Long-Read Sequencing Reveals Rapid Evolution of Immunity- and Cancer-Related Genes in Bats.

Bats are exceptional among mammals for their powered flight, extended lifespans, and robust immune systems and therefore have been of particular interest in comparative genomics. Using the Oxford Nanopore Technologies long-read platform, we sequenced the genomes of two bat species with key phylogenetic positions, the Jamaican fruit bat (Artibeus jamaicensis) and the Mesoamerican mustached bat (Pteronotus mesoamericanus), and carried out a comprehensive comparative genomic analysis with a diverse collection of bats and other mammals. The high-quality, long-read genome assemblies revealed a contraction of interferon (IFN)-α at the immunity-related type I IFN locus in bats, resulting in a shift in relative IFN-ω and IFN-α copy numbers. Contradicting previous hypotheses of constitutive expression of IFN-α being a feature of the bat immune system, three bat species lost all IFN-α genes. This shift to IFN-ω could contribute to the increased viral tolerance that has made bats a common reservoir for viruses that can be transmitted to humans. Antiviral genes stimulated by type I IFNs also showed evidence of rapid evolution, including a lineage-specific duplication of IFN-induced transmembrane genes and positive selection in IFIT2. In addition, 33 tumor suppressors and 6 DNA-repair genes showed signs of positive selection, perhaps contributing to increased longevity and reduced cancer rates in bats. The robust immune systems of bats rely on both bat-wide and lineage-specific evolution in the immune gene repertoire, suggesting diverse immune strategies. Our study provides new genomic resources for bats and sheds new light on the extraordinary molecular evolution in this critically important group of mammals.

Assemblies of the genomes of parasitic wasps using meta-assembly and scaffolding with genetic linkage.

Safe, effective biological-control introductions against invasive pests depend on narrowly host-specific natural enemies with the ability to adapt to a changing environment. As part of a project on the genetic architectures of these traits, we assembled and annotated the genomes of two aphid parasitoids, Aphelinus atriplicis and Aphelinus certus. We report here several assemblies of A. atriplicis made with Illumina and PacBio data, which we combined into a meta-assembly. We scaffolded the meta-assembly with markers from a genetic map of hybrids between A. atriplicis and A. certus. We used this genetic-linkage scaffolded (GLS) assembly of A. atriplicis to scaffold a de novo assembly of A. certus. The de novo assemblies of A. atriplicis differed in contiguity, and the meta-assembly of these assemblies was more contiguous than the best de novo assembly. Scaffolding with genetic-linkage data allowed chromosomal-level assembly of the A. atriplicis genome and scaffolding a de novo assembly of A. certus with this GLS assembly, greatly increased the contiguity of the A. certus assembly to the point where it was also at the chromosomal-level. However, completeness of the A. atriplicis assembly, as measured by percent complete, single-copy BUSCO hymenopteran genes, varied little among de novo assemblies and was not increased by meta-assembly or genetic scaffolding. Furthermore, the greater contiguity of the meta-assembly and GLS assembly had little or no effect on the numbers of genes identified, the proportions with homologs or functional annotations. Increased contiguity of the A. certus assembly provided modest improvement in assembly completeness, as measured by percent complete, single-copy BUSCO hymenopteran genes. The total genic sequence increased, and while the number of genes declined, gene length increased, which together suggest greater accuracy of gene models. More contiguous assemblies provide uses other than gene annotation, for example, identifying the genes associated with quantitative trait loci and understanding of chromosomal rearrangements associated with speciation.

Whole mitochondrial genome phylogeny of Drosophilidae.

A total of 241 mitochondrial genomes were assembled and annotated from the SRA database to reconstruct a mtDNA genome phylogeny for the genus Drosophila, the family Drosophilidae, and close relatives. The resulting mtDNA genome phylogeny is largely congruent with previous higher-level analyses of Drosophila species with the exception of the relationships between the melanogaster, montium, anannassae, saltans and obscura groups. Although relationships within these species groups are congruent between nuclear and mtDNA studies, the mtDNA genome phylogeny of the groups is different when compared to earlier studies. Monophyly of known species groups within the genus Drosophila are highly supported and, as in previous work, the genera Lordiphosa, Hirtodrosophila, Zaprionus and Scaptomya are all imbedded within the genus Drosophila. Incongruence and partitioned support analyses indicate that DNA sequences are better at resolving the phylogeny than their translated protein sequences. Such analyses also indicate that genes on the minus strand of the circular molecule (Lrrna, Srrna, ND4, ND4L and ND5) provide most of the support for the overall phylogenetic hypothesis.

Genome assembly of a Mesoamerican derived variety of lima bean: a foundational cultivar in the Mid-Atlantic USA.

