Evolution of the Insect PPK Gene Family.

Insect pickpocket (PPK) receptors mediate diverse functions, among them the detection of mechano- and chemo-sensory stimuli. Notwithstanding their relevance, studies on their evolution only focused on Drosophila. We have analyzed the genomes of 26 species of eight orders including holometabolous and hemimetabolous insects (Blattodea, Orthoptera, Hemiptera, Phthiraptera, Hymenoptera, Lepidoptera, Coleoptera, and Diptera), to characterize the evolution of this gene family. PPKs were detected in all genomes analyzed, with 578 genes distributed in seven subfamilies. According to our phylogeny, ppk17 is the most divergent member, composing the new subfamily VII. PPKs evolved under a gene birth-and-death model that generated lineage-specific expansions usually located in clusters, while purifying selection affected several orthogroups. Subfamily V was the largest, including a mosquito-specific expansion that can be considered a new target for pest control. PPKs present a high gene turnover generating considerable variation. On one hand, Musca domestica (59), Aedes albopictus (51), Culex quinquefasciatus (48), and Blattella germanica (41) presented the largest PPK repertoires. On the other hand, Pediculus humanus (only ppk17), bees, and ants (6-9) had the smallest PPK sets. A subset of prevalent PPKs was identified, indicating very conserved functions for these receptors. Finally, at least 20% of the sequences presented calmodulin-binding motifs, suggesting that these PPKs may amplify sensory responses similarly as proposed for Drosophila melanogaster ppk25. Overall, this work characterized the evolutionary history of these receptors revealing relevant unknown gene sequence features and clade-specific expansions.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Comparative analysis of tissue-specific transcriptomes in the funnel-web spider Macrothele calpeiana (Araneae, Hexathelidae).

The funnel-web spider Macrothele calpeiana is a charismatic Mygalomorph with a great interest in basic, applied and translational research. Nevertheless, current scarcity of genomic and transcriptomic data of this species clearly limits the research in this non-model organism. To overcome this limitation, we launched the first tissue-specific enriched RNA-seq analysis in this species using a subtractive hybridization approach, with two main objectives, to characterize the specific transcriptome of the putative chemosensory appendages (palps and first pair of legs), and to provide a new set of DNA markers for further phylogenetic studies. We have characterized the set of transcripts specifically expressed in putative chemosensory tissues of this species, much of them showing features shared by chemosensory system genes. Among specific candidates, we have identified some members of the iGluR and NPC2 families. Moreover, we have demonstrated the utility of these newly generated data as molecular markers by inferring the phylogenetic position M. calpeina in the phylogenetic tree of Mygalomorphs. Our results provide novel resources for researchers interested in spider molecular biology and systematics, which can help to expand our knowledge on the evolutionary processes underlying fundamental biological questions, as species invasion or biodiversity origin and maintenance.

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

In the wake of invasion: tracing the historical biogeography of the South American cricetid radiation (Rodentia, Sigmodontinae).

The Great American Biotic Interchange (GABI) was greatly influenced by the completion of the Isthmus of Panama and impacted the composition of modern faunal assemblages in the Americas. However, the contribution of preceding events has been comparatively less explored, even though early immigrants in the fossil records are evidence for waif dispersals. The cricetid rodents of the subfamily Sigmodontinae are a classic example of a species-rich South American radiation resulting from an early episode of North American invasion. Here, we provide a temporal and spatial framework to address key aspects of the historical biogeography and diversification of this diverse mammal group by using mitochondrial and nuclear DNA datasets coupled with methods of divergence time estimation, ancestral area reconstruction and comparative phylogenetics. Relaxed-clock time estimates indicate that divergence of the Sigmodontinae began in the middle-late Miocene (ca. 12-9 Ma). Dispersal-vicariance analyses point to the arrival of a single lineage of northern invaders with a widespread ancestral distribution and imply that the initial differentiation between Central and South America gave rise to the most basal groups within the subfamily. These two major clades diversified in the late Miocene followed by the radiation of main tribes until the early Pliocene. Within the Oryzomyalia, tribes diverged initially in eastern South America whereas multiple dispersals into the Andes promoted further diversification of the majority of modern genera. A comparatively uniform background tempo of diversification explains the species richness of sigmodontines across most nodes, except for two akodontine genera with recent increases in diversification rates. The bridging of the Central American seaway and episodes of low sea levels likely facilitated the invasion of South America long before the onset of the post-Isthmian phase of the GABI.

