Impacts of Sex Ratio Meiotic Drive on Genome Structure and Function in a Stalk-Eyed Fly.

Stalk-eyed flies in the genus Teleopsis carry selfish genetic elements that induce sex ratio (SR) meiotic drive and impact the fitness of male and female carriers. Here, we assemble and describe a chromosome-level genome assembly of the stalk-eyed fly, Teleopsis dalmanni, to elucidate patterns of divergence associated with SR. The genome contains tens of thousands of transposable element (TE) insertions and hundreds of transcriptionally and insertionally active TE families. By resequencing pools of SR and ST males using short and long reads, we find widespread differentiation and divergence between XSR and XST associated with multiple nested inversions involving most of the SR haplotype. Examination of genomic coverage and gene expression data revealed seven X-linked genes with elevated expression and coverage in SR males. The most extreme and likely drive candidate involves an XSR-specific expansion of an array of partial copies of JASPer, a gene necessary for maintenance of euchromatin and associated with regulation of TE expression. In addition, we find evidence for rapid protein evolution between XSR and XST for testis expressed and novel genes, that is, either recent duplicates or lacking a Dipteran ortholog, including an X-linked duplicate of maelstrom, which is also involved in TE silencing. Overall, the evidence suggests that this ancient XSR polymorphism has had a variety of impacts on repetitive DNA and its regulation in this species.

Allelic resolution of insect and spider silk genes reveals hidden genetic diversity.

Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation-both in terms of length and repeat motif order-between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 Mya: the insect clade containing caddisflies and butterflies and spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests common mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation.

Relating spidroin motif prevalence and periodicity to the mechanical properties of major ampullate spider silks.

Spider dragline fibers exhibit incredible mechanical properties, outperforming many synthetic polymers in toughness assays, and possess desirable properties for medical and other human applications. These qualities make dragline fibers popular subjects for biomimetics research. The enormous diversity of spiders presents both an opportunity for the development of new bioinspired materials and a challenge for the identification of fundamental design principles, as the mechanical properties of dragline fibers show both intraspecific and interspecific variations. In this regard, the stress-strain curves of draglines from different species have been shown to be effectively compared by the α* parameter, a value derived from maximum-supercontracted silk fibers. To identify potential molecular mechanisms impacting α* values, here we analyze spider fibroin (spidroin) sequences of the Western black widow (Latrodectus hesperus) and the black and yellow garden spider (Argiope aurantia). This study serves as a primer for investigating the molecular properties of spidroins that underlie species-specific α* values. Initial findings are that while overall motif composition was similar between species, certain motifs and higher level periodicities of glycine-rich region lengths showed variation, notably greater distances between poly-A motifs in A. aurantia sequences. In addition to increased period lengths, A. aurantia spidroins tended to have an increased prevalence of charged and hydrophobic residues. These increases may impact the number and strength of hydrogen bond networks within fibers, which have been implicated in conformational changes and formation of nanocrystals, contributing to the greater extensibility of A. aurantia draglines compared to those of L. hesperus.

Differences in the Elastomeric Behavior of Polyglycine-Rich Regions of Spidroin 1 and 2 Proteins.

Two different polyglycine-rich fragments were selected as representatives of major ampullate gland spidroins (MaSp) 1 and 2 types, and their behavior in a water-saturated environment was simulated within the framework of molecular dynamics (MD). The selected fragments are found in the sequences of the proteins MaSp1a and MaSp2.2a of Argiope aurantia with respective lengths of 36 amino acids (MaSp1a) and 50 amino acids (MaSp2.2s). The simulation took the fully extended ß-pleated conformation as reference, and MD was used to determine the equilibrium configuration in the absence of external forces. Subsequently, MD were employed to calculate the variation in the distance between the ends of the fragments when subjected to an increasing force. Both fragments show an elastomeric behavior that can be modeled as a freely jointed chain with links of comparable length, and a larger number of links in the spidroin 2 fragment. It is found, however, that the maximum recovery force recorded from the spidroin 2 peptide (Fmax ≈ 400 pN) is found to be significantly larger than that of the spidroin 1 (Fmax ≈ 250 pN). The increase in the recovery force of the spidroin 2 polyglycine-rich fragment may be correlated with the larger values observed in the strain at breaking of major ampullate silk fibers spun by Araneoidea species, which contain spidroin 2 proteins, compared to the material produced by spider species that lack these spidroins (RTA-clade).

