Effects of gene flow on phenotype matching between two varieties of Joshua tree (Yucca brevifolia; Agavaceae) and their pollinators.

In animal-pollinated plants, local adaptation to pollinator behaviour or morphology can restrict gene flow among plant populations; but gene flow may also prevent divergent adaptation. Here, we examine possible effects of gene flow on plant-pollinator trait matching in two varieties of Joshua tree (Agavaceae: Yucca brevifolia). The two varieties differ in strikingly in floral morphology, which matches differences in the morphology of their pollinators. However, this codivergence is not present at a smaller scale: within the two varieties of Joshua tree, variation in floral morphology between demes is not correlated with differences in moth morphology. We use population genetic data for Joshua tree and its pollinators to test the hypotheses that gene flow between Joshua tree populations is structured by pollinator specificity, and that gene flow within the divergent plant-pollinator associations 'swamps' fine-scale coadaptation. Our data show that Joshua tree populations are structured by pollinator association, but the two tree varieties are only weakly isolated - meaning that their phenotypic differences are maintained in the face of significant gene flow. Coalescent analysis of gene flow between the two Joshua tree types suggests that it may be shaped by asymmetric pollinator specificity, which has been observed in a narrow zone of sympatry. Finally, we find evidence suggesting that gene flow among Joshua tree sites may shape floral morphology within one plant-pollinator association, but not the other.

Absence of population-level phenotype matching in an obligate pollination mutualism.

Coevolution is thought to promote evolutionary change between demes that ultimately results in speciation. If this is the case, then we should expect to see similar patterns of trait matching and phenotypic divergence between populations and between species in model systems for coevolution. As measures of divergence are frequently only available at one scale (population level or taxon level), this contention is rarely tested directly. Here, we use the case of co-divergence between different varieties of Joshua tree Yucca brevifolia (Agavaceae) and their obligate pollinators, two yucca moths (Tegeticula spp. Prodoxidae), to test for trait matching between taxa and among populations. Using model selection, we show that there is trait matching between mutualists at the taxon level, but once we account for differences between taxa, there is no indication of trait matching in local populations. This result differs from similar studies in other coevolving systems. We hypothesize that this discrepancy arises because coevolution in obligate mutualisms favours divergence less strongly than coevolution in other systems, such as host­parasite interactions.

Host-associated divergence and incipient speciation in the yucca moth Prodoxus coloradensis (Lepidoptera: Prodoxidae) on three species of host plants.

A wide range of evolutionary processes have been implicated in the diversification of yuccas and yucca moths, which exhibit ecological relationships that extend from obligate plant-pollinator mutualisms to commensalist herbivory. Prodoxus coloradensis (Lepidoptera: Prodoxidae) is a yucca moth, which feeds on the flowering stalks of three Yucca species as larvae, but does not provide pollination service. To test for evidence of host-associated speciation, we examined the genetic structure of P. coloradensis using mitochondrial (cytochrome oxidase I) and nuclear (elongation factor 1 alpha) DNA sequence data. Multilocus coalescent simulations indicate that moths on different host plant species are characterized by recent divergence and low levels of effective migration, with large effective population sizes and considerable retention of shared ancestral polymorphism. Although geographical distance explains a proportion of the mitochondrial and nuclear DNA variation among moths on different species of Yucca, the effect of host specificity on genetic distance remains significant after accounting for spatial isolation. The results of this study indicate that differentiation within P. coloradensis is consistent with the evolution of incipient species affiliated with different host plants, potentially influenced by sex-biased dispersal and female philopatry.

Patterns of genetic structure among populations of an oligophagous pollinating yucca moth (Tegeticula yuccasella).

Plant-insect associations have served as models for investigations of coevolution and the influence of biotic interactions on diversification. The pollination association between yuccas and yucca moths is a classic example of an obligate mutualism often suggested to have been affected by coevolution. Recent work has shown high host specificity in pollinating yucca moths, and here we use Tegeticula yuccasella, the species with the widest diet breadth, to ask how host specificity and isolation by distance contribute to specialization. Isolation by distance at a regional scale was observed in nucleotide variation within the mitochondrial gene cytochrome oxidase I (COI) (r =.294; P =.003). Host-related genetic structure (F(ct) = 0.08) was found to be slightly lower than the level of structure observed between eastern and western moth populations (F(ct) = 0.096). However, 56% of the COI haplotypes sampled from moths on Yucca filamentosa mapped to a host-specific clade in the haplotype network. Taken together, these results suggest that differentiation among T. yuccasella populations on alternative hosts is slight, but gene flow is influenced by both host association and geographic distance.

Phylogeographic structure in the bogus yucca moth Prodoxus quinquepunctellus (Prodoxidae): comparisons with coexisting pollinator yucca moths.

