Rapid experimental evolution of reproductive isolation from a single natural population.

Ecological speciation occurs when local adaptation generates reproductive isolation as a by-product of natural selection. Although ecological speciation is a fundamental source of diversification, the mechanistic link between natural selection and reproductive isolation remains poorly understood, especially in natural populations. Here, we show that experimental evolution of parasite body size over 4 y (approximately 60 generations) leads to reproductive isolation in natural populations of feather lice on birds. When lice are transferred to pigeons of different sizes, they rapidly evolve differences in body size that are correlated with host size. These differences in size trigger mechanical mating isolation between lice that are locally adapted to the different sized hosts. Size differences among lice also influence the outcome of competition between males for access to females. Thus, body size directly mediates reproductive isolation through its influence on both intersexual compatibility and intrasexual competition. Our results confirm that divergent natural selection acting on a single phenotypic trait can cause reproductive isolation to emerge from a single natural population in real time.

Phylogenomics using Target-Restricted Assembly Resolves Intrageneric Relationships of Parasitic Lice (Phthiraptera: Columbicola).

Parasitic "wing lice" (Phthiraptera: Columbicola) and their dove and pigeon hosts are a well-recognized model system for coevolutionary studies at the intersection of micro- and macroevolution. Selection on lice in microevolutionary time occurs as pigeons and doves defend themselves against lice by preening. In turn, behavioral and morphological adaptations of the lice improve their ability to evade host defense. Over macroevolutionary time wing lice tend to cospeciate with their hosts; yet, some species of Columbicola have switched to new host species. Understanding the ecological and evolutionary factors that influence coadaptation and codiversification in this system will substantially improve our understanding of coevolution in general. However, further work is hampered by the lack of a robust phylogenetic framework for Columbicola spp. and their hosts. Previous attempts to resolve the phylogeny of Columbicola based on sequences from a few genes provided limited support. Here, we apply a new approach, target restricted assembly, to assemble 977 orthologous gene sequences from whole-genome sequence data generated from very small, ethanol-preserved specimens, representing up to 61 species of wing lice. Both concatenation and coalescent methods were used to estimate the species tree. These two approaches yielded consistent and well-supported trees with 90% of all relationships receiving 100% support, which is a substantial improvement over previous studies. We used this new phylogeny to show that biogeographic ranges are generally conserved within clades of Columbicola wing lice. Limited inconsistencies are probably attributable to intercontinental dispersal of hosts, and host switching by some of the lice. [aTRAM; coalescent; coevolution; concatenation; species tree.].

Does allopreening control avian ectoparasites?

For birds, the first line of defence against ectoparasites is preening. The effectiveness of self-preening for ectoparasite control is well known. By contrast, the ectoparasite control function of allopreening-in which one birds preens another-has not been rigorously tested. We infested captive pigeons with identical numbers of parasitic lice, and then compared rates of allopreening to the abundance of lice on the birds over time. We documented a negative relationship between rates of allopreening and the number of lice on birds. Moreover, we found that allopreening was a better predictor of louse abundance than self-preening. Our data suggest that allopreening may be a more important means of ectoparasite defence than self-preening when birds live in groups. Our results have important implications for the evolution of social behaviour.

Description of two new species of Hymenolepis Weinland, 1858 (Cestoda: Hymenolepididae) from rodents on Luzon Island, Philippines.

Our helminthological examination of murid rodents on Luzon Island, Philippines, revealed a remarkable diversity of Hymenolepis Weinland, 1858. Here we describe two new species based on specimens from murid rodents Rattus everetti (Günther) and Apomys datae (Meyer) collected from Luzon Island. Hymenolepis alterna n. sp. differs from all known species of Hymenolepis in having irregularly alternating genital pores. This feature has not been reported from any previously known member of Hymenolepis. Additionally, Hymenolepis alterna n. sp. also differs from other Hymenolepis spp. in the relative position of both poral and antiporal dorsal osmoregulatory canals which are shifted towards the middle of the proglottis in relation to the ventral canals on both sides of the proglottides, and in having curved or twisted external seminal vesicle, covered externally by a dense layer of intensely stained cells. Hymenolepis bilaterala n. sp. differs from all known species of Hymenolepis in the relative position of both poral and antiporal dorsal osmoregulatory canals, which are shifted bilaterally towards the margins of proglottides in relation to the ventral canals, and in possession of testes situated in a triangle and eggs with very thin outer coat. A total of seven species of Hymenolepis are known from the Philippine archipelago. This total includes the cosmopolitan species Hymenolepis diminuta (Rudolphi, 1819), which was likely introduced to the island with invasive rats. Strikingly, all seven known species occur on the island of Luzon alone. By comparison, only six Hymenolepis spp. are known from the whole Palaearctic and seven from the Nearctic despite a much better level of knowledge of rodent helminths in these zoogeographical regions, as well as vast territories, diverse landscapes and very rich rodent fauna. This suggests that Hymenolepis spp. may have undergone an unusually active radiation in the Philippines. Possible explanations of this phenomenon are discussed.