Rapid molecular diversification and homogenization of clustered major ampullate silk genes in Argiope garden spiders.

The evolutionary diversification of orb-web weaving spiders is closely tied to the mechanical performance of dragline silk. This proteinaceous fiber provides the primary structural framework of orb web architecture, and its extraordinary toughness allows these structures to absorb the high energy of aerial prey impact. The dominant model of dragline silk molecular structure involves the combined function of two highly repetitive, spider-specific, silk genes (spidroins)-MaSp1 and MaSp2. Recent genomic studies, however, have suggested this framework is overly simplistic, and our understanding of how MaSp genes evolve is limited. Here we present a comprehensive analysis of MaSp structural and evolutionary diversity across species of Argiope (garden spiders). This genomic analysis reveals the largest catalog of MaSp genes found in any spider, driven largely by an expansion of MaSp2 genes. The rapid diversification of Argiope MaSp genes, located primarily in a single genomic cluster, is associated with profound changes in silk gene structure. MaSp2 genes, in particular, have evolved complex hierarchically organized repeat units (ensemble repeats) delineated by novel introns that exhibit remarkable evolutionary dynamics. These repetitive introns have arisen independently within the genus, are highly homogenized within a gene, but diverge rapidly between genes. In some cases, these iterated introns are organized in an alternating structure in which every other intron is nearly identical in sequence. We hypothesize that this intron structure has evolved to facilitate homogenization of the coding sequence. We also find evidence of intergenic gene conversion and identify a more diverse array of stereotypical amino acid repeats than previously recognized. Overall, the extreme diversification found among MaSp genes requires changes in the structure-function model of dragline silk performance that focuses on the differential use and interaction among various MaSp paralogs as well as the impact of ensemble repeat structure and different amino acid motifs on mechanical behavior.

The evolutionary history of cribellate orb-weaver capture thread spidroins.

BACKGROUND: Spiders have evolved two types of sticky capture threads: one with wet adhesive spun by ecribellate orb-weavers and another with dry adhesive spun by cribellate spiders. The evolutionary history of cribellate capture threads is especially poorly understood. Here, we use genomic approaches to catalog the spider-specific silk gene family (spidroins) for the cribellate orb-weaver Uloborus diversus. RESULTS: We show that the cribellar spidroin, which forms the puffy fibrils of cribellate threads, has three distinct repeat units, one of which is conserved across cribellate taxa separated by ~ 250 Mya. We also propose candidates for a new silk type, paracribellar spidroins, which connect the puffy fibrils to pseudoflagelliform support lines. Moreover, we describe the complete repeat architecture for the pseudoflagelliform spidroin (Pflag), which contributes to extensibility of pseudoflagelliform axial fibers. CONCLUSIONS: Our finding that Pflag is closely related to Flag, supports homology of the support lines of cribellate and ecribellate capture threads. It further suggests an evolutionary phase following gene duplication, in which both Flag and Pflag were incorporated into the axial lines, with subsequent loss of Flag in uloborids, and increase in expression of Flag in ecribellate orb-weavers, explaining the distinct mechanical properties of the axial lines of these two groups.

Characterization of the genome and silk-gland transcriptomes of Darwin's bark spider (Caerostris darwini).

