Showing papers in "Frontiers in Zoology in 2013"

A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

Abstract: The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.

Dispersal and gene flow in free-living marine nematodes

Abstract: Dispersal and gene flow determine connectivity among populations, and can be studied through population genetics and phylogeography. We here review the results of such a framework for free-living marine nematodes. Although field experiments have illustrated substantial dispersal in nematodes at ecological time scales, analysis of the genetic diversity illustrated the importance of priority effects, founder effects and genetic bottlenecks for population structuring between patches <1 km apart. In contrast, only little genetic structuring was observed within an estuary (<50 km), indicating that these small scale fluctuations in genetic differentiation are stabilized over deeper time scales through extensive gene flow. Interestingly, nematode species with contrasting life histories (extreme colonizers vs persisters) or with different habitat preferences (algae vs sediment) show similar, low genetic structuring. Finally, historical events have shaped the genetic pattern of marine nematodes and show that gene flow is restricted at large geographical scales. We also discuss the presence of substantial cryptic diversity in marine nematodes, and end with highlighting future important steps to further unravel nematode evolution and diversity.

How to describe a cryptic species? Practical challenges of molecular taxonomy

Abstract: Background Molecular methods of species delineation are rapidly developing and widely considered as fast and efficient means to discover species and face the 'taxonomic impediment’ in times of biodiversity crisis. So far, however, this form of DNA taxonomy frequently remains incomplete, lacking the final step of formal species description, thus enhancing rather than reducing impediments in taxonomy. DNA sequence information contributes valuable diagnostic characters and –at least for cryptic species – could even serve as the backbone of a taxonomic description. To this end solutions for a number of practical problems must be found, including a way in which molecular data can be presented to fulfill the formal requirements every description must meet. Multi-gene barcoding and a combined molecular species delineation approach recently revealed a radiation of at least 12 more or less cryptic species in the marine meiofaunal slug genus Pontohedyle (Acochlidia, Heterobranchia). All identified candidate species are well delimited by a consensus across different methods based on mitochondrial and nuclear markers.

Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species

Abstract: Invertebrate communities are central to many environmental monitoring programs. In freshwater ecosystems, aquatic macroinvertebrates are collected, identified and then used to infer ecosystem condition. Yet the key step of species identification is often not taken, as it requires a high level of taxonomic expertise, which is lacking in most organizations, or species cannot be identified as they are morphologically cryptic or represent little known groups. Identifying species using DNA sequences can overcome many of these issues; with the power of next generation sequencing (NGS), using DNA sequences for routine monitoring becomes feasible. In this study, we test if NGS can be used to identify species from field-collected samples in an important bioindicator group, the Chironomidae. We show that Cytochrome oxidase I (COI) and Cytochrome B (CytB) sequences provide accurate DNA barcodes for chironomid species. We then develop a NGS analysis pipeline to identifying species using megablast searches of high quality sequences generated using 454 pyrosequencing against comprehensive reference libraries of Sanger-sequenced voucher specimens. We find that 454 generated COI sequences successfully identified up to 96% of species in samples, but this increased up to 99% when combined with CytB sequences. Accurate identification depends on having at least five sequences for a species; below this level species not expected in samples were detected. Incorrect incorporation of some multiplex identifiers (MID’s) used to tag samples was a likely cause, and most errors could be detected when using MID tags on forward and reverse primers. We also found a strong quantitative relationship between the number of 454 sequences and individuals showing that it may be possible to estimate the abundance of species from 454 pyrosequencing data. Next generation sequencing using two genes was successful for identifying chironomid species. However, when detecting species from 454 pyrosequencing data sets it was critical to include known individuals for quality control and to establish thresholds for detecting species. The NGS approach developed here can lead to routine species-level diagnostic monitoring of aquatic ecosystems.

Integrative taxonomy on the fast track - towards more sustainability in biodiversity research.

Abstract: A so called “taxonomic impediment” has been recognized as a major obstacle to biodiversity research for the past two decades. Numerous remedies were then proposed. However, neither significant progress in terms of formal species descriptions, nor a minimum standard for descriptions have been achieved so far. Here, we analyze the problems of traditional taxonomy which often produces keys and descriptions of limited practical value. We suggest that phylogenetics and phenetics had a subtle and so far unnoticed effect on taxonomy leading to inflated species descriptions. The term “turbo-taxonomy” was recently coined for an approach combining cox1 sequences, concise morphological descriptions by an expert taxonomist, and high-resolution digital imaging to streamline the formal description of larger numbers of new species. We propose a further development of this approach which, together with open access web-publication and automated pushing of content from journal into a wiki, may create the most efficient and sustainable way to conduct taxonomy in the future. On demand, highly concise descriptions can be gradually updated or modified in the fully versioned wiki-framework we use. This means that the visibility of additional data is not compromised, while the original species description -the first version- remains preserved in the wiki, and of course in the journal version. A DNA sequence database with an identification engine replaces an identification key, helps to avoid synonyms and has the potential to detect grossly incorrect generic placements. We demonstrate the functionality of a species-description pipeline by naming 101 new species of hyperdiverse New Guinea Trigonopterus weevils in the open-access journal ZooKeys. Fast track taxonomy will not only increase speed, but also sustainability of global species inventories. It will be of great practical value to all the other disciplines that depend on a usable taxonomy and will change our perception of global biodiversity. While this approach is certainly not suitable for all taxa alike, it is the tool that will help to tackle many hyperdiverse groups and pave the road for more sustainable comparative studies, e.g. in community ecology, phylogeography and large scale biogeographic studies.

