Predator-induced collapse of niche structure and species coexistence.

Biological invasions are both a pressing environmental challenge and an opportunity to investigate fundamental ecological processes, such as the role of top predators in regulating biodiversity and food-web structure. In whole-ecosystem manipulations of small Caribbean islands on which brown anole lizards (Anolis sagrei) were the native top predator, we experimentally staged invasions by competitors (green anoles, Anolis smaragdinus) and/or new top predators (curly-tailed lizards, Leiocephalus carinatus). We show that curly-tailed lizards destabilized the coexistence of competing prey species, contrary to the classic idea of keystone predation. Fear-driven avoidance of predators collapsed the spatial and dietary niche structure that otherwise stabilized coexistence, which intensified interspecific competition within predator-free refuges and contributed to the extinction of green-anole populations on two islands. Moreover, whereas adding either green anoles or curly-tailed lizards lengthened food chains on the islands, adding both species reversed this effect-in part because the apex predators were trophic omnivores. Our results underscore the importance of top-down control in ecological communities, but show that its outcomes depend on prey behaviour, spatial structure, and omnivory. Diversity-enhancing effects of top predators cannot be assumed, and non-consumptive effects of predation risk may be a widespread constraint on species coexistence.

The generality of cryptic dietary niche differences in diverse large-herbivore assemblages.

Ecological niche differences are necessary for stable species coexistence but are often difficult to discern. Models of dietary niche differentiation in large mammalian herbivores invoke the quality, quantity, and spatiotemporal distribution of plant tissues and growth forms but are agnostic toward food plant species identity. Empirical support for these models is variable, suggesting that additional mechanisms of resource partitioning may be important in sustaining large-herbivore diversity in African savannas. We used DNA metabarcoding to conduct a taxonomically explicit analysis of large-herbivore diets across southeastern Africa, analyzing ∼4,000 fecal samples of 30 species from 10 sites in seven countries over 6 y. We detected 893 food plant taxa from 124 families, but just two families-grasses and legumes-accounted for the majority of herbivore diets. Nonetheless, herbivore species almost invariably partitioned food plant taxa; diet composition differed significantly in 97% of pairwise comparisons between sympatric species, and dissimilarity was pronounced even between the strictest grazers (grass eaters), strictest browsers (nongrass eaters), and closest relatives at each site. Niche differentiation was weakest in an ecosystem recovering from catastrophic defaunation, indicating that food plant partitioning is driven by species interactions, and was stronger at low rainfall, as expected if interspecific competition is a predominant driver. Diets differed more between browsers than grazers, which predictably shaped community organization: Grazer-dominated trophic networks had higher nestedness and lower modularity. That dietary differentiation is structured along taxonomic lines complements prior work on how herbivores partition plant parts and patches and suggests that common mechanisms govern herbivore coexistence and community assembly in savannas.

Grazing herbivores reduce herbaceous biomass and fire activity across African savannas.

Fire and herbivory interact to alter ecosystems and carbon cycling. In savannas, herbivores can reduce fire activity by removing grass biomass, but the size of these effects and what regulates them remain uncertain. To examine grazing effects on fuels and fire regimes across African savannas, we combined data from herbivore exclosure experiments with remotely sensed data on fire activity and herbivore density. We show that, broadly across African savannas, grazing herbivores substantially reduce both herbaceous biomass and fire activity. The size of these effects was strongly associated with grazing herbivore densities, and surprisingly, was mostly consistent across different environments. A one-zebra increase in herbivore biomass density (~100 kg/km2 of metabolic biomass) resulted in a ~53 kg/ha reduction in standing herbaceous biomass and a ~0.43 percentage point reduction in burned area. Our results indicate that fire models can be improved by incorporating grazing effects on grass biomass.

Large herbivores suppress liana infestation in an African savanna.

