150 Years of Coevolution Research: Evolution and Ecology of Yucca Moths (Prodoxidae) and Their Hosts.

Yucca moths (Tegeticula and Parategeticula) are specialized pollinators of yucca plants, possessing unique, tentacle-like mouthparts used to actively collect pollen and deposit it onto the flowers of their hosts. The moths' larvae feed on the developing seeds and fruit tissue. First described in 1873, the yucca-yucca moth pollination system is now considered the archetypical example of a coevolved intimate mutualism. Research conducted over the past three decades has transformed our understanding of yucca moth diversity and host plant interactions. We summarize the current understanding of the diversity, ecology, and evolution of this group, review evidence for coevolution of the insects and their hosts, and describe how the nature of the interaction varies across evolutionary time and ecological contexts. Finally, we identify unresolved questions and areas for future research.

Genome-scale data resolves the timing of divergence in Joshua trees.

PREMISE: Joshua trees (Yucca brevifolia and Y. jaegeriana) and their yucca moth pollinators (Tegeticula synthetica and T. antithetica) are a model system for studies of plant-pollinator coevolution and, they are thought to be one of the only cases in which there is compelling evidence for cospeciation driven by coevolution. Previous work attempted to evaluate whether divergence between the plant and their pollinators was contemporaneous. That work concluded that the trees diverged more than 5 million years ago-well before the pollinators. However, clear inferences were hampered by a lack of data from the nuclear genome and low genetic variation in chloroplast genes. As a result, divergence times in the trees could not be confidently estimated. METHODS: We present an analysis of whole chloroplast genome sequence data and RADseq data from >5000 loci in the nuclear genome. We developed a molecular clock for the Asparagales and the Agavoideae using multiple fossil calibration points. Using Bayesian inference, we produced new estimates for the age of the genus Yucca and for Joshua trees. We used calculated summary statistics describing genetic variation and used coalescent-based methods to estimate population genetic parameters. RESULTS: We find that the Joshua trees are moderately genetically differentiated, but that they diverged quite recently (~100-200 kya), and much more recently than their pollinators. CONCLUSIONS: The results argue against the notion that coevolution directly contributed to speciation in this system, suggesting instead that coevolution with pollinators may have reinforced reproductive isolation following initial divergence in allopatry.

Mitochondrial Divergence between Western and Eastern Great Bustards: Implications for Conservation and Species Status.

The great bustard is the heaviest bird capable of flight and an iconic species of the Eurasian steppe. Populations of both currently recognized subspecies are highly fragmented and critically small in Asia. We used DNA sequence data from the mitochondrial cytochrome b gene and the mitochondrial control region to estimate the degree of mitochondrial differentiation and rates of female gene flow between the subspecies. We obtained genetic samples from 51 individuals of Otis tarda dybowskii representing multiple populations, including the first samples from Kazakhstan and Mongolia and samples from near the Altai Mountains, the proposed geographic divide between the subspecies, allowing for better characterization of the boundary between the 2 subspecies. We compared these with existing sequence data (n = 66) from Otis tarda tarda. Our results suggest, though do not conclusively prove, that O. t. dybowskii and O. t. tarda may be distinct species. The geographic distribution of haplotypes, phylogenetic analysis, analyses of molecular variance, and coalescent estimation of divergence time and female migration rates indicate that O. t. tarda and O. t. dybowskii are highly differentiated in the mitochondrial genome, have been isolated for approximately 1.4 million years, and exchange much less than 1 female migrant per generation. Our findings indicate that the 2 forms should at least be recognized and managed as separate evolutionary units. Populations in Xinjiang, China and Khövsgöl and Bulgan, Mongolia exhibited the highest levels of genetic diversity and should be prioritized in conservation planning.

First Recorded Observations of Pollination and Oviposition Behavior in Tegeticula antithetica (Lepidoptera: Prodoxidae) Suggest a Functional Basis for Coevolution With Joshua Tree (Yucca) Hosts.