The pollination mutualism between yucca moths and yuccas highlights the potential importance of host plant specificity in insect diversification. Historically, one pollinator moth species, Tegeticula yuccasella, was believed to pollinate most yuccas. Recent phylogenetic studies have revealed that it is a complex of at least 13 distinct species, eight of which are specific to one yucca species. Moths in the closely related genus Prodoxus also specialize on yuccas, but they do not pollinate and their larvae feed on different plant parts. Previous research demonstrated that the geographically widespread Prodoxus quinquepunctellus can rapidly specialize to its host plants and may harbor hidden species diversity. We examined the phylogeographic structure of P. quinquepunctellus across its range to compare patterns of diversification with six coexisting pollinator yucca moth species. Morphometric and mtDNA cytochrome oxidase I sequence data indicated that P. quinquepunctellus as currently described contains two species. There was a deep division between moth populations in the eastern and the western United States, with limited sympatry in central Texas; these clades are considered separate species and are redescribed as P. decipiens and P. quinquepunctellus (sensu stricto), respectively. Sequence data also showed a lesser division within P. quinquepunctellus s.s. between the western populations on the Colorado Plateau and those elsewhere. The divergence among the three emerging lineages corresponded with major biogeographic provinces, whereas AMOVA indicated that host plant specialization has been relatively unimportant in diversification. In comparison, the six pollinator species comprise three lineages, one eastern and two western. A pollinator species endemic to the Colorado Plateau has evolved in both of the western lineages. The east-west division and the separate evolution of two Colorado Plateau pollinator species suggest that similar biogeographic factors have influenced diversification in both Tegeticula and Prodoxus. For the pollinators, however, each lineage has produced a monophagous species, a pattern not seen in P. quinquepunctellus.

Phylogeography of the yucca moth Tegeticula maculata: the role of historical biogeography in reconciling high genetic structure with limited speciation.

Tegeticula maculata is one of the most ancient and morphologically variable lineages within the yucca moths, yet has apparently undergone little diversification in comparison with much younger yucca moth lineages that have rapidly diversified. A phylogeographic approach was used to determine the number of independent lineages within T. maculata and to examine whether these patterns corresponded with morphological differences between its subspecies maculata and extranea. Phylogenetic analysis of mitochondrial DNA sequence variation indicated that the two subspecies are in separate clades, but there was also an equally deep split within subspecies maculata. There was no evidence for gene flow among regions and there was considerable substructure within clades. The phylogeographic structure of moth populations among and within subspecies can be explained in part by historical biogeographic boundaries and increasingly patchy postglacial distribution of the exclusive host plant, Hesperoyucca whipplei. Local specialization and co-adaptation would be possible in the absence of apparent gene flow, yet gross morphological divergence is limited to the very old split between the subspecies. Sorting of ancient mitochondrial lineages followed by local genetic differentiation may explain the pattern of high genetic structure with limited speciation.

Pollen dispersal in Yucca filamentosa (Agavaceae): the paradox of self-pollination behavior by Tegeticula yuccasella (Prodoxidae).

We investigated pollen dispersal in an obligate pollination mutualism between Yucca filamentosa and Tegeticula yuccasella. Yucca moths are the only documented pollinator of yuccas, and moth larvae feed solely on developing yucca seeds. The quality of pollination by a female moth affects larval survival because flowers receiving small amounts of pollen or self-pollen have a high abscission probability, and larvae die in abscised flowers. We tested the prediction that yucca moths primarily perform outcross pollinations by using fluorescent dye to track pollen dispersal in five populations of Y. filamentosa. Dye transfers within plants were common in all populations (mean ± 1 SE, 55 ± 3.0%), indicating that moths frequently deposit self-pollen. Distance of dye transfers ranged from 0 to 50 m, and the mean number of flowering plants between the pollen donor and recipient was 5 (median = 0), suggesting that most pollen was transferred among near neighbors. A multilocus genetic estimate of outcrossing based on seedlings matured from open-pollinated fruits at one site was 94 ± 6% (mean ± 1 SD). We discuss why moths frequently deposit self-pollen to the detriment of their offspring and compare the yucca-yucca moth interaction with other obligate pollinator mutualisms in which neither pollinator nor plant benefit from self-pollination.

Forty million years of mutualism: evidence for eocene origin of the yucca-yucca moth association.