Family size evolution in Drosophila chemosensory gene families: a comparative analysis with a critical appraisal of methods.

Gene turnover rates and the evolution of gene family sizes are important aspects of genome evolution. Here, we use curated sequence data of the major chemosensory gene families from Drosophila-the gustatory receptor, odorant receptor, ionotropic receptor, and odorant-binding protein families-to conduct a comparative analysis among families, exploring different methods to estimate gene birth and death rates, including an ad hoc simulation study. Remarkably, we found that the state-of-the-art methods may produce very different rate estimates, which may lead to disparate conclusions regarding the evolution of chemosensory gene family sizes in Drosophila. Among biological factors, we found that a peculiarity of D. sechellia's gene turnover rates was a major source of bias in global estimates, whereas gene conversion had negligible effects for the families analyzed herein. Turnover rates vary considerably among families, subfamilies, and ortholog groups although all analyzed families were quite dynamic in terms of gene turnover. Computer simulations showed that the methods that use ortholog group information appear to be the most accurate for the Drosophila chemosensory families. Most importantly, these results reveal the potential of rate heterogeneity among lineages to severely bias some turnover rate estimation methods and the need of further evaluating the performance of these methods in a more diverse sampling of gene families and phylogenetic contexts. Using branch-specific codon substitution models, we find further evidence of positive selection in recently duplicated genes, which attests to a nonneutral aspect of the gene birth-and-death process.

Each flying fox on its own branch: a phylogenetic tree for Pteropus and related genera (Chiroptera: Pteropodidae).

Pteropodidae is a diverse Old World family of non-echolocating, frugivorous and nectarivorous bats that includes the flying foxes (genus Pteropus) and allied genera. The subfamily Pteropodinae includes the largest living bats and is distributed across an immense geographic range from islands in East Africa to the Cook Islands of Polynesia. These bats are keystone species in their ecosystems and some carry zoonotic diseases that are increasingly a focus of interest in biomedical research. Here we present a comprehensive phylogeny for pteropodines focused on Pteropus. The analyses included 50 of the ∼63 species of Pteropus and 11 species from 7 related genera. We obtained sequences of the cytochrome b and the 12S rRNA mitochondrial genes for all species and sequences of the nuclear RAG1, vWF, and BRCA1 genes for a subsample of taxa. Some of the sequences of Pteropus were obtained from skin biopsies of museum specimens including that of an extinct species, P. tokudae. The resulting trees recovered Pteropus as monophyletic, although further work is needed to determine whether P. personatus belongs in the genus. Monophyly of the majority of traditionally-recognized Pteropus species groups was rejected, but statistical support was strong for several clades on which we based a new classification of the Pteropus species into 13 species groups. Other noteworthy results emerged regarding species status of several problematic taxa, including recognition of P. capistratus and P. ennisae as distinct species, paraphyly of the P. hypomelanus complex, and conspecific status of P. pelewensis pelewensis and P. p. yapensis. Relationships among the pteropodine genera were not completely resolved with the current dataset. Divergence time analysis suggests that Pteropus originated in the Miocene and that two independent bursts of diversification occurred in the Pleistocene in different regions of the Indo-Pacific realm.

Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group.

We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With ca. 100 species endemic to the New World, the repleta species group represents one of the major species radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the group were also recovered, despite poorly resolved relationships between these clades. Divergence time estimates suggested that the repleta group split from its sister group about 21millionyears ago (Mya), although diversification of the crown group began ca. 16Mya. Character mapping of patterns of host plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host use from Opuntia to the more chemically complex columnar cacti occurred several times independently during the history of this group. Although some species retained the use of Opuntia after acquiring the use of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these dates are consistent with the suggested times when the Opuntioideae originated in South America. We discuss the generally accepted South American origin of the repleta group.

Evolutionary relationships of the old world fruit bats (Chiroptera, Pteropodidae): another star phylogeny?