Yucca moths (Tegeticula spp.) are the exclusive pollinators of Joshua trees (Yucca brevifolia s. l.). The moths actively pollinate the Joshua tree flowers and lay their eggs in the style. Recent studies have revealed that the plants commonly known as Joshua trees include two distinct, sister-species of plant: Yucca brevifolia Engelm. and Yucca jaegeriana McKelvey, each pollinated by two sister-species of yucca moth Tegeticula synthetica Riley and Tegeticula antithetica Pellmyr, respectively. A number of studies have argued that the moths have coevolved with their hosts, producing a pattern of phenotype matching between moth ovipositor length and floral style length. However, the only known descriptions of yucca moth pollination and oviposition behavior on Joshua trees are observations of T. synthetica made in 1893. The behavior of T. antithetica has never been observed before. We produced the first video recordings of the behavior of T. antithetica, and measured the points of oviposition and egg placement within the floral style. We found a number of differences between the behaviors of T. antithetica and T. synthetica, which appear to be a consequence of differences in floral morphology between Y. jaegeriana and Y. brevifolia. We also found that variation in floral style length strongly influences the placement of eggs within the flower, which may explain patterns of phenotype matching described previously. However, unlike in other yucca moths, we find that the mode of oviposition is unlikely to wound the floral ovules, and thus that oviposition by T. antithetica is unlikely to prompt floral abscission.

Population genomics of divergence within an obligate pollination mutualism: Selection maintains differences between Joshua tree species.

PREMISE OF THE STUDY: Speciation is a complex process that can be shaped by many factors, from geographic isolation to interspecific interactions. In Joshua trees, selection from pollinators on style length has been hypothesized to contribute to the maintenance of differentiation between two hybridizing sister species. We used population genomics approaches to measure the extent of genetic differentiation between these species, test whether selection maintains differences between them, and determine whether genetic variants associated with style length show signatures of selection. METHODS: Using restriction-site-associated DNA (RAD)-sequencing, we identified 9516 single nucleotide polymorphisms (SNPs) across the Joshua tree genome. We characterized the genomic composition of trees in a narrow hybrid zone and used genomic scans to search for signatures of selection acting on these SNPs. We used a genome-wide association study to identify SNPs associated with variation in phenotypic traits, including style length, and asked whether those SNPs were overrepresented among the group under selection. KEY RESULTS: The two species were highly genetically differentiated (FST = 0.25), and hybrids were relatively rare in the hybrid zone. Approximately 20% of SNPs showed evidence of selection maintaining divergence. While SNPs associated with style length were overrepresented among those under selection (P << 0.0001), the same was true for SNPs associated with highly differentiated vegetative traits. CONCLUSIONS: The two species of Joshua tree are clearly genetically distinct, and selection is maintaining differences between them. We found that loci associated with differentiated traits were likely to be under selection. However, many traits other than style length appeared to be under selection. Together with the dearth of intermediate hybrids, these findings reveal that these taxa are more strongly diverged than previously suspected and that selection, likely on many targets, is maintaining separation where the two species meet and hybridize.

Historical biogeography: the new synthesis.

Since Darwin first noted the impact of past ice ages on the distribution of organisms, biogeographers have debated whether Pleistocene glaciations shaped evolutionary patterns. A new synthesis of population genetics and palaeoclimatology promises unprecedented insights into Pleistocene evolutionary history.

Strong Selection Against Early Generation Hybrids in Joshua Tree Hybrid Zone Not Explained by Pollinators Alone.

Coevolution frequently plays an important role in diversification, but the role of obligate pollination mutualisms in the maintenance of hybrid zones has rarely been investigated. Like most members of the genus Yucca, the two species of Joshua tree (Yucca brevifolia and Yucca jaegeriana) are involved in a tightly coevolved mutualism with yucca moths. There is strong evidence of a history of coevolution between Joshua trees and their moth pollinators. We use a geographic clines approach in the Joshua tree hybrid zone to ask if selection by the moths may currently contribute to maintaining separation between these species. We compare genomic, phenotypic, and pollinator frequency clines to test whether pollinators maintain the hybrid zone or follow it as passive participants. The results reveal dramatic overlapping genomic and pollinator clines, consistent with a narrow hybrid zone maintained by strong selection. Wider phenotypic clines and a chloroplast genomic cline displaced opposite the expected direction suggest that pollinators are not the main source of selection maintaining the hybrid zone. Rather, it seems that high levels of reproductive isolation, likely acting through multiple barriers and involving many parts of the genome, keep the hybrid zone narrow.

