Dr Saad Arif

Current rates of habitat degradation and climate change are causing unprecedented declines in global biodiversity. Studies on vertebrates highlight how conservation genomics can be effective in identifying and managing threatened populations, but it is unclear how vertebrate-derived metrics of genomic erosion translate to invertebrates, with their markedly different population sizes and life histories. The Black-veined White butterfly (Aporia crataegi) was extirpated from Britain in the 1920s. Here, we sequenced historical DNA from 17 specimens collected between 1854 and 1924 to reconstruct demography and compare levels of genomic erosion between extirpated British and extant European mainland populations. We contrast these results using modern samples of the Common Blue butterfly (Polyommatus icarus); a species with relatively stable demographic trends in Great Britain. We provide evidence for bottlenecks in both these species around the period of post-glacial colonization of the British Isles. Our results reveal different demographic histories and Ne for both species, consistent with their fates in Britain, likely driven by differences in life history, ecology and genome size. Despite a difference, by an order of magnitude, in historical effective population sizes (Ne), reduction in genome-wide heterozygosity in A. crataegi was comparable to that in P. icarus. Symptomatic of A. crataegi's disappearance were marked increases in runs-of-homozygosity (RoH), potentially indicative of recent inbreeding, and accumulation of putatively mildly and weakly deleterious variants. Our results provide a rare glimpse of genomic erosion in a regionally extinct insect and support the potential use of genomic erosion metrics in identifying invertebrate populations or species in decline.

We present a genome assembly from an individual male Aporia crataegi (the black-veined white; Arthropoda; Insecta; Lepidoptera; Pieridae). The genome sequence is 230 megabases in span. The complete assembly is scaffolded into 26 chromosomal pseudomolecules, with the Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 10,860 protein coding genes.

Whole-genome duplications (WGDs) have occurred multiple times during animal evolution, including in lineages leading to vertebrates, teleosts, horseshoe crabs, and arachnopulmonates. These dramatic events initially produce a wealth of new genetic material, generally followed by extensive gene loss. It appears, however, that developmental genes such as homeobox genes, signaling pathway components and microRNAs are frequently retained as duplicates (so-called ohnologs) following WGD. These not only provide the best evidence for WGD, but an opportunity to study its evolutionary consequences. Although these genes are well studied in the context of vertebrate WGD, similar comparisons across the extant arachnopulmonate orders are patchy. We sequenced embryonic transcriptomes from two spider species and two amblypygid species and surveyed three important gene families, Hox, Wnt, and frizzled, across these and 12 existing transcriptomic and genomic resources for chelicerates. We report extensive retention of putative ohnologs, further supporting the ancestral arachnopulmonate WGD. We also found evidence of consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of the six arachnopulmonate orders, with interorder variation in the retention of specific paralogs. We identified variation between major clades in spiders and are better able to reconstruct the chronology of gene duplications and losses in spiders, amblypygids, and scorpions. These insights shed light on the evolution of the developmental toolkit in arachnopulmonates, highlight the importance of the comparative approach within lineages, and provide substantial new transcriptomic data for future study.

The paradigm of isolation in southern refugia during glacial periods followed by expansions during interglacials, producing limited genetic differentiation in northern areas, dominates European phylogeography. However, the existence of complex structured populations in formerly glaciated areas, and islands connected to mainland areas during glacial maxima, call for alternative explanations. We reconstructed the mtDNA phylogeography of the widespread Polyommatus icarus butterfly with an emphasis on the formerly glaciated and connected British Isles. We found distinct geographical structuring of CO1 haplogroups, with an ancient lineage restricted to the marginal European areas, including Northern Scotland and Outer Hebrides. Population genomic analyses, using ddRADSeq genomic markers, also reveal substantial genetic structuring within Britain. However, there is negligble mito-nuclear concordance consistent with independent demographic histories of mitochondrial vs. nuclear DNA. While mtDNA-Wolbachia associations in northern Britain could account for the geographic structuring of mtDNA across most of the British Isles, for nuclear DNA markers (derived from ddRADseq data) butterflies from France cluster between northern and southern British populations – an observation consistent with a scenario of multiple recolonisation. Taken together our results suggest that contemporary mtDNA structuring in the British Isles (and potentially elsewhere in Europe) largely results from Wolbachia infections, however, nuclear genomic structuring suggests a history of at least two distinct colonisations. This two-stage colonisation scenario has previously been put forth to explain genetic diversity and structuring in other British flora and fauna. Additionally, we also present preliminary evidence for potential Wolbachia-induced feminization in the Outer Hebrides.

