Natural experiments and long-term monitoring are critical to understand and predict marine host-microbe ecology and evolution.

Marine multicellular organisms host a diverse collection of bacteria, archaea, microbial eukaryotes, and viruses that form their microbiome. Such host-associated microbes can significantly influence the host's physiological capacities; however, the identity and functional role(s) of key members of the microbiome ("core microbiome") in most marine hosts coexisting in natural settings remain obscure. Also unclear is how dynamic interactions between hosts and the immense standing pool of microbial genetic variation will affect marine ecosystems' capacity to adjust to environmental changes. Here, we argue that significantly advancing our understanding of how host-associated microbes shape marine hosts' plastic and adaptive responses to environmental change requires (i) recognizing that individual host-microbe systems do not exist in an ecological or evolutionary vacuum and (ii) expanding the field toward long-term, multidisciplinary research on entire communities of hosts and microbes. Natural experiments, such as time-calibrated geological events associated with well-characterized environmental gradients, provide unique ecological and evolutionary contexts to address this challenge. We focus here particularly on mutualistic interactions between hosts and microbes, but note that many of the same lessons and approaches would apply to other types of interactions.

The evolution of parasitism from mutualism in wasps pollinating the fig, Ficus microcarpa, in Yunnan Province, China.

Theory identifies factors that can undermine the evolutionary stability of mutualisms. However, theory's relevance to mutualism stability in nature is controversial. Detailed comparative studies of parasitic species that are embedded within otherwise mutualistic taxa (e.g., fig pollinator wasps) can identify factors that potentially promote or undermine mutualism stability. We describe results from behavioral, morphological, phylogenetic, and experimental studies of two functionally distinct, but closely related, Eupristina wasp species associated with the monoecious host fig, Ficus microcarpa, in Yunnan Province, China. One (Eupristina verticillata) is a competent pollinator exhibiting morphologies and behaviors consistent with observed seed production. The other (Eupristina sp.) lacks these traits, and dramatically reduces both female and male reproductive success of its host. Furthermore, observations and experiments indicate that individuals of this parasitic species exhibit greater relative fitness than the pollinators, in both indirect competition (individual wasps in separate fig inflorescences) and direct competition (wasps of both species within the same fig). Moreover, phylogenetic analyses suggest that these two Eupristina species are sister taxa. By the strictest definition, the nonpollinating species represents a "cheater" that has descended from a beneficial pollinating mutualist. In sharp contrast to all 15 existing studies of actively pollinated figs and their wasps, the local F. microcarpa exhibit no evidence for host sanctions that effectively reduce the relative fitness of wasps that do not pollinate. We suggest that the lack of sanctions in the local hosts promotes the loss of specialized morphologies and behaviors crucial for pollination and, thereby, the evolution of cheating.

Extraordinarily precise nematode sex ratios: adaptive responses to vanishingly rare mating opportunities.

Sex ratio theory predicts both mean sex ratio and variance under a range of population structures. Here, we compare two genera of phoretic nematodes (Parasitodiplogaster and Ficophagus spp.) associated with 12 fig pollinating wasp species in Panama. The host wasps exhibit classic local mate competition: only inseminated females disperse from natal figs, and their offspring form mating pools that consist of scores of the adult offspring contributed by one or a few foundress mothers. By contrast, in both nematode genera, only sexually undifferentiated juveniles disperse and their mating pools routinely consist of 10 or fewer adults. Across all mating pool sizes, the sex ratios observed in both nematode genera are consistently female-biased (approx. 0.34 males), but markedly less female-biased than is often observed in the host wasps (approx. 0.10 males). In further contrast with their hosts, variances in nematode sex ratios are also consistently precise (significantly less than binomial). The constraints associated with predictably small mating pools within highly subdivided populations appear to select for precise sex ratios that contribute both to the reproductive success of individual nematodes, and to the evolutionary persistence of nematode species. We suggest that some form of environmental sex determination underlies these precise sex ratios.

Genome-wide sequence data show no evidence of hybridization and introgression among pollinator wasps associated with a community of Panamanian strangler figs.