Specialized acquisition behaviors maintain reliable environmental transmission in an insect-microbial mutualism.

Understanding how horizontally transmitted mutualisms are maintained is a major focus of symbiosis research.1,2,3,4 Unlike vertical transmission, hosts that rely on horizontal transmission produce symbiont-free offspring that must find and acquire their beneficial microbes from the environment. This transmission strategy is inherently risky since hosts may not obtain the right symbiont every generation. Despite these potential costs, horizontal transmission underlies stable mutualisms involving a large diversity of both plants and animals.5,6,7,8,9 One largely unexplored way horizontal transmission is maintained is for hosts to evolve sophisticated mechanisms to consistently find and acquire specific symbionts from the environment. Here, we examine this possibility in the squash bug Anasa tristis, an insect pest that requires bacterial symbionts in the genus Caballeronia10 for survival and development.11 We conduct a series of behavioral and transmission experiments that track strain-level transmission in vivo among individuals in real-time. We demonstrate that nymphs can accurately find feces from adult bugs in both the presence and absence of those adults. Once nymphs locate the feces, they deploy feeding behavior that results in nearly perfect symbiont acquisition success. We further demonstrate that nymphs can locate and feed on isolated, cultured symbionts in the absence of feces. Finally, we show this acquisition behavior is highly host specific. Taken together, our data describe not only the evolution of a reliable horizontal transmission strategy, but also a potential mechanism that drives patterns of species-specific microbial communities among closely related, sympatric host species.

EXPERIMENTAL INFECTION WITH A NATURALLY OCCURRING PROTOZOAN PARASITE REDUCES MONARCH BUTTERFLY (DANAUS PLEXIPPUS) MATING SUCCESS.

Parasitic infection is known to drive sexual selection in persuasive mating systems, where parasites influence the secondary sexual characteristics that underlie mate choice. However, comparatively little is known about their effects on animals that use coercive mating behavior. We use a tractable system consisting of monarch butterflies and their naturally occurring parasite Ophryocystis elektroscirrha to test how parasites influence host mating dynamics when males force females to copulate. Monarchs were placed in mating cages where all, half, or no individuals were experimentally infected with O. elektroscirrha. We found that parasites reduce a male's mating success such that infected males were not only less likely to copulate but obtained fewer lifetime copulations as well. This reduction in mating success was due primarily to the fact that infected males attempt to mate significantly less than uninfected males. However, we found that O. elektroscirrha did not influence male mate choice. Males chose to mate with both infected and uninfected females at similar rates, regardless of their infection status. Overall, our data highlight how mating dynamics in coercive systems are particularly vulnerable to parasites.

Lack of inbreeding avoidance during mate selection in migratory monarch butterflies.

Inbreeding is generally thought to have negative consequences for organismal health. However, despite the potential fitness effects, it remains surprisingly common among wild populations. In many cases, the complex factors that underlie mating dynamics make predicting whether individuals should or do avoid inbreeding quite challenging. One reason inbreeding may persist among species is that the likelihood of encountering relatives can be rare. Thus, even if inbreeding has severe consequences, selection to avoid mating with kin will be weak in species that are highly dispersed. Here we investigated if migratory monarch butterflies (Danaus plexippus), which are famous for their dispersal ability, actively avoid inbreeding. We found that neither female nor male monarchs choose mates based on relatedness. These results support the hypothesis that movement ecology can mask the deleterious effects of inbreeding and relax selection for active inbreeding avoidance behaviors. Overall, our data add to the growing list of studies showing that inbreeding avoidance is not the behavioral "default" for most species. We also highlight the implications that inbreeding may have on the declining populations of this iconic butterfly.

Crowding does not affect monarch butterflies' resistance to a protozoan parasite.

