Fidelity in co-diversified symbiosis

nature communications

Fidelity in co-diversified symbiosis

Obligate co-dependence can arise in symbiosis, yielding heritable partnerships. These interactions are considered to be highly specific, but partner fidelity is difficult to quantify owing to the experimental constraints of symbiont exchange between host species. Here, we overcome this challenge by leveraging the unique transmission dynamics of Stammera capleta, the obligate digestive symbiont of tortoise beetles. Despite its extracellular localization, S. capleta possesses a drastically reduced genome (approximately zero point two five megabases) and is vertically transmitted through egg-associated spheres. Manipulating these spheres allowed us to experimentally exchange S. capleta between beetle species to determine their impact on host development. We show that non-native S. capleta can successfully colonize the symbiotic organs of a novel host, but that the interaction outcome correlates with genetic relatedness to the native symbiont. Genetically distant species trigger a more pronounced transcriptional response and can only partially rescue host development. While more closely related symbionts proliferate similarly to the native one and induce a comparable host response, they fail to propagate to the next generation, underscoring how transmission fidelity, host-symbiont compatibility, and local adaptation can further specificity within a Paleocene-aged partnership.

The metabolic intimacy of symbiosis demands the work of specialists, each evolving distinct yet complementary roles to sustain the partnership. Obligate co-dependence can arise within these interactions, necessitating adaptations that ensure the recognition, regulation, and persistence of mutualisms. The resulting partnerships often feature heritable endosymbionts that are intertwined in the evolutionary history of their hosts. As symbiont and host co-diversify, they exhibit parallel lineage splitting over evolutionary timescales.

Among animals, obligately beneficial symbionts are typically acquired early in development. For insects that host endosymbionts within specialized cells (i.e., bacteriocytes) and organs, microbial colonization can take place during embryogenesis or oogenesis. For example, aphids acquire their nutritional endosymbiont, Buchnera aphidicola, during embryo development through calibrated cycles of exocytosis and endocytosis. Endosymbiont cells released from maternal symbiotic organs colonize cells fated to become bacteriocytes in the embryo. Ants similarly integrate their endosymbiont, Blochmannia floridanus, during embryogenesis through a shared regulatory network that is coopted by the microbe to facilitate infection. Such models, and others, continue to offer striking insights into the cellular mechanisms guiding symbiont colonization and proliferation. But our inability to cultivate most obligate symbionts in pure culture, compounded by the brief developmental window that their hosts spend aposymbiotically, constrains the experimental exchange of symbionts and the ability to test fidelity within co-diversified partnerships.

In contrast to the experimental challenges posed by heritable endosymbionts, partnerships with beneficial microbes acquired from the environment have provided crucial insights into the mechanisms that govern specificity in animal-microbe symbioses. For example, the Hawaiian bobtail squid (Euprymna scolopes) can differentiate its bioluminescent symbiont Vibrio fischeri from thousands of marine microbial species through an intricately honed process that relies on mechanical, chemical, immunological, and developmental exclusion. Similarly, the bean bug (Riptortus pedestris) acquires its crypt-associated gut symbiont Caballeronia insecticola every generation from the soil. Despite a horizontal acquisition mode, several factors contribute to a highly selective recognition process, including a microbial-sorting structure within the gut, the competitive exclusion of non-symbiotic strains, and, finally, the morphological differentiation and closure of the symbiotic organ following passage by C. insecticola. While horizontally transmitted symbionts lack evolutionary histories that mirror their hosts, the partnerships between the squid-V. fischeri and bean bug-C. insecticola display striking adaptations for partner recognition at the genetic and molecular levels. Whether these features extend to heritable endosymbionts, however, remains largely unexplored across study systems.

Here, we investigated the specificity of a co-diversified symbiosis by examining the partnership between tortoise leaf beetles (Chrysomelidae: Cassidinae) and their digestive bacterial symbiont, Candidatus Stammera capleta. The tortoise beetle-S. capleta partnership offers a tractable experimental framework to test fidelity in an obligate symbiosis given the microbe's unique transmission route and colonization dynamics.

