TAXONOMIC REASSESSMENT AND MORPHOLOGICAL REDESCRIPTION OF NEMATOSTRIGEA SERPENS ANNULATA (DIGENEA: STRIGEIDAE) FROM OSPREY IN NORTH AMERICA.

Digenean trematodes in the strigeid genus Nematostrigea are parasites of various birds, most often ospreys (Pandion haliaetus). Nematostrigea currently contains 2 species, Nematostrigea serpens and Nematostigea hepatica. Nematostrigea serpens is divided into 2 subspecies: N. serpens serpens from the Palearctic and Indomalayan realms and N. serpens annulata from the Nearctic realm. In the present work, we studied the type material of N. serpens annulata and collected new specimens from an osprey in Wisconsin close to the type locality. The original description and illustration of N. serpens annulata were incomplete. Herein we provide a detailed redescription and new illustrations of N. serpens annulata on the basis of the type material as well as newly collected and sequenced specimens. Nematostrigea serpens annulata has a distinct tegumental thickening near the mid-length of the genital cone, which is absent in its congeners. Partial sequences of the large ribosomal subunit (28S) DNA and cytochrome c oxidase subunit 1 (COI) mitochondrial DNA gene sequences were generated from our specimens of N. serpens annulata. Comparisons with previously published sequences of N. serpens serpens from the Palearctic demonstrated 2.2% difference in 28S and 3.7% in COI. On the basis of morphological and molecular comparisons, we elevate N. serpens annulata to species status and recognize it as Nematostrigea annulata Dubois and Rausch, 1948.

Atypical life cycle does not lead to inbreeding or selfing in parasites despite clonemate accumulation in intermediate hosts.

Many parasites utilize asexual and sexual reproduction and multiple hosts to complete their life cycles. How these taxa avoid inbreeding is an essential question for understanding parasite evolution and ecology. Aquatic trematodes that require multiple host species may benefit from diverse genetic parasite assemblages accumulating within second intermediate hosts prior to sexual reproduction in definitive hosts. However, Cotylurus species are able to utilize the same snail species as first and second intermediate hosts, potentially resulting in the accumulation of genetically identical clones (clonemates) prior to sexual reproduction. In this study, we developed and analysed novel microsatellite loci to determine if clones are accumulating within snail hosts prior to ingestion by bird hosts and the effects this could have on parasite inbreeding. Contrary to previous studies of aquatic trematodes, significantly large numbers of clonemates were present within snails, but full-sibs were not. Genetic structure was present over a relatively small geographical scale despite the use of vagile definitive hosts. Phylogenetic analysis identified the Cotylurus sp. clones as belonging to a single species. Despite the presence of clones within snails, mating between clones/selfing was not common and heterozygosity is maintained within individuals. Potential issues with clones mating may be mitigated by the presence of snails with numerous clones, the consumption of many snails by bird hosts and parasite clone recognition/avoidance. Use of the same host species for multiple life stages may have advantages when parasites are able to avoid inbreeding and the required hosts are common.

Molecular phylogeny of Diplostomum, Tylodelphys, Austrodiplostomum and Paralaria (Digenea: Diplostomidae) necessitates systematic changes and reveals a history of evolutionary host switching events.

The Diplostomidae Poirier, 1886 is a large, globally distributed family of digeneans parasitic in intestines of their definitive hosts. Diplostomum and Tylodelphys spp. are broadly distributed, commonly reported, and the most often sequenced diplostomid genera. The majority of published DNA sequences from these genera originated from larval stages only, which typically cannot be identified to the species level based on morphology alone. We generated partial large ribosomal subunit (28S) rRNA and cytochrome c oxidase subunit 1 (cox1) mtDNA gene sequences from 14 species/species-level lineages of Diplostomum, six species/species-level lineages of Tylodelphys, two species/species-level lineages of Austrodiplostomum, one species previously assigned to Paralaria, two species/species-level lineages of Dolichorchis and one unknown diplostomid. Our DNA sequences of 11 species/species-level lineages of Diplostomum (all identified to species), four species/species-level lineages of Tylodelphys (all identified to species), Austrodiplostomum compactum, Paralaria alarioides and Dolichorchis lacombeensis originated from adult specimens. 28S sequences were used for phylogenetic inference to demonstrate the position of Paralaria alarioides and Dolichorchis spp. within the Diplostomoidea and study the interrelationships of Diplostomum, Tylodelphys and Austrodiplostomum. Our results demonstrate that two diplostomids from the North American river otter (P. alarioides and a likely undescribed taxon) belong within Diplostomum. Further, our results demonstrate the non-monophyly of Tylodelphys due to the position of Austrodiplostomum spp., based on our phylogenetic analyses and morphology. Furthermore, the results of phylogenetic analysis of 28S confirmed the status of Dolichorchis as a separate genus. The phylogenies suggest multiple definitive host-switching events (birds to otters and among major avian groups) and a New World origin of Diplostomum and Tylodelphys spp. Our DNA sequences from adult digeneans revealed identities of 10 previously published lineages of Diplostomum and Tylodelphys, which were previously identified to genus only. The novel DNA data from this work provide opportunities for future comparisons of larval diplostomines collected in ecological studies.

