Horizontal Symbiont Transmission Research Articles

Social transmission of bacterial symbionts homogenizes the microbiome within and across generations of group-living spiders

Disentangling modes and fidelity of symbiont transmission are key for understanding host–symbiont associations in wild populations. In group-living animals, social transmission may evolve to ensure high-fidelity transmission of symbionts, since non-reproducing helpers constitute a dead-end for vertical transmission. We investigated symbiont transmission in the social spider Stegodyphus dumicola, which lives in family groups where the majority of females are non-reproducing helpers, females feed offspring by regurgitation, and individuals feed communally on insect prey. Group members share temporally stable microbiomes across generations, while distinct variation in microbiome composition exists between groups. We hypothesized that horizontal transmission of symbionts is enhanced by social interactions, and investigated transmission routes within (horizontal) and across (vertical) generations using bacterial 16S rRNA gene amplicon sequencing in three experiments: (i) individuals were sampled at all life stages to assess at which life stage the microbiome is acquired. (ii) a cross-fostering design was employed to test whether offspring carry the microbiome from their natal nest, or acquire the microbiome of the foster nest via social transmission. (iii) adult spiders with different microbiome compositions were mixed to assess whether social transmission homogenizes microbiome composition among group members. We demonstrate that offspring hatch symbiont-free, and bacterial symbionts are transmitted vertically across generations by social interactions with the onset of regurgitation feeding by (foster)mothers in an early life stage. Social transmission governs horizontal inter-individual mixing and homogenization of microbiome composition among nest mates. We conclude that temporally stable host–symbiont associations in social species can be facilitated and maintained by high-fidelity social transmission.

A genetic linkage map and improved genome assembly of the termite symbiont Termitomyces cryptogamus

BackgroundThe termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect.MethodsTo explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle.ResultsOur linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi.ConclusionsThe apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate.

Latitudinal patterns of egg size and maternal investment trade‐offs in reef corals

AbstractAimThe aims were to test the role of temperature in latitudinal patterns of egg size and investigate maternal investment trade‐offs among coral taxa.LocationGlobal, from 34° S to 34° N.Time period1981–2020.Major taxa studiedReef coral species from the order Scleractinia.MethodsWe compiled a comprehensive geo‐referenced global dataset of egg sizes (diameter or volume) and fecundity (number of eggs per area) for colonial corals (Scleractinia; 123 species, 5359 observations and 39 localities), substantially enhanced by new field collections (>88% of observations). We used Bayesian phylogenetic multilevel models to test for Rass' rule (a hypothesized negative relationship between egg size and temperature); we also included other environmental variables and life history traits. We also tested whether a trade‐off exists between egg size and fecundity in broadcast spawning hermaphroditic corals with horizontal symbiont transmission (HHT).ResultsWe found a significant relationship between coral egg size and symbiont transmission. Eggs from coral species with vertical symbiont transmission were c. 18.8% smaller than those from species with horizontal symbiont transmission. We also found non‐significant relationships between egg size and sea surface temperature (SST) for broadcast spawning corals and between egg size and fecundity specifically for HHT species.Main conclusionsContrary to recognized latitudinal patterns of egg size across taxa, our study does not provide support for Rass' rule in corals. Additionally, our findings do not support a maternal investment trade‐off between egg size and fecundity for HHT species. Our study used a phylogenetic framework that should be a standard practice when studying interspecific variation, including investigation of maternal investment and identification of the influence of multiple predictors on larval fitness (egg size), in addition to trade‐offs affecting propagule influx (fecundity). Both these functional traits are vital and have direct consequences for population maintenance and connectivity in sessile organisms, such as corals.

Patterns of transmission and horizontal gene transfer in the Dioscorea sansibarensis leaf symbiosis revealed by whole-genome sequencing

Leaves of the wild yam species Dioscorea sansibarensis display prominent forerunner or "drip" tips filled with extracellular bacteria of the species Orrella dioscoreae.1 This species of yam is native to Madagascar and tropical Africa and reproduces mainly asexually through aerial bulbils and underground tubers, which also contain a small population of O.dioscoreae.2,3 Despite apparent vertical transmission, the genome of O.dioscoreae does not show any of the hallmarks of genome erosion often found in hereditary symbionts (e.g., small genome size and accumulation of pseudogenes).4-6 We investigated here the range and distribution of leaf symbiosis between D.sansibarensis and O.dioscoreae using preserved leaf samples from herbarium collections that were originally collected from various locations in Africa. We recovered DNA from the extracellular symbiont in all samples, showing that the symbiosis is widespread throughout continental Africa and Madagascar. Despite the degraded nature of this DNA, we constructed 17 symbiont genomes using de novo methods without relying on a reference. Phylogenetic and genomic analyses revealed that horizontal transmission of symbionts and horizontal gene transfer have shaped the evolution of the symbiont. These mechanisms could help explain lack of signs of reductive genome evolution despite an obligate host-associated lifestyle. Furthermore, phylogenetic analysis of D.sansibarensis based on plastid genomes revealed a strong geographical clustering of samples and provided evidence that the symbiosis originated at least 13 mya, earlier than previously estimated.3.

