Evolution of the Spider Homeobox Gene Repertoire by Tandem and Whole Genome Duplication.

Gene duplication generates new genetic material that can contribute to the evolution of gene regulatory networks and phenotypes. Duplicated genes can undergo subfunctionalization to partition ancestral functions and/or neofunctionalization to assume a new function. We previously found there had been a whole genome duplication (WGD) in an ancestor of arachnopulmonates, the lineage including spiders and scorpions but excluding other arachnids like mites, ticks, and harvestmen. This WGD was evidenced by many duplicated homeobox genes, including two Hox clusters, in spiders. However, it was unclear which homeobox paralogues originated by WGD versus smaller-scale events such as tandem duplications. Understanding this is a key to determining the contribution of the WGD to arachnopulmonate genome evolution. Here we characterized the distribution of duplicated homeobox genes across eight chromosome-level spider genomes. We found that most duplicated homeobox genes in spiders are consistent with an origin by WGD. We also found two copies of conserved homeobox gene clusters, including the Hox, NK, HRO, Irx, and SINE clusters, in all eight species. Consistently, we observed one copy of each cluster was degenerated in terms of gene content and organization while the other remained more intact. Focussing on the NK cluster, we found evidence for regulatory subfunctionalization between the duplicated NK genes in the spider Parasteatoda tepidariorum compared to their single-copy orthologues in the harvestman Phalangium opilio. Our study provides new insights into the relative contributions of multiple modes of duplication to the homeobox gene repertoire during the evolution of spiders and the function of NK genes.

Single-cell atlases of two lophotrochozoan larvae highlight their complex evolutionary histories.

Pelagic larval stages are widespread across animals, yet it is unclear whether larvae were present in the last common ancestor of animals or whether they evolved multiple times due to common selective pressures. Many marine larvae are at least superficially similar; they are small, swim through the beating of bands of cilia, and sense the environment with an apical organ. To understand these similarities, we have generated single-cell atlases for marine larvae from two animal phyla and have compared their cell types. We found clear similarities among ciliary band cells and between neurons of the apical organ in the two larvae pointing to possible homology of these structures, suggesting a single origin of larvae within Spiralia. We also find several clade-specific innovations in each larva, including distinct myocytes and shell gland cells in the oyster larva. Oyster shell gland cells express many recently evolved genes that have made previous gene age estimates for the origin of trochophore larvae too young.

FGF signalling is involved in cumulus migration in the common house spider Parasteatoda tepidariorum.

Cell migration is a fundamental component during the development of most multicellular organisms. In the early spider embryo, the collective migration of signalling cells, known as the cumulus, is required to set the dorsoventral body axis. Here, we show that FGF signalling plays an important role during cumulus migration in the spider Parasteatoda tepidariorum. Spider embryos with reduced FGF signalling show reduced or absent cumulus migration and display dorsoventral patterning defects. Our study reveals that the transcription factor Ets4 regulates the expression of several FGF signalling components in the cumulus. In conjunction with a previous study, we show that the expression of fgf8 in the germ-disc is regulated via the Hedgehog signalling pathway. We also demonstrate that FGF signalling influences the BMP signalling pathway activity in the region around cumulus cells. Finally, we show that FGFR signalling might also influence cumulus migration in basally branching spiders and we propose that fgf8 might act as a chemo-attractant to guide cumulus cells towards the future dorsal pole of the spider embryo.

Genome Assembly of the Polyclad Flatworm Prostheceraeus crozieri.

Polyclad flatworms are widely thought to be one of the least derived of the flatworm classes and, as such, are well placed to investigate evolutionary and developmental features such as spiral cleavage and larval diversification lost in other platyhelminths. Prostheceraeus crozieri, (formerly Maritigrella crozieri), is an emerging model polyclad flatworm that already has some useful transcriptome data but, to date, no sequenced genome. We have used high molecular weight DNA extraction and long-read PacBio sequencing to assemble the highly repetitive (67.9%) P. crozieri genome (2.07 Gb). We have annotated 43,325 genes, with 89.7% BUSCO completeness. Perhaps reflecting its large genome, introns were considerably larger than other free-living flatworms, but evidence of abundant transposable elements suggests genome expansion has been principally via transposable elements activity. This genome resource will be of great use for future developmental and phylogenomic research.

Lack of support for Deuterostomia prompts reinterpretation of the first Bilateria.

The bilaterally symmetric animals (Bilateria) are considered to comprise two monophyletic groups, Protostomia (Ecdysozoa and the Lophotrochozoa) and Deuterostomia (Chordata and the Xenambulacraria). Recent molecular phylogenetic studies have not consistently supported deuterostome monophyly. Here, we compare support for Protostomia and Deuterostomia using multiple, independent phylogenomic datasets. As expected, Protostomia is always strongly supported, especially by longer and higher-quality genes. Support for Deuterostomia, however, is always equivocal and barely higher than support for paraphyletic alternatives. Conditions that cause tree reconstruction errors-inadequate models, short internal branches, faster evolving genes, and unequal branch lengths-coincide with support for monophyletic deuterostomes. Simulation experiments show that support for Deuterostomia could be explained by systematic error. The branch between bilaterian and deuterostome common ancestors is, at best, very short, supporting the idea that the bilaterian ancestor may have been deuterostome-like. Our findings have important implications for the understanding of early animal evolution.