African savannas are the last stronghold of diverse large-mammal communities, and a major focus of savanna ecology is to understand how these animals affect the relative abundance of trees and grasses. However, savannas support diverse plant life-forms, and human-induced changes in large-herbivore assemblages-declining wildlife populations and their displacement by livestock-may cause unexpected shifts in plant community composition. We investigated how herbivory affects the prevalence of lianas (woody vines) and their impact on trees in an East African savanna. Although scarce (<2% of tree canopy area) and defended by toxic latex, the dominant liana, Cynanchum viminale (Apocynaceae), was eaten by 15 wild large-herbivore species and was consumed in bulk by native browsers during experimental cafeteria trials. In contrast, domesticated ungulates rarely ate lianas. When we experimentally excluded all large herbivores for periods of 8 to 17 y (simulating extirpation), liana abundance increased dramatically, with up to 75% of trees infested. Piecewise exclusion of different-sized herbivores revealed functional complementarity among size classes in suppressing lianas. Liana infestation reduced tree growth and reproduction, but herbivores quickly cleared lianas from trees after the removal of 18-y-old exclosure fences (simulating rewilding). A simple model of liana contagion showed that, without herbivores, the long-term equilibrium could be either endemic (liana-tree coexistence) or an all-liana alternative stable state. We conclude that ongoing declines of wild large-herbivore populations will disrupt the structure and functioning of many African savannas in ways that have received little attention and that may not be mitigated by replacing wildlife with livestock.

The Difficulty of Predicting Evolutionary Change in Response to Novel Ecological Interactions: A Field Experiment with Anolis Lizards.

AbstractDetermining whether and how evolution is predictable is an important goal, particularly as anthropogenic disturbances lead to novel species interactions that could modify selective pressures. Here, we use a multigeneration field experiment with brown anole lizards (Anolis sagrei) to test hypotheses about the predictability of evolution. We manipulated the presence/absence of predators and competitors of A. sagrei across 16 islands in the Bahamas that had preexisting brown anole populations. Before the experiment and again after roughly five generations, we measured traits related to locomotor performance and habitat use by brown anoles and used double-digest restriction enzyme-associated DNA sequencing to estimate genome-wide changes in allele frequencies. Although previous work showed that predators and competitors had characteristic effects on brown anole behavior, diet, and population sizes, we found that evolutionary change at both phenotypic and genomic levels was difficult to forecast. Phenotypic changes were contingent on sex and habitat use, whereas genetic change was unpredictable and not measurably correlated with phenotypic changes, experimental treatments, or other environmental factors. Our work shows how differences in ecological context can alter evolutionary outcomes over short timescales and underscores the difficulty of forecasting evolutionary responses to multispecies interactions in natural conditions, even in a well-studied system with ample supporting ecological information.

Host phylogeny and functional traits differentiate gut microbiomes in a diverse natural community of small mammals.

Differences in the bacterial communities inhabiting mammalian gut microbiomes tend to reflect the phylogenetic relatedness of their hosts, a pattern dubbed phylosymbiosis. Although most research on this pattern has compared the gut microbiomes of host species across biomes, understanding the evolutionary and ecological processes that generate phylosymbiosis requires comparisons across phylogenetic scales and under similar ecological conditions. We analysed the gut microbiomes of 14 sympatric small mammal species in a semi-arid African savanna, hypothesizing that there would be a strong phylosymbiotic pattern associated with differences in their body sizes and diets. Consistent with phylosymbiosis, microbiome dissimilarity increased with phylogenetic distance among hosts, ranging from congeneric sets of mice and hares that did not differ significantly in microbiome composition to species from different taxonomic orders that had almost no gut bacteria in common. While phylosymbiosis was detected among just the 11 species of rodents, it was substantially weaker at this scale than in comparisons involving all 14 species together. In contrast, microbiome diversity and composition were generally more strongly correlated with body size, dietary breadth, and dietary overlap in comparisons restricted to rodents than in those including all lineages. The starkest divides in microbiome composition thus reflected the broad evolutionary divergence of hosts, regardless of body size or diet, while subtler microbiome differences reflected variation in ecologically important traits of closely related hosts. Strong phylosymbiotic patterns arose deep in the phylogeny, and ecological filters that promote functional differentiation of cooccurring host species may disrupt or obscure this pattern near the tips.