Divergence in an obligate mutualism is not explained by divergent climatic factors.

Adaptation to divergent environments creates and maintains biological diversity, but we know little about the importance of different agents of ecological divergence. Coevolution in obligate mutualisms has been hypothesized to drive divergence, but this contention has rarely been tested against alternative ecological explanations. Here, we use a well-established example of coevolution in an obligate pollination mutualism, Yucca brevifolia and its two pollinating yucca moths, to test the hypothesis that divergence in this system is the result of mutualists adapting to different abiotic environments as opposed to coevolution between mutualists. We used a combination of principal component analyses and ecological niche modeling to determine whether varieties of Y. brevifolia associated with different pollinators specialize on different environments. Yucca brevifolia occupies a diverse range of climates. When the two varieties can disperse to similar environments, they occupy similar habitats. This suggests that the two varieties have not specialized on distinct habitats. In turn, this suggests that nonclimatic factors, such as the biotic interaction between Y. brevifolia and its pollinators, are responsible for evolutionary divergence in this system.

Host specificity and reproductive success of yucca moths (Tegeticula spp. Lepidoptera: Prodoxidae) mirror patterns of gene flow between host plant varieties of the Joshua tree (Yucca brevifolia: Agavaceae).

Coevolution between flowering plants and their pollinators is thought to have generated much of the diversity of life on Earth, but the population processes that may have produced these macroevolutionary patterns remain unclear. Mathematical models of coevolution in obligate pollination mutualisms suggest that phenotype matching between plants and their pollinators can generate reproductive isolation. Here, we test this hypothesis using a natural experiment that examines the role of natural selection on phenotype matching between yuccas and yucca moths (Tegeticula spp.) in mediating reproductive isolation between two varieties of Joshua tree (Yucca brevifolia var. brevifolia and Y. brevifolia var. jaegeriana). Using passive monitoring techniques, DNA barcoding, microsatellite DNA genotyping, and sibship reconstruction, we track host specificity and the fitness consequences of host choice in a zone of sympatry. We show that the two moth species differ in their degree of host specificity and that oviposition on a foreign host plant results in the production of fewer offspring. This difference in host specificity between the two moth species mirrors patterns of chloroplast introgression from west to east between host varieties, suggesting that natural selection acting on pollinator phenotypes mediates gene flow and reproductive isolation between Joshua-tree varieties.

Distinguishing coevolution from covicariance in an obligate pollination mutualism: asynchronous divergence in Joshua tree and its pollinators.

Obligate pollination mutualisms--in which both plants and their pollinators are reliant upon one another for reproduction--represent some of the most remarkable coevolutionary interactions in the natural world. The intimacy and specificity of these interactions have led to the prediction that the plants and their pollinators may be prone to cospeciation driven by coevolution. Several studies have identified patterns of phylogenetic congruence that are consistent with this prediction, but it is difficult to determine the evolutionary process that underlies these patterns. Phylogenetic congruence might also be produced by extrinsic factors, such as a shared biogeographic history. We examine the biogeographic history of a putative case of codivergence in the obligate pollination mutualism between Joshua trees (Yucca brevifolia) and two sister species of pollinating yucca moths (Tegeticula spp.) We employ molecular phylogenetic methods and coalescent-based approaches, in combination with relaxed-clock estimates of absolute rates of molecular evolution, to analyze multi-locus sequence data from more than 30 populations of Y. brevifolia and its pollinators. The results indicate that the moth species diverged significantly (p < 0.01) more recently than their corresponding host populations, suggesting that the apparent codivergence is not an artifact of a shared biogeographic history.

Coevolution and divergence in the Joshua tree/yucca moth mutualism.