Continent-Scale Sampling Reveals Fine-Scale Turnover in a Beneficial Bug Symbiont.

Many members of animal-associated microbial communities, including the gut flora, are acquired from their host’s environment. While many of these communities are species rich, some true bugs (Hemiptera) in the superfamilies Lygaeoidea and Coreidae allow only ingested Burkholderia to colonize and reproduce in a large portion of the midgut. We studied the spatial structuring of Burkholderia associated with a widespread omnivorous bug genus, Jalysus (Berytidae). We sampled Wickham’s stilt bug, Jalysus wickhami, across the United States and performed limited sampling of its sister species, the spined stilt bug Jalysus spinosus. We asked: (1) What Burkholderia strains are hosted by Jalysus at different locations? (2) Does host insect species, host plant species, or location influence the strain these insects acquire? (3) How does Burkholderia affect the development and reproductive fitness of J. wickhami? We found: (1) Sixty-one Burkholderia strains were present across a sample of 352 individuals, but one strain dominated, accounting for almost half of all symbiont reads. Most strains were closely related to other hemipteran Burkholderia symbionts. (2) Many individuals hosted more than one strain of Burkholderia. (3) J. wickhami and J. spinosus did not differ in the strains they hosted. (4) Insects that fed on different plant species tended to host different Burkholderia, but this accounted for only 4% of the variation in strains hosted. In contrast, the location at which an insect was collected explained 27% of the variation in symbiont strains. (5) Burkholderia confers important fitness benefits to J. wickhami. In laboratory experiments, aposymbiotic (Burkholderia-free) insects developed more slowly and laid fewer eggs than symbiotic (Burkholderia-colonized) insects. (6) In the lab, nymphs sometimes acquired Burkholderia via indirect exposure to adults, indicating that horizontal symbiont transmission can occur via adult insect-mediated enrichment of Burkholderia in the local environment – a phenomenon not previously reported in bug-Burkholderia relationships. Taken together, the results suggest that for these bugs, critical nutritional requirements are outsourced to a highly diverse and spatially structured collection of Burkholderia strains acquired from the environment and, occasionally, from conspecific adults.

The microbiota of diapause: How host-microbe associations are formed after dormancy in an aquatic crustacean.

A critical question in symbiosis research is where and how organisms obtain beneficial microbial symbionts in different ecological contexts. Microbiota of juveniles are often derived directly from their mother or from the immediate environment. The origin of beneficial symbionts, however, is less obvious in organisms with diapause and dispersal stages, such as plants with dormant seeds and animals in ephemeral or strongly seasonal habitats. In these cases, parents and offspring are separated in time and space, which may affect opportunities for both vertical and horizontal transmission of symbionts. The planktonic crustacean Daphnia produces long-lasting resting eggs to endure winter freezing and summer droughts and requires microbiota for growth and reproduction. It is unknown how hatchlings from resting stages form associations with microbial consorts after diapause. Using natural samples of D.magna resting eggs after several years of storage, we show that the total bacterial community derived from both the exterior and interior of the eggs' ephippial cases is sufficiently beneficial to ensure normal Daphnia functioning in otherwise bacteria-free conditions. We do not find direct evidence that the required bacteria are of maternal origin, though sequencing reveals that the resting stage is accompanied by bacterial taxa previously found in association with adult animals. These findings suggest that although Daphnia are strongly dependent on environmental bacteria for normal functioning, host-bacteria associations are somewhat general and availability of specific bacteria is not a strong constraint on host ecology. Nevertheless, animals and microbes may be ecologically linked through co-dispersal.

Draft Genome Sequence of Pedobacter sp. Strain Hv1, an Isolate from Medicinal Leech Mucosal Castings.

The Pedobacter sp. Hv1 strain was isolated from the medicinal leech, Hirudo verbana, mucosal castings. These mucosal sheds have been demonstrated to play a role in horizontal symbiont transmission. Here, we report the draft 4.9 Mbp genome sequence of Pedobacter sp. strain Hv1.

