Ecological genomics of tropical trees: how local population size and allelic diversity of resistance genes relate to immune responses, cosusceptibility to pathogens, and negative density dependence.

In tropical forests, rarer species show increased sensitivity to species-specific soil pathogens and more negative effects of conspecific density on seedling survival (NDD). These patterns suggest a connection between ecology and immunity, perhaps because small population size disproportionately reduces genetic diversity of hyperdiverse loci such as immunity genes. In an experiment examining seedling roots from six species in one tropical tree community, we found that smaller populations have reduced amino acid diversity in pathogen resistance (R) genes but not the transcriptome in general. Normalized R gene amino acid diversity varied with local abundance and prior measures of differences in sensitivity to conspecific soil and NDD. After exposure to live soil, species with lower R gene diversity had reduced defence gene induction, more cosusceptibility of maternal cohorts to colonization by potentially pathogenic fungi, reduced root growth arrest (an R gene-mediated response) and their root-associated fungi showed lower induction of self-defence (antioxidants). Local abundance was not related to the ability to induce immune responses when pathogen recognition was bypassed by application of salicylic acid, a phytohormone that activates defence responses downstream of R gene signalling. These initial results support the hypothesis that smaller local tree populations have reduced R gene diversity and recognition-dependent immune responses, along with greater cosusceptibility to species-specific pathogens that may facilitate disease transmission and NDD. Locally rare species may be less able to increase their equilibrium abundance without genetic boosts to defence via immigration of novel R gene alleles from a larger and more diverse regional population.

Quantitative and evolutionary biology of alternative splicing: how changing the mix of alternative transcripts affects phenotypic plasticity and reaction norms.

Alternative splicing (AS) of pre-messenger RNA is a common phenomenon that creates different transcripts from a single gene, and these alternative transcripts affect phenotypes. The majority of AS research has examined tissue and developmental specificity of expression of particular AS transcripts, how this specificity affects cell function, and how aberrant AS is related to disease. Few studies have examined quantitative between-individual variation in AS within a cell or tissue type, or in relation to phenotypes, but the results are compelling: quantitative variation in AS affects plastic traits such as stress, anxiety, fear, egg production, muscle performance, energetics and plant growth. Genomic analyses of AS are also at a nascent stage, but have revealed a number of significant evolutionary patterns. Growing knowledge of upstream genes and kinases that regulate AS provides the as-yet little explored potential to examine how these genes and pathways respond to environmental and genotype variables. Research in this area can provide glimpses of a labyrinth of genetic architectures that have rarely been considered in evolutionary and organismal biology, or in quantitative genetics. The scarcity of contribution to knowledge about AS from these fields is illustrated by the fact that heritability of quantitative variation in AS has not yet been determined for any gene in any organism. New research tactics that incorporate quantitative analyses of AS will allow organismal and evolutionary biologists to attain a fuller mechanistic understanding of many of the traits they study, and may lead to more rapid discovery of functionally important polymorphisms.

Alternative splicing, muscle contraction and intraspecific variation: associations between troponin T transcripts, Ca(2+) sensitivity and the force and power output of dragonfly flight muscles during oscillatory contraction.

The flight muscles of Libellula pulchella dragonflies contain a mixture of six alternatively spliced transcripts of a single troponin T (TnT) gene. Here, we examine how intraspecific variation in the relative abundance of different TnT transcripts affects the Ca(2+) sensitivity of skinned muscle fibers and the performance of intact muscles during work-loop contraction regimes that approximate in vivo conditions during flight. The relative abundance of one TnT transcript, or the pooled relative abundance of two TnT transcripts, showed a positive correlation with a 10-fold range of variation in Ca(2+) sensitivity of skinned fibers (r(2)=0.77, P<0.0001) and a threefold range in peak specific force (r(2)=0.74, P<0.0001), specific work per cycle (r(2)=0.54; P<0.0001) and maximum specific power output (r(2)=0.48, P=0.0005) of intact muscle. Using these results to reanalyze previously published data for wing kinematics during free flight, we show that the relative abundances of these particular transcripts are also positively correlated with wingbeat frequency and amplitude. TnT variation alone may be responsible for these effects, or TnT variation may be a marker for changes in a suite of co-regulated molecules. Dragonflies from two ponds separated by 16 km differed significantly in both TnT transcript composition and muscle contractile performance, and within each population there are two distinct morphs that showed different maturational trajectories of TnT transcript composition and muscle contractility. Thus, there is broad intraspecific variability and a high degree of population structure for contractile performance phenotypes, TnT ribotypes and ontogenetic patterns involving these traits that affect locomotor performance.

Mite not make it home: tracheal mites reduce the safety margin for oxygen delivery of flying honeybees.

