Evidence of adult male scarcity associated with female-skewed offspring sex ratios in sea turtles.

Climate change is a clear and present threat to species survival. For species with temperature-dependent sex determination, including all sea turtles, it has been hypothesised that climate change may drive the creation of sex-ratio biases leading to population extinctions1. Through a global analysis across multiple species, we present the first direct empirical evidence for a demographic consequence of male scarcity in sea turtle populations, with a lower incidence of multiple paternity being found in populations with more extreme female-biased hatchling sex-ratio skews. For green turtles, when the female bias in hatchling sex ratio was >90%, the incidence of multiple paternity was low compared to other nesting sites, being 24.5% in the eastern Mediterranean (Cyprus), 36.4% on Redang Island (Malaysia) and 15.4% on the southern Great Barrier Reef (Heron Island, Australia) compared to higher values (range 61.1-91.7%) at other sites globally. These results suggest that a low incidence of multiple paternity may serve as a harbinger of future problems with egg fertility if males become even scarcer. Assessments of the incidence of multiple paternity at sites where adult males are expected to become scarce, such as Raine Island on the northern Great Barrier Reef in Australia, may help to identify when a lack of males raises the threat of local extinctions. In such cases, intervention to increase the production of male hatchlings may be needed.

A Review of Patterns of Multiple Paternity Across Sea Turtle Rookeries.

Why females would mate with multiple partners and have multiple fathers for clutches or litters is a long-standing enigma. There is a broad dichotomy in hypotheses ranging from polyandry having benefits to simply being an unavoidable consequence of a high incidence of male-female encounters. If females simply give in to mating when it is too costly to avoid being harassed by males (convenience polyandry), then there should be a higher rate of mating as density increases. However, if females actively seek males because they benefit from multiple mating, then mating frequency, and consequently the incidence of multiple paternity of clutches, should be high throughout. To explore these competing explanations, here we review the incidence of multiple paternity for sea turtles nesting around the World. Across 30 rookeries, including all 7 species of sea turtle, the incidence of multiple paternity was only weakly linked to rookery size (r2=0.14). However, using high resolution at-sea GPS tracking we show that the specifics of movement patterns play a key role in driving packing density and hence the likely rate of male-female encounters. When individuals use the same focal areas, packing density could be 100× greater than when assuming individuals move independently. Once the extent of adult movements in the breeding season was considered so that movements and abundance could be combined to produce a measure of density, then across rookeries we found a very tight relationship (r2=0.96) between packing density and the incidence of multiple paternity. These findings suggest that multiple paternity in sea turtles may have no benefit, but is simply a consequence of the incidence of male-female encounters.

Genetic isolation in an endemic African habitat specialist.

The Chestnut-banded Plover Charadrius pallidus is a Near-Threatened shorebird species endemic to mainland Africa. We examined levels of genetic differentiation between its two morphologically and geographically distinct subspecies, C. p. pallidus in southern Africa (population size 11 000-16 000) and C. p. venustus in eastern Africa (population size 6500). In contrast to other plover species that maintain genetic connectivity over thousands of kilometres across continental Africa, we found profound genetic differences between remote sampling sites. Phylogenetic network analysis based on four nuclear and two mitochondrial gene regions, and population genetic structure analyses based on 11 microsatellite loci, indicated strong genetic divergence, with 2.36% mitochondrial sequence divergence between individuals sampled in Namibia (southern Africa) and those of Kenya and Tanzania (eastern Africa). This distinction between southern and eastern African populations was also supported by highly distinct genetic clusters based on microsatellite markers (global FST  = 0.309, GST' = 0.510, D = 0.182). Behavioural factors that may promote genetic differentiation in this species include habitat specialization, monogamous mating behaviour and sedentariness. Reliance on an extremely small number of saline lakes for breeding and limited dispersal between populations are likely to promote reproductive and genetic isolation between eastern and southern Africa. We suggest that the two Chestnut-banded Plover subspecies may warrant elevation to full species status. To assess this distinction fully, additional sample collection will be needed, with analysis of genetic and phenotypic traits from across the species' entire breeding range.