Bacterial endosymbionts in field-collected samples of Trialeurodes sp. nr. abutiloneus (Hemiptera: Aleyrodidae)

Facultative bacterial endosymbionts are common, influential associates of arthropods, yet their movement among host species has not been well documented. Plant-mediated transmission of Rickettsia has been shown for the whitefly Bemisia tabaci. Bemisia tabaci in USA cotton fields harbors the secondary symbionts Rickettsia and Hamiltonella, and co-occurs with Trialeurodes sp. nr. abutiloneus whiteflies. To determine whether symbionts may be shared, the microbial diversity of these whiteflies on cotton across the USA was analyzed. Trialeurodes sp. nr. abutiloneus bore Portiera, Pseudomonas, Serratia, Arsenophonus and Wolbachia. No Rickettsia or Hamiltonella were detected. These results provide no evidence for horizontal transmission of symbionts between these whitefly genera.

Parasitoids as vectors of facultative bacterial endosymbionts in aphids

Heritable bacterial endosymbionts play an important role in aphid ecology. Sequence-based evidence suggests that facultative symbionts such as Hamiltonella defensa or Regiella insecticola also undergo horizontal transmission. Other than through male-to-female transfer during the sexual generation in autumn, the routes by which this occurs remain largely unknown. Here, we tested if parasitoids or ectoparasitic mites can act as vectors for horizontal transfer of facultative symbionts. Using symbiont-specific primers for diagnostic PCR, we demonstrate for the first time, to our knowledge, that parasitoids can indeed transfer H. defensa and R. insecticola by sequentially stabbing infected and uninfected individuals of their host, Aphis fabae, establishing new, heritable infections. Thus, a natural route of horizontal symbiont transmission is also available during the many clonal generations of the aphid life cycle. No transmissions by ectoparasitic mites were observed, nor did parasitoids that emerged from symbiont-infected aphids transfer any symbionts in our experiments.

Ontogenetic change in the lipid and fatty acid composition of scleractinian coral larvae

Some scleractinian coral larvae have an extraordinary capacity to delay metamorphosis, and this is reflected in the large geographic range of many species. Coral eggs typically contain a high proportion of wax esters, which have been hypothesized to provide a source of energy for long-distance dispersal. To better understand the role of lipids in the dispersal of broadcast spawning coral larvae, ontogenetic changes in the lipid and fatty acid composition of Goniastrea retiformis were measured from the eggs until larvae were 30 days old. Egg biomass was 78.8 ± 0.5% lipids, 86.3 ± 0.2% of which were wax esters, 9.3 ± 0.0% polar lipids, 4.1 ± 0.2% sterols, and 0.3 ± 0.1% triacylglycerols. The biomass of wax esters declined significantly through time, while polar lipids, sterols and triacylglycerols remained relatively constant, suggesting that wax esters are the prime source of energy for development. The most prevalent fatty acid in the eggs was palmitic acid, a marker of the dinoflagellate Symbiodinium, highlighting the importance of symbiosis in coral reproductive ecology. The proportion of polyunsaturated fatty acids declined through time, suggesting that they are essential for larval development. Interestingly, triacylglycerols are only abundant in the propagules that contain Symbiodinium, suggesting important differences in the energetic of dispersal among species with vertical and horizontal transmission of symbionts.

Farming termites determine the genetic population structure ofTermitomycesfungal symbionts

Symbiotic interactions between macrotermitine termites and their fungal symbionts have a moderate degree of specificity. Consistent with horizontal symbiont transmission, host switching has been frequent over evolutionary time so that single termite species can often be associated with several fungal symbionts. However, even in the few termite lineages that secondarily adopted vertical symbiont transmission, the fungal symbionts are not monophyletic. We addressed this paradox by studying differential transmission of fungal symbionts by alate male and female reproductives, and the genetic population structure of Termitomyces fungus gardens across 74 colonies of Macrotermes bellicosus in four west and central African countries. We confirm earlier, more limited, studies showing that the Termitomyces symbionts of M. bellicosus are normally transmitted vertically and clonally by dispersing males. We also document that the symbionts associated with this termite species belong to three main lineages that do not constitute a monophyletic group. The most common lineage occurs over the entire geographical region that we studied, including west, central and southern Africa, where it is also associated with the alternative termite hosts Macrotermes subhyalinus and Macrotermes natalensis. While Termitomyces associated with these alternative hosts are horizontally transmitted and recombine freely, the genetic population structure of the same Termitomyces associated with M. bellicosus is consistent with predominantly clonal reproduction and only occasional recombination. This implies that the genetic population structure of Termitomyces is controlled by the termite host and not by the Termitomyces symbiont.