Many physiological systems appear to have safety margins, with excess capacity relative to normal functional needs, but the significance of such excess capacity remains controversial. In this study, we investigate the effects of parasitic tracheal mites (Acarapis woodi) on the safety margin for oxygen delivery and flight performance of honeybees. Tracheal mites did not affect the flight metabolic rate of honeybees in normoxic (21% oxygen) or hyperoxic (40% oxygen) air, but did reduce their metabolic rate relative to uninfected bees when flying in hypoxic air (5 or 10% oxygen), demonstrating that mites reduced the safety margin for tracheal oxygen delivery. The negative effects of mites on flight metabolic rate in hypoxic atmospheres were graded with the number of mites per trachea. For example, in 10% oxygen atmospheres, flight metabolic rate was reduced by 20% by moderate mite infection and by 40% by severe mite infection. Thus, the safety margin for oxygen delivery in honeybees allows them to retain normal flight metabolic rate and behavior despite tracheal mite infection under most conditions. However, the reduction in tracheal gas-exchange capacity may constrain activities requiring the highest metabolic rates, such as flying in cool weather. In support of this hypothesis, bees that were unable to return to the hive during late-winter flights showed significantly higher levels of mite infection than bees that returned safely.

Aerial performance of stalk-eyed flies that differ in eye span.

Stalk-eyed flies have eyes placed laterally away from the head on elongated peduncles. The elongation of eye span may increase the energetic cost of flight, reduce flight performance via aerodynamic effects or via increased load, or necessitate compensatory changes in other body dimensions. Body mass and body dimensions were measured to test the hypothesis that elongation of eye span is correlated with increased head mass in two closely related species of stalk-eyed flies. Cyrtodiopsis whitei is sexually dimorphic, with the eye span of larger males exceeding body length. Cyrtodiopsis quinqueguttata is sexually monomorphic with eye span substantially less than body length. Although eye span was significantly longer in C. whitei, head mass did not differ between species after accounting for differences in body mass. C. whitei males had longer wings, heavier thoraxes, and lighter abdomens in relation to body mass than did female C. whitei or C. quinqueguttata of either sex. Three-dimensional tracking of flight paths showed that path velocity and the horizontal component of velocity did not differ according to species or sex, but the long-eyed C. whitei males showed reduced overall aerial performance by flying at shallower ascent angles and reduced vertical velocity. Although increased mass loading does not occur in C. whitei males, increased drag, aerodynamic effects from the wake of the eye stalks, and constrained visual processing are possible mechanisms which could cause their reduced performance.

Surface-skimming stoneflies and mayflies: the taxonomic and mechanical diversity of two-dimensional aerodynamic locomotion.

The best supported hypothesis for the evolutionary origin of insect wings is that they evolved from articulated, leg-derived respiratory structures of aquatic ancestors. However, there are no fossils of the immediate ancestors of winged insects, and it is difficult to imagine how a functional transition from gills to wings could have occurred. Recent studies of surface-skimming locomotion in stoneflies and mayflies offer a plausible solution by showing how rudimentary wings and muscle power can be used to accomplish two-dimensional aerodynamic locomotion on the surface of water. Here we extend that line of research by examining the phylogenetic distribution and mechanistic diversity of surface skimming in stoneflies, along with a limited examination of mayflies. These investigations reveal both a broad taxonomic occurrence and a fine gradation of mechanically distinct forms. Distinct forms of wing-flapping surface skimming include (1) stoneflies that flap their wings weakly while maintaining their body in contact with the water and undulating their abdomen laterally in a swimming-like motion, (2) stoneflies that skim while elevating their body above the water and maintaining all six legs on the surface, (3) stoneflies and mayflies that skim with only four legs on the water surface, (4) stoneflies that skim with only their two hind legs on the surface, and (5) stoneflies that, beginning with a series of leg motions nearly identical to hind-leg skimmers, use their hind legs to jump from the water into the air to initiate flapping flight. Comparisons across these forms of skimming show that wing-beat amplitude, horizontal velocity, and the verticality of aerodynamic force production increase as the body orientation becomes more upright and contact with the water is minimized. These behaviors illustrate a mechanical pathway by which flying insects could have evolved from swimming ancestors via a series of finely graded intermediate stages. The phylogenetic distribution of skimming and flight in stoneflies does not indicate any clear directionality toward either greater or lesser aerodynamic abilities; however, the broad and apparently basal phylogenetic distribution of skimming taxa supports the hypothesis that the common ancestor of stoneflies was a surface skimmer. This may also be true for the common ancestor of stoneflies and mayflies, that is, the first winged insects. We combine these data with fossil evidence to form a synthetic model for the evolution of flying insects from surface skimmers.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior.

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Variability in the size, composition, and function of insect flight muscles.