Theory suggests that coevolution drives diversification in obligate pollination mutualism, but it has been difficult to disentangle the effects of coevolution from other factors. We test the hypothesis that differential selection by two sister species of pollinating yucca moths (Tegeticula spp.) drove divergence between two varieties of the Joshua tree (Yucca brevifolia) by comparing measures of differentiation in floral and vegetative features. We show that floral features associated with pollination evolved more rapidly than vegetative features extrinsic to the interaction and that a key floral feature involved in the mutualism is more differentiated than any other and matches equivalent differences in the morphology of the pollinating moths. A phylogenetically based, ancestral states reconstruction shows that differences in moth morphology arose in the time since they first became associated with Joshua trees. These results suggest that coevolution, rather than extrinsic environmental factors, has driven divergence in this obligate pollination mutualism.

Pattern and timing of diversification in Yucca (Agavaceae): specialized pollination does not escalate rates of diversification.

The yucca-yucca moth interaction is one of the most well-known and remarkable obligate pollination mutualisms, and is an important study system for understanding coevolution. Previous research suggests that specialist pollinators can promote rapid diversification in plants, and theoretical work has predicted that obligate pollination mutualism promotes cospeciation between plants and their pollinators, resulting in contemporaneous, parallel diversification. However, a lack of information about the age of Yucca has impeded efforts to test these hypotheses. We used analyses of 4322 AFLP markers and cpDNA sequence data representing six non-protein-coding regions (trnT-trnL, trnL, trnL intron, trnL-trnF, rps16 and clpP intron 2) from all 34 species to recover a consensus organismal phylogeny, and used penalized likelihood to estimate divergence times and speciation rates in Yucca. The results indicate that the pollination mutualism did not accelerate diversification, as Yucca diversity (34 species) is not significantly greater than that of its non-moth-pollinated sister group, Agave sensu latissimus (240 species). The new phylogenetic estimates also corroborate the suggestion that the plant-moth pollination mutualism has at least two origins within the Agavaceae. Finally, age estimates show significant discord between the age of Yucca (ca 6-10Myr) and the current best estimates for the age of their pollinators (32-40Myr).

Comparative phylogeography of a coevolved community: concerted population expansions in Joshua trees and four yucca moths.

Comparative phylogeographic studies have had mixed success in identifying common phylogeographic patterns among co-distributed organisms. Whereas some have found broadly similar patterns across a diverse array of taxa, others have found that the histories of different species are more idiosyncratic than congruent. The variation in the results of comparative phylogeographic studies could indicate that the extent to which sympatrically-distributed organisms share common biogeographic histories varies depending on the strength and specificity of ecological interactions between them. To test this hypothesis, we examined demographic and phylogeographic patterns in a highly specialized, coevolved community--Joshua trees (Yucca brevifolia) and their associated yucca moths. This tightly-integrated, mutually interdependent community is known to have experienced significant range changes at the end of the last glacial period, so there is a strong a priori expectation that these organisms will show common signatures of demographic and distributional changes over time. Using a database of >5000 GPS records for Joshua trees, and multi-locus DNA sequence data from the Joshua tree and four species of yucca moth, we combined paleaodistribution modeling with coalescent-based analyses of demographic and phylgeographic history. We extensively evaluated the power of our methods to infer past population size and distributional changes by evaluating the effect of different inference procedures on our results, comparing our palaeodistribution models to Pleistocene-aged packrat midden records, and simulating DNA sequence data under a variety of alternative demographic histories. Together the results indicate that these organisms have shared a common history of population expansion, and that these expansions were broadly coincident in time. However, contrary to our expectations, none of our analyses indicated significant range or population size reductions at the end of the last glacial period, and the inferred demographic changes substantially predate Holocene climate changes.

How to become a yucca moth: Minimal trait evolution needed to establish the obligate pollination mutualism.

The origins of obligate pollination mutualisms, such as the classic yucca-yucca moth association, appear to require extensive trait evolution and specialization. To understand the extent to which traits truly evolved as part of establishing the mutualistic relationship, rather than being preadaptations, we used an expanded phylogenetic estimate with improved sampling of deeply-diverged groups to perform the first formal reconstruction of trait evolution in pollinating yucca moths and their non-pollinating relatives. Our analysis demonstrates that key life history traits of yucca moths, including larval feeding in the floral ovary and the associated specialized cutting ovipositor, as well as colonization of woody monocots in xeric habitats, may have been established before the obligate mutualism with yuccas. Given these preexisting traits, novel traits in the mutualist moths are limited to the active pollination behaviors and the tentacular appendages that facilitate pollen collection and deposition. These results suggest that a highly specialized obligate mutualism was built on the foundation of preexisting interactions between early Prodoxidae and their host plants, and arose with minimal trait evolution.