Urban-like night illumination reduces melatonin release in European blackbirds (Turdus merula): implications of city life for biological time-keeping of songbirds

Abstract: Introduction Artificial light-at-night is known to affect a broad array of behaviours and physiological processes. In urbanized bird species, light-at-night advances important biological rhythms such as daily cycles of activity/rest and timing of reproduction, but our knowledge of the underlying physiological mechanisms is limited. Given its role as chronobiological signal, melatonin is a strong candidate for mediating the effects of light-at-night.

A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

Abstract: In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system. The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations.

Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing

Abstract: In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata). Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse. Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

Tracing glacial refugia of Triturus newts based on mitochondrial DNA phylogeography and species distribution modeling

Abstract: The major climatic oscillations during the Quaternary Ice Age heavily influenced the distribution of species and left their mark on intraspecific genetic diversity. Past range shifts can be reconstructed with the aid of species distribution modeling and phylogeographical analyses. We test the responses of the different members of the genus Triturus (i.e. the marbled and crested newts) as the climate shifted from the previous glacial period (the Last Glacial Maximum, ~21 Ka) to the current interglacial. We present the results of a dense mitochondrial DNA phylogeography (visualizing genetic diversity within and divergence among populations) and species distribution modeling (using two different climate simulations) for the nine Triturus species on composite maps. The combined use of species distribution modeling and mitochondrial phylogeography provides insight in the glacial contraction and postglacial expansion of Triturus. The combined use of the two independent techniques yields a more complete understanding of the historical biogeography of Triturus than both approaches would on their own. Triturus newts generally conform to the ‘southern richness and northern purity’ paradigm, but we also find more intricate patterns, such as the absence of genetic variation and suitable area at the Last Glacial Maximum (T. dobrogicus), an ‘extra-Mediterranean’ refugium in the Carpathian Basin (T. cristatus), and areas where species displaced one another postglacially (e.g. T. macedonicus and western T. karelinii). We provide a biogeographical scenario for Triturus, showing the positions of glacial refugia, the regions that were postglacially colonized and the areas where species displaced one another as they shifted their ranges.

Effects of social disruption in elephants persist decades after culling

Abstract: Background: Multi-level fission-fusion societies, characteristic of a number of large brained mammal species including some primates, cetaceans and elephants, are among the most complex and cognitively demanding animal social systems. Many free-ranging populations of these highly social mammals already face severe human disturbance, which is set to accelerate with projected anthropogenic environmental change. Despite this, our understanding of how such disruption affects core aspects of social functioning is still very limited. Results: We now use novel playback experiments to assess decision-making abilities integral to operating successfully within complex societies, and provide the first systematic evidence that fundamental social skills may be significantly impaired by anthropogenic disruption. African elephants (Loxodonta africana) that had experienced separation from family members and translocation during culling operations decades previously performed poorly on systematic tests of their social knowledge, failing to distinguish between callers on the basis of social familiarity. Moreover, elephants from the disrupted population showed no evidence of discriminating between callers when age-related cues simulated individuals on an increasing scale of social dominance, in sharp contrast to the undisturbed population where this core social ability was well developed. Conclusions: Key decision-making abilities that are fundamental to living in complex societies could be significantly altered in the long-term through exposure to severely disruptive events (e.g. culling and translocation). There is an assumption that wildlife responds to increasing pressure from human societies only in terms of demography, however our study demonstrates that the effects may be considerably more pervasive. These findings highlight the potential long-term negative consequences of acute social disruption in cognitively advanced species that live in close-knit kin-based societies, and alter our perspective on the health and functioning of populations that have been subjected to anthropogenic disturbance.