BACKGROUND: The family Pteropodidae comprises bats commonly known as megabats or Old World fruit bats. Molecular phylogenetic studies of pteropodids have provided considerable insight into intrafamilial relationships, but these studies have included only a fraction of the extant diversity (a maximum of 26 out of the 46 currently recognized genera) and have failed to resolve deep relationships among internal clades. Here we readdress the systematics of pteropodids by applying a strategy to try to resolve ancient relationships within Pteropodidae, while providing further insight into subgroup membership, by 1) increasing the taxonomic sample to 42 genera; 2) increasing the number of characters (to >8,000 bp) and nuclear genomic representation; 3) minimizing missing data; 4) controlling for sequence bias; and 5) using appropriate data partitioning and models of sequence evolution. RESULTS: Our analyses recovered six principal clades and one additional independent lineage (consisting of a single genus) within Pteropodidae. Reciprocal monophyly of these groups was highly supported and generally congruent among the different methods and datasets used. Likewise, most relationships within these principal clades were well resolved and statistically supported. Relationships among the 7 principal groups, however, were poorly supported in all analyses. This result could not be explained by any detectable systematic bias in the data or incongruence among loci. The SOWH test confirmed that basal branches' lengths were not different from zero, which points to closely-spaced cladogenesis as the most likely explanation for the poor resolution of the deep pteropodid relationships. Simulations suggest that an increase in the amount of sequence data is likely to solve this problem. CONCLUSIONS: The phylogenetic hypothesis generated here provides a robust framework for a revised cladistic classification of Pteropodidae into subfamilies and tribes and will greatly contribute to the understanding of character evolution and biogeography of pteropodids. The inability of our data to resolve the deepest relationships of the major pteropodid lineages suggests an explosive diversification soon after origin of the crown pteropodids. Several characteristics of pteropodids are consistent with this conclusion, including high species diversity, great morphological diversity, and presence of key innovations in relation to their sister group.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses.

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

The phylogenetic relationships of cynopterine fruit bats (Chiroptera: Pteropodidae: Cynopterinae).

The subfamily Cynopterinae comprises ca. 24 species of pteropodid bats (Family Pteropodidae) distributed exclusively in South and Southeast Asia. Although some studies have supported monophyly of the subfamily, molecular analyses have produced contradictory results and there has been little agreement on relationships of cynopterines to other megabat groups. However, no previous studies have included a complete sampling of cynopterine genera. Here we describe a phylogenetic analysis of Cynopterinae based on more than 6000 bp from six different genes sampled in representatives of all 14 recognized genera. Our results support the monophyly of Cynopterinae but refute a close relationship of cynopterines with Nyctimeninae. Within Cynopterinae, our analyses consistently recovered two monophyletic clades, which we recommend be recognized formally as tribes: Cynopterini and Balionycterini. Biogeographic analyses indicate a Sundaland origin of the Cynopterinae and divergence date estimates suggest different timing of diversification of the two major cynopterine clades.

Orthology, function and evolution of accessory gland proteins in the Drosophila repleta group.

The accessory gland proteins (Acps) of Drosophila have become a model for the study of reproductive protein evolution. A major step in the study of Acps is to identify biological causes and consequences of the observed patterns of molecular evolution by comparing species groups with different biology. Here we characterize the Acp complement of Drosophila mayaguana, a repleta group representative. Species of this group show important differences in ecology and reproduction as compared to other Drosophila. Our results show that the extremely high rates of Acp evolution previously found are likely to be ubiquitous among species of the repleta group. These evolutionary rates are considerably higher than the ones observed in other Drosophila groups' Acps. This disparity, however, is not accompanied by major differences in the estimated number of Acps or in the functional categories represented as previously suggested. Among the genes expressed in accessory glands of D. mayaguana almost half are likely products of recent duplications. This allowed us to test predictions of the neofunctionalization model for gene duplication and paralog evolution in a more or less constrained timescale. We found that positive selection is a strong force in the early divergence of these gene pairs.

Evidence of adaptive evolution of accessory gland proteins in closely related species of the Drosophila repleta group.

Accessory gland proteins (Acps) are part of the seminal fluid of Drosophila species. These proteins have important reproductive functions, being responsible for the proper functioning of several steps of the fertilization process. Acps also contribute indirectly for the reproductive success of males by modulating female behavior. Evidence that Acps participate in sperm competition and sexual conflict includes findings that, on average, Acps have fast evolutionary rates, suggestive of adaptive evolution. This is especially true in species of the Drosophila repleta group. Nevertheless, only in a few occasions have robust statistical tests been used to determine whether observed evolutionary rates are in fact due to positive selection on amino acid substitutions between related species. Here we apply maximum likelihood tests for positive selection on 14 Acps of the D. repleta group. To increase statistical robustness, we use at least 8 sequences, all belonging to species of the Drosophila mulleri complex, for each gene analyzed. We found significant evidence of adaptive evolution for 10 of the tested genes. Among these, the ones with a conserved protein domain had positively selected sites within the functional region of the sequence. We also detected one instance of lineage-specific adaptive evolution in a clade formed by 2 sister species.