Tortoise beetles maintain S. capleta in foregut symbiotic organs to facilitate folivory and in ovary-associated glands to ensure transmission. Despite possessing a drastically reduced genome (approximately zero point two to zero point three megabases), S. capleta encodes and exports several plant cell wall-degrading enzymes, including polygalacturonase, a pectinase that underpins convergent nutritional symbioses across leaf beetles. Tortoise beetles vertically transmit S. capleta by depositing an individual caplet at the anterior pole of each egg. This transmission mode is reflected in the strict co-cladogenesis between symbiont and host. The caplet is populated with symbiont-bearing spheres where the microbe is embedded extracellularly. Embryos acquire S. capleta late in development by piercing the caplet membrane and consuming the spheres, which initiates infection.

Removing the egg caplet disrupts symbiont transmission, yielding S. capleta-free (aposymbiotic) larvae that exhibit a diminished digestive phenotype and drastically reduced survivorship. However, reapplying the spheres to aposymbiotic eggs experimentally reconstitutes the symbiosis, highlighting a potential experimental mechanism to exchange the microbe across different species of tortoise beetles by leveraging these structures.

In this study, we capitalized on the conserved symbiont transmission route shared by tortoise beetles to demonstrate that: (i) heritable endosymbionts can be exchanged between host species, (ii) non-native symbionts can colonize and differentially restore survivorship in a novel host, with outcomes ranging from full fitness recovery to reduced survival depending on genetic divergence among symbionts, (iii) but that, ultimately, a high level of fidelity governs this partnership during host development and the symbiont's propagation to the next generation. Our findings highlight the key, complementary roles that partner recognition, transmission fidelity, and local adaptation all play to ensure specificity and stabilize an ancient, co-diversified symbiosis.

Results and discussion

Reciprocal symbiont exchange in tortoise beetles

Symbiont acquisition in tortoise beetles is governed by a strict developmental window. Although the foregut symbiotic organs form three days before larval emergence from the egg, we previously identified the final twenty-four hours of embryogenesis as the critical period for S. capleta colonization. Reapplying symbiont-bearing spheres during this period can restore S. capleta infection in aposymbiotic embryos. Given this, we explored whether the same approach could facilitate the exchange of S. capleta between different beetle species.

Using the tortoise beetle Chelymorpha alternans as a model, we validated this method through four experimental treatments: (a) untreated control, (b) eggs with caplets removed (aposymbiotic), (c) eggs with caplets removed but re-supplied with their original symbiont-bearing spheres at the anterior pole (re-infected), and (d) eggs with caplets removed but instead supplied with spheres collected from Chelymorpha gressoria, a closely related species (cross-infected).

Consistent with previous studies, caplet removal disrupted S. capleta transmission, yielding aposymbiotic embryos, in contrast to the untreated and re-infected controls. Notably, cross-infected embryos (i.e., supplied with spheres from C. gressoria) were successfully colonized by non-native S. capleta. The symbiont occupied the foregut symbiotic organs of its new host, in line with the colonization dynamics observed for the native S. capleta of C. alternans.

To determine whether symbiont exchange is reciprocal across tortoise beetle species, we repeated these assays using C. gressoria as a host. The same four experimental treatments were applied: (a) untreated control, (b) aposymbiotic, (c) re-infected, and (d) cross-infected (i.e., aposymbiotic C. gressoria supplied with spheres collected from C. alternans). The colonization dynamics mirrored the findings observed in C. alternans. Aposymbiotic C. gressoria lacked their native S. capleta, in contrast to the untreated and re-infected controls. On the other hand, cross-infected embryos were successfully colonized by the symbiont of C. alternans, indicating that the cross-infection protocol was reciprocal.

To confirm whether these colonization dynamics persist beyond the embryo stage, we evaluated the localization of each symbiont in five-day-old larvae of both C. alternans and C. gressoria. The same patterns were observed across both beetle species: aposymbiotic insects remained uncolonized, while untreated and re-infected controls maintained the native S. capleta of each species. Finally, non-native symbionts continued to occupy the foregut symbiotic organs of their new hosts. These findings demonstrate that obligate, heritable symbionts can be experimentally exchanged between tortoise beetle species, and that reciprocal cross-infection persists beyond larval eclosion. With this established, we next asked: how do non-native symbionts influence the development of their novel hosts?