Dogs are sensitive to small variations of the Earth’s magnetic field

Abstract: Several mammalian species spontaneously align their body axis with respect to the Earth’s magnetic field (MF) lines in diverse behavioral contexts. Magnetic alignment is a suitable paradigm to scan for the occurrence of magnetosensitivity across animal taxa with the heuristic potential to contribute to the understanding of the mechanism of magnetoreception and identify further functions of magnetosensation apart from navigation. With this in mind we searched for signs of magnetic alignment in dogs. We measured the direction of the body axis in 70 dogs of 37 breeds during defecation (1,893 observations) and urination (5,582 observations) over a two-year period. After complete sampling, we sorted the data according to the geomagnetic conditions prevailing during the respective sampling periods. Relative declination and intensity changes of the MF during the respective dog walks were calculated from daily magnetograms. Directional preferences of dogs under different MF conditions were analyzed and tested by means of circular statistics. Dogs preferred to excrete with the body being aligned along the North–South axis under calm MF conditions. This directional behavior was abolished under unstable MF. The best predictor of the behavioral switch was the rate of change in declination, i.e., polar orientation of the MF. It is for the first time that (a) magnetic sensitivity was proved in dogs, (b) a measurable, predictable behavioral reaction upon natural MF fluctuations could be unambiguously proven in a mammal, and (c) high sensitivity to small changes in polarity, rather than in intensity, of MF was identified as biologically meaningful. Our findings open new horizons in magnetoreception research. Since the MF is calm in only about 20% of the daylight period, our findings might provide an explanation why many magnetoreception experiments were hardly replicable and why directional values of records in diverse observations are frequently compromised by scatter.

Tadpole transport logistics in a Neotropical poison frog: indications for strategic planning and adaptive plasticity in anuran parental care

Abstract: Introduction Individuals should aim to adjust their parental behaviours in order to maximize the success of their offspring but minimize associated costs. Plasticity in parental care is well documented from various bird, mammal and fish species, whereas amphibians were traditionally assumed as being highly instinct-bound. Therefore, little is known about ‘higher’ cognitive abilities of amphibians, such as strategic planning and behavioural flexibility. Dendrobatid frogs have evolved a remarkable diversity of parental behaviours. The most noticeable of these behaviours is tadpole transport, which is obligatory in almost all species. Nonetheless, there is limited knowledge about spatial and temporal patterns of tadpole transport and the possible existence of behavioural plasticity on the individual level. In this study, we investigated correlates of tadpole transport behaviour in a natural population of the dendrobatid frog Allobates femoralis during five years.

Linking melanism to brain development: expression of a melanism-related gene in barn owl feather follicles covaries with sleep ontogeny

Abstract: Intra-specific variation in melanocyte pigmentation, common in the animal kingdom, has caught the eye of naturalists and biologists for centuries. In vertebrates, dark, eumelanin pigmentation is often genetically determined and associated with various behavioral and physiological traits, suggesting that the genes involved in melanism have far reaching pleiotropic effects. The mechanisms linking these traits remain poorly understood, and the potential involvement of developmental processes occurring in the brain early in life has not been investigated. We examined the ontogeny of rapid eye movement (REM) sleep, a state involved in brain development, in a wild population of barn owls (Tyto alba) exhibiting inter-individual variation in melanism and covarying traits. In addition to sleep, we measured melanistic feather spots and the expression of a gene in the feather follicles implicated in melanism (PCSK2). As in mammals, REM sleep declined with age across a period of brain development in owlets. In addition, inter-individual variation in REM sleep around this developmental trajectory was predicted by variation in PCSK2 expression in the feather follicles, with individuals expressing higher levels exhibiting a more precocial pattern characterized by less REM sleep. Finally, PCSK2 expression was positively correlated with feather spotting. We demonstrate that the pace of brain development, as reflected in age-related changes in REM sleep, covaries with the peripheral activation of the melanocortin system. Given its role in brain development, variation in nestling REM sleep may lead to variation in adult brain organization, and thereby contribute to the behavioral and physiological differences observed between adults expressing different degrees of melanism.

Stopover optimization in a long-distance migrant: the role of fuel load and nocturnal take-off time in Alaskan northern wheatears (Oenanthe oenanthe)

Abstract: In long-distance migrants, a considerably higher proportion of time and energy is allocated to stopovers rather than to flights. Stopover duration and departure decisions affect consequently subsequent flight stages and overall speed of migration. In Arctic nocturnal songbird migrants the trade-off between a relatively long migration distance and short nights available for travelling may impose a significant time pressure on migrants. Therefore, we hypothesize that Alaskan northern wheatears (Oenanthe oenanthe) use a time-minimizing migration strategy to reach their African wintering area 15,000 km away. We estimated the factors influencing the birds’ daily departure probability from an Arctic stopover before crossing the Bering Strait by using a Cormack-Jolly-Seber model. To identify in which direction and when migration was resumed departing birds were radio-tracked. Here we show that Alaskan northern wheatears did not behave as strict time minimizers, because their departure fuel load was unrelated to fuel deposition rate. All birds departed with more fuel load than necessary for the sea crossing. Departure probability increased with stopover duration, evening fuel load and decreasing temperature. Birds took-off towards southwest and hence, followed in general the constant magnetic and geographic course but not the alternative great circle route. Nocturnal departure times were concentrated immediately after sunset. Although birds did not behave like time-minimizers in respect of the optimal migration strategies their surplus of fuel load clearly contradicted an energy saving strategy in terms of the minimization of overall energy cost of transport. The observed low variation in nocturnal take-off time in relation to local night length compared to similar studies in the temperate zone revealed that migrants have an innate ability to respond to changes in the external cue of night length. Likely, birds maximized their potential nightly flight range by taking off early in the night which in turn maximizes their overall migration speed. Hence, nocturnal departure time may be a crucial parameter shaping the speed of migration indicating the significance of its integration in future migration models.