Do New World pitvipers "scale-down" at high elevations? Macroecological patterns of scale characters and body size.

Bergmann's rule describes the macroecological pattern of increasing body size in response to higher latitudes and elevations. This pattern is extensively documented in endothermic vertebrates, within and among species; however, studies involving ectotherms are less common and suggest no consistent pattern for amphibians and reptiles. Moreover, adaptive traits, such as epidermal features like scales, have not been widely examined in conjunction with Bergmann's rule, even though these traits affect physiological processes, such as thermoregulation, which are hypothesized as underlying mechanisms for the pattern. Here, we investigate how scale characters correlate with elevation among 122 New World pitviper species, representing 15 genera. We found a contra-Bergmann's pattern, where body size is smaller at higher elevations. This pattern was mainly driven by the presence of small-bodied clades at high elevations and large-bodied clades at low elevations, emphasizing the importance of taxonomic scope in studying macroecological patterns. Within a subset of speciose clades, we found that only Crotalus demonstrated a significant negative relationship between body size and elevation, perhaps because of its wide elevational range. In addition, we found a positive correlation between scale counts and body size but no independent effect of elevation on scale numbers. Our study increases our knowledge of Bergmann's rule in reptiles by specifically examining characters of squamation and suggests a need to reexamine macroecological patterns for this group.

A revised molecular phylogeny reveals polyphyly in Schistura (Teleostei: Cypriniformes: Nemacheilidae).

There is a general consensus that the genus Schistura (Nemacheilidae), currently with 241 species, is not monophyletic. However, weak morphological synapomorphies and a lack of genetic data for most species of Schistura and their presumptive relatives have prevented meaningful diagnoses of species groups within this genus. To aid in deciphering evolutionary relationships, sequence data from two mitochondrial genes (cytochrome b and D-loop) were implemented in phylogenetic analyses for species of Schistura and other nemacheilids for which data from earlier studies and recently collected material were available. This analysis of 67 nemacheilid species, including 28 species of Schistura, provides the most comprehensive phylogeny of Nemacheilidae to date. In the phylogenetic tree for the combined data set, species of Schistura clustered in three clades. One clade contained 14 species of Schistura and Sectoria heterognathos and was sister to Homatula. A second clade of 11 species of Schistura was in a larger clade with Turcinoemacheilus kosswigi and Nemacheilus corica. The third clade contained three species, all from the Mae Khlong basin of Thailand. Taxonomic implications of these results are discussed; however, a more taxon-rich dataset and nuclear sequence data are needed before making taxonomic changes.

Experimental investigation of alternative transmission functions: Quantitative evidence for the importance of nonlinear transmission dynamics in host-parasite systems.

Understanding pathogen transmission is crucial for predicting and managing disease. Nonetheless, experimental comparisons of alternative functional forms of transmission remain rare, and those experiments that are conducted are often not designed to test the full range of possible forms. To differentiate among 10 candidate transmission functions, we used a novel experimental design in which we independently varied four factors-duration of exposure, numbers of parasites, numbers of hosts and parasite density-in laboratory infection experiments. We used interactions between amphibian hosts and trematode parasites as a model system and all candidate models incorporated parasite depletion. An additional manipulation involving anaesthesia addressed the effects of host behaviour on transmission form. Across all experiments, nonlinear transmission forms involving either a power law or a negative binomial function were the best-fitting models and consistently outperformed the linear density-dependent and density-independent functions. By testing previously published data for two other host-macroparasite systems, we also found support for the same nonlinear transmission forms. Although manipulations of parasite density are common in transmission studies, the comprehensive set of variables tested in our experiments revealed that variation in density alone was least likely to differentiate among competing transmission functions. Across host-pathogen systems, nonlinear functions may often more accurately represent transmission dynamics and thus provide more realistic predictions for infection.