Natural silks crafted by spiders comprise some of the most versatile materials known. Artificial silks-based on the sequences of their natural brethren-replicate some desirable biophysical properties and are increasingly utilized in commercial and medical applications today. To characterize the repertoire of protein sequences giving silks their biophysical properties and to determine the set of expressed genes across each unique silk gland contributing to the formation of natural silks, we report here draft genomic and transcriptomic assemblies of Darwin's bark spider, Caerostris darwini, an orb-weaving spider whose dragline is one of the toughest known biomaterials on Earth. We identify at least 31 putative spidroin genes, with expansion of multiple spidroin gene classes relative to the golden orb-weaver, Trichonephila clavipes. We observed substantial sharing of spidroin repetitive sequence motifs between species as well as new motifs unique to C. darwini. Comparative gene expression analyses across six silk gland isolates in females plus a composite isolate of all silk glands in males demonstrated gland and sex-specific expression of spidroins, facilitating putative assignment of novel spidroin genes to classes. Broad expression of spidroins across silk gland types suggests that silks emanating from a given gland represent composite materials to a greater extent than previously appreciated. We hypothesize that the extraordinary toughness of C. darwini major ampullate dragline silk may relate to the unique protein composition of major ampullate spidroins, combined with the relatively high expression of stretchy flagelliform spidroins whose union into a single fiber may be aided by novel motifs and cassettes that act as molecule-binding helices. Our assemblies extend the catalog of sequences and sets of expressed genes that confer the unique biophysical properties observed in natural silks.

Connecting materials, performance and evolution: a case study of the glue of moth-catching spiders (Cyrtarachninae).

Morphological structures and extended phenotypes are made possible by materials that are encoded by the genome. Nearly all biomaterials are viscoelastic, which means that to understand performance, one must understand the strain rate-dependent properties of these materials in relevant ecological interactions, as the behavior of a material can vary dramatically and rapidly. Spider silks are an example of materials whose properties vary substantially intra- and inter-specifically. Here, we focus on aggregate silk, which functions as a biological adhesive. As a case study to understand how a material manifests from genome through organism to ecology, we highlight moth-specialist spiders, the Cyrtarachninae, and their glues as an ideal experimental system to investigate the relationship between genomics and ecologically variable performance of a biological material. There is a clear eco-evolutionary innovation that Cyrtarachne akirai and related species have evolved, a unique trait not found in other spiders, a glue which overcomes the scales of moths. By examining traditional orb-weavers, C. akirai and other subfamily members using biomechanical testing and genomic analysis, we argue that we can track the evolution of this novel bioadhesive and comment on the selection pressures influencing prey specialization. The importance of the ecological context of materials testing is exemplified by the poor performance of C. akirai glue on glass and the exceptional spreading ability and adhesive strength on moths. The genetic basis for these performance properties is experimentally tractable because spider silk genes are minimally pleiotropic and advances in genomic technologies now make possible the discovery of complete silk gene sequences.

Expression of spidroin proteins in the silk glands of golden orb-weaver spiders.

The expression of spidroins in the major ampullate, minor ampullate, flagelliform, and tubuliform silk glands of Trichonephila clavipes spiders was analyzed using proteomics analysis techniques. Spidroin peptides were identified and assigned to different gene products based on sequence concurrence when compared with the whole genome of the spider. It was found that only a relatively low proportion of the spidroin genes are expressed as proteins in any of the studied glands. In addition, the expression of spidroin genes in different glands presents a wide range of patterns, with some spidroins being found in a single gland exclusively, while others appear in the content of several glands. The combination of precise genomics, proteomics, microstructural, and mechanical data provides new insights both on the design principles of these materials and how these principles might be translated for the production of high-performance bioinspired artificial fibers.

Gene expression profiling reveals candidate genes for defining spider silk gland types.