Atlantic cod actively avoid CO2 and predator odour, even after long-term CO2 exposure

Abstract: Introduction The rising atmospheric CO2 level is continuously driving the dissolution of more CO2 into the oceans, and some emission scenarios project that the surface waters may reach 1000 μatm by the end of the century. It is not known if fish can detect moderately elevated CO2 levels, and if they avoid areas with high CO2. If so, avoidance behaviour to water with high CO2 could affect movement patterns and migrations of fish in the future. It is also being increasingly recognized that fish behaviour can be altered by exposure to CO2. Therefore this study investigated how long-term exposure to elevated pCO2 affects predator avoidance and CO2 avoidance in juvenile Atlantic cod (Gadus morhua). The fish were exposed to control water or CO2-enriched water (1000 μatm) for six weeks before being subjected to tests of behaviour.

Sexual size dimorphism in anurans fails to obey Rensch’s rule

Abstract: Background: Sexual size dimorphism (SSD) is related to ecology, behaviour and life history of organisms. Rensch’s rule states that SSD increases with overall body size in species where males are the larger sex, while decreasing with body size when females are larger. To test this rule, we analysed literature as well as own data on male and female body size in anurans (39 species and 17 genera). We also tested the hypothesis that SSD is largely a function of age difference between the sexes. Results: Our data set encompassed 36 species with female-biased SSD, and three species with male-biased SSD. All considered species failed to support Rensch’s rule, also when the analyses were phylogenetically corrected. However, SSD was significantly correlated with Sexual Age Difference (SAD) across species. We also found a significant correlation between SSD contrasts and SAD contrasts. Conclusions: Our study suggests that Rensch’s rule does not accurately describe macroevolutionary patterns of SSD in anurans. That SAD can explain most of the variation in SSD among species when controlling for phylogenetic effects suggests that phylogeny is not responsible for the broad relationship between age and size across the sexes.

More than royal food - Major royal jelly protein genes in sexuals and workers of the honeybee Apis mellifera

Abstract: Background In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Evolutionary history of relict Congeria (Bivalvia: Dreissenidae): unearthing the subterranean biodiversity of the Dinaric Karst

Abstract: Patterns of biodiversity in the subterranean realm are typically different from those encountered on the Earth’s surface. The Dinaric karst of Croatia, Slovenia and Bosnia and Herzegovina is a global hotspot of subterranean biodiversity. How this was achieved and why this is so remain largely unresolved despite a long tradition of research. To obtain insights into the colonisation of the Dinaric Karst and the effects of the subterranean realm on its inhabitants, we studied the tertiary relict Congeria, a unique cave-dwelling bivalve (Dreissenidae), using a combination of biogeographical, molecular, morphological, and paleontological information. Phylogenetic and molecular clock analyses using both nuclear and mitochondrial markers have shown that the surviving Congeria lineage has actually split into three distinct species, i.e., C. kusceri, C. jalzici sp. nov. and C. mulaomerovici sp. nov., by vicariant processes in the late Miocene and Pliocene. Despite millions of years of independent evolution, analyses have demonstrated a great deal of shell similarity between modern Congeria species, although slight differences in hinge plate structure have enabled the description of the two new species. Ancestral plesiomorphic shell forms seem to have been conserved during the processes of cave colonisation and subsequent lineage isolation. In contrast, shell morphology is divergent within one of the lineages, probably due to microhabitat differences. Following the turbulent evolution of the Dreissenidae during the Tertiary and major radiations in Lake Pannon, species of Congeria went extinct. One lineage survived, however, by adopting a unique life history strategy that suited it to the underground environment. In light of our new data, an alternative scenario for its colonisation of the karst is proposed. The extant Congeria comprises three sister species that, to date, have only been found to live in 15 caves in the Dinaric karst. Inter-specific morphological stasis and intra-specific ecophenotypic plasticity of the congerid shell demonstrate the contrasting ways in which evolution in the underground environments shapes its inhabitants.

