complexes. The hydrothermal vent scale worms Branchipolynoe symmytilida and Branchipolynoe seepensis express an extracellular tetra-domain hemoglobin (Hb) that is unique in annelids. We sequenced the gene for the single-domain and tetra-domain globins in these two species. The single-domain gene codes for a mature protein of 137 amino acids, and the tetra-domain gene codes for a mature protein of 552 amino acids. The single-domain gene has a typical three exon/two intron structure, with introns located at their typical positions (B12.2 and G7.0). This structure is repeated four times in the tetra-domain gene, with no bridge introns or linker sequences between domains. The phylogenetic position of Branchipolynoe globins among known annelid globins revealed that, although extracellular, they cluster within the annelid intracellular globins clade, suggesting that the extracellular state of these Hbs is the result of convergent evolution. The tetra-domain structure likely resulted from two tandem duplications, domain 1 giving rise to domain 2 and after this the two-domain gene duplicated to produce domains 3 and 4. The high O(2) affinity of Branchipolynoe extracellular globins may be explained by the two key residues (B10Y and E7Q) in the heme pocket in each of the domains of the single and tetra-domain globins, which have been shown to be essential in the oxygen-avid Hb from the nematode Ascaris suum. This peculiar globin evolutionary path seems to be very different from other annelid extracellular globins and is most likely the product of evolutionary tinkering associated with the strong selective pressure to adapt to chronic hypoxia that characterizes hydrothermal vents.