DNA amplification in the field: move over PCR, here comes LAMP.

It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR-based methods has been the necessity for a generally non-portable, laboratory setting to undertake the time-consuming thermocycling protocols. LAMP (loop-mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno-Cuadros et al. () demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in 'real-time', LAMP promises to revolutionize how molecular ecology is practised in the field.

North or south? Phylogenetic and biogeographic origins of a globally distributed avian clade.

Establishing phylogenetic relationships within a clade can help to infer ancestral origins and indicate how widespread species reached their current biogeographic distributions. The small plovers, genus Charadrius, are cosmopolitan shorebirds, distributed across all continents except Antarctica. Here we present a global, species-level molecular phylogeny of this group based on four nuclear (ADH5, FIB7, MYO2 and RAG1) and two mitochondrial (COI and ND3) genes, and use the phylogeny to examine the biogeographic origin of the genus. A Bayesian multispecies coalescent approach identified two major clades (CRD I and CRD II) within the genus. Clade CRD I contains three species (Thinornis novaeseelandiae, Thinornis rubricollis and Eudromias morinellus), and CRD II one species (Anarhynchus frontalis), that were previously placed outside the Charadrius genus. In contrast to earlier work, ancestral area analyses using parsimony and Bayesian methods supported an origin of the Charadrius plovers in the Northern hemisphere. We propose that major radiations in this group were associated with shifts in the range of these ancestral plover species, leading to colonisation of the Southern hemisphere.

Ontogenic differences in sexual size dimorphism across four plover populations.

Sexual size dimorphism (SSD) among adults is commonly observed in animals and is considered to be adaptive. However, the ontogenic emergence of SSD, i.e. the timing of divergence in body size between males and females, has only recently received attention. It is widely acknowledged that the ontogeny of SSD may differ between species, but it remains unclear how variable the ontogeny of SSD is within species. Kentish Plovers Charadrius alexandrinus and Snowy Plovers C. nivosus are closely related wader species that exhibit similar, moderate (c. 4%), male-biased adult SSD. To assess when SSD emerges we recorded tarsus length variation among 759 offspring in four populations of these species. Tarsus length of chicks was measured on the day of hatching and up to three times on recapture before fledging. In one population (Mexico, Snowy Plovers), males and females differed in size from the day of hatching, whereas growth rates differed between the sexes in two populations (Turkey and United Arab Emirates, both Kentish Plovers). In contrast, a fourth population (Cape Verde, Kentish Plovers) showed no significant SSD in juveniles. Our results suggest that adult SSD can emerge at different stages of development (prenatal, postnatal and post-juvenile) in different populations of the same species. We discuss the proximate mechanisms that may underlie these developmental differences.

Multi-decadal range changes vs. thermal adaptation for north east Atlantic oceanic copepods in the face of climate change.

Populations may potentially respond to climate change in various ways including moving to new areas or alternatively staying where they are and adapting as conditions shift. Traditional laboratory and mesocosm experiments last days to weeks and thus only give a limited picture of thermal adaptation, whereas ocean warming occurring over decades allows the potential for selection of new strains better adapted to warmer conditions. Evidence for adaptation in natural systems is equivocal. We used a 50-year time series comprising of 117 056 samples in the NE Atlantic, to quantify the abundance and distribution of two particularly important and abundant members of the ocean plankton (copepods of the genus Calanus) that play a key trophic role for fisheries. Abundance of C. finmarchicus, a cold-water species, and C. helgolandicus, a warm-water species, were negatively and positively related to sea surface temperature (SST) respectively. However, the abundance vs. SST relationships for neither species changed over time in a manner consistent with thermal adaptation. Accompanying the lack of evidence for thermal adaptation there has been an unabated range contraction for C. finmarchicus and range expansion for C. helgolandicus. Our evidence suggests that thermal adaptation has not mitigated the impacts of ocean warming for dramatic range changes of these key species and points to continued dramatic climate induced changes in the biology of the oceans.