Duplication and expression of Sox genes in spiders.

BACKGROUND: The Sox family of transcription factors is an important part of the genetic 'toolbox' of all metazoans examined to date and is known to play important developmental roles in vertebrates and insects. However, outside the commonly studied Drosophila model little is known about the repertoire of Sox family transcription factors in other arthropod species. Here we characterise the Sox family in two chelicerate species, the spiders Parasteatoda tepidariorum and Stegodyphus mimosarum, which have experienced a whole genome duplication (WGD) in their evolutionary history. RESULTS: We find that virtually all of the duplicate Sox genes have been retained in these spiders after the WGD. Analysis of the expression of Sox genes in P. tepidariorum embryos suggests that it is likely that some of these genes have neofunctionalised after duplication. Our expression analysis also strengthens the view that an orthologue of vertebrate Group B1 genes, SoxNeuro, is implicated in the earliest events of CNS specification in both vertebrates and invertebrates. In addition, a gene in the Dichaete/Sox21b class is dynamically expressed in the spider segment addition zone, suggestive of an ancient regulatory mechanism controlling arthropod segmentation as recently suggested for flies and beetles. Together with the recent analysis of Sox gene expression in the embryos of other arthropods, our findings support the idea of conserved functions for some of these genes, including a potential role for SoxC and SoxD genes in CNS development and SoxF in limb development. CONCLUSIONS: Our study provides a new chelicerate perspective to understanding the evolution and function of Sox genes and how the retention of duplicates of such important tool-box genes after WGD has contributed to different aspects of spider embryogenesis. Future characterisation of the function of these genes in spiders will help us to better understand the evolution of the regulation of important developmental processes in arthropods and other metazoans including neurogenesis and segmentation.

A SoxB gene acts as an anterior gap gene and regulates posterior segment addition in a spider.

Sox genes encode a set of highly conserved transcription factors that regulate many developmental processes. In insects, the SoxB gene Dichaete is the only Sox gene known to be involved in segmentation. To determine if similar mechanisms are used in other arthropods, we investigated the role of Sox genes during segmentation in the spider Parasteatoda tepidariorum. While Dichaete does not appear to be involved in spider segmentation, we found that the closely related Sox21b-1 gene acts as a gap gene during formation of anterior segments and is also part of the segmentation clock for development of the segment addition zone and sequential addition of opisthosomal segments. Thus, we have found that two different mechanisms of segmentation in a non-mandibulate arthropod are regulated by a SoxB gene. Our work provides new insights into the function of an important and conserved gene family, and the evolution of the regulation of segmentation in arthropods.

Homeobox Gene Duplication and Divergence in Arachnids.

Homeobox genes are key toolkit genes that regulate the development of metazoans and changes in their regulation and copy number have contributed to the evolution of phenotypic diversity. We recently identified a whole genome duplication (WGD) event that occurred in an ancestor of spiders and scorpions (Arachnopulmonata), and that many homeobox genes, including two Hox clusters, appear to have been retained in arachnopulmonates. To better understand the consequences of this ancient WGD and the evolution of arachnid homeobox genes, we have characterized and compared the homeobox repertoires in a range of arachnids. We found that many families and clusters of these genes are duplicated in all studied arachnopulmonates (Parasteatoda tepidariorum, Pholcus phalangioides, Centruroides sculpturatus, and Mesobuthus martensii) compared with nonarachnopulmonate arachnids (Phalangium opilio, Neobisium carcinoides, Hesperochernes sp., and Ixodes scapularis). To assess divergence in the roles of homeobox ohnologs, we analyzed the expression of P. tepidariorum homeobox genes during embryogenesis and found pervasive changes in the level and timing of their expression. Furthermore, we compared the spatial expression of a subset of P. tepidariorum ohnologs with their single copy orthologs in P. opilio embryos. We found evidence for likely subfunctionlization and neofunctionalization of these genes in the spider. Overall our results show a high level of retention of homeobox genes in spiders and scorpions post-WGD, which is likely to have made a major contribution to their developmental evolution and diversification through pervasive subfunctionlization and neofunctionalization, and paralleling the outcomes of WGD in vertebrates.

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BACKGROUND: The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. RESULTS: We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication. CONCLUSIONS: Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.

Arthropod evolution and development: recent insights from chelicerates and myriapods.

Research on arthropod genetics and development has added much to our understanding of animal evolution. While this work has mainly focused on insects, a growing body of research on the less studied myriapods and chelicerates is providing important new insights into arthropod genomics and development. Multiple chelicerate lineages have a high incidence of gene duplication, which is suggestive of large-scale and even whole genome duplications. Furthermore, the duplication and divergence of genes is associated with the evolution of appendage morphology and other phenotypes in chelicerates and myriapods. Recent studies of these arthropods have also helped to understand the evolution and development of segmented bodies. Further research on chelicerate and myriapod models as well as species from other orders of these subphyla has great potential to expand our understanding of the evolution of animal genomes and development.