The specificity of pollinator host choice influences opportunities for reproductive isolation in their host plants. Similarly, host plants can influence opportunities for reproductive isolation in their pollinators. For example, in the fig and fig wasp mutualism, offspring of fig pollinator wasps mate inside the inflorescence that the mothers pollinate. Although often host specific, multiple fig pollinator species are sometimes associated with the same fig species, potentially enabling hybridization between wasp species. Here, we study the 19 pollinator species (Pegoscapus spp.) associated with an entire community of 16 Panamanian strangler fig species (Ficus subgenus Urostigma, section Americanae) to determine whether the previously documented history of pollinator host switching and current host sharing predicts genetic admixture among the pollinator species, as has been observed in their host figs. Specifically, we use genome-wide ultraconserved element (UCE) loci to estimate phylogenetic relationships and test for hybridization and introgression among the pollinator species. In all cases, we recover well-delimited pollinator species that contain high interspecific divergence. Even among pairs of pollinator species that currently reproduce within syconia of shared host fig species, we found no evidence of hybridization or introgression. This is in contrast to their host figs, where hybridization and introgression have been detected within this community, and more generally, within figs worldwide. Consistent with general patterns recovered among other obligate pollination mutualisms (e.g. yucca moths and yuccas), our results suggest that while hybridization and introgression are processes operating within the host plants, these processes are relatively unimportant within their associated insect pollinators.

Host-associated microbiomes drive structure and function of marine ecosystems.

The significance of symbioses between eukaryotic hosts and microbes extends from the organismal to the ecosystem level and underpins the health of Earth's most threatened marine ecosystems. Despite rapid growth in research on host-associated microbes, from individual microbial symbionts to host-associated consortia of significantly relevant taxa, little is known about their interactions with the vast majority of marine host species. We outline research priorities to strengthen our current knowledge of host-microbiome interactions and how they shape marine ecosystems. We argue that such advances in research will help predict responses of species, communities, and ecosystems to stressors driven by human activity and inform future management strategies.

Host affinity of endophytic fungi and the potential for reciprocal interactions involving host secondary chemistry.

PREMISE: Interactions between fungal endophytes and their host plants present useful systems for identifying important factors affecting assembly of host-associated microbiomes. Here we investigated the role of secondary chemistry in mediating host affinity of asymptomatic foliar endophytic fungi using Psychotria spp. and Theobroma cacao (cacao) as hosts. METHODS: First, we surveyed endophytic communities in Psychotria species in a natural common garden using culture-based methods. Then we compared differences in endophytic community composition with differences in foliar secondary chemistry in the same host species, determined by liquid chromatography-tandem mass spectrometry. Finally, we tested how inoculation with live and heat-killed endophytes affected the cacao chemical profile. RESULTS: Despite sharing a common environment and source pool for endophyte spores, different Psychotria host species harbored strikingly different endophytic communities that reflected intrinsic differences in their leaf chemical profiles. In T. cacao, inoculation with live and heat-killed endophytes produced distinct cacao chemical profiles not found in uninoculated plants or pure fungal cultures, suggesting that endophytes, like pathogens, induce changes in secondary chemical profiles of their host plant. CONCLUSIONS: Collectively our results suggest at least two potential processes: (1) Plant secondary chemistry influences assembly and composition of fungal endophytic communities, and (2) host colonization by endophytes subsequently induces changes in the host chemical landscape. We propose a series of testable predictions based on the possibility that reciprocal chemical interactions are a general property of plant-endophyte interactions.

Active pollination drives selection for reduced pollen-ovule ratios.