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.

Almost There: Transmission Routes of Bacterial Symbionts between Trophic Levels

Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts – Rickettsia and Hamiltonella – from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed.

Establishment and Vertical Passage of Enterobacter (Pantoea) Agglomerans and Klebsiella pneumoniae through All Life Stages of the Mediterranean Fruit Fly (Diptera: Tephritidae)

Abstract We investigated the fate of ingested Enterobacter (Pantoea) agglomerans and Klebsiella pneumoniae within adult Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in a mass rearing facility. This examination revealed the establishment of both bacterial strains as biofilms within the adult intestines, on the apical end of developing and developed eggs, and throughout all subsequent life stages. The bacteria were detected in adults through two generations. Irradiation treatment for the sterile insect technique did not disrupt the vertical transmission of E. (P.) agglomerans or K. pneumoniae. This is the first demonstration of maternal spread of Enterobacter/ Pantoea spp. and Klebsiella spp. through populations of C. capitata. A mixed pattern of vertical and horizontal transmission of symbionts associated with tephritids may be one explanation for the difficulty in defining the symbiotic associations of tephritids.

Molecular Microbial Diversity Survey of Sponge Reproductive Stages and Mechanistic Insights into Vertical Transmission of Microbial Symbionts

Many marine sponges, hereafter termed high-microbial-abundance (HMA) sponges, harbor large and complex microbial consortia, including bacteria and archaea, within their mesohyl matrices. To investigate vertical microbial transmission as a strategy to maintain these complex associations, an extensive phylogenetic analysis was carried out with the 16S rRNA gene sequences of reproductive (n = 136) and adult (n = 88) material from five different Caribbean species, as well as all published 16S rRNA gene sequences from sponge offspring (n = 116). The overall microbial diversity, including members of at least 13 bacterial phyla and one archaeal phylum, in sponge reproductive stages is high. In total, 28 vertical-transmission clusters, defined as clusters of phylotypes that are found both in adult sponges and their offspring, were identified. They are distributed among at least 10 bacterial phyla and one archaeal phylum, demonstrating that the complex adult microbial community is collectively transmitted through reproductive stages. Indications of host-species specificity and cospeciation were not observed. Mechanistic insights were provided using a combined electron microscopy and fluorescence in situ hybridization analysis, and an indirect mechanism of vertical transmission via nurse cells is proposed for the oviparous sponge Ectyoplasia ferox. Based on these phylogenetic and mechanistic results, we suggest the following symbiont transmission model: entire microbial consortia are vertically transmitted in sponges. While vertical transmission is clearly present, additional environmental transfer between adult individuals of the same and even different species might obscure possible signals of cospeciation. We propose that associations of HMA sponges with highly sponge-specific microbial communities are maintained by this combination of vertical and horizontal symbiont transmission.

A Gene’s‐Eye View of Symbiont Transmission

Symbiotic associations between species are ubiquitous, but we only poorly understand why some symbioses evolve to be mutualistic and others to be parasitic. One prominent hypothesis holds that vertical transmission of symbionts from host parents to their offspring selects for symbionts that are benign or beneficial, while horizontal transmission of symbionts among unrelated hosts selects for symbionts that are less beneficial or outright harmful. A long-standing challenge to this hypothesis, however, is the existence of selfish genetic elements (SGEs). SGEs are passed exclusively from parent to offspring and are able to spread and persist in populations despite reducing the fitness of their hosts. Here I show that SGEs are in fact consistent with the transmission mode hypothesis if one measures transmission from the perspective of host genes instead of host organisms. Both meiotic drive genes and cytoplasmic sex ratio distorters require horizontal transmission, in the form of outbred sex, to spread as parasites. Transmission from parent to offpsring does not constrain SGEs to evolve toward mutualism. The gene-centered perspective I present here is applicable to symbioses at all levels of selection and brings closer together our understandings of cooperation within and between species.