Effects of Echinostoma trivolvis metacercariae infection during development and metamorphosis of the wood frog (Lithobates sylvaticus).

Many organisms face energetic trade-offs between defense against parasites and other host processes that may determine overall consequences of infection. These trade-offs may be particularly evident during unfavorable environmental conditions or energetically demanding life history stages. Amphibian metamorphosis, an ecologically important developmental period, is associated with drastic morphological and physiological changes and substantial energetic costs. Effects of the trematode parasite Echinostoma trivolvis have been documented during early amphibian development, but effects during later development and metamorphosis are largely unknown. Using a laboratory experiment, we examined the energetic costs of late development and metamorphosis coupled with E. trivolvis infection in wood frogs, Lithobates [=Rana] sylvaticus. Echinostoma infection intensity did not differ between tadpoles examined prior to and after completing metamorphosis, suggesting that metacercariae were retained through metamorphosis. Infection with E. trivolvis contributed to a slower growth rate and longer development period prior to the initiation of metamorphosis. In contrast, E. trivolvis infection did not affect energy expenditure during late development or metamorphosis. Possible explanations for these results include the presence of parasites not interfering with pronephros degradation during metamorphosis or the mesonephros compensating for any parasite damage. Overall, the energetic costs of metamorphosis for wood frogs were comparable to other species with similar life history traits, but differed from a species with a much shorter duration of metamorphic climax. Our findings contribute to understanding the possible role of energetic trade-offs between parasite defense and host processes by considering parasite infection with simultaneous energetic demands during a sensitive period of development.

Population-specific toxicity of six insecticides to the trematode Echinoparyphium sp.

The ubiquitous use of pesticides has increased concerns over their direct and indirect effects on disease dynamics. While studies examining the effects of pesticides on host-parasite interactions have largely focused on how pesticides influence the host, few studies have considered the effects of pesticides on parasites. We investigated the toxicity of six common insecticides at six environmentally-relevant concentrations to cercariae of the trematode Echinoparyphium from two populations. All six insecticides reduced the survival of cercariae (overall difference between mortality in control vs pesticide exposure = 86·2 ± 8·7%) but not in a predictable dose-dependent manner. These results suggest that Echinoparyphium are sensitive to a broad range of insecticides commonly used in the USA. The lack of a clear dose-dependent response in Echinoparyphium highlights the potential limitations of toxicity assays in predicting pesticide toxicity to parasites. Finally, population-level variation in cercarial susceptibility to pesticides underscores the importance of accounting for population variation as overlooking this variation can limit our ability to predict toxicity in nature. Collectively, this work demonstrates that consideration of pesticide toxicity to parasites is important to understanding how pesticides ultimately shape disease dynamics in nature.

It's a predator-eat-parasite world: how characteristics of predator, parasite and environment affect consumption.

Understanding the effects of predation on disease dynamics is increasingly important in light of the role ecological communities can play in host-parasite interactions. Surprisingly, however, few studies have characterized direct predation of parasites. Here we used an experimental approach to show that consumption of free-living parasite stages is highly context dependent, with significant influences of parasite size, predator size and foraging mode, as well as environmental condition. Among the four species of larval trematodes and two types of predators (fish and larval damselflies) studied here, parasites with larger infective stages (size >1,000 µm) were most vulnerable to predation by fish, while small-bodied fish and damselflies (size <10 mm) consumed the most infectious stages. Small parasite species (size approx. 500 µm) were less frequently consumed by both fish and larval damselflies. However, these results depended strongly on light availability; trials conducted in the dark led to significantly fewer parasites consumed overall, especially those with a size of <1,000 µm, emphasizing the importance of circadian shedding times of parasite free-living stages for predation risk. Intriguingly, active predation functioned to help limit fishes' infection by directly penetrating parasite species. Our results are consistent with established theory developed for predation on zooplankton that emphasizes the roles of body size, visibility and predation modes and further suggest that consumer-resource theory may provide a predictive framework for when predators should significantly influence parasite transmission. These results contribute to our understanding of transmission in natural systems, the role of predator-parasite links in food webs and the evolution of parasite morphology and behavior.