Non-native symbionts spur a gradient of mutualistic outcomes. Numerous insect traits are endowed through symbiosis, with obligate dependence shaping interactions across diverse clades such as bugs, ants, termites, bees, and beetles. Insects specializing on nutritionally imbalanced diets continue to offer important insights into these obligate interactions and the extent to which host development is stunted in their absence. For example, aposymbiotic aphids deprived of Buchnera-and the essential amino acids it supplements suffer significantly reduced survivorship and complete loss of fecundity. Similarly, bedbugs, unable to balance the nutritional deficiencies of their bloodmeal, are developmentally constrained in the absence of their B vitamin-supplementing endosymbiont. Such findings are consistent with consequences of aposymbiosis in tortoise beetles, where the experimental loss of S. capleta impairs the insects' ability to process a leafy diet, resulting in low survivorship and failure to reach adulthood. Given these pronounced effects, we asked whether non-native S. capleta could rescue aposymbiotic mortality. And if so, how do these beetles develop compared to larvae colonized by the native symbiont?

We pursued the above assays using six species of tortoise beetles: the aforementioned C. alternans and C. gressoria, and in addition, Chelymorpha bullata, Acromis sparsa, Aspidimorpha quinquefasciata, and Cassida rubiginosa. Each of these beetles species harbors a single, genetically distinct species of S.

capleta within its foregut symbiotic organs, which is transmitted extracellularly via egg-associated spheres.

Using C. alternans as a host, we assessed the colonization efficiency of different S. capleta species in a novel host and quantified their effects on larval survivorship. We structured our bioassays around eight experimental treatments: (a) untreated control, (b) aposymbiotic eggs, (c) eggs whose caplets were removed but re-infected with the native symbiont, or (d-h) eggs whose caplets were removed but cross-infected with S. capleta-bearing spheres collected from each of C. bullata, C. gressoria, A. sparsa, A. quinquefasciata, and C. rubiginosa.

Five-day-old larvae were dissected to assess their symbiotic status. Experimental manipulation significantly influenced S. capleta colonization. As demonstrated earlier, removal of the egg caplet disrupted symbiont transmission relative to the untreated and re-infected controls.

Given the consistent cross-infection of S. capleta stemming from six donor beetle species, we further quantified symbiont titre to determine whether infection success was reflected in S. capeta abundance. Quantitative analysis revealed treatment-dependent variation in symbiont titer. S. capleta abundances were statistically comparable between larvae harboring their native symbiont and those cross-infected with C. bullata, C. gressoria, and A. sparsa symbionts. Conversely, larvae cross-infected with A. quinquefasciata and C. rubiginosa symbionts contained lower symbiont titers than the untreated larvae. Quantitative polymerase chain reaction amplification in aposymbiotic individuals was indistinguishable from background levels, indicating that the reported copy numbers are likely overestimations and that aposymbiotic larvae are devoid of symbionts, as corroborated by the diagnostic PCR results.