The Sox family of transcription factors regulate many processes during metazoan development, including stem cell maintenance and nervous system specification. Characterising the repertoires and roles of these genes can therefore provide important insights into animal evolution and development. We further characterised the Sox repertoires of several arachnid species with and without an ancestral whole genome duplication (WGD), and compared their expression between the spider Parasteatoda tepidariorum and the harvestman Phalangium opilio. We also found that most Sox families have been retained as ohnologs after WGD and evidence for potential subfunctionalisation and/or neofunctionalization events. Our results also suggest that Sox21b-1 likely regulated segmentation ancestrally in arachnids, playing a similar role to the closely related SoxB gene, Dichaete, in insects. We previously showed that Sox21b-1 is required for the simultaneous formation of prosomal segments and sequential addition of opisthosomal segments in P. tepidariorum. We studied the expression and function of Sox21b-1 further in this spider and found that while this gene regulates the generation of both prosomal and opisthosomal segments, it plays different roles in the formation of these tagmata reflecting their contrasting modes of segmentation and deployment of gene regulatory networks with different architectures.

In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, we have limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. Here, we used new D. simulans/D. mauritiana introgression lines on chromosome 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. We then carried out RNA-seq on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed us to test the function of expressed positional candidates during genital development in D. melanogaster. We identified several new genes involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, we discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, our results provide new insights into the regulation of genital development and how this has evolved between species.

The compound eyes of insects exhibit striking variation in size, reflecting adaptation to different lifestyles and habitats. However, the genetic and developmental bases of variation in insect eye size is poorly understood, which limits our understanding of how these important morphological differences evolve. To address this, we further explored natural variation in eye size within and between four species of the Drosophila melanogaster species subgroup. We found extensive variation in eye size among these species, and flies with larger eyes generally had a shorter inter-ocular distance and vice versa. We then carried out quantitative trait loci (QTL) mapping of intra-specific variation in eye size and inter-ocular distance in both D. melanogaster and D. simulans. This revealed that different genomic regions underlie variation in eye size and inter-ocular distance in both species, which we corroborated by introgression mapping in D. simulans. This suggests that although there is a trade-off between eye size and inter-ocular distance, variation in these two traits is likely to be caused by different genes and so can be genetically decoupled. Finally, although we detected QTL for intra-specific variation in eye size at similar positions in D. melanogaster and D. simulans, we observed differences in eye fate commitment between strains of these two species. This indicates that different developmental mechanisms and therefore, most likely, different genes contribute to eye size variation in these species. Taken together with the results of previous studies, our findings suggest that the gene regulatory network that specifies eye size has evolved at multiple genetic nodes to give rise to natural variation in this trait within and among species.

Ectothermic species such as insects are particularly vulnerable to climatic fluctuations. Nevertheless, many insects that evolved and diversified in the tropics have successfully colonized temperate regions all over the globe. To shed light on the genetic basis of cold tolerance in such species, we conducted a quantitative trait locus (QTL) mapping experiment for chill coma recovery time (CCRT) in Drosophila ananassae, a cosmopolitan species that has expanded its range from tropical to temperate regions. We created a mapping population of recombinant inbred advanced intercross lines (RIAILs) from two founder strains with diverging CCRT phenotypes. The RIAILs were phenotyped for their CCRT and, together with the founder strains, genotyped for polymorphic markers with double-digest restriction site-associated DNA (ddRAD) sequencing. Using a hierarchical mapping approach that combined standard interval mapping and a multiple-QTL model, we mapped three QTL which altogether explained 64% of the phenotypic variance. For two of the identified QTL, we found evidence of epistasis. To narrow down the list of cold tolerance candidate genes, we cross-referenced the QTL intervals with genes that we previously identified as differentially expressed in response to cold in D. ananassae, and with thermotolerance candidate genes of D. melanogaster. Among the 58 differentially expressed genes that were contained within the QTL, GF15058 showed a significant interaction of the CCRT phenotype and gene expression. Further, we identified the orthologs of four D. melanogaster thermotolerance candidate genes, MtnA, klarsicht, CG5246 (D.ana/GF17132) and CG10383 (D.ana/GF14829) as candidates for cold tolerance in D. ananassae.

Microtrichia or trichomes are non-sensory actin protrusions produced by the epidermal cells of many insects. Studies of trichome formation in Drosophila have over the last 30 years provided key insights towards our understanding of gene regulation, gene regulatory networks (GRNs), development, the genotype to phenotype map, and the evolution of these processes. Here we review classic studies that have used trichome formation as a model to shed light on Drosophila development as well as recent research on the architecture of the GRN underlying trichome formation. This includes the findings that both small peptides and microRNAs play important roles in the regulation and evolution of this network. In addition, we review research on the evolution of trichome patterns that has provided novel insights into the function and architecture of cis-regulatory modules, and into the genetic basis of morphological change. We conclude that further research on these apparently simple and often functionally enigmatic structures will continue to provide new and important knowledge about development and evolution.