Large-herbivore nemabiomes: patterns of parasite diversity and sharing.

Amidst global shifts in the distribution and abundance of wildlife and livestock, we have only a rudimentary understanding of ungulate parasite communities and parasite-sharing patterns. We used qPCR and DNA metabarcoding of fecal samples to characterize gastrointestinal nematode (Strongylida) community composition and sharing among 17 sympatric species of wild and domestic large mammalian herbivore in central Kenya. We tested a suite of hypothesis-driven predictions about the role of host traits and phylogenetic relatedness in describing parasite infections. Host species identity explained 27-53% of individual variation in parasite prevalence, richness, community composition and phylogenetic diversity. Host and parasite phylogenies were congruent, host gut morphology predicted parasite community composition and prevalence, and hosts with low evolutionary distinctiveness were centrally positioned in the parasite-sharing network. We found no evidence that host body size, social-group size or feeding height were correlated with parasite composition. Our results highlight the interwoven evolutionary and ecological histories of large herbivores and their gastrointestinal nematodes and suggest that host identity, phylogeny and gut architecture-a phylogenetically conserved trait related to parasite habitat-are the overriding influences on parasite communities. These findings have implications for wildlife management and conservation as wild herbivores are increasingly replaced by livestock.

Evidence-based strategies to navigate complexity in dietary DNA metabarcoding: A reply.

It is clearly beneficial to eliminate low-abundance sequences that arise in error during dietary DNA metabarcoding studies, but to purge all low-abundance sequences is to risk eliminating real sequences and complicating ecological analyses. Our prior literature review noted that DNA sequence relative read abundance (RRA) thresholds can help ameliorate false-positive taxon occurrences, but that historical emphasis on this utility has fostered uncertainty about the associated risk of inflating the false-negative rate (Littleford-Colquhoun et al., 2022). To address this, we combined a simulation study and an empirical data set to both illustrate the issue and provide blueprints for simulation studies and sensitivity analyses that can help investigators avoid overcorrecting and thereby bolster confidence in ecological inferences. Awareness of both the costs and the benefits of abundance-filtering is needed because accurately characterizing dietary distributions can be critically important for understanding animal diets, nutrition and trophic networks. Highlighting the need to raise awareness, a critique of our paper emphasized the misleading notion that "false positive interactions between species can present fundamentally incorrect network structures in network ecology, whereas false negatives will provide a correct but incomplete version of the network" (Tercel & Cuff, 2022). Asserting that the reliability of results will be eroded by false positives but resilient to the omission of true positives is risky and runs counter to evidence. Unfortunately, abundance-filtering methods can introduce false negatives at higher rates than they eliminate false positives and thereby undermine the analysis of otherwise reliable sequencing data. Overcorrecting can qualitatively alter and ultimately undermine ecological interpretations.

The precautionary principle and dietary DNA metabarcoding: Commonly used abundance thresholds change ecological interpretation.

Dietary DNA metabarcoding enables researchers to identify and characterize trophic interactions with a high degree of taxonomic precision. It is also sensitive to sources of bias and contamination in the field and laboratory. One of the earliest and most common strategies for dealing with such sensitivities has been to remove all low-abundance sequences and conduct ecological analyses based on the presence or absence of food taxa. Although this step is now often perceived to be necessary, evidence of its sufficiency is lacking and more attention to the risk of introducing other errors is needed. Using computer simulations, we demonstrate that common strategies to remove low-abundance sequences can erroneously eliminate true dietary sequences in ways that impact downstream inferences. Using real data from well-studied wildlife populations in Yellowstone National Park, we further show how these strategies can markedly alter the composition of dietary profiles in ways that scale-up to obscure ecological interpretations about dietary generalism, specialism, and composition. Although the practice of removing low-abundance sequences may continue to be a useful strategy to address research questions that focus on a subset of relatively abundant foods, its continued widespread use risks generating misleading perceptions about the structure of trophic networks. Researchers working with dietary DNA metabarcoding data-or similar data such as environmental DNA, microbiomes, or pathobiomes-should be aware of drawbacks and consider alternative bioinformatic, experimental, and statistical solutions.