We next quantified how these non-native symbionts shaped the development of C. alternans larvae and found that their effects on host survival varied, yielding a gradient of mutualistic outcomes. While all cross-infected treatments consistently outpaced aposymbiotic insects, only two non-native S. capleta species completely rescued larval survivorship to levels mirroring to the untreated and re-infected controls. These symbionts stem from C. bullata and C. gressoria, two beetle species that are congeneric (Chelymorpha) to the recipient host, C. alternans. In contrast, the three S. capleta species associated with A. sparsa, A. quinquefasciata, and C. rubiginosa could only partially rescue the survivorship of their novel host, underscoring the diverse developmental consequences of colonization by a non-native symbiont. Collectively, this indicates that the observed mortality is only partly quasi-Poisson, D F equals seven, P is less than point zero zero one, X squared equals one hundred nineteen point three two; post hoc comparisons with Bonferroni correction. Larval survivorship to adult eclosion across treatments. Line colors correspond to experimental treatments: dotted lines represent untreated control (seven rep N equals sixty-one) and aposymbiotic (eight rep N equals one hundred nine) treatments, while solid lines indicate caplet-free eggs re-infected with the native symbiont (magenta; seven rep N equals thirty-two) or cross-infected with non-native symbionts from five different beetle species (green gradient; C. bullata, seven rep N equals sixty-four; C. gressoria, ten rep N equals fifty-seven; A. sparsa, five rep N equals fifty-three; A. quinquefasciata, seven rep N equals thirty-one; and C. rubiginosa, five rep N equals twenty-seven). Beetle survival was monitored until metamorphosis; truncated lines denote surviving individuals censored from the assay. Letters denote significant differences between treatments (Mixed effects Cox regression model, D F equals seven, P is less than point zero zero one, X squared equals two hundred twenty-nine point zero four four post hoc comparisons with Bonferroni correction). D, E Correlations between larval survivorship and pairwise genome-wide comparisons of S. capleta average nucleotide identity and collinearity scores. Squares represent the untreated control treatment, while circles indicate caplet-free eggs re-infected with the native symbiont (magenta) or cross-infected with non-native symbionts (green gradient). Spearman correlation coefficients (R) and their significance (P) are shown in each panel. Source data are provided as a Source Data file.

explained by a reduction in symbiont abundance, as seen in the cross-infection with the A. quinquefasciata symbiont. However, this pattern does not hold for all high-mortality cross-infections, such as those involving the A. sparsa symbiont, where symbiont abundance is comparable to those of the native S. capleta. This variation suggests that host survival following our cross-infections may be influenced by factors beyond symbiont abundance, including reduced symbiont activity and/or the degree of compatibility between host and microbe.

To explore this possibility, we examined whether the mutualistic potential of S. capleta in a novel host correlates with its evolutionary relatedness to the native symbiont. S. capleta that are closely related to the original symbiont of C. alternans were more likely to rescue larval survivorship than more distantly related species. Using genome-wide pairwise comparisons of average nucleotide identity (ANI) and collinearity as measures of genetic distance, we observed a strong positive correlation between symbiont relatedness and host survivorship (Spearman's rank correlations: ANI, R equals zero point eight nine, P equals zero point zero zero seven one; collinearity score, R equals zero point nine two, P equals zero point zero zero three four).

How do different symbiont genotypes influence the survivorship of a novel host? Despite strong metabolic conservation across the S. capleta pangenome in categories related to informational processing (e.g., transcription, translation, and replication), different species encode and supplement two iterations of host-beneficial factors to tortoise beetles. All genomes of S. capleta species sequenced to date encode polygalacturonase (PG), a pectinase that breaks down homogalacturonan, the most abundant pectic substrate. However, different symbiont species also produce rhamnogalacturonan lyase (RL), a secondary pectinase that degrades the heteropolymer rhamnogalacturonan one, or alpha-glucuronidase (AG), a xylanase involved in hemicellulose digestion.

The six S. capleta species used in our study encompass these two configurations of host-beneficial factors: PG and RL, or PG and AG. The symbiont of C. alternans, along with those of C. gressoria, C. bullata, and A. sparsa, all encode PG and AG. In contrast, the symbionts of C. rubiginosa and A. quinquefasciata provide their beetle hosts with PG and RL. Since the latter two species only partially restored survivorship in a novel beetle host that typically depends on PG and AG from its native symbiont, we hypothesized that differences in host-beneficial factors might explain the observed variation in mutualistic outcomes. However, this is unlikely to be the sole explanation, since the cross-infected symbiont from A. sparsa encodes the same set of plant cell wall-degrading enzymes (PG and

AG) as the native S. capleta in C. alternans yet fails to restore larval survivorship relative to the untreated and re-infected controls. It is conceivable that differences in the expression or regulation of these host-beneficial factors could contribute to the observed variation. Alternatively, the mutualistic potential of S. capleta in a novel host may be constrained by other features of its genotype rather than the digestive enzymes it supplements. To explore this further, we investigated the role that partner recognition plays in shaping the symbiotic outcome between tortoise beetles and S. capleta. Specifically, we asked: are genetically distant species of S. capleta less likely to be recognized as symbionts by their host?