Living on the edge: how philopatry maintains adaptive potential.

Without genetic variation, species cannot cope with changing environments, and evolution does not proceed. In endangered species, adaptive potential may be eroded by decreased population sizes and processes that further reduce gene flow such as philopatry and local adaptations. Here, we focused on the philopatric and endangered loggerhead sea turtle (Caretta caretta) nesting in Cape Verde as a model system to investigate the link between adaptive potential and philopatry. We produced a dataset of three complementary genomic regions to investigate female philopatric behaviour (mitochondrial DNA), male-mediated gene flow (microsatellites) and adaptive potential (major histocompatibility complex, MHC). Results revealed genetically distinct nesting colonies, indicating remarkably small-scale philopatric behaviour of females. Furthermore, these colonies also harboured local pools of MHC alleles, especially at the margins of the population's distribution, which are therefore important reserves of additional diversity for the population. Meanwhile, directional male-mediated gene flow from the margins of distribution sustains the adaptive potential for the entire rookery. We therefore present the first evidence for a positive association between philopatry and locally adapted genomic regions. Contrary to expectation, we propose that philopatry conserves a high adaptive potential at the margins of a distribution, while asymmetric gene flow maintains genetic connectivity with the rest of the population.

Identification of genetically and oceanographically distinct blooms of jellyfish.

Reports of nuisance jellyfish blooms have increased worldwide during the last half-century, but the possible causes remain unclear. A persistent difficulty lies in identifying whether blooms occur owing to local or regional processes. This issue can be resolved, in part, by establishing the geographical scales of connectivity among locations, which may be addressed using genetic analyses and oceanographic modelling. We used landscape genetics and Lagrangian modelling of oceanographic dispersal to explore patterns of connectivity in the scyphozoan jellyfish Rhizostoma octopus, which occurs en masse at locations in the Irish Sea and northeastern Atlantic. We found significant genetic structure distinguishing three populations, with both consistencies and inconsistencies with prevailing physical oceanographic patterns. Our analyses identify locations where blooms occur in apparently geographically isolated populations, locations where blooms may be the source or result of migrants, and a location where blooms do not occur consistently and jellyfish are mostly immigrant. Our interdisciplinary approach thus provides a means to ascertain the geographical origins of jellyfish in outbreaks, which may have wide utility as increased international efforts investigate jellyfish blooms.

Lost at sea: genetic, oceanographic and meteorological evidence for storm-forced dispersal.

For many species, there is broad-scale dispersal of juvenile stages and/or long-distance migration of individuals and hence the processes that drive these various wide-ranging movements have important life-history consequences. Sea turtles are one of these paradigmatic long-distance travellers, with hatchlings thought to be dispersed by ocean currents and adults often shuttling between distant breeding and foraging grounds. Here, we use multi-disciplinary oceanographic, atmospheric and genetic mixed stock analyses to show that juvenile turtles are encountered 'downstream' at sites predicted by currents. However, in some cases, unusual occurrences of juveniles are more readily explained by storm events and we show that juvenile turtles may be displaced thousands of kilometres from their expected dispersal based on prevailing ocean currents. As such, storms may be a route by which unexpected areas are encountered by juveniles which may in turn shape adult migrations. Increased stormy weather predicted under climate change scenarios suggests an increasing role of storms in dispersal of sea turtles and other marine groups with life-stages near the ocean surface.

Conflict between genetic and phenotypic differentiation: the evolutionary history of a 'lost and rediscovered' shorebird.