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble, and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa, and a resource for the identification of potential resistance genes. Combined efforts of sequencing, linkage, and comparative analysis resulted in a 623 Mb annotated assembly for lima bean, as well as a better understanding of an evolutionarily dynamic resistance locus in legumes.

Whole Genome Sequencing and Assembly of the Asian Honey Bee Apis dorsata.

The Asian honey bee (Apis dorsata) is distinct from its more widely distributed cousin Apis mellifera by a few key characteristics. Most prominently, A. dorsata, nest in the open by forming a colony clustered around the honeycomb, whereas A. mellifera nest in concealed cavities. Additionally, the worker and reproductive castes are all of the same size in A. dorsata. In order to investigate these differences, we performed whole genome sequencing of A. dorsata using a hybrid Oxford Nanopore and Illumina approach. The 223 Mb genome has an N50 of 35 kb with the largest scaffold of 302 kb. We have found that there are many genes in the dorsata genome that are distinct from other hymenoptera and also large amounts of transposable elements, and we suggest some candidate genes for A. dorsata's exceptional level of defensive aggression.

Counties not countries: Variation in host specificity among populations of an aphid parasitoid.

Parasitic wasps are among the most species-rich groups on Earth. A major cause of this diversity may be local adaptation to host species. However, little is known about variation in host specificity among populations within parasitoid species. Not only is such knowledge important for understanding host-driven speciation, but because parasitoids often control pest insects and narrow host ranges are critical for the safety of biological control introductions, understanding variation in specificity and how it arises are crucial applications in evolutionary biology. Here, we report experiments on variation in host specificity among 16 populations of an aphid parasitoid, Aphelinus certus. We addressed several questions about local adaptation: Do parasitoid populations differ in host ranges or in levels of parasitism of aphid species within their host range? Are differences in parasitism among parasitoid populations related to geographical distance, suggesting clinal variation in abundances of aphid species? Or do nearby parasitoid populations differ in host use, as would be expected if differences in aphid abundances, and thus selection, were mosaic? Are differences in parasitism among parasitoid populations related to genetic distances among them? To answer these questions, we measured parasitism of a taxonomically diverse group of aphid species in laboratory experiments. Host range was the same for all the parasitoid populations, but levels of parasitism varied among aphid species, suggesting adaptation to locally abundant aphids. Differences in host specificity did not correlate with geographical distances among parasitoid populations, suggesting that local adaption is mosaic rather than clinal, with a spatial scale of less than 50 kilometers. We sequenced and assembled the genome of A. certus, made reduced-representation libraries for each population, analyzed for single nucleotide polymorphisms, and used these polymorphisms to estimate genetic differentiation among populations. Differences in host specificity correlated with genetic distances among the parasitoid populations.

Good Citizenship Made Easy: A Step-by-Step Guide to Submitting RNA-Seq Data to NCBI.

The analysis of transcriptome data from non-model organisms contributes to our understanding of diverse aspects of evolutionary biology, including developmental processes, speciation, adaptation, and extinction. Underlying this diversity is one shared feature, the generation of enormous amounts of sequence data. Data availability requirements in most journals oblige researchers to make their raw transcriptome data publicly available, and the databases housed at the National Center for Biotechnology Information (NCBI) are a popular choice for data deposition. Unfortunately, the successful submission of raw sequences to the Sequence Read Archive (SRA) and transcriptome assemblies to the Transcriptome Shotgun Assembly (TSA) can be challenging for novice users, significantly delaying data availability and publication. Here we present two comprehensive protocols for submitting RNA-Seq data to NCBI databases, accompanied by an easy-to-use website that facilitates the timely submission of data by researchers of any experience level. © 2018 by John Wiley & Sons, Inc.

Transcriptome profiling with focus on potential key genes for wing development and evolution in Megaloprepus caerulatus, the damselfly species with the world's largest wings.

The evolution, development and coloration of insect wings remains a puzzling subject in evolutionary research. In basal flying insects such as Odonata, genomic research regarding bauplan evolution is still rare. Here we focus on the world's largest odonate species-the "forest giant" Megaloprepus caerulatus, to explore its potential for looking deeper into the development and evolution of wings. A recently discovered cryptic species complex in this genus previously considered monotypic is characterized by morphological differences in wing shape and color patterns. As a first step toward understanding wing pattern divergence and pathways involved in adaptation and speciation at the genomic level, we present a transcriptome profiling of M. caerulatus using RNA-Seq and compare these data with two other odonate species. The de novo transcriptome assembly consists of 61,560 high quality transcripts and is approximately 93% complete. For almost 75% of the identified transcripts a possible function could be assigned: 48,104 transcripts had a hit to an InterPro protein family or domain, and 28,653 were mapped to a Gene Ontology term. In particular, we focused on genes related to wing development and coloration. The comparison with two other species revealed larva-specific genes and a conserved 'core' set of over 8,000 genes forming orthologous clusters with Ischnura elegans and Ladona fulva. This transcriptome may provide a first point of reference for future research in odonates addressing questions surrounding the evolution of wing development, wing coloration and their role in speciation.