Identifying the genetic mechanisms underlying phenotypic change is essential to understanding how gene regulatory networks and ultimately the genotype-to-phenotype map evolve. It is recognized that microRNAs (miRNAs) have the potential to facilitate evolutionary change [1, 2and3]; however, there are no known examples of natural morphological variation caused by evolutionary changes in miRNA expression. Therefore, the contribution of miRNAs to evolutionary change remains unknown [1and4]. Drosophila melanogaster subgroup species display a portion of trichome-free cuticle on the femur of the second leg called the "naked valley." It was previously shown that Ultrabithorax (Ubx) is involved in naked valley variation between D.melanogaster and D.simulans [ 5and6]. However, naked valley size also varies among populations of D.melanogaster, ranging from 1,000 up to 30,000μm2. We investigated the genetic basis of intraspecific differences in the naked valley in D.melanogaster and found that neither Ubx nor shavenbaby (svb) [ 7and8] contributes to this morphological difference. Instead, we show that changes in mir-92a expression underlie the evolution of naked valley size in D.melanogaster through repression of shavenoid (sha) [9]. Therefore, our results reveala novel mechanism for morphological evolution and suggest that modulation of the expression of miRNAs potentially plays a prominent role in generating organismal diversity.

Eye and head morphology vary considerably among insects and even between closely related species of Drosophila. Species of the D. melanogaster subgroup, and other Drosophila species, exhibit a negative correlation between eye size and face width (FW); for example, D. mauritiana generally has bigger eyes composed of larger ommatidia and conversely a narrower face than its sibling species. To better understand the evolution of eye and head morphology, we investigated the genetic and developmental basis of differences in eye size and FW between male D. mauritiana and D. simulans. QTL mapping of eye size and FW showed that the major loci responsible for the interspecific variation in these traits are localized to different genomic regions. Introgression of the largest effect QTL underlying the difference in eye size resulted in flies with larger eyes but no significant difference in FW. Moreover, introgression of a QTL region on the third chromosome that contributes to the FW difference between these species affected FW, but not eye size. We also observed that this difference in FW is detectable earlier in the development of the eye-antennal disc than the difference in the size of the retinal field. Our results suggest that different loci that act at different developmental stages underlie changes in eye size and FW. Therefore, while there is a negative correlation between these traits in Drosophila, we show genetically that they also have the potential to evolve independently and this may help to explain the evolution of these traits in other insects.

A striking diversity of compound eye size and shape has evolved among insects. The number of ommatidia and their size are major determinants of the visual sensitivity and acuity of the compound eye. Each ommatidium is composed of eight photoreceptor cells that facilitate the discrimination of different colours via the expression of various light sensitive Rhodopsin proteins. It follows that variation in eye size, shape, and opsin composition is likely to directly influence vision. We analyzed variation in these three traits in D. melanogaster, D. simulans and D. mauritiana. We show that D. mauritiana generally has larger eyes than its sibling species, which is due to a combination of larger ommatidia and more ommatidia. In addition, intra- and inter-specific differences in eye size among D. simulans and D. melanogaster strains are mainly caused by variation in ommatidia number. By applying a geometric morphometrics approach to assess whether the formation of larger eyes influences other parts of the head capsule, we found that an increase in eye size is associated with a reduction in the adjacent face cuticle. Our shape analysis also demonstrates that D. mauritiana eyes are specifically enlarged in the dorsal region. Intriguingly, this dorsal enlargement is associated with enhanced expression of rhodopsin 3 in D. mauritiana. In summary, our data suggests that the morphology and functional properties of the compound eyes vary considerably within and among these closely related Drosophila species and may be part of coordinated morphological changes affecting the head capsule.

Among the various types of evolutionary changes in morphology, the origin of novel structures may be the most rare and intriguing. Here we show statistically that the origins of different novel structures may be correlated and phylogenetically clustered into "hot spots" of evolutionary novelty, in a case study involving skull elements in treefrogs. We reconstruct phylogenetic relationships within a clade of Middle American treefrogs based on data from 10 nuclear and four mitochondrial genes and then analyze morphological evolution across this tree. New cranial elements are rare among anurans and tetrapods in general, but three novel elements have evolved within this clade, with a 40% increase in the number of skull roof elements in some species. Two of these elements also evolved in a related clade of treefrogs, and these two novel elements may have each evolved repeatedly within one or both clades. The molecular phylogeny suggests striking homoplasy in cranial morphology and shows that parsimony and Bayesian analyses of the morphological data have produced misleading results with strong statistical support. The origins of the novel elements are associated with an overall increase in the ossification of dermal skull roof elements (suggesting peramorphosis) and with the evolution of a novel adaptive behavior. Our study may be the first to statistically document significant phylogenetic clustering and correlation in the origins of novel structures, and to demonstrate the strongly misleading effects of peramorphosis on phylogenetic analysis.