Covariation of diet and gut microbiome in African megafauna.

A major challenge in biology is to understand how phylogeny, diet, and environment shape the mammalian gut microbiome. Yet most studies of nonhuman microbiomes have relied on relatively coarse dietary categorizations and have focused either on individual wild populations or on captive animals that are sheltered from environmental pressures, which may obscure the effects of dietary and environmental variation on microbiome composition in diverse natural communities. We analyzed plant and bacterial DNA in fecal samples from an assemblage of 33 sympatric large-herbivore species (27 native, 6 domesticated) in a semiarid East African savanna, which enabled high-resolution assessment of seasonal variation in both diet and microbiome composition. Phylogenetic relatedness strongly predicted microbiome composition (r = 0.91) and was weakly but significantly correlated with diet composition (r = 0.20). Dietary diversity did not significantly predict microbiome diversity across species or within any species except kudu; however, diet composition was significantly correlated with microbiome composition both across and within most species. We found a spectrum of seasonal sensitivity at the diet-microbiome nexus: Seasonal changes in diet composition explained 25% of seasonal variation in microbiome composition across species. Species' positions on (and deviations from) this spectrum were not obviously driven by phylogeny, body size, digestive strategy, or diet composition; however, domesticated species tended to exhibit greater diet-microbiome turnover than wildlife. Our results reveal marked differences in the influence of environment on the degree of diet-microbiome covariation in free-ranging African megafauna, and this variation is not well explained by canonical predictors of nutritional ecology.

Experimental evidence that effects of megaherbivores on mesoherbivore space use are influenced by species' traits.

The extinction of 80% of megaherbivore (>1,000 kg) species towards the end of the Pleistocene altered vegetation structure, fire dynamics and nutrient cycling world-wide. Ecologists have proposed (re)introducing megaherbivores or their ecological analogues to restore lost ecosystem functions and reinforce extant but declining megaherbivore populations. However, the effects of megaherbivores on smaller herbivores are poorly understood. We used long-term exclusion experiments and multispecies hierarchical models fitted to dung counts to test (a) the effect of megaherbivores (elephant and giraffe) on the occurrence (dung presence) and use intensity (dung pile density) of mesoherbivores (2-1,000 kg), and (b) the extent to which the responses of each mesoherbivore species was predictable based on their traits (diet and shoulder height) and phylogenetic relatedness. Megaherbivores increased the predicted occurrence and use intensity of zebras but reduced the occurrence and use intensity of several other mesoherbivore species. The negative effect of megaherbivores on mesoherbivore occurrence was stronger for shorter species, regardless of diet or relatedness. Megaherbivores substantially reduced the expected total use intensity (i.e. cumulative dung density of all species) of mesoherbivores, but only minimally reduced the expected species richness (i.e. cumulative predicted occurrence probabilities of all species) of mesoherbivores (by <1 species). Simulated extirpation of megaherbivores altered use intensity by mesoherbivores, which should be considered during (re)introductions of megaherbivores or their ecological proxies. Species' traits (in this case shoulder height) may be more reliable predictors of mesoherbivores' responses to megaherbivores than phylogenetic relatedness, and may be useful for predicting responses of data-limited species.

Multiple dimensions of dietary diversity in large mammalian herbivores.