PREMISE: Variation in pollen-ovule ratios is thought to reflect the degree of pollen transfer efficiency-the more efficient the process, the fewer pollen grains needed. Few studies have directly examined the relationship between pollen-ovule ratio and pollen transfer efficiency. For active pollination in the pollination brood mutualisms of yuccas and yucca moths, figs and fig wasps, senita and senita moths, and leafflowers and leafflower moths, pollinators purposefully collect pollen and place it directly on the stigmatic surface of conspecific flowers. The tight coupling of insect reproductive interests with pollination of the flowers in which larvae develop ensures that pollination is highly efficient. METHODS: We used the multiple evolutionary transitions between passive pollination and more efficient active pollination to test if increased pollen transfer efficiency leads to reduced pollen-ovule ratios. We collected pollen and ovule data from a suite of plant species from each of the pollination brood mutualisms and used phylogenetically controlled tests and sister-group comparisons to examine whether the shift to active pollination resulted in reduced pollen-ovule ratios. RESULTS: Across all transitions between passive and active pollination in plants, actively pollinated plants had significantly lower pollen-ovule ratios than closely related passively pollinated taxa. Phylogenetically corrected comparisons demonstrated that actively pollinated plant species had an average 76% reduction in the pollen-ovule ratio. CONCLUSIONS: The results for active pollination systems support the general utility of pollen-ovule ratios as indicators of pollination efficiency and the central importance of pollen transfer efficiency in the evolution of pollen-ovule ratio.

Foliar endophytic fungi alter patterns of nitrogen uptake and distribution in Theobroma cacao.

Colonization by foliar endophytic fungi can affect the expression of host plant defenses and other ecologically important traits. However, whether endophyte colonization affects the uptake or redistribution of resources within and among host plant tissues remains unstudied. We inoculated leaves of Theobroma cacao with four common colonizers that range in their effect from protective to pathogenic (Colletotrichum tropicale, Pestalotiopsis sp., Colletotrichum theobromicola, or Phytophthora palmivora). We pulsed the soil with nitrogen-15 (15 N) and then traced 15 N uptake and its subsequent distribution to whole plants and individual leaves. At a whole-plant level, C. tropicale-inoculated plants showed significantly greater 15 N uptake than endophyte-free plants did in the same pot. Among leaves within plants, younger leaves were particularly enriched in 15 N, but endophyte inoculation at the individual leaf level did not alter 15 N distribution within plants. However, leaves co-inoculated with pathogenic Phytophthora and protective C. tropicale experienced significantly elevated 15 N content as pathogen damage increased, compared with leaves inoculated only with the pathogen. Further, endophyte-pathogen co-infection also increased total plant biomass. Our results indicate that colonization by foliar endophytes significantly affects N uptake and distribution among and within host plants in ways that appear to be context dependent on other microbiome components.

The Response of Litter-Associated Myxomycetes to Long-Term Nutrient Addition in a Lowland Tropical Forest.

Myxomycetes (plasmodial slime molds) are abundant protist predators that feed on bacteria and other microorganisms, thereby playing important roles in terrestrial nutrient cycling. Despite their significance, little is known about myxomycete communities and the extent to which they are affected by nutrient availability. We studied the influence of long-term addition of N, P, and K on the myxomycete community in a lowland forest in the Republic of Panama. In a previous study, microbial biomass increased with P but not N or K addition at this site. We hypothesized that myxomycetes would increase in abundance in response to P but that they would not respond to the sole addition of N or K. Moist chamber cultures of leaf litter and small woody debris were used to quantify myxomycete abundance. We generated the largest myxomycete dataset (3,381 records) for any single locality in the tropics comprised by 91 morphospecies. In line with our hypothesis, myxomycete abundance increased in response to P addition but did not respond to N or K. Community composition was unaffected by nutrient treatments. This work represents one of very few large-scale and long-term field studies to include a heterotrophic protist highlighting the feasibility and value in doing so.

Exposure to the leaf litter microbiome of healthy adults protects seedlings from pathogen damage.

It is increasingly recognized that microbiota affect host health and physiology. However, it is unclear what factors shape microbiome community assembly in nature, and how microbiome assembly can be manipulated to improve host health. All plant leaves host foliar endophytic fungi, which make up a diverse, environmentally acquired fungal microbiota. Here, we experimentally manipulated assembly of the cacao tree (Theobroma cacao) fungal microbiome in nature and tested the effect of assembly outcome on host health. Using next-generation sequencing, as well as culture-based methods coupled with Sanger sequencing, we found that manipulating leaf litter exposure and location within the forest canopy significantly altered microbiome composition in cacao. Exposing cacao seedlings to leaf litter from healthy conspecific adults enriched the seedling microbiome with Colletotrichum tropicale, a fungal endophyte known to enhance pathogen resistance of cacao seedlings by upregulating host defensive pathways. As a result, seedlings exposed to healthy conspecific litter experienced reduced pathogen damage. Our results link processes that affect the assembly and composition of microbiome communities to their functional consequences for host success, and have broad implications for understanding plant-microbe interactions. Deliberate manipulation of the plant-fungal microbiome also has potentially important applications for cacao production and other agricultural systems in general.