Is ecological speciation a major trend in aphids? Insights from a molecular phylogeny of the conifer-feeding genus Cinara

Abstract: Introduction: In the past decade ecological speciation has been recognized as having an important role in the diversification of plant-feeding insects. Aphids are host-specialised phytophagous insects that mate on their host plants and, as such, they are prone to experience reproductive isolation linked with host plant association that could ultimately lead to species formation. The generality of such a scenario remains to be tested through macroevolutionary studies. To explore the prevalence of host-driven speciation in the diversification of the aphid genus Cinara and to investigate alternative modes of speciation, we reconstructed a phylogeny of this genus based on mitochondrial, nuclear and Buchnera aphidicola DNA sequence fragments and applied a DNA-based method of species delimitation. Using a recent software (PhyloType), we explored evolutionary transitions in host-plant genera, feeding sites and geographic distributions in the diversification of Cinara and investigated how transitions in these characters have accompanied speciation events. Results: The diversification of Cinara has been constrained by host fidelity to conifer genera sometimes followed by sequential colonization onto different host species and by feeding-site specialisation. Nevertheless, our analyses suggest that, at the most, only half of the speciation events were accompanied by ecological niche shifts. The contribution of geographical isolation in the speciation process is clearly apparent in the occurrence of species from two continents in the same clades in relatively terminal positions in our phylogeny. Furthermore, in agreement with predictions from scenarios in which geographic isolation accounts for speciation events, geographic overlap between species increased significantly with time elapsed since their separation. Conclusions: The history of Cinara offers a different perspective on the mode of speciation of aphids than that provided by classic models such as the pea aphid. In this genus of aphids, the role of climate and landscape history has probably been as important as host-plant specialisation in having shaped present-day diversity.

Offspring pay sooner, parents pay later: experimental manipulation of body mass reveals trade-offs between immune function, reproduction and survival.

Abstract: Introduction: Life-history theory predicts that organisms trade off survival against reproduction. However, the time scales on which various consequences become evident and the physiology mediating the cost of reproduction remain poorly understood. Yet, explaining not only which mechanisms mediate this trade-off, but also how fast or slow the mechanisms act, is crucial for an improved understanding of life-history evolution. We investigated three time scales on which an experimental increase in body mass could affect this trade-off: within broods, within season and between years. We handicapped adult skylarks (Alauda arvensis) by attaching extra weight during first broods to both adults of a pair. We measured body mass, immune function and return rates in these birds. We also measured nest success, feeding rates, diet composition, nestling size, nestling immune function and recruitment rates. Results: When nestlings of first broods fledged, parent body condition had not changed, but experimental birds experienced higher nest failure. Depending on the year, immune parameters of nestlings from experimental parents were either higher or lower than of control nestlings. Later, when parents were feeding their second brood, the balance between self-maintenance and nest success had shifted. Control and experimental adults differed in immune function, while mass and immune function of their nestlings did not differ. Although weights were removed after breeding, immune measurements during the second brood had the capacity to predict return rates to the next breeding season. Among birds that returned the next year, body condition and reproductive performance a year after the experiment did not differ between treatment groups. Conclusions: We conclude that the balance between current reproduction and survival shifts from affecting nestlings to affecting parents as the reproductive season progresses. Furthermore, immune function is apparently one physiological mechanism involved in this trade-off. By unravelling a physiological mechanism underlying the trade-offs between current and future reproduction and by demonstrating the different time scales on which it acts, our study represents an important step in understanding a central theory of life-history evolution.

At the limits of a successful body plan – 3D microanatomy, histology and evolution of Helminthope (Mollusca: Heterobranchia: Rhodopemorpha), the most worm-like gastropod

Abstract: Gastropods are among the most diverse animal clades, and have successfully colonized special habitats such as the marine sand interstitial. Specialized meiofaunal snails and slugs are tiny and worm-shaped. They combine regressive features – argued to be due to progenetic tendencies – with convergent adaptations. Microscopic size and concerted convergences make morphological examination non-trivial and hamper phylogenetic reconstructions. The enigmatic turbellarian-like Rhodopemorpha are a small group that has puzzled systematists for over a century. A preliminary molecular framework places the group far closer to the root of Heterobranchia – one of the major gastropod groups – than previously suggested. The poorly known meiofaunal Helminthope psammobionta Salvini-Plawen, 1991 from Bermuda is the most worm-shaped free-living gastropod and shows apparently aberrant aspects of anatomy. Its study may give important clues to understand the evolution of rhodopemorphs among basal heterobranchs versus their previously thought origin among ‘higher’ euthyneuran taxa. We describe the 3D-microanatomy of H. psammobionta using three-dimensional digital reconstruction based on serial semithin histological sections. The new dataset expands upon the original description and corrects several aspects. Helminthope shows a set of typical adaptations and regressive characters present in other mesopsammic slugs (called ‘meiofaunal syndrome’ herein). The taxonomically important presence of five separate visceral loop ganglia is confirmed, but considerable further detail of the complex nervous system are corrected and revealed. The digestive and reproductive systems are simple and modified to the thread-like morphology of the animal; the anus is far posterior. There is no heart; the kidney resembles a protonephridium. Data on all organ systems are compiled and compared to Rhodope. Helminthope is related to Rhodope sharing unique apomorphies. We argue that the peculiar kidney, configuration of the visceral loop and simplicity or lack of other organs in Rhodopemorpha are results of progenesis. The posterior shift of the anus in Helminthope is interpreted as a peramorphy, i.e. hypertrophy of body length early in ontogeny. Our review of morphological and molecular evidence is consistent with an origin of Rhodopemorpha slugs among shelled ‘lower Heterobranchia’. Previously thought shared ‘diagnostic’ features such as five visceral ganglia are either plesiomorphic or convergent, while euthyneury and a double-rooted cerebral nerve likely evolved independently in Rhodopemorpha and Euthyneura.