Theory predicts that trophic specialization (i.e. low dietary diversity) should make consumer populations sensitive to environmental disturbances. Yet diagnosing specialization is complicated both by the difficulty of precisely quantifying diet composition and by definitional ambiguity: what makes a diet 'diverse'? We sought to characterize the relationship between taxonomic dietary diversity (TDD) and phylogenetic dietary diversity (PDD) in a species-rich community of large mammalian herbivores in a semi-arid East African savanna. We hypothesized that TDD and PDD would be positively correlated within and among species, because taxonomically diverse diets are likely to include plants from many lineages. By using DNA metabarcoding to analyse 1,281 faecal samples collected across multiple seasons, we compiled high-resolution diet profiles for 25 sympatric large-herbivore species. For each of these populations, we calculated TDD and PDD with reference to a DNA reference library for local plants. Contrary to our hypothesis, measures of TDD and PDD were either uncorrelated or negatively correlated with each other. Thus, these metrics reflect distinct dimensions of dietary specialization both within and among species. In general, grazers and ruminants exhibited greater TDD, but lower PDD, than did browsers and non-ruminants. We found significant seasonal variation in TDD and/or PDD for all but four species (Grevy's zebra, buffalo, elephant, Grant's gazelle); however, the relationship between TDD and PDD was consistent across seasons for all but one of the 12 best-sampled species (plains zebra). Our results show that taxonomic generalists can be phylogenetic specialists, and vice versa. These two dimensions of dietary diversity suggest contrasting implications for efforts to predict how consumers will respond to climate change and other environmental perturbations. For example, populations with low TDD may be sensitive to phylogenetically 'random' losses of food species, whereas populations with low PDD may be comparatively more sensitive to environmental changes that disadvantage entire plant lineages-and populations with low dietary diversity in both taxonomic and phylogenetic dimensions may be most vulnerable of all.

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumer's diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi-quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population-level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.

Phylogeography of the Neotropical epiphytic orchid, Brassavola nodosa: evidence for a secondary contact zone in northwestern Costa Rica.

Spatial patterns of genetic variation can reveal otherwise cryptic evolutionary and landscape processes. In northwestern Costa Rica, an approximately concordant genetic discontinuity occurs among populations of several plant species. We conducted phylogeographic analyses of an epiphytic orchid, Brassavola nodosa, to test for genetic discontinuity and to explore its underlying causes. We genotyped 18 populations with 19 nuclear loci and two non-coding chloroplast sequence regions. We estimated genetic diversity and structure, relative importance of pollen and seed dispersal, and divergence time to understand how genetic diversity was spatially partitioned. Nuclear genetic diversity was high with little differentiation among populations (GSTn = 0.065). In contrast, chloroplast haplotypes were highly structured (GSTc = 0.570) and reveal a discontinuity between northwestern and southeastern populations within Costa Rica. Haplotype differences suggest two formerly isolated lineages that diverged ~10,000-100,000 YBP. Haplotype mixing and greater genetic diversity occur in an intermediate transition zone. Patterns of nuclear and chloroplast data were consistent. Different levels of genetic differentiation for the two genomes reflect the relative effectiveness of biotic versus abiotic dispersers of pollen and seeds, respectively. Isolation of the two lineages likely resulted from the complex environmental and geophysical history of the region. Our results suggest a recent cryptic seed dispersal barrier and/or zone of secondary contact. We hypothesize that powerful northeasterly trade winds hinder movement of wind-borne seeds between the two regions, while the multi-directional dispersal of pollen by strong-flying sphinx moths resulted in lower differentiation of nuclear loci.

DNA metabarcoding illuminates dietary niche partitioning by African large herbivores.

Niche partitioning facilitates species coexistence in a world of limited resources, thereby enriching biodiversity. For decades, biologists have sought to understand how diverse assemblages of large mammalian herbivores (LMH) partition food resources. Several complementary mechanisms have been identified, including differential consumption of grasses versus nongrasses and spatiotemporal stratification in use of different parts of the same plant. However, the extent to which LMH partition food-plant species is largely unknown because comprehensive species-level identification is prohibitively difficult with traditional methods. We used DNA metabarcoding to quantify diet breadth, composition, and overlap for seven abundant LMH species (six wild, one domestic) in semiarid African savanna. These species ranged from almost-exclusive grazers to almost-exclusive browsers: Grass consumption inferred from mean sequence relative read abundance (RRA) ranged from >99% (plains zebra) to <1% (dik-dik). Grass RRA was highly correlated with isotopic estimates of % grass consumption, indicating that RRA conveys reliable quantitative information about consumption. Dietary overlap was greatest between species that were similar in body size and proportional grass consumption. Nonetheless, diet composition differed between all species-even pairs of grazers matched in size, digestive physiology, and location-and dietary similarity was sometimes greater across grazing and browsing guilds than within them. Such taxonomically fine-grained diet partitioning suggests that coarse trophic categorizations may generate misleading conclusions about competition and coexistence in LMH assemblages, and that LMH diversity may be more tightly linked to plant diversity than is currently recognized.

