A Veritable Menagerie of Heritable Bacteria from Ants, Butterflies, and Beyond: Broad Molecular Surveys and a Systematic Review

Maternally transmitted bacteria have been important players in the evolution of insects and other arthropods, affecting their nutrition, defense, development, and reproduction. Wolbachia are the best studied among these and typically the most prevalent. While several other bacteria have independently evolved a heritable lifestyle, less is known about their host ranges. Moreover, most groups of insects have not had their heritable microflora systematically surveyed across a broad range of their taxonomic diversity. To help remedy these shortcomings we used diagnostic PCR to screen for five groups of heritable symbionts—Arsenophonus spp., Cardinium hertigii, Hamiltonella defensa, Spiroplasma spp., and Wolbachia spp.—across the ants and lepidopterans (focusing, in the latter case, on two butterfly families—the Lycaenidae and Nymphalidae). We did not detect Cardinium or Hamiltonella in any host. Wolbachia were the most widespread, while Spiroplasma (ants and lepidopterans) and Arsenophonus (ants only) were present at low levels. Co-infections with different Wolbachia strains appeared especially common in ants and less so in lepidopterans. While no additional facultative heritable symbionts were found among ants using universal bacterial primers, microbes related to heritable enteric bacteria were detected in several hosts. In summary, our findings show that Wolbachia are the dominant heritable symbionts of ants and at least some lepidopterans. However, a systematic review of symbiont frequencies across host taxa revealed that this is not always the case across other arthropods. Furthermore, comparisons of symbiont frequencies revealed that the prevalence of Wolbachia and other heritable symbionts varies substantially across lower-level arthropod taxa. We discuss the correlates, potential causes, and implications of these patterns, providing hypotheses on host attributes that may shape the distributions of these influential bacteria.Organismic and Evolutionary Biolog

Phylogeny and palaeoecology of Polyommatus blue butterflies show Beringia was a climate-regulated gateway to the New World

Transcontinental dispersals by organisms usually represent improbable events that constitute a major challenge for biogeographers. By integrating molecular phylogeny, historical biogeography and palaeoecology, we test a bold hypothesis proposed by Vladimir Nabokov regarding the origin of Neotropical Polyommatus blue butterflies, and show that Beringia has served as a biological corridor for the dispersal of these insects from Asia into the New World. We present a novel method to estimate ancestral temperature tolerances using distribution range limits of extant organisms, and find that climatic conditions in Beringia acted as a decisive filter in determining which taxa crossed into the New World during five separate invasions over the past 11 Myr. Our results reveal a marked effect of the Miocene–Pleistocene global cooling, and demonstrate that palaeoclimatic conditions left a strong signal on the ecology of present-day taxa in the New World. The phylogenetic conservatism in thermal tolerances that we have identified may permit the reconstruction of the palaeoecology of ancestral organisms, especially mobile taxa that can easily escape from hostile environments rather than adapt to them