The obligate mutualism between yuccas and yucca moths is a major model system for the study of coevolving species interactions. Exploration of the processes that have generated current diversity and associations within this mutualism requires robust phylogenies and timelines for both moths and yuccas. Here we establish a molecular clock for the moths based on mtDNA and use it to estimate the time of major life history events within the yucca moths. Colonization of yuccas had occurred by 41.5 +/- 9.8 million years ago (Mya), with rapid life history diversification and the emergence of pollinators within 0-6 My after yucca colonization. A subsequent burst of diversification 3.2 +/- 1.8 Mya coincided with evolution of arid habitats in western North America. Derived nonpollinating cheater yucca moths evolved 1.26 +/- 0.96 Mya. The estimated age of the moths far predates the host fossil record, but is consistent with suggested host age based on paleobotanical, climatological, biogeographical, and geological data, and a tentative estimation from an rbcL-based molecular clock for yuccas. The moth data are used to establish three alternative scenarios of how the moths and plants have coevolved. They yield specific predictions that can be tested once a robust plant phylogeny becomes available.

Phylogeography and host association in a pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae).

Inferring the historical context of ecological diversification is an important step in understanding the way that population-level processes result in a diversity of species and interactions in communities. We performed a phylogeographic analysis of mitochondrial DNA haplotypes from the pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae) in order to determine the degree to which populations were structured according to geographical location and host-plant association. Ninety-eight individuals were sampled from 29 locations ranging from southern California to western Idaho. Restriction-site variation in 87 individuals (27 populations) was screened by digestion with 11 endonucleases, followed by Southern blotting; 38 restriction-site positions were mapped by double digests. Haplotypes were further defined by generating fragments 251 bases in length via PCR, screening them for sequence variation using denaturing gel gradient electrophoresis (DGGE), and sequencing the resulting variants. Parsimony analysis of the resulting 12 restriction-site and 15 sequence haplotypes indicated strong geographical structuring of populations: (i) most populations were monomorphic for haplotype; (ii) haplotypes from California and the Pacific Northwest (Oregon, Washington and Idaho) formed robust monophyletic groups. Population structure was significant both within and between the two regions, as reflected by NST. Patterns of host-plant association and haplotype phylogeny suggest that populations have recently undergone host-plant shifts in many different parts of the species range, although the direction and number of host shifts cannot be determined at the present level of sampling resolution.

Genetic consequences of specialization: yucca moth behavior and self-pollination in yuccas.

Reciprocal specialization in interspecific interactions, such as plant-pollinator mutualisms, increases the probability that either party can have detrimental effects on the other without the interaction being dissolved. This should be particularly apparent in obligate mutualisms, such as those that exist between yucca and yucca moths. Female moths collect pollen from yucca flowers, oviposit into floral ovaries, and then pollinate those flowers. Yucca moths, which are the sole pollinators of yuccas, impose a cost in the form of seed consumption by the moth larvae. Here we ask whether there also is a genetic cost through selfish moth behavior that may lead to high levels of self fertilization in the yuccas. Historically, it has been assumed that females leave a plant immediately after collecting pollen, but few data are available. Observations of a member of the Tegeticula yuccasella complex on Yucca filamentosa revealed that females remained on the plant and oviposited in 66% of all instances after observed pollen collections, and 51% of all moths were observed to pollinate the same plant as well. Manual cross and self pollinations showed equal development and retention of fruits. Subsequent trials to assess inbreeding depression by measuring seed weight, germination date, growth rate, and plant mass at 5 months revealed significant negative effects on seed weight and germination frequency in selfed progeny arrays. Cumulative inbreeding depression was 0.475, i.e., fitness of selfed seeds was expected to be less than half that of outcrossed seeds. Single and multilocus estimates of outcrossing rates based on allozyme analyses of open-pollinated progeny arrays did not differ from 1.0. The discrepancy between high levels of behavioral self-pollination by the moths and nearly complete outcrossing in mature seeds can be explained through selective foreign pollen use by the females, or, more likely, pollen competition or selective abortion of self-pollinated flowers during early stages of fruit development. Thus, whenever the proportion of pollinated flowers exceeds the proportion that can be matured to ripe fruit based on resource availability, the potential detrimental genetic effects imposed through geitonogamous pollinations can be avoided in the plants. Because self-pollinated flowers have a lower probability of retention, selection should act on female moths to move among plants whenever moth density is high enough to trigger abortion.

Non-mutualistic yucca moths and their evolutionary consequences.

Interspecific mutualisms are regarded as having evolved from antagonistic or commensalistic interactions, with most mutualisms remaining facultative but some having coevolved into obligate reciprocal dependency. Underlying mutualism is an intrinsic conflict between the parties, in that each is under selection for increased exploitation of the other. Theoretical models suggest that this conflict is a source of evolutionary instability, and that evolution of 'cheating' by one party may lead to reciprocal extinction. Here we present phylogenic evidence for reversal of an obligate mutualism: within the yucca moth complex, distinct cheater species derived from obligate pollinators inflict a heavy cost on their yucca hosts by laying their eggs but not pollinating the yucca. Phylogenetic data show the cheaters to have existed for a long time. Coexisting pollinators and cheaters are not sister taxa, supporting predictions that evolution of cheating within a single pollinator is evolutionarily unstable. Several lines of evidence support a hypothesis that host shifts preceded the reversal of obligate mutualism. Host or partner shifts is a mechanism that can provide a route of evolutionary escape among obligate mutualists in general.