Evolutionary origins of ultrasonic hearing and laryngeal echolocation in bats inferred from morphological analyses of the inner ear

Abstract: Many mammals have evolved highly adapted hearing associated with ecological specialisation. Of these, bats possess the widest frequency range of vocalisations and associated hearing sensitivities, with frequencies of above 200 kHz in some lineages that use laryngeal echolocation. High frequency hearing in bats appears to have evolved via structural modifications of the inner ear, however, studying these minute features presents considerable challenges and hitherto few such attempts have been made. To understand these adaptations more fully, as well as gain insights into the evolutionary origins of ultrasonic hearing and echolocation in bats, we undertook micro-computed tomography (μCT) scans of the cochleae of representative bat species from 16 families, encompassing their broad range of ecological diversity. To characterise cochlear gross morphology, we measured the relative basilar membrane length and number of turns, and compared these values between echolocating and non-echolocating bats, as well as other mammals. We found that hearing and echolocation call frequencies in bats correlated with both measures of cochlear morphology. In particular, relative basilar membrane length was typically longer in echolocating species, and also correlated positively with the number of cochlear turns. Ancestral reconstructions of these parameters suggested that the common ancestor of all extant bats was probably capable of ultrasonic hearing; however, we also found evidence of a significant decrease in the rate of morphological evolution of the basilar membrane in multiple ancestral branches within the Yangochiroptera suborder. Within the echolocating Yinpterochiroptera, there was some evidence of an increase in the rate of basilar membrane evolution in some tips of the tree, possibly associated with reported shifts in call frequency associated with recent speciation events. The two main groups of echolocating bat were found to display highly variable inner ear morphologies. Ancestral reconstructions and rate shift analyses of ear morphology point to a complex evolutionary history, with the former supporting ultrasonic hearing in the common bat ancestor but the latter suggesting that morphological changes associated with echolocation might have occurred later. These findings are consistent with theories that sophisticated laryngeal echolocation, as seen in modern lineages, evolved following the divergence of the two main suborders.

Only distance matters - non-choosy females in a poison frog population.

Abstract: Background: Females have often been shown to exhibit preferences for certain male traits. However, little is known about behavioural rules females use when searching for mates in their natural habitat. We investigated mate sampling tactics and related costs in the territorial strawberry poison frog (Oophaga pumilio) possessing a lek-like mating system, where both sequential and simultaneous sampling might occur. We continuously monitored the sampling pattern and behaviour of females during the complete period between two successive matings. Results: We found no evidence that females compared males by visiting them. Instead females mated with the closest calling male irrespective of his acoustic and physical traits, and territory size. Playback experiments in the natural home ranges of receptive females revealed that tested females preferred the nearest speaker and did not discriminate between low and high call rates or dominant frequencies. Conclusions: Our results suggest that females of O. pumilio prefer the closest calling male in the studied population. We hypothesize that the sampling tactic in this population is affected by 1) a strongly female biased sex ratio and 2) a low variance in traits of available males due to strong male-male competition, preventing low quality males from defending a territory and mating.

Winner and loser effects are modulated by hormonal states.

Abstract: Introduction: Many animals use information acquired from recent experiences to modify their responses to new situations. Animals’ decisions in contests also depend on their previous experience: after recent victories individuals tend to behave more aggressively and after defeats more submissively. Although these winner and/or loser effects have been reported for animals of different taxa, they have only recently been shown to be flexible traits, which can be influenced by extrinsic factors. In a mangrove killifish (Kryptolebias marmoratus), for instance, individuals which lost an earlier contest were more likely than others to alter contest decisions after a recent win/loss. This result suggests that individuals perceiving themselves to have worse fighting abilities are more inclined to adjust contest strategy based on new information. If this is the case, an individual’s propensity to modify behaviour after a win/loss might also be modulated by intrinsic mechanisms related to its ability to fight. Stress and sex steroid hormones are often associated with an individual’s contest behaviour and performance, so, in this study, we tested the hypothesis that an individual’s propensity to change behaviour after wins or losses also depends on its hormonal state. Results: Our results show that an individual’s propensity to adjust contest decisions after wins and losses does depend on its hormonal state: individuals with lower levels of cortisol (F), testosterone (T) and 11-ketotestosterone (KT) are more receptive than others to the influence of recent contest experiences, especially losing experiences, and the influences last longer. Furthermore, although winning and losing experiences resulted in significant changes in behaviour, they did not bring about a significant change in the levels of F, T, KT or oestradiol (E2). Conclusions: This study shows that an individual’s receptivity to the influence of recent wins and losses is modulated by its internal state, as well as by extrinsic factors. Individuals with hormonal profiles corresponding to lower aggressiveness and a reduced likelihood of winning were more likely to alter contest decisions after a recent win/loss. The results also suggest that F, T, KT and E2 are not the primary physiological mechanisms mediating winner-loser effects in this fish.