Evolution of Chemosensation in Herbivorous Drosophilidae

Plants and the insects that feed on them dominate diversity in terrestrial ecosystems: half of all named species are contained within these two groups. Herbivorous insects (herbivores) are abundant and diverse, yet paradoxically, two thirds of insect orders contain no major lineages of herbivores, implying barriers to the evolution of this trophic interaction. How herbivory evolves and why herbivores are so diverse are questions that are key to understanding the processes that have shaped global biodiversity. Yet, most lineages of herbivores are ancient with sister groups either absent or too divergent for a comparative genomic analysis to yield a mechanistic understanding of both their origin and diversification. Many of the exceptions to this pattern are among the Diptera, where lineages such as the leaf-mining drosophilids in the genus Scaptomyza have emerged within the last 10 million years. Scaptomyza is particularly well-suited for identifying the adaptations associated with the evolution of herbivory because it is embedded within the paraphyletic genus Drosophila, which contains species with 25 sequenced genomes, and is closely related to D. melanogaster, the genetic model, and a taxon with one of the most well-studied nervous systems. Behavior is thought to be one of the earliest adaptations during the evolution of herbivory and niche shifts in general. Insects undergoing a niche shift likely lose their preferences for their ancestral diet, and also evolve an attraction to novel cues indicative of their new oviposition substrate. Once females lay eggs in a new environment, herbivores must consume the new diet, despite the fact that it may contain aversive chemicals and a different balance of macronutrients compared to the ancestral diet. Using the herbivorous Scaptomyza flava as a model system, the primary aim of my dissertation was to use methods in comparative genomics, chemical ecology, ethology, and neural imaging to characterize the mechanistic basis of behavioral changes associated with the evolution of herbivory in insects. Using a comparative genomics approach, I found that targeted gain- and loss-of-function mutations were associated with the evolution of herbivory in the genus Scaptomyza. First, four Odorant (Olfactory) Receptor (OR) genes were lost in herbivorous species of Scaptomyza, which are deeply conserved among microbe-feeding drosophilids. The OR genes lost code for receptors that detect yeast-volatiles and are known to stimulate oviposition, feeding and attraction behaviors in Drosophila species. Consistent with these losses was also a loss of detection sensitivity to ligands of these ORs, specifically short-chain aliphatic esters such as ethyl and propyl acetate, major yeast-produced odorants. S. flava female flies were also unresponsive to volatiles produced by active yeast cultures, in contrast to D. melanogaster flies. In contrast to some other specialized lineages of Drosophila, I found no evidence of increased or mass chemosensory gene loss, with one interesting and novel exception. The majority of the genes encoding the Plus-C subfamily of Odorant Binding-like proteins (OBPs) are deleted or pseudogenized in Scaptomyza. Additional conserved cysteine residues that form disulfide bonds that stabilize the tertiary structure characterize this subfamily. Interestingly the extra disulfide bonds in Plus-C OBPs are known to be vulnerable to attack by toxic breakdown products of glucosinolates, isothiocyanates, chemicals that are characteristic of S. flava’s host plants in the mustard family. Other than the loss of OBPs, I found S. flava to have multiple duplications of genes encoding ORs, OBPs, gustatory receptors (GRs) and ionotropic receptors (IRs), some of which showed evidence for positive selection (Or67b, Obp49a, Gr33a, Ir67a and Ir76a). Among receptors expressed in the gustatory system, losses, duplications and genes with selection regime changes were more often orthologs of genes expressed in bitter gustatory neurons in D. melanogaster, especially gustatory sensory neurons with a broad expression of gustatory receptor genes. Changes, such as deletions, duplications and increased amino acid substitution rates, were also found among genes encoding receptors implicated in reproductive behavior including the loss of an anti-aphrodisiac receptor, Gr68a, which could be associated with a switch from males chemically guarding mated females with anti-aphrodisiacs to physical guarding behavior where males remain on the backs of females post-mating. Another gain-of-function mutation I found was a unique triplication of the gene encoding odorant receptor Or67b, which is present only in single-copy in non-herbivorous Scaptomyza and Drosophila. Homologs of Or67b in D. melanogaster detect a wide array of compounds, but also respond to green leaf volatiles, chemicals produced by leaves when damaged physically or by herbivores. Paralogs of Or67b in S. flava showed signatures of episodic positive selection. Using both gas chromatography-coupled electroantennographic detection (GC-EAD) and GC-MS methods I discovered that S. flava antennae respond to green leaf volatiles, particularly both cis- and trans-3-Hexen-1-yl acetate. Previous antennography experiments also found that S. flava responds to crushed leaf volatiles of host plants. Having such a large detectable antennographic response to a single volatile is predictable given the results imaging S. flava’s primary olfactory processing centers: the antennal lobes. Confocal imaging of S. flava and other herbivorous and non-herbivorous congeners showed that herbivorous Scaptomyza had structures known as macroglomeruli that form when there is an over-abundance of one olfactory sensory neuron type converging on the antennal lobes. This is consistent with my electroantennographic (EAG) results showing a single large deflection indicative of a large population of cells depolarizing in reaction to cis- and trans-3-Hexen-1-yl acetate. Given the function of homologs of Or67b in D. melanogaster detecting this compound and evidence for positive selection in paralogs of this gene in S. flava, Or67b genes are candidates for the receptor expressed in macroglomerular olfactory sensory neurons and are likely involved in host-substrate choice. I successfully cloned these genes in S. flava and a non-herbivorous relative: S. pallida. Future work expressing these Scaptomyza genes in the D. melanogaster empty neuron line will allow me to functionally characterize these receptors and determine if they are involved in green leaf volatile detection