Evolutionary consequences of dispersal ability in cactus-feeding insects.

Although gene flow is an important determinant of evolutionary change, the role of ecological factors such as specialization in determining migration and gene flow has rarely been explored empirically. To examine the consequences of dispersal ability and habitat patchiness on gene flow, migration rates were compared in three cactophagous longhorn beetles using coalescent analyses of mtDNA sequences. Analyses of covariance were used to identify the roles of dispersal ability and habitat distribution in determining migration patterns. Dispersal ability was a highly significant predictor of gene flow (p< 0.001), and was more important than any other factor. These findings predict that dispersal ability may be an import factor shaping both microevolutionary and macroevolutionary patterns; this prediction is borne out by comparisons of species diversity in cactus-feeding groups.

Phylogeography of the longhorn cactus beetle Moneilema appressum LeConte (Coleoptera: Cerambycidae): was the differentiation of the Madrean sky islands driven by Pleistocene climate changes?

Although it has been suggested that Pleistocene climate changes drove population differentiation and speciation in many groups of organisms, population genetic evidence in support of this scenario has been ambiguous, and it has often been difficult to distinguish putative vicariance from simple isolation by distance. The sky island communities of the American Southwest present an ideal system in which to compare late Pleistocene range fragmentations documented by palaeoenvironmental studies with population genetic data from organisms within these communities. In order to elucidate the impact of Pleistocene climate fluctuations on these environments, biogeographic patterns in the flightless longhorn cactus beetle, Moneilema appressum were examined using mitochondrial DNA sequence data. Gene tree relationships between haplotypes were inferred using parsimony, maximum-likelihood, and Bayesian analysis. Nested clade analysis, Mantel tests, and coalescent modelling were employed to examine alternative biogeographic scenarios, and to test the hypothesis that Pleistocene climate changes drove population differentiation in this species. The program mdiv was used to estimate migration and divergence times between populations, and to measure the statistical support for isolation over ongoing migration. These analyses showed significant geographic structure in genetic relationships, and implicated topography as a key determinant of isolation. However, although the coalescent analyses suggested that a history of past habitat fragmentation underlies the observed geographic patterns, the nested clade analysis indicated that the pattern was consistent with isolation by distance. Estimated divergence times indicated that range fragmentation in M. appressum is considerably older than the end of the most recent glacial, but coincided with earlier interglacial warming events and with documented range expansions in other, desert-dwelling species of Moneilema.

Range expansions in the flightless longhorn cactus beetles, Moneilema gigas and Moneilema armatum, in response to Pleistocene climate changes.

Pollen cores and plant and animal fossils suggest that global climate changes at the end of the last glacial period caused range expansions in organisms indigenous to the North American desert regions, but this suggestion has rarely been investigated from a population genetic perspective. In order to investigate the impact of Pleistocene climate changes and glacial/interglacial cycling on the distribution and population structure of animals in North American desert communities, biogeographical patterns in the flightless, warm-desert cactus beetles, Moneilema gigas and Moneilema armatum, were examined using mitochondrial DNA (mtDNA) sequence data from the cytochrome oxidase I (COI) gene. Gene tree relationships between haplotypes were inferred using parsimony, maximum-likelihood, and Bayesian analysis. Nested clade analysis and coalescent modelling using the programs mdiv and fluctuate were used to identify demographically independent populations, and to test the hypothesis that Pleistocene climate changes caused recent range expansions in these species. A sign test was used to evaluate the probability of observing concerted population growth across multiple, independent populations. The phylogeographical and nested clade analyses reveal a history of northward expansion in both of these species, as well as a history of past range fragmentation, followed by expansion from refugia. The coalescent analyses provide highly significant evidence for independent range expansions from multiple refugia, but also identify biogeographical patterns that predate the most recent glacial period. The results indicate that widespread desert environments are more ancient than has been suggested in the past.