The Wnt and Delta-Notch signalling pathways interact to direct pair-rule gene expression via caudal during segment addition in the spider Parasteatoda tepidariorum.

In short-germ arthropods, posterior segments are added sequentially from a segment addition zone (SAZ) during embryogenesis. Studies in spiders such as Parasteatoda tepidariorum have provided insights into the gene regulatory network (GRN) underlying segment addition, and revealed that Wnt8 is required for dynamic Delta (Dl) expression associated with the formation of new segments. However, it remains unclear how these pathways interact during SAZ formation and segment addition. Here, we show that Delta-Notch signalling is required for Wnt8 expression in posterior SAZ cells, but represses the expression of this Wnt gene in anterior SAZ cells. We also found that these two signalling pathways are required for the expression of the spider orthologues of even-skipped (eve) and runt-1 (run-1), at least in part via caudal (cad). Moreover, it appears that dynamic expression of eve in this spider does not require a feedback loop with run-1, as is found in the pair-rule circuit of the beetle Tribolium Taken together, our results suggest that the development of posterior segments in Parasteatoda is directed by dynamic interactions between Wnt8 and Delta-Notch signalling that are read out by cad, which is necessary but probably not sufficient to regulate the expression of eve and run-1 Our study therefore provides new insights towards better understanding the evolution and developmental regulation of segmentation in other arthropods, including insects.

Pervasive microRNA Duplication in Chelicerates: Insights from the Embryonic microRNA Repertoire of the Spider Parasteatoda tepidariorum.

MicroRNAs are small (∼22 nt) noncoding RNAs that repress translation and therefore regulate the production of proteins from specific target mRNAs. microRNAs have been found to function in diverse aspects of gene regulation within animal development and many other processes. Among invertebrates, both conserved and novel, lineage specific, microRNAs have been extensively studied predominantly in holometabolous insects such as Drosophila melanogaster However little is known about microRNA repertoires in other arthropod lineages such as the chelicerates. To understand the evolution of microRNAs in this poorly sampled subphylum, we characterized the microRNA repertoire expressed during embryogenesis of the common house spider Parasteatoda tepidariorum We identified a total of 148 microRNAs in P. tepidariorum representing 66 families. Approximately half of these microRNA families are conserved in other metazoans, while the remainder are specific to this spider. Of the 35 conserved microRNAs families 15 had at least two copies in the P. tepidariorum genome. A BLAST-based approach revealed a similar pattern of duplication in other spiders and a scorpion, but not among other chelicerates and arthropods, with the exception of a horseshoe crab. Among the duplicated microRNAs we found examples of lineage-specific tandem duplications, and the duplication of entire microRNA clusters in three spiders, a scorpion, and in a horseshoe crab. Furthermore, we found that paralogs of many P. tepidariorum microRNA families exhibit arm switching, which suggests that duplication was often followed by sub- or neofunctionalization. Our work shows that understanding the evolution of microRNAs in the chelicerates has great potential to provide insights into the process of microRNA duplication and divergence and the evolution of animal development.

Sexual dimorphism and natural variation within and among species in the Drosophila retinal mosaic.

BACKGROUND: Insect compound eyes are composed of ommatidia, which contain photoreceptor cells that are sensitive to different wavelengths of light defined by the specific rhodopsin proteins that they express. The fruit fly Drosophila melanogaster has several different ommatidium types that can be localised to specific retinal regions, such as the dorsal rim area (DRA), or distributed stochastically in a mosaic across the retina, like the 'pale' and 'yellow' types. Variation in these ommatidia patterns very likely has important implications for the vision of insects and could underlie behavioural and environmental adaptations. However, despite the detailed understanding of ommatidia specification in D. melanogaster, the extent to which the frequency and distribution of the different ommatidium types vary between sexes, strains and species of Drosophila is not known. RESULTS: We investigated the frequency and distribution of ommatidium types based on rhodopsin protein expression, and the expression levels of rhodopsin transcripts in the eyes of both sexes of different strains of D. melanogaster, D. simulans and D. mauritiana. We found that while the number of DRA ommatidia was invariant, Rh3 expressing ommatidia were more frequent in the larger eyes of females compared to the males of all species analysed. The frequency and distribution of ommatidium types also differed between strains and species. The D. simulans strain ZOM4 has the highest frequency of Rh3 expressing ommatidia, which is associated with a non-stochastic patch of pale and odd-coupled ommatidia in the dorsal-posterior of their eyes. CONCLUSIONS: Our results show that there is striking variation in the frequency and distribution of ommatidium types between sexes, strains and species of Drosophila. This suggests that evolutionary changes in the underlying regulatory mechanisms can alter the distribution of ommatidium types to promote or restrict their expression in specific regions of the eye within and between species, and that this could cause differences in vision among these flies.