The scaling of host density with richness affects the direction, shape, and detectability of diversity-disease relationships.

Pathogen transmission responds differently to host richness and abundance, two unique components of host diversity. However, the heated debate around whether biodiversity generally increases or decreases disease has not considered the relationships between host richness and abundance that may exist in natural systems. Here we use a multi-species model to study how the scaling of total host community abundance with species richness mediates diversity-disease relationships. For pathogens with density-dependent transmission, non-monotonic trends emerge between pathogen transmission and host richness when host community abundance saturates with richness. Further, host species identity drives high variability in pathogen transmission in depauperate communities, but this effect diminishes as host richness accumulates. Using simulation we show that high variability in low richness communities and the non-monotonic relationship observed with host community saturation may reduce the detectability of trends in empirical data. Our study emphasizes that understanding the patterns and predictability of host community composition and pathogen transmission mode will be crucial for predicting where and when specific diversity-disease relationships should occur in natural systems.

Complex life cycles in a pond food web: effects of life stage structure and parasites on network properties, trophic positions and the fit of a probabilistic niche model.

Most food webs use taxonomic or trophic species as building blocks, thereby collapsing variability in feeding linkages that occurs during the growth and development of individuals. This issue is particularly relevant to integrating parasites into food webs because parasites often undergo extreme ontogenetic niche shifts. Here, we used three versions of a freshwater pond food web with varying levels of node resolution (from taxonomic species to life stages) to examine how complex life cycles and parasites alter web properties, the perceived trophic position of organisms, and the fit of a probabilistic niche model. Consistent with prior studies, parasites increased most measures of web complexity in the taxonomic species web; however, when nodes were disaggregated into life stages, the effects of parasites on several network properties (e.g., connectance and nestedness) were reversed, due in part to the lower trophic generality of parasite life stages relative to free-living life stages. Disaggregation also reduced the trophic level of organisms with either complex or direct life cycles and was particularly useful when including predation on parasites, which can inflate trophic positions when life stages are collapsed. Contrary to predictions, disaggregation decreased network intervality and did not enhance the fit of a probabilistic niche model to the food webs with parasites. Although the most useful level of biological organization in food webs will vary with the questions of interest, our results suggest that disaggregating species-level nodes may refine our perception of how parasites and other complex life cycle organisms influence ecological networks.

Does life history mediate changing disease risk when communities disassemble?

Biodiversity loss sometimes increases disease risk or parasite transmission in humans, wildlife and plants. Some have suggested that this pattern can emerge when host species that persist throughout community disassembly show high host competence - the ability to acquire and transmit infections. Here, we briefly assess the current empirical evidence for covariance between host competence and extirpation risk, and evaluate the consequences for disease dynamics in host communities undergoing disassembly. We find evidence for such covariance, but the mechanisms for and variability around this relationship have received limited consideration. This deficit could lead to spurious assumptions about how and why disease dynamics respond to community disassembly. Using a stochastic simulation model, we demonstrate that weak covariance between competence and extirpation risk may account for inconsistent effects of host diversity on disease risk that have been observed empirically. This model highlights the predictive utility of understanding the degree to which host competence relates to extirpation risk, and the need for a better understanding of the mechanisms underlying such relationships.

Biomass and productivity of trematode parasites in pond ecosystems.