In defence of inclusive fitness theory.

Arising from M. A. Nowak, C. E. Tarnita & E. O. Wilson 466, 1057-1062 (2010); Nowak et al. reply. Arguably the defining characteristic of the scientific process is its capacity for self-criticism and correction. Nowak et al. challenge proposed connections between relatedness and the evolution of eusociality, suggest instead that defensible nests and "spring-loaded" traits are key, and present alternative modelling approaches. They then dismiss the utility of Hamilton's insight that relatedness has a profound evolutionary effect, formalized in his widely accepted inclusive fitness theory as Hamilton's rule ("Rise and fall of inclusive fitness theory"). However, we believe that Nowak et al. fail to make their case for logical, theoretical and empirical reasons.

Theobroma cacao L. pathogenesis-related gene tandem array members show diverse expression dynamics in response to pathogen colonization.

BACKGROUND: The pathogenesis-related (PR) group of proteins are operationally defined as polypeptides that increase in concentration in plant tissues upon contact with a pathogen. To date, 17 classes of highly divergent proteins have been described that act through multiple mechanisms of pathogen resistance. Characterizing these families in cacao, an economically important tree crop, and comparing the families to those in other species, is an important step in understanding cacao's immune response. RESULTS: Using publically available resources, all members of the 17 recognized pathogenesis-related gene families in the genome of Theobroma cacao were identified and annotated resulting in a set of ~350 members in both published cacao genomes. Approximately 50 % of these genes are organized in tandem arrays scattered throughout the genome. This feature was observed in five additional plant taxa (three dicots and two monocots), suggesting that tandem duplication has played an important role in the evolution of the PR genes in higher plants. Expression profiling captured the dynamics and complexity of PR genes expression at basal levels and after induction by two cacao pathogens (the oomycete, Phytophthora palmivora, and the fungus, Colletotrichum theobromicola), identifying specific genes within families that are more responsive to pathogen challenge. Subsequent qRT-PCR validated the induction of several PR-1, PR-3, PR-4, and PR-10 family members, with greater than 1000 fold induction detected for specific genes. CONCLUSIONS: We describe candidate genes that are likely to be involved in cacao's defense against Phytophthora and Colletotrichum infection and could be potentially useful for marker-assisted selection for breeding of disease resistant cacao varieties. The data presented here, along with existing cacao-omics resources, will enable targeted functional genetic screening of defense genes likely to play critical functions in cacao's defense against its pathogens.

Host sanctions in Panamanian Ficus are likely based on selective resource allocation.

PREMISE OF THE STUDY: Fig trees and their pollinators, fig wasps, present a powerful model system for studying mutualism stability: both partners depend on each other for reproduction, cooperation levels can be manipulated, and the resulting field-based fitness quantified. Previous work has shown that fig trees can severely reduce the fitness of wasps that do not pollinate by aborting unpollinated figs or reducing the number and size of wasp offspring. Here we evaluated four hypotheses regarding the mechanism of sanctions in four Panamanian fig species. METHODS: We examined wasp and fig samples from field experiments with manipulated levels of pollination. KEY RESULTS: In unpollinated figs, the fig wall and the wasp offspring had a lower dry mass. Unpollinated figs had as many initiated wasp galls as pollinated figs but fewer galls that successfully produced live wasp offspring. Across three experimentally increasing levels of pollination, we found nonlinear increases in fig wall mass, the proportion of wasp galls that develop, and wasp mass. CONCLUSIONS: Our data did not support the hypotheses that lack of pollination prevents gall formation or that fertilized endosperm is required for wasp development. While our data are potentially consistent with the hypothesis that trees produce a wasp-specific toxin in response to lack of pollination, we found the hypothesis that sanctions are a consequence of trees allocating more resources to better-pollinated figs more parsimonious with the aggregate data. Our findings are completely analogous to the selective resource allocation to more beneficial tissues documented in other mutualistic systems.