Host density is an important factor when it comes to parasite transmission and host resistance. Increased host density can increase contact rate between individuals and thus parasite transmission. Host density can also cause physiological changes in the host, which can affect host resistance. Yet, the direction in which host density affects host resistance remains unresolved. It is also unclear whether food limitation plays a role in this effect. We investigated the effect of larval density in monarch butterflies, Danaus plexippus, on the resistance to their natural protozoan parasite Ophryocystis elektroscirrha under both unlimited and limited food conditions. We exposed monarchs to various density treatments as larvae to mimic high densities observed in sedentary populations. Data on infection and parasite spore load were collected as well as development time, survival, wing size, and melanization. Disease susceptibility under either food condition or across density treatments was similar. However, we found high larval density impacted development time, adult survival, and wing morphology when food was limited. This study aids our understanding of the dynamics of environmental parasite transmission in monarch populations, which can help explain the increased prevalence of parasites in sedentary monarch populations compared to migratory populations.

The assembled and annotated genome of the pigeon louse Columbicola columbae, a model ectoparasite.

The pigeon louse Columbicola columbae is a longstanding and important model for studies of ectoparasitism and host-parasite coevolution. However, a deeper understanding of its evolution and capacity for rapid adaptation is limited by a lack of genomic resources. Here, we present a high-quality draft assembly of the C. columbae genome, produced using a combination of Oxford Nanopore, Illumina, and Hi-C technologies. The final assembly is 208 Mb in length, with 12 chromosome-size scaffolds representing 98.1% of the assembly. For gene model prediction, we used a novel clustering method (wavy_choose) for Oxford Nanopore RNA-seq reads to feed into the MAKER annotation pipeline. High recovery of conserved single-copy orthologs (BUSCOs) suggests that our assembly and annotation are both highly complete and highly accurate. Consistent with the results of the only other assembled louse genome, Pediculus humanus, we find that C. columbae has a relatively low density of repetitive elements, the majority of which are DNA transposons. Also similar to P. humanus, we find a reduced number of genes encoding opsins, G protein-coupled receptors, odorant receptors, insulin signaling pathway components, and detoxification proteins in the C. columbae genome, relative to other insects. We propose that such losses might characterize the genomes of obligate, permanent ectoparasites with predictable habitats, limited foraging complexity, and simple dietary regimes. The sequencing and analysis for this genome were relatively low cost, and took advantage of a new clustering technique for Oxford Nanopore RNAseq reads that will be useful to future genome projects.

Host defense triggers rapid adaptive radiation in experimentally evolving parasites.

Adaptive radiation occurs when the members of a single lineage evolve different adaptive forms in response to selection imposed by competitors or predators. Iconic examples include Darwin's finches, Caribbean anoles, and Hawaiian silverswords, all of which live on islands. Although adaptive radiation is thought to be an important generator of biodiversity, most studies concern groups that have already diversified. Here, we take the opposite approach. We experimentally triggered diversification in the descendants of a single population of host-specific parasites confined to different host "islands." We show rapid adaptive divergence of experimentally evolving feather lice in response to preening, which is a bird's main defense against ectoparasites. We demonstrate that host defense exerts strong phenotypic selection for crypsis in lice transferred to different colored rock pigeons (Columba livia). During four years of experimental evolution (∼60 generations), the lice evolved heritable differences in color. Strikingly, the observed color differences spanned the range of phenotypes found among congeneric lice adapted to other species of birds. To our knowledge, this is the first real-time demonstration that microevolution is fast enough to simulate millions of years of macroevolutionary change. Our results further indicate that host-mediated selection triggers rapid divergence in the adaptive radiation of parasites, which are among the most diverse organisms on Earth.

Integrating phylogenomic and population genomic patterns in avian lice provides a more complete picture of parasite evolution.

Parasite diversity accounts for most of the biodiversity on earth, and is shaped by many processes (e.g., cospeciation, host switching). To identify the effects of the processes that shape parasite diversity, it is ideal to incorporate both deep (phylogenetic) and shallow (population) perspectives. To this end, we developed a novel workflow to obtain phylogenetic and population genetic data from whole genome sequences of body lice parasitizing New World ground-doves. Phylogenies from these data showed consistent, highly resolved species-level relationships for the lice. By comparing the louse and ground-dove phylogenies, we found that over long-term evolutionary scales their phylogenies were largely congruent. Many louse lineages (both species and populations) also demonstrated high host-specificity, suggesting ground-dove divergence is a primary driver of their parasites' diversity. However, the few louse taxa that are generalists are structured according to biogeography at the population level. This suggests dispersal among sympatric hosts has some effect on body louse diversity, but over deeper time scales the parasites eventually sort according to host species. Overall, our results demonstrate that multiple factors explain the patterns of diversity in this group of parasites, and that the effects of these factors can vary over different evolutionary scales. The integrative approach we employed was crucial for uncovering these patterns, and should be broadly applicable to other studies.

