Carlos Lopez-Vaamonde

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification. [Biogeography; coevolution; cospeciation; host switching; long-branch attraction; phylogeny.].

Alien Terrestrial Invertebrates of Europe

Unlike other groups of animals and plants, no checklist of alien terrestrial invertebrates was available in any of the European countries until recently. Since 2002, such checklists were successively provided by Austria (Essl and Rabitsch 2002), Germany (Geiter et al. 2002), the Czech Republic (Sefrova and Lastu vka 2005), Scandinavia (NOBANIS 2007), the United Kingdom (Hill et al. 2005), Switzerland (Wittenberg 2006) and Israel (Roll et al. 2007). However, most European regions remained uncovered and, furthermore, comparisons between the existing lists were inherently difficult because they used different definitions of alien. Thus, estimating the importance of terrestrial alien invertebrates at the European level remained impossible, mostly because of poor taxonomic knowledge existed for several groups. By gathering taxonomists and ecologists specialised on most invertebrate taxa together with collaborators working at the national level in 35 European countries, the DAISIE project intended to fill this gap. However, a lack of European expertise in some taxonomic groups did not allow coverage of all the terrestrial invertebrates with the same level of precision. Data on insects were more reliable than those of other taxa, and consequently the analyses presented below will mostly refer to this group.

EVOLUTIONARY DYNAMICS OF HOST-PLANT USE IN A GENUS OF LEAF-MINING MOTHS

Abstract We used nuclear 28S rDNA sequence data to estimate the phylogeny of 77 leaf-mining Phyllonorycter (Gracillariidae) moth species, including all 55 British species, feeding on 44 different plant genera. There was strong support for both the monophyly of Phyllonorycter and the placement of the genus Cameraria as its sister group. Host-plant use was mapped onto the moth phylogeny and investigated statistically in several ways. First, we show that the estimated level of cospeciation between leaf miners and their host plants is not greater than expected by chance, despite the physical intimacy of the association. Nevertheless, the pattern of host-plant use is far from random, with closely related Phyllonorycter species generally feeding on closely related plants. However, although Phyllonorycter species from a given host plant tend to form distinct clades, there is also statistical support for multiple independent colonizations of some host-plant taxa (e.g. the order Rosales and the genus Corylus). Despite numerous host shifts, most Phyllonorycter species feed on trees and the few species that attack shrubs or herbs have mostly acquired these habits independently. There is also limited evidence that host shifts to herbs are more likely from shrubs than from trees. Similarly, most species mine the lower surface of leaves but the few upper-surface miners have each evolved the habit independently. Consequently, these shifts to new adaptive zones have not led to substantial radiations.

Fossil-calibrated molecular phylogenies reveal that leaf-mining moths radiated millions of years after their host plants.

Coevolution has been hypothesized as the main driving force for the remarkable diversity of insect–plant associations. Dating of insect and plant phylogenies allows us to test coevolutionary hypotheses and distinguish between the contemporaneous radiation of interacting lineages vs. insect ‘host tracking’ of previously diversified plants. Here, we used nuclear DNA to reconstruct a molecular phylogeny for 100 species of Phyllonorycter leaf‐mining moths and 36 outgroup taxa. Ages for nodes in the moth phylogeny were estimated using a combination of a penalized likelihood method and a Bayesian approach, which takes into account phylogenetic uncertainty. To convert the relative ages of the moths into dates, we used an absolute calibration point from the fossil record. The age estimates of (a selection of) moth clades were then compared with fossil‐based age estimates of their host plants. Our results show that the principal radiation of Phyllonorycter leaf‐mining moths occurred well after the main radiation of their host plants and may represent the dominant associational mode in the fossil record.

Molecular analysis of parasitoid linkages (MAPL): gut contents of adult parasitoid wasps reveal larval host

Metamorphosing insects often have complex and poorly known life histories. In particular, what they feed on during their larval stages remains unknown for the vast majority of species, and its documentation only results from difficult and time‐intensive field observations, rearing or dissections. Through the application of a DNA analysis of gut contents in adult parasitoid wasps, we were able to selectively sequence a diagnostic DNA marker that permitted the identification of the host used by these wasps during their larval stages. By reproducing these results in species with different life histories, we excluded other potential sources of host DNA, confirming that after ingestion by the parasitoid larva the host DNA can persist through metamorphosis in the abdominal contents of the adult wasp. Our discovery considerably extends the applicability of molecular analysis of gut contents by enabling the documentation of food used by insects during their larval stages and thus increasing the accuracy and precision of food web studies. The 24% success rate of our approach is surprisingly high considering the challenging context for host DNA preservation, and we discuss the factors possibly affecting this rate. We propose molecular analysis of parasitoid linkages (MAPL) as a new method to document host–parasitoid associations at a faster pace and with unrivalled precision. Because of the key regulatory role of parasitoid wasps in ecosystems, which makes them the most commonly used biological control agents, MAPL will have immediate applications in both basic and applied biological sciences.