Molecular studies of the secretory glands involved in spider silk production have revealed candidate genes for silk synthesis and a complicated history of spider silk gene evolution. However, differential gene expression profiles of the multiple silk gland types within an individual orb-web weaving spider are lacking. Each of these gland types produces a functionally distinct silk type. Comparison of gene expression among spider silk gland types would provide insight into the genes that define silk glands generally from non-silk gland tissues, and the genes that define silk glands from each other. Here, we perform 3' tag digital gene expression profiling of the seven silk gland types of the silver garden orb weaver Argiope argentata. Five of these gland types produce silks that are non-adhesive fibers, one silk includes both fibers and glue-like adhesives, and one silk is exclusively glue-like. We identify 1275 highly expressed, significantly upregulated, and tissue specific silk gland specific transcripts (SSTs). These SSTs include seven types of spider silk protein encoding genes known as spidroin genes. We find that the fiber-producing major ampullate and minor ampullate silk glands have more similar expression profiles than any other pair of glands. We also find that a subset of the SSTs is enriched for transmembrane transport and oxidoreductases, and that these transcripts highlight differences and similarities among the major ampullate, minor ampullate, and aggregate silk glands. Furthermore, we show that the wet glue-producing aggregate glands have the most unique SSTs, but still share some SSTs with fiber producing glands. Aciniform glands were the only gland type to share a majority of SSTs with other silk gland types, supporting previous hypotheses that duplication of aciniform glands and subsequent divergence of the duplicates gave rise to the multiple silk gland types within an individual spider.

Protein Composition and Associated Material Properties of Cobweb Spiders' Gumfoot Glue Droplets.

The origin of aggregate silk glands and their production of wet adhesive silks is considered a key innovation of the Araneoidea, a superfamily of spiders that build orb-webs and cobwebs. Orb-web weavers place aggregate glue on an extensible capture spiral, whereas cobweb weavers add it to the ends of strong, stiff fibers, called gumfoot lines. Here we describe the material behavior and quantitative proteomics of the aggregate glues of two cobweb weaving species, the western black widow, Latrodectus hesperus, and the common house spider, Parasteatoda tepidariorum. For each species, respectively, we identified 48 and 33 proteins that were significantly more abundant in the portion of the gumfoot line with glue than in its fibers. These proteins were more highly glycosylated and phosphorylated than proteins found in silk fibers without glue, which likely explains aggregate glue stickiness. Most glue-enriched proteins were of anterior aggregate gland origin, supporting the hypothesis that cobweb weavers' posterior aggregate glue is specialized for another function. We found that cobweb weaver glue droplets are stiffer and tougher than the adhesive of most orb-web weaving species. Attributes of gumfoot glue protein composition that likely contribute to this stiffness include the presence of multiple protein families with conserved cysteine residues, a bimodal distribution of isoelectric points, and families with conserved functions in protein aggregation, all of which should contribute to cohesive protein-protein interactions. House spider aggregate droplets were more responsive to humidity changes than black widow droplets, which could be mediated by differences in protein sequence, post-translational modifications, the non-protein components of the glue droplets, and/or the larger amount of aqueous material that surrounds the adhesive cores of their glue droplets.

Golden orb-weaving spider (Trichonephila clavipes) silk genes with sex-biased expression and atypical architectures.

Spider silks are renowned for their high-performance mechanical properties. Contributing to these properties are proteins encoded by the spidroin (spider fibroin) gene family. Spidroins have been discovered mostly through cDNA studies of females based on the presence of conserved terminal regions and a repetitive central region. Recently, genome sequencing of the golden orb-web weaver, Trichonephila clavipes, provided a complete picture of spidroin diversity. Here, we refine the annotation of T. clavipes spidroin genes including the reclassification of some as non-spidroins. We rename these non-spidroins as spidroin-like (SpL) genes because they have repetitive sequences and amino acid compositions like spidroins, but entirely lack the archetypal terminal domains of spidroins. Insight into the function of these spidroin and SpL genes was then examined through tissue- and sex-specific gene expression studies. Using qPCR, we show that some silk genes are upregulated in male silk glands compared to females, despite males producing less silk in general. We also find that an enigmatic spidroin that lacks a spidroin C-terminal domain is highly expressed in silk glands, suggesting that spidroins could assemble into fibers without a canonical terminal region. Further, we show that two SpL genes are expressed in silk glands, with one gene highly evolutionarily conserved across species, providing evidence that particular SpL genes are important to silk production. Together, these findings challenge long-standing paradigms regarding the evolutionary and functional significance of the proteins and conserved motifs essential for producing spider silks.