1. Ecologists often measure the biomass and productivity of organisms to understand the importance of populations and communities in the flow of energy through ecosystems. Despite the central role of such studies in the advancement of freshwater ecology, there has been little effort to incorporate parasites into studies of freshwater energy flow. This omission is particularly important considering the roles that parasites sometimes play in shaping community structure and ecosystem processes. 2. Using quantitative surveys and dissections of over 1600 aquatic invertebrate and amphibian hosts, we calculated the ecosystem-level biomass and productivity of trematode parasites alongside the biomass of free-living aquatic organisms in three freshwater ponds in California, USA. 3. Snails and amphibian larvae, which are both important intermediate trematode hosts, dominated the dry biomass of free-living organisms across ponds (snails = 3.2 g m(-2); amphibians = 3.1 g m(-2)). An average of 33.5% of mature snails were infected with one of six trematode taxa, amounting to a density of 13 infected snails m(-2) of pond substrate. Between 18% and 33% of the combined host and parasite biomass within each infected snail consisted of larval trematode tissue, which collectively accounted for 87% of the total trematode biomass within the three ponds. Mid-summer trematode dry biomass averaged 0.10 g m(-2), which was equal to or greater than that of the most abundant insect orders (coleoptera = 0.10 g m(-2), odonata = 0.08 g m(-2), hemiptera = 0.07 g m(-2) and ephemeroptera = 0.03 g m(-2)). 4. On average, each trematode taxon produced between 14 and 1660 free-swimming larvae (cercariae) infected snail(-1) 24 h(-1) in mid-summer. Given that infected snails release cercariae for 3-4 months a year, the pond trematode communities produced an average of 153 mg m(-2) yr(-1) of dry cercarial biomass (range = 70-220 mg m(-2) yr(-1)). 5. Our results suggest that a significant amount of energy moves through trematode parasites in freshwater pond ecosystems, and that their contributions to ecosystem energetics may exceed those of many free-living taxa known to play key roles in structuring aquatic communities.

Parasite transmission in complex communities: predators and alternative hosts alter pathogenic infections in amphibians.

While often studied in isolation, host-parasite interactions are typically embedded within complex communities. Other community members, including predators and alternative hosts, can therefore alter parasite transmission (e.g., the dilution effect), yet few studies have experimentally evaluated more than one such mechanism. Here, we used data from natural wetlands to design experiments investigating how alternative hosts and predators of parasites mediate trematode (Ribeiroia ondatrae) infection in a focal amphibian host (Pseudacris regilla). In short-term predation bioassays involving mollusks, zooplankton, fish, larval insects, or newts, four of seven tested species removed 62-93% of infectious stages. In transmission experiments, damselfly nymphs (predators) and newt larvae (alternative hosts) reduced infection in P. regilla tadpoles by -50%, whereas mosquitofish (potential predators and alternative hosts) did not significantly influence transmission. Additional bioassays indicated that predators consumed parasites even in the presence of alternative prey. In natural wetlands, newts had similar infection intensities as P. regilla, suggesting that they commonly function as alternative hosts despite their unpalatability to downstream hosts, whereas mosquitofish had substantially lower infection intensities and are unlikely to function as hosts. These results underscore the importance of studying host-parasite interactions in complex communities and of broadly linking research on predation, biodiversity loss, and infectious diseases.

Macroparasite infections of amphibians: what can they tell us?

Understanding linkages between environmental changes and disease emergence in human and wildlife populations represents one of the greatest challenges to ecologists and parasitologists. While there is considerable interest in drivers of amphibian microparasite infections and the resulting consequences, comparatively little research has addressed such questions for amphibian macroparasites. What work has been done in this area has largely focused on nematodes of the genus Rhabdias and on two genera of trematodes (Ribeiroia and Echinostoma). Here, we provide a synopsis of amphibian macroparasites, explore how macroparasites may affect amphibian hosts and populations, and evaluate the significance of these parasites in larger community and ecosystem contexts. In addition, we consider environmental influences on amphibian-macroparasite interactions by exploring contemporary ecological factors known or hypothesized to affect patterns of infection. While some macroparasites of amphibians have direct negative effects on individual hosts, no studies have explicitly examined whether such infections can affect amphibian populations. Moreover, due to their complex life cycles and varying degrees of host specificity, amphibian macroparasites have rich potential as bioindicators of environmental modifications, especially providing insights into changes in food webs. Because of their documented pathologies and value as bioindicators, we emphasize the need for broader investigation of this understudied group, noting that ecological drivers affecting these parasites may also influence disease patterns in other aquatic fauna.

Land use and wetland spatial position jointly determine amphibian parasite communities.