Mitochondrial and microsatellite DNA markers reveal a Balkan origin for the highly invasive horse‐chestnut leaf miner Cameraria ohridella (Lepidoptera, Gracillariidae)

Biological invasions usually start with a small number of founder individuals. These founders are likely to represent a small fraction of the total genetic diversity found in the source population. Our study set out to trace genetically the geographical origin of the horse‐chestnut leafminer, Cameraria ohridella, an invasive microlepidopteran whose area of origin is still unkown. Since its discovery in Macedonia 25 years ago, this insect has experienced an explosive westward range expansion, progressively colonizing all of Central and Western Europe. We used cytochrome oxidase I sequences (DNA barcode fragment) and a set of six polymorphic microsatellites to assess the genetic variability of C. ohridella populations, and to test the hypothesis that C. ohridella derives from the southern Balkans (Albania, Macedonia and Greece). Analysis of mtDNA of 486 individuals from 88 localities allowed us to identify 25 geographically structured haplotypes. In addition, 480 individuals from 16 populations from Europe and the southern Balkans were genotyped for 6 polymorphic microsatellite loci. High haplotype diversity and low measures of nucleotide diversities including a significantly negative Tajima’s D indicate that C. ohridella has experienced rapid population expansion during its dispersal across Europe. Both mtDNA and microsatellites show a reduction in genetic diversity of C. ohridella populations sampled from artificial habitats (e.g. planted trees in public parks, gardens, along roads in urban or sub‐urban areas) across Europe compared with C. ohridella sampled in natural stands of horse‐chestnuts in the southern Balkans. These findings suggest that European populations of C. ohridella may indeed derive from the southern Balkans.

Preservation of hymenopteran specimens for subsequent molecular and morphological study

Some guidelines are presented for long‐term preservation of insects for non‐traditional systematic studies such as DNA sequencing and internal anatomical research. The main criteria for good DNA preservation appear to be the rapidity with which the DNA is protected from enzymatic and chemical breakdown, and subsequent storage conditions. The immediate post mortem treatment of specimens appears particularly important to the preservation of amplifiable DNA. For storage we recommend 70–100% ethanol at low temperature, deep freezing, and critical point or chemical drying direct from alcohol. For internal anatomy, traditional fixatives, 70% ethanol in a refrigerator, critical point drying or chemical drying are recommended. Cold‐storage in 70% ethanol with or without subsequent critical point drying or chemical drying using hexamethyldisilazane allow both molecular and morphological study.

Molecular dating and biogeography of fig-pollinating wasps

Figs and fig-pollinating wasps are obligate mutualists that have coevolved for over 60 million years. But when and where did pollinating fig wasps (Agaonidae) originate? Some studies suggest that agaonids arose in the Late Cretaceous and the current distribution of fig-wasp faunas can be explained by the break-up of the Gondwanan landmass. However, recent molecular-dating studies suggest divergence time estimates that are inconsistent with the Gondwanan vicariance hypothesis and imply that long distance oceanic dispersal could have been an important process for explaining the current distribution of both figs and fig wasps. Here, we use a combination of phylogenetic and biogeographical data to infer the age, the major period of diversification, and the geographic origin of pollinating fig wasps. Age estimates ranged widely depending on the molecular-dating method used and even when using the same method but with slightly different constraints, making it difficult to assess with certainty a Gondwanan origin of agaonids. The reconstruction of ancestral areas suggests that the most recent common ancestor of all extant fig-pollinating wasps was most likely Asian, although a southern Gondwana origin cannot be rejected. Our analysis also suggests that dispersal has played a more important role in the development of the fig-wasp biota than previously assumed.

Increased gene sampling strengthens support for higher-level groups within leaf-mining moths and relatives (Lepidoptera: Gracillariidae)

BackgroundResearchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test.ResultsPartial augmentation of genes led to high support for deep divergences, especially when non-synonymous changes were analyzed alone. Increasing the number of taxa without an increase in number of characters led to lower bootstrap support; increasing the number of characters without increasing the number of taxa generally increased bootstrap support. More than three-quarters of nodes were supported with bootstrap values greater than 80% when all taxa and genes were combined. Gracillariidae, Lithocolletinae + Leucanthiza, and Acrocercops and Parectopa groups were strongly supported in nearly every analysis. Gracillaria group was well supported in some analyses, but less so in others. We find strong evidence for the exclusion of Douglasiidae from Gracillarioidea sensu Davis and Robinson (1998). Our results strongly support the monophyly of a G.B.R.Y. clade, a group comprised of Gracillariidae + Bucculatricidae + Roeslerstammiidae + Yponomeutidae, when analyzed with non-synonymous changes only, but this group was frequently split when synonymous and non-synonymous substitutions were analyzed together.Conclusions1) Partially or fully augmenting a data set with more characters increased bootstrap support for particular deep nodes, and this increase was dramatic when non-synonymous changes were analyzed alone. Thus, the addition of sites that have low levels of saturation and compositional heterogeneity can greatly improve results. 2) Gracillarioidea, as defined by Davis and Robinson (1998), clearly do not include Douglasiidae, and changes to current classification will be required. 3) Gracillariidae were monophyletic in all analyses conducted, and nearly all species can be placed into one of six strongly supported clades though relationships among these remain unclear. 4) The difficulty in determining the phylogenetic placement of Bucculatricidae is probably attributable to compositional heterogeneity at the third codon position. From our tests for compositional heterogeneity and strong bootstrap values obtained when synonymous changes are excluded, we tentatively conclude that Bucculatricidae is closely related to Gracillariidae + Roeslerstammiidae + Yponomeutidae.

Lifetime reproductive success and longevity of queens in an annual social insect.

Although central to understanding life‐history evolution, the relationship between lifetime reproductive success and longevity remains uncertain in many organisms. In social insects, no studies have reported estimates of queens’ lifetime reproductive success and longevity within populations, despite the importance of understanding how sociality and associated within‐group conflict affect life‐history traits. To address this issue, we studied two samples of colonies of the annual bumblebee, Bombus terrestris audax, reared from wild‐caught queens from a single population. In both samples, queens’ lifetime reproductive success, measured as either queens’ inclusive fitness or as total biomass of queen‐produced sexuals (new queens and males), was significantly positively associated with queen longevity, measured from the day the first worker was produced. We suggest that a positive relationship between reproductive success and longevity was inherited from nonsocial ancestors showing parental care and maintained, at least in part, because the presence of workers buffers queens against extrinsic mortality.