A Gene's-Eye View of Symbiont Transmission

Symbiotic associations between species are ubiquitous, but we only poorly understand why some symbioses evolve to be mutualistic and others to be parasitic. One prominent hypothesis holds that vertical transmission of symbionts from host parents to their offspring selects for symbionts that are benign or beneficial, while horizontal transmission of symbionts among unrelated hosts selects for symbionts that are less beneficial or outright harmful. A long‐standing challenge to this hypothesis, however, is the existence of selfish genetic elements (SGEs). SGEs are passed exclusively from parent to offspring and are able to spread and persist in populations despite reducing the fitness of their hosts. Here I show that SGEs are in fact consistent with the transmission mode hypothesis if one measures transmission from the perspective of host genes instead of host organisms. Both meiotic drive genes and cytoplasmic sex ratio distorters require horizontal transmission, in the form of outbred sex, to spread as parasites. Transmission from parent to offpsring does not constrain SGEs to evolve toward mutualism. The gene‐centered perspective I present here is applicable to symbioses at all levels of selection and brings closer together our understandings of cooperation within and between species.

Cryptic sex and many-to-one coevolution in the fungus-growing ant symbiosis.

The fungus-growing ants have long provided a spectacular example of coevolutionary integration. Their ecological success is thought to depend largely on the evolutionary alignment of reproductive interests between ants and fungi after vertical transmission and the ancient suppression of fungal sexuality. In the present study we test these assumptions and provide the first evidence of recombination in attine cultivars, contradicting widely held perceptions of obligate clonality. In addition, we document long-distance horizontal transmission of symbionts between leaf-cutter ant species on mainland Central America and South America and those endemic to Cuba, suggesting both lack of pairwise coevolutionary specificity in ant/cultivar interactions and dispersal of symbionts independent of their ant hosts. The coevolution between leaf-cutters and their fungal symbionts is thus not reciprocally pairwise. Rather, a single widespread and sexual fungal symbiont species is engaged in multiple interactions with divergent ant lineages. Strict fungal clonality and vertical transmission evidently have not played a critical role in the long-term evolutionary or ecological success of this well known mutualism.

Wolbachia infection in crustaceans: novel hosts and potential routes for horizontal transmission

Abstract Thirty-five percent of isopods are estimated to be infected by Wolbachia, an intracellular maternally inherited α-Proteobacterium. Previous studies have indicated that horizontal transfer of Wolbachia strains may occur, although the mechanisms are unclear. The wsp gene was sequenced from 17 Wolbachia strains harboured by crustacean host species and three from their associated predators and parasites. Two major clades of Wolbachia were found in crustacean, with relatives also found in insects, the other restricted to crustaceans. Highly divergent Wolbachia strains were found in a woodlouse-eating spider and its prey, suggesting no intertaxon bacterial exchange via the predator–prey route. The phylogenetic proximity of Wolbachia from parasitoid flies or phoretic mites to those from isopods suggests that horizontal symbiont transmission may have occurred between those taxa. Two distant Wolbachia strains were detected in two intertidal amphipods; these strains were closely related to different coastal isopod symbionts, suggesting Wolbachia transmission may occur between distantly related crustacean hosts living under the same ecological conditions.

Cospeciation between bacterial endosymbionts (Buchnera) and a recent radiation of aphids (Uroleucon) and pitfalls of testing for phylogenetic congruence.

Previous studies of phylogenetic congruence between aphids and their symbiotic bacteria (Buchnera) supported long-term vertical transmission of symbionts. However, those studies were based on distantly related aphids and would not have revealed horizontal transfer of symbionts among closely related hosts. Aphid species of the genus Uroleucon are closely related phylogenetically and overlap in geographic ranges, habitats, and parasitoids. To examine support for congruence of phylogenies of Buchnera and Uroleucon, sequences from four mitochondrial, one nuclear, and one endosymbiont gene (trpB) were obtained. Congruence of phylogenies based on pooled aphid genes with phylogenies based on trpB was highly significant: Most nodes resolved by trpB corresponded to nodes resolved by the pooled aphid genes. Furthermore, no nodes were both inconsistent between the trees and strongly supported in both trees. Two kinds of analyses testing the null hypothesis of perfect congruence between pairwise combinations of datasets and tree topologies were performed: the Kishino-Hasegawa test and the likelihood-ratio test. Both tests indicated significant disagreement among most pairwise combinations of mitochondrial, nuclear, and symbiont datasets. Because rampant recombination among mitochondrial genomes of different aphid species is unlikely, inaccurate assumptions in the evolutionary models underlying these tests appear to be causing the hypothesis of a shared history to be incorrectly rejected. Moreover, trpB was more consistent with the aphid genes as a set than any single aphid gene was with the others, suggesting that the symbionts show the same phylogeny as the aphids. Overall, analyses support the interpretation that symbionts and aphids have undergone strict cospeciation, with no horizontal transmission of symbionts even among closely related, ecologically similar aphid hosts.