Ultrastructures and Mechanics of Annealed Nephila clavipes Major Ampullate Silk.

The semicrystalline protein structure and impressive mechanical properties of major ampullate (MA) spider silk make it a promising natural alternative to polyacrylonitrile (PAN) fibers for carbon fiber manufacture. However, when annealed using a similar procedure to carbon fiber production, the tensile strength and Young's modulus of MA silk decrease. Despite this, MA silk fibers annealed at 600 °C remain stronger and tougher than similarly annealed PAN but have a lower Young's modulus. Although MA silk and PAN graphitize to similar extents, annealing disrupts the hydrogen bonding that controls crystal alignment within MA silk. Consequently, unaligned graphite crystals form in annealed MA silk, causing it to weaken, while graphite crystals in PAN maintain alignment along the fiber axis, strengthening the fibers. These shortcomings of spider silk when annealed provide insights into the selection and design of future alternative carbon fiber precursors.

Phylogeny of the orb-weaving spider family Araneidae (Araneae: Araneoidea).

We present a new phylogeny of the spider family Araneidae based on five genes (28S, 18S, COI, H3 and 16S) for 158 taxa, identified and mainly sequenced by us. This includes 25 outgroups and 133 araneid ingroups representing the subfamilies Zygiellinae Simon, 1929, Nephilinae Simon, 1894, and the typical araneids, here informally named the "ARA Clade". The araneid genera analysed here include roughly 90% of all currently named araneid species. The ARA Clade is the primary focus of this analysis. In taxonomic terms, outgroups comprise 22 genera and 11 families, and the ingroup comprises three Zygiellinae and four Nephilinae genera, and 85 ARA Clade genera (ten new). Within the ARA Clade, we recognize ten informal groups that contain at least three genera each and are supported under Bayesian posterior probabilities (≥ 0.95): "Caerostrines" (Caerostris, Gnolus and Testudinaria), "Micrathenines" (Acacesia, Micrathena, Ocrepeira, Scoloderus and Verrucosa), "Eriophorines" (Acanthepeira, Alpaida, Eriophora, Parawixia and Wagneriana), "Backobourkiines" (Acroaspis, Backobourkia, Carepalxis, Novakiella, Parawixia, Plebs, Singa and three new genera), "Argiopines" (Arachnura, Acusilas, Argiope, Cyrtophora, Gea, Lariniaria and Mecynogea), "Cyrtarachnines" (Aranoethra, Cyrtarachne, Paraplectana, Pasilobus and Poecilopachys), "Mastophorines" (Celaenia, Exechocentrus and Mastophora,), "Nuctenines" (Larinia, Larinioides and Nuctenea), "Zealaraneines" (Colaranea, Cryptaranea, Paralarinia, Zealaranea and two new genera) and "Gasteracanthines" (Augusta, Acrosomoides, Austracantha, Gasteracantha, Isoxya, Macracantha, Madacantha, Parmatergus and Thelacantha). Few of these groups are currently corroborated by morphology, behaviour, natural history or biogeography. We also include the large genus Araneus, along with Aculepeira, Agalenatea, Anepsion, Araniella, Cercidia, Chorizopes, Cyclosa, Dolophones, Eriovixia, Eustala, Gibbaranea, Hingstepeira, Hypognatha, Kaira, Larinia, Mangora, Metazygia, Metepeira, Neoscona, Paraplectanoides, Perilla, Poltys, Pycnacantha, Spilasma and Telaprocera, but the placement of these genera was generally ambiguous, except for Paraplectanoides, which is strongly supported as sister to traditional Nephilinae. Araneus, Argiope, Eriophora and Larinia are polyphyletic, Araneus implying nine new taxa of genus rank, and Eriophora and Larinia two each. In Araneus and Eriophora, polyphyly was usually due to north temperate generic concepts being used as dumping grounds for species from southern hemisphere regions, e.g. South-East Asia, Australia or New Zealand. Although Araneidae is one of the better studied spider families, too little natural history and/or morphological data are available across these terminals to draw any strong evolutionary conclusions. However, the classical orb web is reconstructed as plesiomorphic for Araneidae, with a single loss in "cyrtarachnines"-"mastophorines". Web decorations (collectively known as stabilimenta) evolved perhaps five times. Sexual dimorphism generally results from female body size increase with few exceptions; dimorphic taxa are not monophyletic and revert to monomorphism in a few cases.