Elephants in the understory: opposing direct and indirect effects of consumption and ecosystem engineering by megaherbivores.

Positive indirect effects of consumers on their resources can stabilize food webs by preventing overexploitation, but the coupling of trophic and non-trophic interactions remains poorly integrated into our understanding of community dynamics. Elephants engineer African savanna ecosystems by toppling trees and breaking branches, and although their negative effects on trees are well documented, their effects on small-statured plants remain poorly understood. Using data on 117 understory plant taxa collected over 7 yr within 36 1-ha experimental plots in a semi-arid Kenyan savanna, we measured the strength and direction of elephant impacts on understory vegetation. We found that elephants had neutral effects on most (83-89%) species, with a similar frequency of positive and negative responses among the remainder. Overall, estimated understory biomass was 5-14% greater in the presence of elephants across a range of rainfall levels. Whereas direct consumption likely accounts for the negative effects, positive effects are presumably indirect. We hypothesized that elephants create associational refuges for understory plants by damaging tree canopies in ways that physically inhibit feeding by other large herbivores. As predicted, understory biomass and species richness beneath elephant-damaged trees were 55% and 21% greater, respectively, than under undamaged trees. Experimentally simulated elephant damage increased understory biomass by 37% and species richness by 49% after 1 yr. Conversely, experimentally removing elephant damaged branches decreased understory biomass by 39% and richness by 30% relative to sham-manipulated trees. Camera-trap surveys revealed that elephant damage reduced the frequency of herbivory by 71%, whereas we detected no significant effect of damage on temperature, light, or soil moisture. We conclude that elephants locally facilitate understory plants by creating refuges from herbivory, which countervails the direct negative effects of consumption and enhances larger-scale biomass and diversity by promoting the persistence of rare and palatable species. Our results offer a counterpoint to concerns about the deleterious impacts of elephant "overpopulation" that should be considered in debates over wildlife management in African protected areas: understory species comprise the bulk of savanna plant biodiversity, and their responses to elephants are buffered by the interplay of opposing consumptive and non-consumptive interactions.

Heterogeneity of clonal patterns among patches of kudzu, Pueraria montana var. lobata, an invasive plant.

BACKGROUND AND AIMS: Viny species are among the most serious invasive plants, and better knowledge of how vines grow to dominate landscapes is needed. Patches may contain a single genotype (i.e. genet), a competitively dominant genet or many independent but interacting genets, yet the clonal structure of vining species is often not apparent. Molecular markers can discriminate among the genetic identities of entwined vines to reveal the number and spatial distribution of genets. This study investigated how genets are spatially distributed within and among discrete patches of the invasive vine kudzu, Pueraria montana var. lobata, in the United States. It was expected that ramets of genets would be spatially clustered within patches, and that an increase in the number of genets within a patch would be associated with a decrease in the average size of each genet. METHODS: Six discrete kudzu patches were sampled across 2 years, and 1257 samples were genotyped at 21 polymorphic allozyme loci. Variation in genotypic and genetic diversity among patches was quantified and patterns of genet interdigitation were analysed. KEY RESULTS: Substantial genotypic and genetic variation occurred within and among patches. As few as ten overlapping genets spanned up to 68 m(2) in one patch, while >90 % of samples were genetically unique in another patch. Genotypic diversity within patches increased as mean clone size decreased, although spatially widespread genets did not preclude interdigitation. Eight genets were shared across ≥2 patches, suggesting that vegetative dispersal can occur among patches. CONCLUSIONS: Genetically unique kudzu vines are highly interdigitated. Multiple vegetative propagules have become established in spatially discrete patches, probably through the movement of highway construction or maintenance machinery. The results suggest that common methods for controlling invasive vines (e.g. mowing) may inadvertently increase genotypic diversity. Thus, understanding vine architecture and growth has practical implications.