In order to fly, insects require flight muscles that constitute at least 12 to 16% of their total mass, and flight performance increases as this percentage increases. However, flight muscles are energetically and materially expensive to build and maintain, and investment in flight muscles constrains other aspects of function, particularly female fecundity. This review examines ways in which insects vary the size of their flight muscles, and how variation in the relative size and composition of flight muscles affects flight performance. Sources of variability in flight muscle size and composition include genetic differences within and between species, individual phenotypic responses to environmental stimuli, and maturational changes that occur before and during the adult stage. Insects have evolved a wide variety of ways to adjust flight muscle size and contractile performance in order to meet demands imposed by variation in life history and ecology.

Alternative splicing, muscle calcium sensitivity, and the modulation of dragonfly flight performance.

Calcium sensitivity of myosin cross-bridge activation in striated muscles commonly varies during ontogeny and in response to alterations in muscle usage, but the consequences for whole-organism physiology are not well known. Here we show that the relative abundances of alternatively spliced transcripts of the calcium regulatory protein troponin T (TnT) vary widely in flight muscle of Libellula pulchella dragonflies, and that the mixture of TnT splice variants explains significant portions of the variation in muscle calcium sensitivity, wing-beat frequency, and an index of aerodynamic power output during free flight. Two size-distinguishable morphs differ in their maturational pattern of TnT splicing, yet they show the same relationship between TnT transcript mixture and calcium sensitivity and between calcium sensitivity and aerodynamic power output. This consistency of effect in different developmental and physiological contexts strengthens the hypothesis that TnT isoform variation modulates muscle calcium sensitivity and whole-organism locomotor performance. Modulating muscle power output appears to provide the ecologically important ability to operate at different points along a tradeoff between performance and energetic cost.

Aerial performance of Drosophila melanogaster from populations selected for upwind flight ability.

A computerized system for three-dimensional tracking of large numbers of individual free-flying insects was used to assess the performance of Drosophila melanogaster from populations that had undergone 160 generations of selection for upwind flight ability. Compared with control lines, the selected lines showed significant increases in mean flight velocity, decreases in angular trajectory and a significant change in the interaction between velocity and angular trajectory. Maximal flight velocity was apparent as a sharply defined upper boundary of the distribution of horizontal and vertical velocity as a function of angular trajectory; this upper bound (0.85 ms-1) differed little between the selected and control lines, although individuals from the selected lines attained maximal performance levels much more frequently. Maximum induced power output was calculated directly from the product of maximum vertical velocity and body weight. This measure (28 W kg-1 muscle) was closely predicted by a scaling relationship derived from the load-lifting limits of larger insects and vertebrates, as well as tethered D. melanogaster stimulated via their optomotor reflex to produce maximal lift. These results indicate that selection for flight performance can readily alter the relative effort and/or the frequency of phenotypes capable of attaining population-wise maximal performance levels, but shows little ability to increase population-wise maximal performance.

Surface-skimming stoneflies: a possible intermediate stage in insect flight evolution.

Insect wings appear to have evolved from gills used by aquatic forms for ventilation and swimming, yet the nature of intermediate stages remains a mystery. Here a form of nonflying aerodynamic locomotion used by aquatic insects is described, called surface skimming, in which thrust is provided by wing flapping while continuous contact with the water removes the need for total aerodynamic weight support. Stoneflies surface skim with wing areas and muscle power output severely reduced, which indicates that surface skimming could have been an effective form of locomotion for ancestral aquatic insects with small protowings and low muscle power output.

From damselflies to pterosaurs: how burst and sustainable flight performance scale with size.

Recent empirical data for short-burst lift and power production of flying animals indicate that mass-specific lift and power output scale independently (lift) or slightly positively (power) with increasing size. These results contradict previous theory, as well as simple observation, which argues for degradation of flight performance with increasing size. Here, empirical measures of lift and power during short-burst exertion are combined with empirically based estimates of maximum muscle power output in order to predict how burst and sustainable performance scale with body size. The resulting model is used to estimate performance of the largest extant flying birds and insects, along with the largest flying animals known from fossils. These estimates indicate that burst flight performance capacities of even the largest extinct fliers (estimated mass 250 kg) would allow takeoff from the ground; however, limitations on sustainable power output should constrain capacity for continuous flight at body sizes exceeding 0.003-1.0 kg, depending on relative wing length and flight muscle mass.

Intrapopulation variation in nectar secretion in Impatiens capensis.

Variation in nectar secretion rates within a population of Impatiens capensis was examined. There was no evidence for phenotypic variation between plants in nectar secretion rates. There was significant daily variation in secretion rates, which appeared to be caused by environmental factors. Individual flowers showed variable rates of secretion over consecutive hours, and these fluctuations in secretion occurred independently among the many flowers on any single plant. Time of day, plant size, and flower age (as indicated by sexual phase) showed no relationship to nectar secretion rates.