De Novo characterization of transcriptomes from two North American Papaipema stem-borers (Lepidoptera: Noctuidae).

Stem-borers in the genus Papaipema (Lepidoptera: Noctuidae) range from highly polyphagous agricultural pests to specialists on more than 20 families of flowering plants, many of them highly toxic. Papaipema is the largest genus of noctuids endemic to North America and provides an excellent study system for the evolution of noctuid host plant use. To improve the availability of genomic resources for such investigations, we performed de novo transcriptome sequencing and assembly for two specialist Papaipema with unusual larval hosts: P. speciosissima, which is associated with ferns, and the undescribed P. "sp. 4," which is associated with bamboo. The resulting transcriptomes were similar in terms of completeness, gene count, and gene identity, but we identified some 8,000 genes (~17% of each transcriptome) not shared between the two species. While some of these have identifiable orthologs in other Lepidoptera, ~5% of each transcriptome consists of species-specific genes. We examine the function of these genes and find that almost half have retrotransposon-related functional domains. The potential role of species-specific genes is discussed, and the expansion of certain retrotransposon families in Papaipema is examined.

The genetic architecture of ecological adaptation: intraspecific variation in host plant use by the lepidopteran crop pest Chloridea virescens.

Intraspecific variation in ecologically important traits is a cornerstone of Darwin's theory of evolution by natural selection. The evolution and maintenance of this variation depends on genetic architecture, which in turn determines responses to natural selection. Some models suggest that traits with complex architectures are less likely to respond to selection than those with simple architectures, yet rapid divergence has been observed in such traits. The simultaneous evolutionary lability and genetic complexity of host plant use in the Lepidopteran subfamily Heliothinae suggest that architecture may not constrain ecological adaptation in this group. Here we investigate the response of Chloridea virescens, a generalist that feeds on diverse plant species, to selection for performance on a novel host, Physalis angulata (Solanaceae). P. angulata is the preferred host of Chloridea subflexa, a narrow specialist on the genus Physalis. In previous experiments, we found that the performance of C. subflexa on P. angulata depends on many loci of small effect distributed throughout the genome, but whether the same architecture would be involved in the generalist's adoption of P. angulata was unknown. Here we report a rapid response to selection in C. virescens for performance on P. angulata, and establish that the genetic architecture of intraspecific variation is quite similar to that of the interspecific differences in terms of the number, distribution, and effect sizes of the QTL involved. We discuss the impact of genetic architecture on the ability of Heliothine moths to respond to varying ecological selection pressures.

Genome content analysis yields new insights into the relationship between the human malaria parasite Plasmodium falciparum and its anopheline vectors.

BACKGROUND: The persistent and growing gap between the availability of sequenced genomes and the ability to assign functions to sequenced genes led us to explore ways to maximize the information content of automated annotation for studies of anopheline mosquitos. Specifically, we use genome content analysis of a large number of previously sequenced anopheline mosquitos to follow the loss and gain of protein families over the evolutionary history of this group. The importance of this endeavor lies in the potential for comparative genomic studies between Anopheles and closely related non-vector species to reveal ancestral genome content dynamics involved in vector competence. In addition, comparisons within Anopheles could identify genome content changes responsible for variation in the vectorial capacity of this family of important parasite vectors. RESULTS: The competence and capacity of P. falciparum vectors do not appear to be phylogenetically constrained within the Anophelinae. Instead, using ancestral reconstruction methods, we suggest that a previously unexamined component of vector biology, anopheline nucleotide metabolism, may contribute to the unique status of anophelines as P. falciparum vectors. While the fitness effects of nucleotide co-option by P. falciparum parasites on their anopheline hosts are not yet known, our results suggest that anopheline genome content may be responding to selection pressure from P. falciparum. Whether this response is defensive, in an attempt to redress improper nucleotide balance resulting from P. falciparum infection, or perhaps symbiotic, resulting from an as-yet-unknown mutualism between anophelines and P. falciparum, is an open question that deserves further study. CONCLUSIONS: Clearly, there is a wealth of functional information to be gained from detailed manual genome annotation, yet the rapid increase in the number of available sequences means that most researchers will not have the time or resources to manually annotate all the sequence data they generate. We believe that efforts to maximize the amount of information obtained from automated annotation can help address the functional annotation deficit that most evolutionary biologists now face, and here demonstrate the value of such an approach.

