Buchnera has changed flatmate but the repeated replacement of co-obligate symbionts is not associated with the ecological expansions of their aphid hosts.

Symbiotic associations with bacteria have facilitated important evolutionary transitions in insects and resulted in long-term obligate interactions. Recent evidence suggests that these associations are not always evolutionarily stable and that symbiont replacement, and/or supplementation of an obligate symbiosis by an additional bacterium, has occurred during the history of many insect groups. Yet, the factors favouring one symbiont over another in this evolutionary dynamic are not well understood; progress has been hindered by our incomplete understanding of the distribution of symbionts across phylogenetic and ecological contexts. While many aphids are engaged into an obligate symbiosis with a single Gammaproteobacterium, Buchnera aphidicola, in species of the Lachninae subfamily, this relationship has evolved into a 'ménage à trois', in which Buchnera is complemented by a cosymbiont, usually Serratia symbiotica. Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. Most species host both Serratia and Buchnera but, in several clades, endosymbionts related to Sodalis, Erwinia or an unnamed member of the Enterobacteriaceae have replaced Serratia. Endosymbiont genome sequences from four aphid species confirm that these coresident symbionts fulfil essential metabolic functions not ensured by Buchnera. We further demonstrate through comparative phylogenetic analyses that cosymbiont replacement is not associated with the adaptation of aphids to new ecological conditions. We propose that symbiont succession was driven by factors intrinsic to the phenomenon of endosymbiosis, such as rapid genome deterioration or competitive interactions between bacteria with similar metabolic capabilities.

Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus.

The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecological studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each specimen studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences. With this approach, we show that (i) contaminants accounted for a negligible proportion of the bacteria identified in our samples; (ii) the taxonomic composition of our samples and the relative abundance of reads assigned to a taxon were very similar across PCR and DNA replicates for each aphid sample; in particular, bacterial DNA concentration had no impact on the results. Furthermore, by analysing the distribution of unique sequences across samples rather than aggregating them into operational taxonomic units (OTUs), we gained insight into the specificity of endosymbionts for their hosts. Our results confirm that Serratia symbiotica is often present in Cinara species, in addition to the primary symbiont, Buchnera aphidicola. Furthermore, our findings reveal new symbiotic associations with Erwinia- and Sodalis-related bacteria. We conclude with suggestions for generating and analysing 16S rRNA gene sequences for arthropod-endosymbiont studies.

Regular or covert sex defines two lineages and worldwide superclones within the leaf-curl plum aphid (Brachycaudus helichrysi, Kaltenbach).

Asexual reproduction occurs widely in plants and animals, particularly in insects. Aphid species usually reproduce by cyclic parthenogenesis, but many species include obligate asexual lineages. We recently showed that the leaf-curl plum aphid, Brachycaudus helichrysi, actually encompasses two lineages, B. helichrysi H1 and H2. Ecological data suggest that these lineages have different life cycles. We conducted a large population genetics study, based on 14 microsatellite loci, to infer their respective life cycles and investigate their population structure and geographical distribution. Brachycaudus helichrysi H1 displayed the genetic signature of cyclical parthenogenesis, using plum trees as primary hosts for sexual reproduction, as classically described for B. helichrysi. This global survey showed that the Central Asian population of H1 was clearly differentiated from American-European populations. By contrast, B. helichrysi H2 displayed the typical signature of obligate asexual reproduction. H2 encompassed at least eight highly successful genotypes or superclones. This lack of ability to undergo sexual reproduction was confirmed for one of the superclones by sex induction experiments. We found only one B. helichrysi H2 population that underwent sexual reproduction, which was collected from peach trees, in Northern India. Our results confirm that H1 and H2 have different life cycles. Brachycaudus helichrysi H1 is clearly heteroecious using plum trees as primary hosts, while B. helichrysi H2 encompasses several anholocyclic lineages, and some heteroecious populations that until now have only been found associated with peach trees as primary hosts. We discuss implications of these findings for the pest status of B. helichrysi lineages.

Inferring the origin of populations introduced from a genetically structured native range by approximate Bayesian computation: case study of the invasive ladybird Harmonia axyridis.