Understanding and resolving conflicts between phenotypic and genetic differentiation is central to evolutionary research. While phenotypically monomorphic species may exhibit deep genetic divergences, some morphologically distinct taxa lack notable genetic differentiation. Here we conduct a molecular investigation of an enigmatic shorebird with a convoluted taxonomic history, the White-faced Plover (Charadrius alexandrinus dealbatus), widely regarded as a subspecies of the Kentish Plover (C. alexandrinus). Described as distinct in 1863, its name was consistently misapplied in subsequent decades until taxonomic clarification ensued in 2008. Using a recently proposed test of species delimitation, we reconfirm the phenotypic distinctness of dealbatus. We then compare three mitochondrial and seven nuclear DNA markers among 278 samples of dealbatus and alexandrinus from across their breeding range and four other closely related plovers. We fail to find any population genetic differentiation between dealbatus and alexandrinus, whereas the other species are deeply diverged at the study loci. Kentish Plovers join a small but growing list of species for which low levels of genetic differentiation are accompanied by the presence of strong phenotypic divergence, suggesting that diagnostic phenotypic characters may be encoded by few genes that are difficult to detect. Alternatively, gene expression differences may be crucial in producing different phenotypes whereas neutral differentiation may be lagging behind.

Microsatellite markers characterized in the barn owl (Tyto alba) and of high utility in other owls (Strigiformes: AVES).

We have identified 15 polymorphic microsatellite loci for the barn owl (Tyto alba), five from testing published owl loci and 10 from testing non-owl loci, including loci known to be of high utility in passerines and shorebirds. All 15 loci were sequenced in barn owl, and new primer sets were designed for eight loci. The 15 polymorphic loci displayed two to 26 alleles in 56-58 barn owls. When tested in 10 other owl species (n = 1-6 individuals), between four and nine loci were polymorphic per species. These loci are suitable for studies of population structure and parentage in owls.

New and improved molecular sexing methods for museum bird specimens.

We present two new avian molecular sexing techniques for nonpasserine and passerine birds (Neognathae), which are more suitable for use with museum specimens than earlier methods. The technique for nonpasserines is based on a new primer (M5) which, in combination with the existing P8 primer, targets a smaller amplicon in the CHD1 sex-linked gene than previously. Primers targeting ATP5A1, an avian sex-linked gene not previously used for sex identification, were developed for passerines. Comprehensive testing across species demonstrated that both primer pairs sex a range of different species within their respective taxonomic groups. Rigorous evaluation of each method within species showed that these permitted sexing of specimens dating from the 1850s. For corn bunting museum specimens, the ATP5A1 method sexed 98% of 63 samples (1857-1966). The M5/P8 CHD1 method was similarly successful, sexing 90% of 384 moorhen specimens from six different museum collections (1855-2001). In contrast, the original P2/P8 CHD1 sexing method only identified the sex of less than half of 111 museum moorhen samples. In addition to dried skin samples, these methods may be useful for other types of material that yield degraded or damaged DNA, and are hence potential new sexing tools for avian conservation genetics, population management and wildlife forensics.

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA.

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176-bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the 'DNA barcode' region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full 'barcode' of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176-bp target compared with a larger 288-bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR-based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).

Detecting female precise natal philopatry in green turtles using assignment methods.

It is well established that sea turtles return to natal rookeries to mate and lay their eggs, and that individual females are faithful to particular nesting sites within the rookery. Less certain is whether females are precisely returning to their natal beach. Attempts to demonstrate such precise natal philopatry with genetic data have had mixed success. Here we focused on the green turtles of three nesting sites in the Ascension Island rookery, separated by 5-15 km. Our approach differed from previous work in two key areas. First, we used male microsatellite data (five loci) reconstructed from samples collected from their offspring (N = 17) in addition to data for samples taken directly from females (N = 139). Second, we employed assignment methods in addition to the more traditional F-statistics. No significant genetic structure could be demonstrated with F(ST). However, when average assignment probabilities of females were examined, those for nesting populations in which they were sampled were indeed significantly higher than their probabilities for other populations (Mann-Whitney U-test: P < 0.001). Further evidence was provided by a significant result for the mAI(C) test (P < 0.001), supporting greater natal philopatry for females compared with males. The results suggest that female natal site fidelity was not sufficient for significant genetic differentiation among the nesting populations within the rookery, but detectable with assignment tests.