Spidroins and Silk Fibers of Aquatic Spiders.

Spiders are commonly found in terrestrial environments and many rely heavily on their silks for fitness related tasks such as reproduction and dispersal. Although rare, a few species occupy aquatic or semi-aquatic habitats and for them, silk-related specializations are also essential to survive in aquatic environments. Most spider silks studied to date are from cob-web and orb-web weaving species, leaving the silks from many other terrestrial spiders as well as water-associated spiders largely undescribed. Here, we characterize silks from three Dictynoidea species: the aquatic spiders Argyroneta aquatica and Desis marina as well as the terrestrial Badumna longinqua. From silk gland RNA-Seq libraries, we report a total of 47 different homologs of the spidroin (spider fibroin) gene family. Some of these 47 spidroins correspond to known spidroin types (aciniform, ampullate, cribellar, pyriform, and tubuliform), while other spidroins represent novel branches of the spidroin gene family. We also report a hydrophobic amino acid motif (GV) that, to date, is found only in the spidroins of aquatic and semi-aquatic spiders. Comparison of spider silk sequences to the silks from other water-associated arthropods, shows that there is a diversity of strategies to function in aquatic environments.

Sex-biased expression between guppies varying in the presence of ornamental coloration.

Sex-biased gene expression provides a means to achieve sexual dimorphism across a genome largely shared by both sexes. Trinidadian guppies are ideal to examine questions of sex-bias as they exhibit sexual dimorphism in ornamental coloration with male only expression. Here we use RNA-sequencing to quantify whole transcriptome gene expression differences, with a focus on differential expression of color genes between the sexes. We determine whether males express genes positively correlated with coloration at higher levels than females. We find that all the differentially expressed color genes were more highly expressed by males. Males also expressed all known black melanin synthesis genes at higher levels than females, regardless of whether the gene was significantly differentially expressed in the analysis. These differences correlated with the visual color differences between sexes at the stage sampled, as all males had ornamental black coloration apparent. We propose that sexual dimorphism in ornamental coloration is caused by male-biased expression of color genes.

Convergent evolution of coloration in experimental introductions of the guppy (Poecilia reticulata).

Despite the multitude of examples of evolution in action, relatively fewer studies have taken a replicated approach to understand the repeatability of evolution. Here, we examine the convergent evolution of adaptive coloration in experimental introductions of guppies from a high-predation (HP) environment into four low-predation (LP) environments. LP introductions were replicated across 2 years and in two different forest canopy cover types. We take a complementary approach by examining both phenotypes and genetics. For phenotypes, we categorize the whole color pattern on the tail fin of male guppies and analyze evolution using a correspondence analysis. We find that coloration in the introduction sites diverged from the founding Guanapo HP site. Sites group together based on canopy cover, indicating convergence in response to light environment. However, the axis that explains the most variation indicates a lack of convergence. Therefore, evolution may proceed along similar phenotypic trajectories, but still maintain unique variation within sites. For the genetics underlying the divergent phenotypes, we examine expression levels of color genes. We find no evidence for differential expression, indicating that the genetic basis for the color changes remains undetermined.

Silk genes and silk gene expression in the spider Tengella perfuga (Zoropsidae), including a potential cribellar spidroin (CrSp).