Correct identification of the source population of an invasive species is a prerequisite for testing hypotheses concerning the factors responsible for biological invasions. The native area of invasive species may be large, poorly known and/or genetically structured. Because the actual source population may not have been sampled, studies based on molecular markers may generate incorrect conclusions about the origin of introduced populations. In this study, we characterized the genetic structure of the invasive ladybird Harmonia axyridis in its native area using various population genetic statistics and methods. We found that native area of H. axyridis most probably consisted of two geographically distinct genetic clusters located in eastern and western Asia. We then performed approximate Bayesian computation (ABC) analyses on controlled simulated microsatellite data sets to evaluate (i) the risk of selecting incorrect introduction scenarios, including admixture between sources, when the populations of the native area are genetically structured and sampling is incomplete and (ii) the ability of ABC analysis to minimize such risks by explicitly including unsampled populations in the scenarios compared. Finally, we performed additional ABC analyses on real microsatellite data sets to retrace the origin of biocontrol and invasive populations of H. axyridis, taking into account the possibility that the structured native area may have been incompletely sampled. We found that the invasive population in eastern North America, which has served as the bridgehead for worldwide invasion by H. axyridis, was probably formed by an admixture between the eastern and western native clusters. This admixture may have facilitated adaptation of the bridgehead population.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010.

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Phylogeny of the genus Aphis Linnaeus, 1758 (Homoptera: Aphididae) inferred from mitochondrial DNA sequences.

Aphis is the largest aphid genus in the world and contains several of the most injurious aphid pests. It is also the most reluctant aphid genus to any comprehensive taxonomic treatment: while most species are easily classified into "species groups" that form well defined entities, numerous species within these groups are difficult to tell apart morphologically and identification keys remain ambiguous and mostly rely on host plant affiliation. In this paper, we used partial sequences of COI/COII and CytB genes to reconstruct the first phylogeny of Aphis and discuss the present systematics. The monophyly of the subgenus Bursaphis and of the tree major species groups, Black aphid, Black backed aphid and frangulae-like species was recovered by all phylogenetic analyses. However our data suggested that the nominal subgenus was not monophyletic. Relationships between major species groups were often ambiguous but "Black" and "Black backed" species groups appeared as sister clades. The most striking result of this study was that our molecular data met the same limits as the morphological characters used in classifications: mitochondrial DNA did not allow the differentiation of species that are difficult to identify. Further, interspecies relationships within groups of species for which taxonomic treatment is difficult stayed unresolved. This suggests that species delineation in the genus Aphis is often ambiguous and that diversification might have been a rapid process.

Patterns of diversification of Afrotropical Otiteselline fig wasps: phylogenetic study reveals a double radiation across host figs and conservatism of host association.

We studied the phylogenetic relationships of Otiteselline fig waSPS associated with Ficus in the Afrotropical region using rDNA sequences. African fig species usually host two species of Otiteselline fig waSPS. Phylogenetic analyses reveal that this pattern of association results from the radiation of two clades of waSPS superimposed on the fig system. Within each clade, wasp species generally cluster according to their host classification. The phylogenies of the two clades are also generally more congruent than expected by chance. Together these results suggest that Otiteselline wasp speciation is largely constrained by the diversification of their hosts. Finally, we show a difference in ovipositor length between the two Otiteselline species coexisting in the same Ficus species, which probably corresponds to ecological differences. The diversification of ecological niches within the fig is probably, with cospeciation, one of the key factors explaining the diversification and maintenance of species of parasites of the fig/pollinator system.

Pollination mode in fig wasps: the predictive power of correlated traits.

The over 700 species of Ficus are thought to have co-speciated with their obligate pollinators (family Agaonidae). Some of these wasp species pollinate figs actively, while others are passive pollinators. Based on direct observations of mode of pollination in 88 species, we show that mode of pollination can confidently be predicted from fig traits only (anther-to-ovule ratio) or from wasp traits only (presence of coxal combs). The presence of pollen pockets is not a predictor of mode of pollination. Data, direct and indirect, on 142 species, demonstrate numerous cases of the loss of active pollination and suggest one or few origins of active pollination. Hence, active pollination, an impressive example of the sophisticated traits that may result from mutualistic coevolution, depends on selective forces that can be overcome in some species, allowing reversions. Despite frequent loss, active pollination remains the predominant mode of pollination in Ficus.

Phylogenetic relationships, historical biogeography and character evolution of fig-pollinating wasps.

Nucleotide sequences from the cytochrome oxidase I (COI) gene were used to reconstruct phylogenetic relationships among 15 genera of fig-pollinating wasps. We present evidence supporting broad-level co-cladogenesis with respect to most but not all of the corresponding groups of figs. Using fossil evidence for calibrating a molecular clock for these data, we estimated the origin of the fig-wasp mutualism to have occurred ca. 90 million years ago. The estimated divergence times among the pollinator genera and their current geographical distributions corresponded well with several features of the break-up of the southern continents during the Late Cretaceous period. We then explored the evolutionary trajectories of two characteristics that hold profound consequences for both partners in the mutualism: the breeding system of the host (monoecious or dioecious) and pollination behaviour of the wasp (passive or active). The fig wasp mutualism exhibits extraordinarily long-term evolutionary stability despite clearly identifiable conflicts of interest between the interactors, which are reflected by the very distinct variations found on the basic mutualistic theme.