Land use change is one of the most commonly cited contributing factors to infectious disease emergence, yet the mechanisms responsible for such changes and the spatial scales at which they operate are rarely identified. The distributions of parasites with complex life cycles depend on interactions between multiple host species, suggesting the net effects of land use on infection patterns may be difficult to predict a priori. Here, we used an information-theoretic approach to evaluate the importance of land use and spatial scale (local, watershed, and regional) in determining the presence and abundance of multi-host trematodes of amphibians. Among 40 wetlands and 160 hosts sampled, trematode abundance, species richness, and the presence and abundance of pathogenic species were strongly influenced by variables at the watershed and regional scales. Based on model averaging results, overall parasite richness and abundance were higher in forested wetlands than in agricultural areas; however, this pattern was influenced by a wetland's proximity to the Mississippi Flyway at the regional scale. These patterns likely reflect the activity of trematode definitive hosts, such as mammals and especially birds, such that infections decreased with increasing distance from the Mississippi River. Interestingly, despite lower mean infections, agricultural wetlands had higher variances and maximum infections. At the wetland scale, phosphorus concentrations and the abundances of intermediate hosts, such as snails and larval amphibians, positively affected parasite distributions. Taken together, these results contribute to our understanding of how altered landscapes affect parasite communities and inform further research on the environmental drivers of amphibian parasite infections.

When parasites become prey: ecological and epidemiological significance of eating parasites.

Recent efforts to include parasites in food webs have drawn attention to a previously ignored facet of foraging ecology: parasites commonly function as prey within ecosystems. Because of the high productivity of parasites, their unique nutritional composition and their pathogenicity in hosts, their consumption affects both food-web topology and disease risk in humans and wildlife. Here, we evaluate the ecological, evolutionary and epidemiological significance of feeding on parasites, including concomitant predation, grooming, predation on free-living stages and intraguild predation. Combining empirical data and theoretical models, we show that consumption of parasites is neither rare nor accidental, and that it can sharply affect parasite transmission and food web properties. Broader consideration of predation on parasites will enhance our understanding of disease control, food web structure and energy transfer, and the evolution of complex life cycles.

Energetics of metamorphic climax in the pickerel frog (Lithobates palustris).

Anuran metamorphosis, the transition from aquatic larvae to terrestrial juveniles, is accompanied by significant morphological, physiological, and behavioral changes. Timing of metamorphosis and final size, which can influence adult fitness, may depend on sufficient energy accumulated during the larval period to support metamorphosis. However, only two species of anurans have been examined for energetic costs of metamorphosis, Rana tigrina and Anaxyrus terrestris. Based on these species, it has been hypothesized that differences in energy expenditure are related to duration of metamorphosis. To compare energetic costs of metamorphosis among species and examine this hypothesis, we quantified the total energy required for metamorphosis of Lithobates palustris tadpoles by measuring oxygen consumption rates over the duration of metamorphic climax using closed-circuit respirometry. Total energy costs for L. palustris were positively related to tadpole mass and duration of metamorphic climax. However, larger tadpoles completed metamorphosis more efficiently because they used proportionally less total energy for metamorphic climax than smaller counterparts. Costs were intermediate to R. tigrina, a larger species with similar metamorphic duration, and A. terrestris, a smaller species with shorter metamorphic climax. The results supported the hypothesis that amphibian species with more slowly developing tadpoles, such as ranids, require more absolute energy for metamorphosis in comparison to more rapidly developing species like bufonids.

Moderate Echinostoma trivolvis infection has no effects on physiology and fitness-related traits of larval pickerel frogs (Rana palustris).

High intensity infections of Echinostoma trivolvis metacercariae decrease survival and growth of young amphibian larvae. However, in nature, parasites are highly aggregated, which results in a large proportion of the amphibian population being only moderately infected. Survival and growth responses at these more-common, low-infection levels remain poorly studied. Thus, we investigated the effects of moderate Echinostoma trivolvis metacercariae infection (following exposure to 0, 10, 30, or 90 cercariae) on the growth and development of pickerel frog (Rana palustris) tadpoles. We measured metabolism to determine whether increased energy expenditure is a potential physiological mechanism underlying previously documented reduced growth. Furthermore, we quantified tadpole intestine size, which can exhibit plasticity in response to changing metabolic demands, and we characterized metacercariae distribution in tadpole kidneys. Metacercariae encysted in the pronephros significantly more than in the mesonephros, but tended to occur equally in right and left kidneys. Two mo post-infection (PI), there were no changes in tadpole survival, development, intestine size, or growth related to metacercariae infection. Similarly, metacercariae did not significantly increase metabolic rates during encystment or at 1 mo PI. Our study demonstrated that modest E. trivolvis infections, representative of a large proportion of the host population, had no detectable effects on fitness-related traits in laboratory isolation from other ecological variables.