Evolution of Chemosensation in Herbivorous Drosophilidae

Plants and the insects that feed on them dominate diversity in terrestrial ecosystems: half of all named species are contained within these two groups. Herbivorous insects (herbivores) are abundant and diverse, yet paradoxically, two thirds of insect orders contain no major lineages of herbivores, implying barriers to the evolution of this trophic interaction. How herbivory evolves and why herbivores are so diverse are questions that are key to understanding the processes that have shaped global biodiversity. Yet, most lineages of herbivores are ancient with sister groups either absent or too divergent for a comparative genomic analysis to yield a mechanistic understanding of both their origin and diversification. Many of the exceptions to this pattern are among the Diptera, where lineages such as the leaf-mining drosophilids in the genus Scaptomyza have emerged within the last 10 million years. Scaptomyza is particularly well-suited for identifying the adaptations associated with the evolution of herbivory because it is embedded within the paraphyletic genus Drosophila, which contains species with 25 sequenced genomes, and is closely related to D. melanogaster , the genetic model, and a taxon with one of the most well-studied nervous systems. Behavior is thought to be one of the earliest adaptations during the evolution of herbivory and niche shifts in general. Insects undergoing a niche shift likely lose their preferences for their ancestral diet, and also evolve an attraction to novel cues indicative of their new oviposition substrate. Once females lay eggs in a new environment, herbivores must consume the new diet, despite the fact that it may contain aversive chemicals and a different balance of macronutrients compared to the ancestral diet. Using the herbivorous Scaptomyza flava as a model system, the primary aim of my dissertation was to use methods in comparative genomics, chemical ecology, ethology, and neural imaging to characterize the mechanistic basis of behavioral changes associated with the evolution of herbivory in insects. Using a comparative genomics approach, I found that targeted gain- and loss-of-function mutations were associated with the evolution of herbivory in the genus Scaptomyza. First, four Odorant (Olfactory) Receptor (OR) genes were lost in herbivorous species of Scaptomyza , which are deeply conserved among microbe-feeding drosophilids. The OR genes lost code for receptors that detect yeast-volatiles and are known to stimulate oviposition, feeding and attraction behaviors in Drosophila species. Consistent with these losses was also a loss of detection sensitivity to ligands of these ORs, specifically short-chain aliphatic esters such as ethyl and propyl acetate, major yeast-produced odorants. S. flava female flies were also unresponsive to volatiles produced by active yeast cultures, in contrast to D. melanogaster flies. In contrast to some other specialized lineages of Drosophila , I found no evidence of increased or mass chemosensory gene loss, with one interesting and novel exception. The majority of the genes encoding the Plus-C subfamily of Odorant Binding-like proteins (OBPs) are deleted or pseudogenized in Scaptomyza. Additional conserved cysteine residues that form disulfide bonds that stabilize the tertiary structure characterize this subfamily. Interestingly the extra disulfide bonds in Plus-C OBPs are known to be vulnerable to attack by toxic breakdown products of glucosinolates, isothiocyanates, chemicals that are characteristic of S. flava's host plants in the mustard family. Other than the loss of OBPs, I found S. flava to have multiple duplications of genes encoding ORs, OBPs, gustatory receptors (GRs) and ionotropic receptors (IRs), some of which showed evidence for positive selection (Or67b, Obp49a, Gr33a, Ir67a and Ir76a). Among receptors expressed in the gustatory system, losses, duplications and genes with selection regime changes were more often orthologs of genes expressed in bitter gustatory neurons in D. melanogaster , especially gustatory sensory neurons with a broad expression of gustatory receptor genes. Changes, such as deletions, duplications and increased amino acid substitution rates, were also found among genes encoding receptors implicated in reproductive behavior including the loss of an anti-aphrodisiac receptor, Gr68a, which could be associated with a switch from males chemically guarding mated females with anti-aphrodisiacs to physical guarding behavior where males remain on the backs of females post-mating. (Abstract shortened by ProQuest.