Is the exhaustion time at maximal aerobic speed an index of aerobic endurance?

The velocity of the last stage of the Montreal Track Test (MTT) has been measured in fifteen well trained runners. This velocity (vMTT) was assumed to be close to maximal aerobic running speed. In three different sessions, the subjects ran up to exhaustion at velocities corresponding to 95, 100 and 105% vMTT. The exhaustion time at 100 % vMTT (tlim100) was assumed to be an estimation of the exhaustion time corresponding to maximal aerobic speed. The relationship between exhaustion time (tlim) and distance (Dlim) in the case of running exercises at constant velocity until exhaustion can be described by a linear relationship (Dlim = D + b*tlim). The slope of the relation corresponds to a velocity (vcrit) which can be sustained for a long time. The values of vcrit were calculated from the results of running exercises performed at 95, 100 and 105% of vMTT. The present study showed that tlim at 100% vMTT (tlim100) was negatively correlated with vMTT and vcrit but that D and ratio vcrit/vMTT were independent of vMTT. A theoretical study based on models previously proposed for oxygen kinetics during supramaximal exercises (exponential model and Margaria's model) demonstrates that this negative relationship between vMTT and tlim100 can be explained by the kinetics of the accumulation of oxygen deficit (O2 def). tlim100 should also depend on VO2max, maximal oxygen deficit (Max O2 def) and the relative importance of anaerobic energy when a VO2 plateau is reached. Moreover, the value of tlim100 largely depends on the accuracy of the assessment of vMTT. Consequently, the exhaustion times corresponding to the different estimations of maximal aerobic speed on a track or a treadmill cannot be considered as valid indices of aerobic endurance.

Critical velocity of continuous and intermittent running exercise. An example of the limits of the critical power concept.

The relationship between exhaustion time (tlim) and distance Dlim for running exercises at constant velocity until exhaustion can be described by a linear relationship (Dlim = a + b tlim) whose slope corresponds to a critical velocity. Seven runners participated to the study which compared the critical velocity of continuous versus intermittent running exercises. The critical velocity for continuous running (Vcritc) was calculated from the results (tlimc and Dlimc) of running exercises performed at 95 and 105% of the final velocity of the Montreal Track Test (vMTT). The intermittent running consisted of repetitions of running exercises performed at 95 and 105% vMTT during a time equal to half the value of the corresponding tlimc. The subjects recovered during a time equal to running time while jogging at a slow pace. The critical velocity for intermittent running (Vcriti) was calculated from the cumulated running distance (Dlimi) and cumulated running time (tlimi) corresponding to 95 and 105% vMTT. Vcriti was equal to Vcritc (4.56 +/- 0.444 m.s-1 vs 4.60 +/- 0.416 m.s-1). Nevertheless, in some subjects, the repetition numbers were very different for the intermittent running exercises at 95 and 105% vMTT. This paradoxical result could be explained by the fact that the value of Vcrit should be theoretically little sensitive to a large error in the value of tlim corresponding to a velocity slightly higher than critical velocity, for intermittent exercises as well as continuous exercises.

The energy cost of running increases with the distance covered.

The net energy cost of running per unit of body mass and distance (Cr, ml O2.kg-1.km-1) was determined on ten amateur runners before and immediately after running 15, 32 or 42 km on an indoor track at a constant speed. The Cr was determined on a treadmill at the same speed and each run was performed twice. The average value of Cr, as determined before the runs, amounted to 174.9 ml O2.kg-1.km-1, SD 13.7. After 15 km, Cr was not significantly different, whereas it had increased significantly after 32 or 42 km, the increase ranging from 0.20 to 0.31 ml O2.kg-1.km-1 per km of distance (D). However, Cr before the runs decreased, albeit at a progressively smaller rate, with the number of trials (N), indicating an habituation effect (H) to treadmill running. The effects of D alone were determined assuming that Cr increased linearly with D, whereas H decreased exponentially with increasing N, i.e. Cr = Cr0 + a D + He-bN. The Cr0, the "true" energy cost of running in nonfatigued subjects accustomed to treadmill running, was assumed to be equal to the average value of Cr before the run for N equal to or greater than 7 (171.1 ml O2.kg-1.km-1, SD 12.7; n = 30).(ABSTRACT TRUNCATED AT 250 WORDS)