Walk or ride? Phoretic behaviour of amblyceran and ischnoceran lice.

Phoresy is a behaviour where one organism hitches a ride on another more mobile organism. This is a common dispersal mechanism amongst relatively immobile species that specialise on patchy resources. Parasites specialise on patchily distributed resources: their hosts. Although host individuals are isolated in space and time, parasites must transmit between hosts or they will die with their hosts. Lice are permanent obligate ectoparasites that complete their entire life cycle on their host. They typically transmit when hosts come into direct contact; however, lice are also capable of transmitting phoretically. Yet, phoresy is rare amongst some groups of lice. Fundamental morphological differences have traditionally been used to explain the phoretic differences amongst different suborders of lice; however, these hypotheses do not fully explain observed patterns. We propose that a more fundamental natural history trait may better explain variation in phoresy. Species able to disperse under their own power should be less likely to engage in phoresy than more immobile species. Here we experimentally tested the relationship between independent louse mobility and phoresy using a system with four species of lice (Phthiraptera: Ischnocera and Amblycera) that all parasitize a single host species, the Rock Pigeon (Columba livia). We quantified the relative ability of all four species of lice to move independently off the host, and we quantified their ability to attach to, and remain attached to, hippoboscid flies (Pseudolynchia canariensis). Our results show that the most mobile louse species is the least phoretic, and the most phoretic species is quite immobile off the host. Our findings were consistent with the hypothesis that phoretic dispersal should be rare amongst species of lice that are capable of independent dispersal; however other factors such as interspecific competition may also play a role.

Diversity of feather mites (Acari: Astigmata) on Darwin's finches.

Feather mites are a diverse group of ectosymbionts that occur on most species of birds. Although Darwin's finches are a well-studied group of birds, relatively little is known about their feather mites. Nearly 200 birds across 9 finch species, and from 2 locations on Santa Cruz Island, Galápagos, were dust-ruffled during the 2009 breeding season. We found 8 genera of feather mites; the most prevalent genus was Mesalgoides (53-55%), followed by Trouessartia (40-45%), Amerodectes and Proctophyllodes (26-33%), Xolalgoides (21-27%), Analges and Strelkoviacarus (0-6%), and Dermoglyphus (2-4%). There was no evidence for microclimatic effects (ambient temperature and relative humidity) on mite diversity. Host body mass was significantly correlated with mean feather mite abundance across 7 of 8 well-sampled species of finches. Certhidea olivacea, the smallest species, did not fit this pattern and had a disproportionately high number of mites for its body mass.

Influence of bill and foot morphology on the ectoparasites of barn owls.

Preening is the principle behavioral defense used by birds to combat ectoparasites. Most birds have a small overhang at the tip of their bills that is used to shear through the tough cuticle of ectoparasitic arthropods, making preening much more efficient. Birds may also scratch with their feet to defend against ectoparasites. This is particularly important for removing ectoparasites on the head, which birds cannot preen. Scratching may be enhanced by the comb-like serrations that are found on the claws of birds in many avian families. We examined the prevalence and intensity of ectoparasites of barn owls (Tyto alba pratincola) in southern Idaho in relation to bill hook length and morphological characteristics of the pectinate claw. The barn owls in our study were infested with 3 species of lice (Phthiraptera: Ischnocera): Colpocephalum turbinatum , Kurodaia subpachygaster, and Strigiphilus aitkeni . Bill hook length was associated with the prevalence of these lice. Owls with longer hooks were more likely to be infested with lice. Conventional wisdom suggests that the bill morphology of raptors has been shaped by selection for efficient foraging; our data suggest that hook morphology may also play a role in ectoparasite defense. The number of teeth on the pectinate claw was also associated with the prevalence of lice. Owls that had claws with more teeth were less likely to be infested with lice, which suggests that larger pectinate claws may offer relatively more protection against ectoparasitic lice. Experiments that manipulate the bill hook and pectinate claw are needed to confirm whether these host characters are involved in ectoparasite defense. Finally, we recovered mammalian ectoparasites from 4 barn owls. We recovered species of mammalian lice (Phthiraptera:Anoplura) and fleas (Siphonaptera) that are commonly found on microtine rodents. The owls probably acquired these parasites from recently eaten prey. This represents 1 of the few documented cases of parasites "straggling" from prey to predator.