Field sampling of fig pollinator wasps across host species and host developmental phase: Implications for host recognition and specificity.

Previous genetic studies of pollinator wasps associated with a community of strangler figs (Ficus subgenus Urostigma, section Americana) in Central Panama suggest that the wasp species exhibit a range in host specificity across their host figs. To better understand factors that might contribute to this observed range of specificity, we used sticky traps to capture fig-pollinating wasp individuals at 13 Ficus species, sampling at different phases of the reproductive cycle of the host figs (e.g., trees with receptive inflorescences, or vegetative trees, bearing only leaves). We also sampled at other tree species, using them as non-Ficus controls. DNA barcoding allowed us to identify the wasps to species and therefore assign their presence and abundance to host fig species and the developmental phase of that individual tree. We found: (1) wasps were only very rarely captured at non-Ficus trees; (2) nonetheless, pollinators were captured often at vegetative individuals of some host species; (3) overwhelmingly, wasp individuals were captured at receptive host fig trees representing the fig species from which they usually emerge. Our results indicate that wasp occurrence is not random either spatially or temporally within the forest and across these hosts, and that wasp specificity is generally high, both at receptive and vegetative host trees. Therefore, in addition to studies that show chemicals produced by receptive fig inflorescences attract pollinator wasps, we suggest that other cues (e.g., chemicals produced by the leaves) can also play a role in host recognition. We discuss our results in the context of recent findings on the role of host shifts in diversification processes in the Ficus genus.

Pollinator and host sharing lead to hybridization and introgression in Panamanian free-standing figs, but not in their pollinator wasps.

Obligate pollination mutualisms, in which plant and pollinator lineages depend on each other for reproduction, often exhibit high levels of species specificity. However, cases in which two or more pollinator species share a single host species (host sharing), or two or more host species share a single pollinator species (pollinator sharing), are known to occur in current ecological time. Further, evidence for host switching in evolutionary time is increasingly being recognized in these systems. The degree to which departures from strict specificity differentially affect the potential for hybridization and introgression in the associated host or pollinator is unclear. We addressed this question using genome-wide sequence data from five sympatric Panamanian free-standing fig species (Ficus subgenus Pharmacosycea, section Pharmacosycea) and their six associated fig-pollinator wasp species (Tetrapus). Two of the five fig species, F. glabrata and F. maxima, were found to regularly share pollinators. In these species, ongoing hybridization was demonstrated by the detection of several first-generation (F1) hybrid individuals, and historical introgression was indicated by phylogenetic network analysis. By contrast, although two of the pollinator species regularly share hosts, all six species were genetically distinct and deeply divergent, with no evidence for either hybridization or introgression. This pattern is consistent with results from other obligate pollination mutualisms, suggesting that, in contrast to their host plants, pollinators appear to be reproductively isolated, even when different species of pollinators mate in shared hosts.

Precision of host sanctions in the fig tree-fig wasp mutualism: consequences for uncooperative symbionts.

Host sanctions that reduce the relative fitness of uncooperative symbionts provide a mechanism that can limit cheating and thus stabilise mutualisms over evolutionary timescales. Sanctions have been demonstrated empirically in several mutualisms. However, if multiple individual symbionts interact with each host, the precision with which individual cheating symbionts are targeted by host sanctions is critical to their short- and long-term effectiveness. No previous empirical study has directly addressed this issue. Here, we report the precision of host sanctions in the mutualism between fig trees and their pollinating wasps. Using field experiments and molecular parentage analyses, we show that sanctions in Ficus nymphaeifolia act at the level of entire figs (syconia), not at the level of the individual flowers within. Such fig-level sanctions allow uncooperative wasps, which do not bring pollen, to avoid sanctions in figs to which other wasps bring pollen. We discuss the relevance of sanction precision to other mutualisms.