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data.

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.

Multiple occurrences of mutualism in the yucca moth lineage.

The complex mutualism between yuccas and the moths that pollinate their flowers is regarded as one of the most obvious cases of coevolution. Studies of related genera show that at least two of the critical behavioral and life history traits suggested to have resulted from coevolved mutualism in yucca moths are plesiomorphic to the family. Another trait, oviposition into flowers, has evolved repeatedly within the family. One species with these traits, Greya politella, feeds on and pollinates plants of a different family, but pollination occurs through a different component of the oviposition behavior than in the yucca moths. Major differences compared with yucca moths and their hosts are that G. politella only passively pollinates its host and that copollinators often contribute to pollination. This analysis suggests that evolution of mutualism between yuccas and yucca moths may have required few behavioral and life history changes in the moths. The truly coevolved features of this interaction appear to be the evolution of active pollination by the moths, the associated morphological structures in the moths for carrying pollen, and the exclusion of copollinators by yuccas.

Evolution of insect pollination and angiosperm diversification.

Insect pollination is a prominent type of plant-insect mutualism that can provide clues to the nature of diffuse coevolution. A long-standing question in this field is to what extent the emergence of insect polination caused rapid diversification in the interacting organisms. Recent progress in systematics and paleontology is providing much of the information needed to address this issue.

Origins of variance in seed number and mass: interaction of sex expression and herbivory in Lomatium salmoniflorum.

As in many plant species, Lomatium salmoniflorum (Umbelliferae) individuals produce many flowers, only a subset of which produce mature seeds that escape seed parasitism and enter the seed bank. The interrelationships between the timing and number of flowers produced, sex expression, seed set, and seed parasitism were studied for their direct and indirect effects on the numbers and masses of viable seeds produced by individual plants. In a sample population of 369 plants that produced 161 386 flowers, 76% of the plants produced some hermaphroditic flowers. The percentage of hermaphroditic flowers increased significantly with the total number of flowers produced by a plant. Seed set was 65-90% in plants producing >600 flowers, but was highly variable in plants producing fewer flowers. Hand-pollinated plants showed the same pattern of seed set, suggesting that variable seed set in small plants may result from insufficient resources for seed development. The majority of schizocarps was produced by only 12% of the plants. Parasites killed 24.5% of the seeds prior to dispersal. Another 14.5% of the seeds lacked endosperm. Hence, the initial 161 386 flowers, which included 25874 hermaphroditic flowers each capable of producing two seeds, produced 42 468 seeds of which an estimated 25906 entered the seed bank as undamaged seeds with fully developed endosperm. Path analysis indicated that the number of hermaphroditic flowers on a plant and the percentage of seeds attacked by seed parasites had the greatest direct effects on the number of viable seeds entering the seed bank. The date at which a plant began flowering and the percentage of flowers setting seed had smaller or only indirect effects on viable seed production. Mean seed mass for plants was not significantly related to any of the factors that affected seed number, but little of the variance in seed mass occurred among plants. Masses of intact seeds in the population ranged 9-fold in both 1987 and 1988. Thirty-five percent of the variance was among seeds within umbels, 46% was among umbels within plants, and only 19% was among plants. The large variation among umbels within plants resulted from a seasonal pattern in which seeds from umbels produced late in the spring had lower mean seed masses than seeds from umbels produced early in the spring. Overall, the results indicate that both direct and indirect interactions between number of flowers, the date of initiation of flowering, seed set, and seed parasitism affect the number of viable seeds entering the seed bank. These interactions strongly bias viable seed output to a small minority of plants that produce many seeds with a wide range of masses over the growing season.

The pollination of zygogynum (winteraceae) by a moth, sabatinca (micropterigidae): an ancient association?

The primitive and vesselless angiosperm Zygogynum (Winteraceae), which is restricted to New Caledonia, is pollinated by a moth, Sabatinca (Micropterigidae). Fossil records of both the moth and the plant families extend to the Early Cretaceous. Adult Sabatinca have grinding mandibles and usually feed on the spores of ferns and on pollen. The insects use the flowers as mating sites and eat the pollen which is immersed in a dense pollenkitt. This mode of pollination in which flowers serve as mating and feeding stations with floral odors acting as cues may have been common in the early evolution of flowering plants.