Phylogenetics and Diversification of Syringa Inferred from Nuclear and Plastid DNA Sequences

Analyses of nuclear ribosomal DNA have suggested the paraphyly of Syringa with respect to Ligustrum, a hypothesis requiring further testing from other sources of data. In addition, relationships among lineages of Syringa have not been resolved. In this study, we used DNA sequences of two nuclear and five plastid regions to better resolve phylogenetic relationships of Syringa and Ligustrum. Our results support the derivation of Ligustrum from within Syringa, thus the paraphyly of Syringa. Six series are recognized in Syringa and their relationships are: (Syringa, (Pinnatifoliae, aigustrae, (Ligustrina, (Pubescentes, Villosae))))). Subgenus Syringa as traditionally defined is paraphyletic, containing subgenus Ligustrina. The Syringa pubescens complex may be paraphyletic with S. pubescens more closely related to the Syringa pinetorum complex than to other species of its own complex. Our estimated times of divergence suggest that series of Syringa have diversified in the middle Miocene (9.65-17.67 mya), each with unique morphology

Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet

Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. We found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. We sequenced the genome of S. flava and characterized this species' odorant receptor repertoire. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires

Asynchronous Diversification in a Specialized Plant-Pollinator Mutualism

Most flowering plants establish mutualistic associations with insect pollinators to facilitate sexual reproduction. However, the evolutionary processes that gave rise to these associations remain poorly understood. We reconstructed the times of divergence, diversification patterns, and interaction networks of a diverse group of specialized orchids and their bee pollinators. In contrast to a scenario of coevolution by race formation, we show that fragrance-producing orchids originated at least three times independently after their fragrance-collecting bee mutualists. Whereas orchid diversification has apparently tracked the diversification of orchids’ bee pollinators, bees appear to have depended on the diverse chemical environment of neotropical forests. We corroborated this apparent asymmetrical dependency by simulating co-extinction cascades in real interaction networks that lacked reciprocal specialization. These results suggest that the diversification of insect-pollinated angiosperms may have been facilitated by the exploitation of preexisting sensory biases of insect pollinators.Organismic and Evolutionary Biolog

Army Ants Harbor a Host-Specific Clade of Entomoplasmatales Bacteria ▿ †

In this article, we describe the distributions of Entomoplasmatales bacteria across the ants, identifying a novel lineage of gut bacteria that is unique to the army ants. While our findings indicate that the Entomoplasmatales are not essential for growth or development, molecular analyses suggest that this relationship is host specific and potentially ancient. The documented trends add to a growing body of literature that hints at a diversity of undiscovered associations between ants and bacterial symbionts

Specialization and Geographic Isolation among Wolbachia Symbionts from Ants and Lycaenid Butterflies

Wolbachia are the most prevalent and influential bacteria described among the insects to date. But despite their significance, we lack an understanding of their evolutionary histories. To describe the evolution of symbioses between Wolbachia and their hosts, we surveyed global collections of two diverse families of insects, the ants and lycaenid butterflies. In total, 54 Wolbachia isolates were typed using a Multi Locus Sequence Typing (MLST) approach, in which five unlinked loci were sequenced and analyzed to decipher evolutionary patterns. AMOVA and phylogenetic analyses demonstrated that related Wolbachia commonly infect related hosts, revealing a pattern of host association that was strongest among strains from the ants. A review of the literature indicated that horizontal transfer is most successful when Wolbachia move between related hosts, suggesting that patterns of host association are driven by specialization on a common physiological background. Aside from providing the broadest and strongest evidence to date for Wolbachia specialization, our findings also reveal that strains from New World ants differ markedly from those in ants from other locations. We, therefore, conclude that both geographic and phylogenetic barriers have promoted evolutionary divergence among these influential symbionts.Organismic and Evolutionary Biolog