Polyandry in a marine turtle: females make the best of a bad job.

The female perspective on reproductive strategies remains one of the most active areas of debate in biology. Even though a single mating is often sufficient to satisfy the fertilization needs of most females and the act of further mating incurs costs, multiple paternity within broods or clutches is a common observation in nature. Direct or indirect advantage to females is the most popular explanation. However, the ubiquity of this explanation is being challenged by an increasing number of cases for which benefits are not evident. For the first time, we test possible fitness correlates of multiple paternity in a marine turtle, an organism that has long attracted attention in this area of research. Contrary to the wide-spread assumption that multiple mating by female marine turtles confers fitness benefits, none were apparent. In this study, the environment played a far stronger role in determining the success of clutches than whether paternity had been single or multiple. A more likely explanation for observations of multiply sired clutches in marine turtles is that these are successful outcomes of male coercion, where females have conceded to superfluous matings as a compromise. Thus, multiple matings by female marine turtles may be a form of damage control as females attempt to make the best of a bad job in response to male harassment.

Widespread local house-sparrow extinctions.

House-sparrow populations have declined sharply in Western Europe in recent decades, but the reasons for this decline have yet to be identified, despite intense public interest in the matter. Here we use a combination of field experimentation, genetic analysis and demographic data to show that a reduction in winter food supply caused by agricultural intensification is probably the principal explanation for the widespread local extinctions of rural house-sparrow populations in southern England. We show that farmland populations exhibit fine-level genetic structuring and that some populations are unable to sustain themselves (sinks), whereas others act as sources.

Sex and death: CHD1Z associated with high mortality in moorhens.

Sex ratios in clutches of moorhens (Gallinula chloropus) in Britain were measured on 83 chicks using the sex-linked CHD1 gene (Chromo-helicase/ATPase-DNA binding protein 1). Among birds, the female is the heterogametic sex (Z and W chromosomes), and the male is homogametic (two copies of the Z chromosome). We report variation among the PCR-amplified fragments of the CHD1Z, and the death of nearly all heterozygous male chicks (92%). In contrast, survivorship among females and homozygote males was 54-60%. Mortality in male heterozygotes was significantly higher than that of male homozygotes (P < 0.001). Chick and egg biometrics were not significantly different between these males. The CHD1Z was unlikely to be directly responsible but may have been hitchhiked by the causal gene(s). The observations appear to follow a classic underdominance (heterozygote inferiority) pattern, but raise the paradoxical question of why one form of the Z chromosome has not been fixed, as is expected from evolutionary theory. We discuss possible explanations and include a survey of British populations based on skin specimens.

Reciprocal Natural Selection on Host-Parasite Phenotypes.

Coevolution is evolution in one species in response to selection imposed by a second species, followed by evolution in the second species in response to reciprocal selection imposed by the first species. Although reciprocal selection is a prerequisite of coevolution, it has seldom been documented in natural populations. We examined the feasibility of reciprocal selection in a simple host-parasite system consisting of feral pigeons (Columba livia) and their Ischnoceran feather lice (Phthiraptera: Insecta). We tested for a selective effect of parasites on hosts with experimentally altered defenses and for a selective effect of host defense on a component of parasite escape. Previous work indicates that pigeons control lice through efficient preening, while lice escape from preening using complex avoidance behavior. Our results show that feral pigeons with impaired preening, owing to slight bill deformities, have higher louse loads than pigeons with normal bills. We use a controlled experiment to show that high louse loads reduce the survival of pigeons, suggesting that lice select for efficient preening and against bill deformities. In a reciprocal experiment, we demonstrate that preening with a normal bill selects for small body size in lice, which may facilitate their escape from preening. The results of this study verify a crucial element of coevolutionary theory by identifying likely targets of reciprocal phenotypic selection between host and parasite.