Examining ancient inter-domain horizontal gene transfer.

Details of the genomic changes that occurred in the ancestors of Eukarya, Archaea and Bacteria are elusive. Ancient interdomain horizontal gene transfer (IDHGT) amongst the ancestors of these three domains has been difficult to detect and analyze because of the extreme degree of divergence of genes in these three domains and because most evidence for such events are poorly supported. In addition, many researchers have suggested that the prevalence of IDHGT events early in the evolution of life would most likely obscure the patterns of divergence of major groups of organisms let alone allow the tracking of horizontal transfer at this level. In order to approach this problem, we mined the E. coli genome for genes with distinct paralogs. Using the 1,268 E. coli K-12 genes with 40% or higher similarity level to a paralog elsewhere in the E. coli genome we detected 95 genes found exclusively in Bacteria and Archaea and 86 genes found in Bacteria and Eukarya. These genes form the basis for our analysis of IDHGT. We also applied a newly developed statistical test (the node height test), to examine the robustness of these inferences and to corroborate the phylogenetically identified cases of ancient IDHGT. Our results suggest that ancient inter domain HGT is restricted to special cases, mostly involving symbiosis in eukaryotes and specific adaptations in prokaryotes. Only three genes in the Bacteria + Eukarya class (Deoxyxylulose-5-phosphate synthase (DXPS), fructose 1,6-phosphate aldolase class II protein and glucosamine-6-phosphate deaminase) and three genes-in the Bacteria + Archaea class (ABC-type FE3+-siderophore transport system, ferrous iron transport protein B, and dipeptide transport protein) showed evidence of ancient IDHGT. However, we conclude that robust estimates of IDHGT will be very difficult to obtain due to the methodological limitations and the extreme sequence saturation of the genes suspected of being involved in IDHGT.

Phylogeny and temporal diversification of Calomys (Rodentia, Sigmodontinae): implications for the biogeography of an endemic genus of the open/dry biomes of South America.

A thorough analysis of character evolution and biogeography of a group is only possible with a comprehensive sampling of its diversity. The sigmodontine genus Calomys is particularly interesting for the study of neotropical biogeography as it occurs exclusively in the dry and grassland biomes: Cerrado, Caatinga, Chaco, Pampas, Venezuelan Llanos, Puna, and a diversity of dry forests biomes. Although Brazil encompasses a large part of the geographic distribution of the genus and at least three endemic species, the last published phylogeny of Calomys included only two specimens (both representing the same species) from a single locality in this country. In the present paper we add complete cytochrome b sequences of Brazilian karyomorphs to previously published sequences in order to provide a phylogenetic hypothesis including most of the genus diversity. The main objectives of this study were to clarify taxonomic issues related to Brazilian karyomorphs and to study the diversification processes of the genus by analyzing its biogeography in combination with cladogenesis dates estimated with a molecular clock. The phylogeny indicates that at least six different species inhabit the Brazilian territory, one of them still undescribed. Date estimates indicate that two sequential basal splits, the first separating Andean from lowland species and the second isolating species north and south of the Amazonia, took place in the Pliocene between 3 and 4 Ma. A large-bodied and speciose clade of lowland species associated with dry forests and ecotones of the core Cerrado and Chaco areas with adjacent biomes diversified in the Pleistocene. This indicates the importance of safeguarding ecotones and the Cerrado at a time where this biome is being rapidly destroyed.

Population structure and genetic variability of mainland and insular populations of the Neotropical water rat, Nectomys squamipes (Rodentia, Sigmodontinae)

Seven microsatellite loci were used to investigate the genetic variability and structure of six mainland and two island populations of the Neotropical water rat Nectomys squamipes, a South American semi-aquatic rodent species with a wide distribution. High levels of variability were found within mainland populations while island populations were less variable but the more differentiated in respect to allele number and frequency. The time of biological divergence between mainland and island populations coincided with geological data. A significant geographic structure was found in mainland populations (q = 0.099; r = 0.086) although the degree of differentiation was relatively low in respect to the distance between surveyed localities (24 to 740 km). Genetic and geographic distances were not positively correlated as previously found with random amplified polymorphic DNA (RAPD) markers. Significant but low genetic differentiation in the mainland and lack of isolation by distance can be explained by large population size and/or recent population expansion. Additionally, the agreement between the age of geologic events (sea level fluctuations) and divergence times for insular populations points to a good reference for molecular clock calibration to associate recent environmental changes and the distribution pattern of small mammals in the Brazilian Atlantic Forest