Anonymous and EST-based microsatellite DNA markers that transfer broadly across the fig tree genus (Ficus, Moraceae).

PREMISE OF THE STUDY: We developed a set of microsatellite markers for broad utility across the species-rich pantropical tree genus Ficus (fig trees). The markers were developed to study population structure, hybridization, and gene flow in neotropical species. METHODS AND RESULTS: We developed seven novel primer sets from expressed sequence tag (EST) libraries of F. citrifolia and F. popenoei (subgen. Urostigma sect. Americana) and optimized five previously developed anonymous loci for cross-species amplification. The markers were successfully tested on four species from the basal subgenus Pharmacosycea sect. Pharmacosycea (F. insipida, F. maxima, F. tonduzii, and F. yoponensis) and seven species of the derived subgenus Urostigma (F. citrifolia, F. colubrinae, F. costaricana, F. nymphaeifolia, F. obtusifolia, F. pertusa, and F. popenoei). The 12 markers amplified consistently and displayed polymorphism in all the species. CONCLUSIONS: This set of microsatellite markers is transferable across the phylogenetic breadth of Ficus, and should therefore be useful for studies of population structure and gene flow in approximately 750 fig species worldwide.

Culture-free survey reveals diverse and distinctive fungal communities associated with developing figs (Ficus spp.) in Panama.

The ancient association of figs (Ficus spp.) and their pollinating wasps (fig wasps; Chalcidoidea, Hymenoptera) is one of the most interdependent plant-insect mutualisms known. In addition to pollinating wasps, a diverse community of organisms develops within the microcosm of the fig inflorescence and fruit. To better understand the multipartite context of the fig-fig wasp association, we used a culture-free approach to examine fungal communities associated with syconia of six species of Ficus and their pollinating wasps in lowland Panama. Diverse fungi were recovered from surface-sterilized flowers of all Ficus species, including gall- and seed flowers at four developmental stages. Fungal communities in syconia and on pollinating wasps were similar, dominated by diverse and previously unknown Saccharomycotina, and distinct from leaf- and stem endophyte communities in the same region. Before pollination, fungal communities were similar between gall- and seed flowers and among Ficus species. However, fungal communities differed significantly in flowers after pollination vs. before pollination, and between anciently diverged lineages of Ficus with active vs. passive pollination syndromes. Within groups of relatively closely related figs, there was little evidence for strict-sense host specificity between figs and particular fungal species. Instead, mixing of fungal communities among related figs, coupled with evidence for possible transfer by pollinating wasps, is consistent with recent suggestions of pollinator mixing within syconia. In turn, changes in fungal communities during fig development and ripening suggest an unexplored role of yeasts in the context of the fig-pollinator wasp mutualism.

Above- and belowground interactions drive habitat segregation between two cryptic species of tropical trees.

In the lowlands of central Panama, the Neotropical pioneer tree Trema micrantha (sensu lato) exists as two cryptic species: "landslide" Trema is restricted to landslides and road embankments, while "gap" Trema occurs mostly in treefall gaps. In this study, we explored the relative contributions of biotic interactions and physical factors to habitat segregation in T. micrantha. Field surveys showed that soils from landslides were significantly richer in available phosphorus and harbored distinct arbuscular mycorrhizal fungal (AMF) communities compared to gap soils. Greenhouse experiments designed to determine the effect of these abiotic and biotic differences showed that: (1) both landslide and gap species performed better in sterilized soil from their own habitat, (2) the availability of phosphorus and nitrogen was limiting in gap and landslide soils, respectively, (3) a standardized AMF inoculum increased performance of both species, but primarily on gap soils, and (4) landslide and gap species performed better when sterilized soils were inoculated with the microbial inoculum from their own habitat. A field experiment confirmed that survival and growth of each species was highest in its corresponding habitat. This experiment also showed that browsing damage significantly decreased survival of gap Trema on landslides. We conclude that belowground interactions with soil microbes and aboveground interactions with herbivores contribute in fundamental ways to processes that may promote and reinforce adaptive speciation.