Most spiders spin multiple types of silk, including silks for reproduction, prey capture, and draglines. Spiders are a megadiverse group and the majority of spider silks remain uncharacterized. For example, nothing is known about the silk molecules of Tengella perfuga, a spider that spins sheet webs lined with cribellar silk. Cribellar silk is a type of adhesive capture thread composed of numerous fibrils that originate from a specialized plate-like spinning organ called the cribellum. The predominant components of spider silks are spidroins, members of a protein family synthesized in silk glands. Here, we use silk gland RNA-Seq and cDNA libraries to infer T. perfuga silks at the protein level. We show that T. perfuga spiders express 13 silk transcripts representing at least five categories of spider silk proteins (spidroins). One category is a candidate for cribellar silk and is thus named cribellar spidroin (CrSp). Studies of ontogenetic changes in web construction and spigot morphology in T. perfuga have documented that after sexual maturation, T. perfuga females continue to make capture webs but males halt web maintenance and cease spinning cribellar silk. Consistent with these observations, our candidate CrSp was expressed only in females. The other four spidroin categories correspond to paralogs of aciniform, ampullate, pyriform, and tubuliform spidroins. These spidroins are associated with egg sac and web construction. Except for the tubuliform spidroin, the spidroins from T. perfuga contain novel combinations of amino acid sequence motifs that have not been observed before in these spidroin types. Characterization of T. perfuga silk genes, particularly CrSp, expand the diversity of the spidroin family and inspire new structure/function hypotheses.

The developmental and genetic trajectory of coloration in the guppy (Poecilia reticulata).

Examining the association between trait variation and development is crucial for understanding the evolution of phenotypic differences. Male guppy ornamental caudal fin coloration is one trait that shows a striking degree of variation within and between guppy populations. Males initially have no caudal fin coloration, then gradually develop it as they reach sexual maturity. For males, there is a trade-off between female preference for caudal fin coloration and increased visibility to predators. This trade-off may reach unique endpoints in males from different predation regimes. Caudal fin coloration includes black melanin, orange/yellow pteridines or carotenoids, and shimmering iridescence. This study examined the phenotypic trajectory and genetics associated with color development. We found that black coloration always developed first, followed by orange/yellow, then iridescence. The ordering and timing of color appearance was the same regardless of predation regime. The increased expression of melanin synthesis genes correlated well with the visual appearance of black coloration, but there was no correlation between carotenoids or pteridine synthesis gene expression and the appearance of orange/yellow. The lack of orange/yellow coloration in earlier male caudal fin developmental stages may be due to reduced expression of genes underlying the development of orange/yellow xanthophores.

Microstructure and mechanical properties of different keratinous horns.

Animal horns play an important role during intraspecific combat. This work investigates the microstructure and mechanical properties of horns from four representative ruminant species: the bighorn sheep (Ovis canadensis), domestic sheep (Ovis aries), mountain goat (Oreamnos americanus) and pronghorn (Antilocapra americana), aiming to understand the relation between evolved microstructures and mechanical properties. Microstructural similarity is found where disc-shaped keratin cells attach edge-to-edge along the growth direction of the horn core (longitudinal direction) forming a lamella; multiple lamellae are layered face to face along the impact direction (radial direction, perpendicular to horn core growth direction), forming a wavy pattern surrounding a common feature, the tubules. Differences among species include the number and shape of the tubules, the orientation of aligned lamellae and the shape of keratin cells. Water absorption tests reveal that the pronghorn horn has the largest water-absorbing ability due to the presence of nanopores in the keratin cells. The loading direction (compressive and tensile) and level of hydration vary among the horns from different species. The differences in mechanical properties among species may relate to their different fighting behaviours: high stiffness and strength in mountain goat to support the forces during stabbing; high tensile strength in pronghorn for interlocked pulling; impact energy absorption properties in domestic and bighorn sheep to protect the skull during butting. These design rules based on evolutionary modifications among species can be applied in synthetic materials to meet different mechanical requirements.