A CRISPR-based strategy for targeted sequencing in biodiversity science.

Many applications in molecular ecology require the ability to match specific DNA sequences from single- or mixed-species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target-specific enrichment capabilities of CRISPR-Cas systems may offer advantages in some applications. We identified 54,837 CRISPR-Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single- and mixed-species samples, which yielded mean chloroplast sequence lengths of 2,530-11,367 bp, depending on the experiment. In comparison to mixed-species experiments, single-species experiments yielded more on-target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed-species experiments yielded sufficient data to provide ≥48-fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL-P6 marker. Prior work developed CRISPR-based enrichment protocols for long-read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short-read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR-based analyses of mixed-species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.

Relative importance of pollen and seed dispersal across a Neotropical mountain landscape for an epiphytic orchid.

Populations of many species are isolated within narrow elevation bands of Neotropical mountain habitat, and how well dispersal maintains genetic connectivity is unknown. We asked whether genetic structure of an epiphytic orchid, Epidendrum firmum, corresponds to gaps between Costa Rican mountain ranges, and how these gaps influence pollen and seed flow. We predicted that significant genetic structure exists among mountain ranges due to different colonization histories and limited gene flow. Furthermore, we predicted that pollen movement contributes more to gene flow than seeds because seeds are released into strong winds perpendicular to the narrow northwest-southeast species distribution, while the likely pollinators are strong fliers. Individuals from 12 populations and three mountain ranges were genotyped with nuclear microsatellites (nDNA) and chloroplast sequences (cpDNA). Genetic diversity was high for both markers, while nDNA genetic structure was low (FSTn  = 0.020) and cpDNA structure was moderate (FSTc  = 0.443). Significant cpDNA barriers occurred within and among mountain ranges, but nDNA barriers were not significant after accounting for geographic distance. Consistent with these contrasting patterns of genetic structure, pollen contributes substantially more to gene flow among populations than seed (mp /ms  = 46). Pollinators mediated extensive gene flow, eroding nDNA colonization footprints, while seed flow was comparatively limited, possibly due to directional prevailing winds across linearly distributed populations. Dispersal traits alone may not accurately inform predictions about gene flow or genetic structure, supporting the need for research into the potentially crucial role of pollinators and landscape context in gene flow among isolated populations.

Highly diverse and spatially heterogeneous mycorrhizal symbiosis in a rare epiphyte is unrelated to broad biogeographic or environmental features.

Symbiotic interactions are common in nature. In dynamic or degraded environments, the ability to associate with multiple partners (i.e. broad specificity) may enable species to persist through fluctuations in the availability of any particular partner. Understanding how species interactions vary across landscapes is necessary to anticipate direct and indirect consequences of environmental degradation on species conservation. We asked whether mycorrhizal symbiosis by populations of a rare epiphytic orchid (Epidendrum firmum) is related to geographic or environmental heterogeneity. The latter would suggest that interactions are governed by environmental conditions rather than historic isolation of populations and/or mycorrhizal fungi. We used DNA-based methods to identify mycorrhizal fungi from eleven E. firmum populations in Costa Rica. We used molecular and phylogenetic analyses to compare associations. Epidendrum firmum exhibited broad specificity, associating with diverse mycorrhizal fungi, including six Tulasnellaceae molecular operational taxonomic units (MOTUs), five Sebacinales MOTUs and others. Notably, diverse mycorrhizal symbioses formed in disturbed pasture and roadside habitats. Mycorrhizal fungi exhibited significant similarity within populations (spatial and phylogenetic autocorrelation) and significant differences among populations (phylogenetic community dissimilarity). However, mycorrhizal symbioses were not significantly associated with biogeographic or environmental features. Such unexpected heterogeneity among populations may result from complex combinations of fine-scale environmental factors and macro-evolutionary patterns of change in mycorrhizal specificity. Thus, E. firmum exhibits broad specificity and the potential for opportunistic associations with diverse fungi. We suggest that these characteristics could confer symbiotic assurance when mycorrhizal fungi are stochastically available, which may be crucial in dynamic or disturbed habitats such as tropical forest canopies.