Intraguild relationships between sympatric predators exposed to lethal control: predator manipulation experiments

Abstract: Terrestrial top-predators are expected to regulate and stabilise food webs through their consumptive and non-consumptive effects on sympatric mesopredators and prey. The lethal control of top-predators has therefore been predicted to inhibit top-predator function, generate the release of mesopredators and indirectly harm native fauna through trophic cascade effects. Understanding the outcomes of lethal control on interactions within terrestrial predator guilds is important for zoologists, conservation biologists and wildlife managers. However, few studies have the capacity to test these predictions experimentally, and no such studies have previously been conducted on the eclectic suite of native and exotic, mammalian and reptilian taxa we simultaneously assess. We conducted a series of landscape-scale, multi-year, manipulative experiments at nine sites spanning five ecosystem types across the Australian continental rangelands to investigate the responses of mesopredators (red foxes, feral cats and goannas) to contemporary poison-baiting programs intended to control top-predators (dingoes) for livestock protection. Short-term behavioural releases of mesopredators were not apparent, and in almost all cases, the three mesopredators we assessed were in similar or greater abundance in unbaited areas relative to baited areas, with mesopredator abundance trends typically either uncorrelated or positively correlated with top-predator abundance trends over time. The exotic mammals and native reptile we assessed responded similarly (poorly) to top-predator population manipulation. This is because poison baits were taken by multiple target and non-target predators and top-predator populations quickly recovered to pre-control levels, thus reducing the overall impact of baiting on top-predators and averting a trophic cascade. These results are in accord with other predator manipulation experiments conducted worldwide, and suggest that Australian populations of native prey fauna at lower trophic levels are unlikely to be negatively affected by contemporary dingo control practices through the release of mesopredators. We conclude that contemporary lethal control practices used on some top-predator populations do not produce the conditions required to generate positive responses from mesopredators. Functional relationships between sympatric terrestrial predators may not be altered by exposure to spatially and temporally sporadic application of non-selective lethal control.

A complete three-dimensional reconstruction of the myoanatomy of Loricifera: comparative morphology of an adult and a Higgins larva stage

Abstract: Loricifera is a group of small, marine animals, with undetermined phylogenetic relationships within Ecdysozoa (molting protostome animals). Despite their well-known external morphology, data on the internal anatomy of loriciferans are still incomplete. Aiming to increase the knowledge of this enigmatic phylum, we reconstruct for the first time the three-dimensional myoanatomy of loriciferans. Adult Nanaloricus sp. and the Higgins larva of Armorloricus elegans were investigated with cytochemical labeling techniques and CLSM. We discuss our findings with reference to other loriciferan species and recently established phylogenies. The somatic musculature of both adult and larval stages is very complex and includes several muscles arranged in three orientations: circular, transverse and longitudinal. In adult Nanaloricus sp., the introvert is characterized by a net-like muscular arrangement, which is composed of five thin circular fibers crossed by several (up to 30) thin longitudinal fibers with bifurcated anterior ends. Two sets of muscles surround the pre-pharyngeal armature: 6 buccal tube retractors arranged 3 × 2 in a conical shaped structure, and 8 mouth cone retractors. Additionally, a thick, circular muscle marks the neck region and a putative anal sphincter is the posteriormost myoanatomical feature. In the Higgins larva of A. elegans, two circular muscles are distinguished anteriorly in the introvert: a dorsal semicircular fiber and a thin ring muscle. The posteriormost region of the body is characterized by an anal sphincter and a triangular muscle. Based on the currently available knowledge, the myoanatomical bodyplan of adult loriciferans includes: (i) 8 mouth cone retractors, (ii) a pharynx bulb composed of transversal fibers arranged radially, (iii) circular muscles of the head and neck, (iv) internal muscles of the spinoscalids, (v) longitudinal muscles spanning all body regions, and (vi) transverse (circular) muscles in the abdomen. Concerning the Higgins larva, the muscle subsets assigned to its myoanatomical ground pattern are the (i) longitudinal retractors of the mouth cone, introvert, and abdomen, (ii) abdominal transverse muscles, and (iii) a pharynx bulb composed of transverse, radial fibers. In a comparison with phyla traditionally regarded as phylogenetically close, our data show that the overall myoanatomy of Loricifera is more similar to Kinorhyncha and Nematomorpha than to Priapulida. However, the head musculature of all these groups is very similar, which supports homology of their introverts and head morphology.