A whole genome gene content phylogenetic analysis of anopheline mosquitoes.

Construction of stringent gene content matrices was accomplished for 21 Anopheline mosquito species and strains and four outgroups species. The presence absence matrix using e-75 as a cutoff in single linkage clustering had over 17,000 ortholog groups. We used the gene content matrix to generate a phylogenetic hypothesis that is in general agreement with gene sequence based phylogenies. In addition to establishing a congruent gene content phylogeny we examined the consistency of three methods for analyzing presence absence data - unweighted parsimony, dollo parsimonly and maximum likelihood using a BINGAMMA model. An examination of the chromosomal location of the gains and losses in the presence absence matrix revealed a low frequency of gains and losses at centromeres and tips of chromosomes.

We can't all be supermodels: the value of comparative transcriptomics to the study of non-model insects.

Insects are the most diverse group of organisms on the planet. Variation in gene expression lies at the heart of this biodiversity and recent advances in sequencing technology have spawned a revolution in researchers' ability to survey tissue-specific transcriptional complexity across a wide range of insect taxa. Increasingly, studies are using a comparative approach (across species, sexes and life stages) that examines the transcriptional basis of phenotypic diversity within an evolutionary context. In the present review, we summarize much of this research, focusing in particular on three critical aspects of insect biology: morphological development and plasticity; physiological response to the environment; and sexual dimorphism. A common feature that is emerging from these investigations concerns the dynamic nature of transcriptome evolution as indicated by rapid changes in the overall pattern of gene expression, the differential expression of numerous genes with unknown function, and the incorporation of novel, lineage-specific genes into the transcriptional profile.

The genetic architecture of a complex ecological trait: host plant use in the specialist moth, Heliothis subflexa.

We used genetic mapping to examine the genetic architecture of differences in host plant use between two species of noctuid moths, Heliothis subflexa, a specialist on Physalis spp., and its close relative, the broad generalist H. virescens. We introgressed H. subflexa chromosomes into the H. virescens background and analyzed 1462 backcross insects. The effects of H. subflexa-origin chromosomes were small when measured as the percent variation explained in backcross populations (0.2-5%), but were larger when considered in relation to the interspecific difference explained (1.5-165%). Most significant chromosomes had effects on more than one trait, and their effects varied between years, sexes, and genetic backgrounds. Different chromosomes could produce similar phenotypes, suggesting that the same trait might be controlled by different chromosomes in different backcross populations. It appears that many loci of small effect contribute to the use of Physalis by H. subflexa. We hypothesize that behavioral changes may have paved the way for physiological adaptation to Physalis by the generalist ancestor of H. subflexa and H. virescens.

Behavioral adaptations increase the value of enemy-free space for Heliothis subflexa, a specialist herbivore.

We investigated the importance of specialized behaviors in the use of enemy-free space by comparing the host-use behavior of two closely related moths, Heliothis subflexa Guenee and H. virescens Fabricius. Heliothis subflexa is a specialist on plants in the genus Physalis, whereas H. virescens is an extreme generalist, feeding on plants in at least 14 families. Heliothis subflexa uses the inflated calyx surrounding Physalis fruits as enemy-free space, and field rates of parasitism for H. subflexa on Physalis are much lower than for H. virescens on tobacco and cotton, common hosts found in the same habitat as Physalis. If Physalis, architecture were solely responsible for H. subflexa's low rates of parasitism on Physalis, we predicted that H. virescens larvae experimentally induced to feed on Physalis would experience parasitism rates similar to those of H. subflexa. We found, however, that specialized host-use and host-acceptance behaviors are integral to the use of enemy-free space on Physalis and strongly augment the effects of the structural refuge. In laboratory assays, we found considerable differences between the larval behavior of the specialist. H. subflexa, and the generalist, H. virescens, and these contributed to H. subflexa's superior use of enemy-free space on Physalis. We tested the importance of these behavioral differences in the field by comparing parasitism of H. virescens on Physalis, H. virescens on tobacco, and H. subflexa on Physalis by Cardiochiles nigriceps Vierick, a specialist braconid parasitoid. For H. virescens, a threefold decrease in parasitism occurred when feeding on Physalis (mean parasitism +/- SEM = 13 +/- 4%) rather than tobacco (43 +/- 4%), a difference we attribute to the structural refuge provided by Physalis. However, parasitism of H. virescens on Physalis was more than ten times as great as that of H. subflexa on Pliv.salis (1 +/- 4%), supporting the hypothesis that specialized behaviors have a substantial impact on use of Physalis as enemy-free space. Behavioral adaptations may be central to the use of enemy-free space by phytophagous insects and may act as an important selective force in the evolution of dietary specialization.