Color plumage polymorphism and predator mimicry in brood parasites.

Abstract: Plumage polymorphism may evolve during coevolution between brood parasites and their hosts if rare morph(s), by contravening host search image, evade host recognition systems better than common variant(s). Females of the parasitic common cuckoo (Cuculus canorus) are a classic example of discrete color polymorphism: gray females supposedly mimic the sparrowhawk (Accipiter nisus), while rufous females are believed to mimic the kestrel (Falco tinnunculus). Despite many studies on host responses to adult cuckoos comprehensive tests of the “hawk mimicry” and “kestrel mimicry” hypotheses are lacking so far. We tested these hypotheses by examining host responses to stuffed dummies of the sparrowhawk, kestrel, cuckoo and the innocuous turtle dove (Streptopelia turtur) as a control at the nest. Our experimental data from an aggressive cuckoo host, the great reed warbler (Acrocephalus arundinaceus), showed low effectiveness of cuckoo-predator mimicry against more aggressive hosts regardless of the type of model and the degree of perfection of the mimic. Specifically, warblers discriminated gray cuckoos from sparrowhawks but did not discriminate rufous cuckoos from kestrels. However, both gray and rufous cuckoos were attacked vigorously and much more than control doves. The ratio of aggression to gray vs. rufous cuckoo was very similar to the ratio between frequencies of gray vs. rufous cuckoo morphs in our study population. Overall, our data combined with previous results from other localities suggest polymorphism dynamics are not strongly affected by local predator model frequencies. Instead, hosts responses and discrimination abilities are proportional, other things being equal, to the frequency with which hosts encounter various cuckoo morphs near their nests. This suggests that female cuckoo polymorphism is a counter-adaptation to thwart a specific host adaptation, namely an ability to not be fooled by predator mimicry. We hypothesize the dangerousness of a particular model predator (sparrowhawks are more dangerous to adult birds than kestrels) may be another important factor responsible for better discrimination between the gray cuckoo and its model rather than between the rufous cuckoo and its model. We also provide a review of relevant existing literature, detailed discussion of plumage polymorphism in cuckoos, methodological recommendations and new ideas for future work.

Vocal communication in a complex multi-level society: constrained acoustic structure and flexible call usage in Guinea baboons

Abstract: Background To understand the evolution of acoustic communication in animals, it is important to distinguish between the structure and the usage of vocal signals, since both aspects are subject to different constraints. In terrestrial mammals, the structure of calls is largely innate, while individuals have a greater ability to actively initiate or withhold calls. In closely related taxa, one would therefore predict a higher flexibility in call usage compared to call structure. In the present study, we investigated the vocal repertoire of free living Guinea baboons (Papio papio) and examined the structure and usage of the animals’ vocal signals. Guinea baboons live in a complex multi-level social organization and exhibit a largely tolerant and affiliative social style, contrary to most other baboon taxa. To classify the vocal repertoire of male and female Guinea baboons, cluster analyses were used and focal observations were conducted to assess the usage of vocal signals in the particular contexts.

Compensatory regulation of Na+ absorption by Na+/H+ exchanger and Na+-Cl- cotransporter in zebrafish (Danio rerio)

Abstract: In mammals, internal Na+ homeostasis is maintained through Na+ reabsorption via a variety of Na+ transport proteins with mutually compensating functions, which are expressed in different segments of the nephrons. In zebrafish, Na+ homeostasis is achieved mainly through the skin/gill ionocytes, namely Na+/H+ exchanger (NHE3b)-expressing H+-ATPase rich (HR) cells and Na+-Cl- cotransporter (NCC)-expressing NCC cells, which are functionally homologous to mammalian proximal and distal convoluted tubular cells, respectively. The present study aimed to investigate whether or not the functions of HR and NCC ionocytes are differentially regulated to compensate for disruptions of internal Na+ homeostasis and if the cell differentiation of the ionocytes is involved in this regulation pathway. Translational knockdown of ncc caused an increase in HR cell number and a resulting augmentation of Na+ uptake in zebrafish larvae, while NHE3b loss-of-function caused an increase in NCC cell number with a concomitant recovery of Na+ absorption. Environmental acid stress suppressed nhe3b expression in HR cells and decreased Na+ content, which was followed by up-regulation of NCC cells accompanied by recovery of Na+ content. Moreover, knockdown of ncc resulted in a significant decrease of Na+ content in acid-acclimated zebrafish. These results provide evidence that HR and NCC cells exhibit functional redundancy in Na+ absorption, similar to the regulatory mechanisms in mammalian kidney, and suggest this functional redundancy is a critical strategy used by zebrafish to survive in a harsh environment that disturbs body fluid Na+ homeostasis.

Anatomy and development of the larval nervous system in Echinococcus multilocularis

Abstract: The metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system. We describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-α and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm. We provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages.