A new method for sexing Oriental White Storks

ABSTRACT.   For species where males and females are monomorphic, or nearly so, determining the sex of individual birds generally requires either capturing birds or collecting samples, such as feathers, for DNA analysis. We developed a new method, involving the use of photographs, to determine the sex of endangered Oriental White Storks ( Ciconia boyciana ). Using photographs, we analyzed the lateral features of the heads of 25 captive storks of known sex (12 males and 13 females) and found differences between males and females in the distance from the bill tip to the nape and the distance from the bill tip to the commissural point. These differences were used to generate a discriminant function that was then tested on 22 captive storks at Hyogo Homeland Park (Toyooka, Japan), and we correctly determined the sex of 18 individuals (82%). In addition, the sex of two wild storks was correctly assigned. Our results suggest that good-quality photographs can be useful for determining the sex of both captive and wild Oriental White Storks and, further, that similar methods may prove useful for determining sex in other species of birds.     

Thermal characteristics of wild and captive Micronesian kingfisher nesting habitats

To provide information for managing the captive population of endangered Guam Micronesian kingfishers (Halcyon cinnamomina cinnamomina), four biologically relevant thermal metrics were compared among captive facilities on the United States mainland and habitats used by wild Micronesian kingfishers on the island of Pohnpei (H. c. reichenbachii), Federated States of Micronesia. Additionally, aviaries where kingfishers laid eggs were compared to those in which birds did not attempt to breed. Compared to aviaries, habitats used by wild Pohnpei kingfishers had 3.2°C higher daily maximum and minimum temperatures and the proportion of time when temperatures were in the birds' thermoneutral zone was 45% greater. No differences were found in the magnitude of temperature fluctuation in captive and wild environments. In captive environments in which birds bred, daily maximum temperatures were 2.1°C higher and temperatures were within the thermoneutral zone 25% more often than in the aviaries where the kingfishers did not breed. No differences were found in the magnitude of temperature fluctuation or the daily minimum temperature. Results suggest that the thermal environment has the potential to influence reproduction, and that consideration should be given to increasing temperatures in captive breeding facilities to improve propagation of the endangered Micronesian kingfisher. Zoo Biol 23:301–308, 2004. Published 2004 Wiley‐Liss, Inc.     

Multiscale Habitat Use and Selection in Cooperatively Breeding Micronesian Kingfishers

Abstract: Information about the interaction between behavior and landscape resources is key to directing conservation management for endangered species. We studied multi-scale occurrence, habitat use, and selection in a cooperatively breeding population of Micronesian kingfishers (Todiramphus cinnamominus) on the island of Pohnpei, Federated States of Micronesia. At the landscape level, point-transect surveys resulted in kingfisher detection frequencies that were higher than those reported in 1994, although they remained 15-40% lower than 1983 indices. Integration of spatially explicit vegetation information with survey results indicated that kingfisher detections were positively associated with the amount of wet forest and grass-urban vegetative cover, and they were negatively associated with agricultural forest, secondary vegetation, and upland forest cover types. We used radiotelemetry and remote sensing to evaluate habitat use by individual kingfishers at the home-range scale. A comparison of habitats in Micronesian kingfisher home ranges with those in randomly placed polygons illustrated that birds used more forested areas than were randomly available in the immediate surrounding area. Further, members of cooperatively breeding groups included more forest in their home ranges than birds in pair-breeding territories, and forested portions of study areas appeared to be saturated with territories. Together, these results suggested that forest habitats were limited for Micronesian kingfishers. Thus, protecting and managing forests is important for the restoration of Micronesian kingfishers to the island of Guam (United States Territory), where they are currently extirpated, as well as to maintaining kingfisher populations on the islands of Pohnpei and Palau. Results further indicated that limited forest resources may restrict dispersal opportunities and, therefore, play a role in delayed dispersal and cooperative behaviors in Micronesian kingfishers.     

Sex determination of three raptor species using morphology and molecular techniques

ABSTRACT.   Accurate identification of sex is important for many raptor studies, but may be difficult to determine in the field for some species. Because of size differences between males and females, morphological measurements have often been used to sex raptors. However, few investigators have examined the accuracy of using measurements of individuals made at one location to sex individuals of the same species at another location. Our objective was to develop more accurate region-specific methods for determining the sex of Red-tailed Hawks ( Buteo jamaicensis ), Red-shouldered Hawks ( B. lineatus ), and Cooper's Hawks ( Accipiter cooperii ) migrating through and wintering in California. We determined sex using a polymerase chain reaction (PCR)-based genetic test and grouped individuals based on sex, age, and geographic area. We did not combine groups due to differences in measurements between age classes and geographic areas. We then compared a suite of morphological measurements between males and females of each combination, and developed both a discriminant function and a flowchart to determine the sex of Red-tailed and Red-shouldered hawks in the field. The flowcharts were more accurate than the functions for both species. We also confirmed the accuracy of the current flowchart used to determine the sex of Cooper's Hawks migrating along the California coast. These region-specific methods for Red-tailed and Cooper's hawks were generally more accurate than published methods, possibly indicating different populations of these species and highlighting the importance of validating sexing methods when using them in different locations.     

Population boundaries and genetic diversity in the endangered Mariana crow (Corvus kubaryi)

The Mariana crow (Corvus kubaryi) is an endangered species that is restricted to the islands of Guam and Rota in the Mariana archipelago. Predation by the introduced brown tree snake (Boiga irregularis) has decimated bird populations on Guam, and the crow population there is the last wild remnant of the endemic forest avifauna. The population on Guam is critically endangered and, despite intensive management, the population has continued to decline. Additional management options include intermixing the Guam and Rota populations, but such options are best evaluated within a population genetics framework. We used three types of molecular markers to assay genetic variation in the Mariana crow: mitochondrial DNA (mtDNA) sequences, minisatellites and microsatellites. The two populations could be differentiated by mtDNA sequencing and they differed in allele frequencies at nuclear markers. Thus, the populations could be designated as evolutionarily significant units. However, the Guam population is genetically more diverse than the Rota population, and its survival probability if managed separately is very low. All markers did indicate that the two populations are closely related and separated by a shallow genealogical division. Intermixing the populations is justified by two rationales. First, the apparent population differences may result from recent human activities. Second, a greater amount of genetic information may be preserved by joint management. The translocation of birds from Rota to Guam has begun, but strategies that will ensure maintenance of the variation in the Guam population warrant further exploration.     

Determination of Sex from Footprint Dimensions in a Ghanaian Population

The present study sought to verify the utility and reliability of footprint dimensions in sex determination in a Ghanaian population. Bilateral footprints were obtained from 126 Ghanaian students (66 males and 60 females) aged 18–30 years at Koforidua Polytechnic using an ink pad and white papers. Seven dimensions–length of each toe (designated T1-T5) from the most anterior point of the toe to the mid-rear heel point, breadth at ball (BAB) and breadth at heel (BAH)–and the heel-ball (HB) index were obtained from each footprint. Some footprint dimensions (i.e. T2, T3, T4 and T5) showed statistically significant bilateral asymmetry in males only. All the footprint dimensions, except HB index, were significantly greater in males than females (p<0.001). Applied singly in discriminant function analysis, the footprint dimensions allowed 69.8%-80.3% of cases to be correctly classified into their sex groups; the accuracy of sex classification was higher using left footprints than right footprints. With all dimensions subjected to stepwise discriminant function analysis 80.3% and 77% of cases could be correctly classified, combining both T5 and BAH for left footprints and T1, BAB and BAH for left footprints respectively. The present study has demonstrated, for the first time among Ghanaian subjects, the utility and reliability of sex determination standards developed from footprint dimensions. The results thus provide the baseline for elaborated studies in the future.     

Bill length: a reliable method for sexing Purple Sandpipers

ABSTRACT.   Purple Sandpipers ( Calidris maritima ) are sexually dimorphic, with females larger than males. We tested the reliability of using bill length to sex individuals and estimate the sex ratio at a stopover site in Iceland in May 2003 and 2005. Feather samples from 65 of 324 captured birds were used for molecular sexing, and generalized linear models (GLMs) were used to discriminate the sexes from body measurements of the molecularly sexed birds. About 3% of the 324 individuals were misclassified by the Harding-Cassie method, and the proportion of males was 0.657 compared to 0.656 according to the best GLM. Our results showed that the reliability of determining the sex and sex ratio of Purple Sandpipers using a Harding-Cassie plot of bill length measurements was high, but that reliability was improved by including other variables in a GLM. The estimate of an uneven sex ratio in the population we studied was not due to a systematic error, and supports the conclusion of earlier studies that Purple Sandpipers exhibit an uneven sex ratio in favor of males.     

A Comparison of Field and Molecular Techniques for Sexing Beavers

Abstract: The traditional method of sex identification in beavers (Castor canadensis) by external palpation can be inaccurate. We tested 2 genetic methods for determining sex in beavers, the zinc-finger DNA marker and the Y chromosome-specific sex determining region (SRY) marker. The SRY marker identified sex correctly in 57 of 67 (85%) beavers, whereas the zinc-finger technique was successful less often in only 48 of 67 (72%) animals. Sex was correctly assigned by palpation for 21 of 27 beavers (78%). Beaver studies in which accurate sex identification is critical may benefit by verifying the sex of individuals using one or both of these molecular markers.     

Relationships between bird morphology and prey selection in two sympatric Great Cormorant Phalacrocorax carbo subspecies during winter

Variation in bird morphology (notably sex size dimorphism) has been suggested to contribute to differences in food use between individuals. We explore the hypothesis of food partitioning (diet overlap and prey size selection) in two sympatric subspecies of the Great Cormorant Phalacrocorax carbo with respect to bird morphology (subspecies and sex) in inland French waters. These areas represent a recent and increasing contact zone used as a common non-territorial winter feeding area by the continental P. c. sinensis and the marine P. c. carbo subspecies. A high dietary overlap between subspecies and sexes was found at the major sites studied. Prey size selection was found to be site-specific and generally related to a gradient of structural size and body mass (male P. c. carbo  > male P. c. sinensis  > female P. c. carbo  > female P. c. sinensis ). With respect to bird morphology, differences in prey size consumed by Great Cormorants were more pronounced between sexes in freshwater habitats than between subspecies. This was reinforced by the fact that P. c. carbo birds entering inland areas were smaller than birds on the coast. These results also suggest that bird morphology is an important determinant of dietary differences. Our study demonstrated that P. c. carbo is an efficient generalist forager in continental areas, and food partitioning in type and size of prey between the two subspecies is low.     

Space use by Great Lakes Piping Plovers during the breeding season

ABSTRACT.   Identifying and protecting breeding habitat for imperiled species requires an understanding of the spatial and temporal movements of breeding individuals. During the 2003 and 2004 breeding seasons, we examined space use by Piping Plovers ( Charadrius melodus ) in the federally endangered Great Lakes population. We used coordinate geometry to estimate home range sizes of individual birds and examined relationships between home range size and breeding stage (incubation versus chick rearing), year, sex, number of locations, minimum plover age, distance to the nearest nest, and human beach use (high, medium, or low). The mean size of home ranges of Piping Plovers that fledged at least one chick was 2.9 ± 0.5 (SE) ha (range = 0.4–11.2 ha), and the mean linear beach distance traversed was 475 ± 53 m (range = 130–1435 m). Individuals used 3 times more beach area and 1.5 times more shoreline distance in 2003 than in 2004. Females used smaller areas than males overall and during chick rearing. Home ranges were smallest on beaches with low public use, suggesting that human disturbance may cause greater movement by individual plovers and that larger protected areas may be warranted on beaches frequented by the public to minimize disturbance to breeding birds. Our results demonstrate that nesting Great Lakes Piping Plovers occupy relatively small ranges and, therefore, that even relatively small areas of suitable habitat can have a high conservation value for this endangered population. However, the total area of habitat used varied substantially among individuals, and this should be considered when protecting habitat for the species.     

A select panel of polymorphic microsatellite loci for individual identification of snow leopards (Panthera uncia)

Snow leopards (Panthera uncia) are elusive endangered carnivores found in remote mountain regions of Central Asia. New methods for identifying and counting snow leopards are needed for conservation and management efforts. To develop molecular genetic tools for individual identification of hair and faecal samples, we screened 50 microsatellite loci developed for the domestic cat (Felis catus) in 19 captive snow leopards. Forty‐eight loci were polymorphic with numbers of alleles per locus ranging from two to 11. The probability of observing matching genotypes for unrelated individuals (2.1 × 10^?11) and siblings (7.5 × 10^?5) using the 10 most polymorphic loci was low, suggesting that this panel would easily discriminate among individuals in the wild.     

Sex determination in Turdus amaurochalinus (Passeriformes: Muscicapidae): morphometrical analysis supported by CHD gene

Sex determination is important for conservation and population studies, particularly for reproduction programs of threatened species and behavioural ecology. Turdus amaurochalinus, Creamy-bellied Thrush, only exhibits sexual dimorphism during the breeding season, when males are considered to show intense yellow bills, and females and immature males show dark brown bills. The objectives of this study were: 1) to determine the sex of individuals using genetic techniques, and 2) to test the hypothesis that sex dimorphism can be detected by morphometry. This study was carried out at Parque Nacional da Restinga de Jurubatiba, a preserved area located on the North coast of Rio de Janeiro State. The birds were captured using ornithological nets, singly marked with metal rings, weighed, measured and had blood samples collected before being released. The sex of 42 T. amaurochalinus individuals was determined using the CHD gene marker. A total of 20 males and 22 females were identified from June to August, with peak capture frequency in June. Turdus amaurochalinus females and males differed significantly in morphometrical measures. The most important traits to distinguish males from females were wing length (Student t-test = 4.34, df = 40, p = 0.0001) and weight (Student t-test = 2.08, d f = 40, p = 0.044): females were heavier and had significantly shorter wing length than males. Females and males were correctly classified in 86% and 75% of cases, respectively, using Discriminant Analysis. The molecular analysis was the most secure method for sex determination in the studied species.     

Environmental sex determination in a splash pool copepod

The sex-determining mechanism has important demographic and genetic consequences by virtue of its effect on the population sex ratio. Here we investigate the effect of temperature dependent sex determination (TSD) on the primary sex ratio of the harpacticoid copepod, Tigriopus californicus . At the two experimental temperatures (15° and 22°C) used in this study, the primary sex ratio is almost always biased in favour of males. Higher temperatures induce masculinization and the change in sex ratio is not caused by differential mortality of the sexes. The mean level of TSD in the population is small (proportion of males increases by ~5% between 15° and 22°C) because only one-third of the families actually exhibit a significant sex-ratio response while the rest of the population is insensitive to temperature. A comparison of the primary sex ratio and the level of TSD between two locations reveals few differences among populations. Finally, individuals still exhibited TSD after having been maintained under constant temperature conditions in the lab for several generations. In addition the proportion of temperature-sensitive individuals remained unchanged. This suggests that the observed level of TSD is not an artefact of testing field-captured individuals in a novel laboratory environment. At this point the adaptive significance of temperature-dependent sex determination in T. californicus remains unknown.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 76 , 511–520.     

Identification of a sex-determining region in Nile tilapia (Oreochromis niloticus) using bulked segregant analysis

Sex determination in the Nile tilapia (Oreochromis niloticus) is thought to be an XX-XY (male heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major locus using bulked segregant analysis. Ten microsatellite markers belonging to linkage group 8 were found to be linked to phenotypic sex. The putative Y-chromosome alleles correctly predict the sex of 95% of male and female individuals in two families. Our results suggest a major sex-determining locus within a few centimorgans of markers UNH995 and UNH104. A third family from the same population showed no evidence for linkage of this region with phenotypic sex, indicating that additional genetic and/or environmental factors regulate sex determination in some families. These markers have immediate utility for studying the strength of different Y chromosome alleles, and for identifying broodstock carrying one or more copies of the Y haplotype.     

Computation vs. cloning: evaluation of two methods for haplotype determination

Nuclear sequence data, often from multiple loci, are increasingly being employed in analyses of population structure and history, yet there has been relatively little evaluation of methods for accurately and efficiently separating the alleles or haplotypes in heterozygous individuals. We compared the performance of a computational method of haplotype reconstruction and standard cloning methods using a highly variable intron (ornithine decarboxylase, intron 6) in three closely related species of dabbling ducks (genus Anas). Cloned sequences from 32 individuals were compared to results obtained from phase 2.1.1 . phase correctly identified haplotypes in 28 of 30 heterozygous individuals when the underlying model assumed no recombination. Haplotypes of the remaining two individuals were also inferred correctly except for unique polymorphisms, the phase of which was appropriately indicated as uncertain (phase probability = 0.5). For a larger set of 232 individuals, results were essentially identical regardless of the recombination model used and haplotypes for all 30 of the tested heterozygotes were correctly inferred, with the exception of uncertain phase for unique polymorphisms in one individual. In contrast, initial sequences of one clone per sample yielded accurate haplotype determination in only 26 of 30 individuals; polymerase chain reaction (PCR)/cloning errors resulting from misincorporation of individual nucleotides could be recognized and avoided by comparison to direct sequences, but errors due to PCR recombination resulted in incorrect haplotype reconstruction in four individuals. The accuracy of haplotypes reconstructed by phase , even when dealing with a relatively small number of samples and numerous variable sites, suggests broad utility of computational approaches for reducing the cost and improving the efficiency of data collection from nuclear sequence loci.     

PRODUCTION OF SECONDARY METABOLITES BY FILAMENTOUS TROPICAL MARINE CYANOBACTERIA: ECOLOGICAL FUNCTIONS OF THE COMPOUNDS

Cyanobacterial blooms are becoming more common in many reef habitats. The broadly acting feeding deterrent compound ypaoamide, produced by a mixed cyanobacterial assemblage, has been linked to bloom formation and mass fish die-offs ( Siganus argenteus and Siganus spinus ) in Guam. Specific metabolites produced by Lyngbya majuscula Gomont act as both feeding attractants to the specialist herbivore Stylocheilus longicauda , and as effective feeding deterrents to generalist fishes. Two-dimensional TLC (2D-TLC) analysis of cyanobacterial crude extracts was used to select chemically distinct populations (chemotypes) of bloom-forming filamentous cyanobacteria for chemical and ecological evaluation. Crude extracts produced by different species, chemotypes, and chemically distinct Micronesian marine cyanobacterial assemblages deter feeding activity of generalist reef herbivores. The ecological function of cyanobacterial secondary metabolites, especially as related to diversity of compound production and the relationship of metabolite production to bloom formation is discussed.     

Enhanced understanding of predator–prey relationships using molecular methods to identify predator species,individual and sex

Predator species identification is an important step in understanding predator‐prey interactions, but predator identifications using kill site observations are often unreliable. We used molecular tools to analyse predator saliva, scat and hair from caribou calf kills in Newfoundland, Canada to identify the predator species, individual and sex. We sampled DNA from 32 carcasses using cotton swabs to collect predator saliva. We used fragment length analysis and sequencing of mitochondrial DNA to distinguish between coyote, black bear, Canada lynx and red fox and used nuclear DNA microsatellite analysis to identify individuals. We compared predator species detected using molecular tools to those assigned via field observations at each kill. We identified a predator species at 94% of carcasses using molecular methods, while observational methods assigned a predator species to 62.5% of kills. Molecular methods attributed 66.7% of kills to coyote and 33.3% to black bear, while observations assigned 40%, 45%, 10% and 5% to coyote, bear, lynx and fox, respectively. Individual identification was successful at 70% of kills where a predator species was identified. Only one individual was identified at each kill, but some individuals were found at multiple kills. Predator sex was predominantly male. We demonstrate the first large‐scale evaluation of predator species, individual and sex identification using molecular techniques to extract DNA from swabs of wild prey carcasses. Our results indicate that kill site swabs (i) can be highly successful in identifying the predator species and individual responsible; and (ii) serve to inform and complement traditional methods.     

Morphological sexing of passerines: not valid over larger geographical scales

Sex determination of birds is important for many ecological studies but is often difficult in species with monomorphic plumage. Morphology often provides a possibility for sex determination, but the characters need to be verified. We tested whether five passerine species can be sexed according to standard morphological measurements applying a forward logistic regression with sex determined by molecular analysis as the dependent variable. Furthermore, we tested whether the results can be used on a larger geographic scale by applying morphological sexing methods gained by similar studies from other regions to our data set. Of the five species of this study only Garden Warblers Sylvia borin could not be sexed morphologically. In the Robin Erithacus rubecula, 87.2% of all individuals were sexed correctly. For Reed Warblers Acrocephalus scirpaceus, Willow Warblers Phylloscopus trochilus and Reed Buntings Emberiza schoeniclus, the respective values were 77.6, 89.4 and 86.4%. When the logistic regression functions from similar studies on Robins and Reed Buntings in Denmark and Scotland were applied to the birds from south-western Germany, they performed less well compared to the original dataset of these studies and compared to the logistic regression function of our own study. The same was the case for Willow Warblers when a wing length criterion used in Great Britain was applied to the birds of our study. These discrepancies may have several explanations: (1) the models are optimised for the dataset from which they were extracted, (2) inter-ringer variation in measurements, (3) the use of different age cohorts, (4) different morphology due to different habitat availability around the study site, or, most likely, (5) different morphology due to different migratory behaviour. We recommend that morphological sex differentiation methods similar to this study (1) be only used population specific, (2) only with one age cohort and (3) to adjust the extracted equations from time to time.     

Sex determination of ancient human skeletons using DNA

A method for determining the sex of human skeletons was developed using molecular genetic techniques. The amelogenin gene, found on the X and Y chromosomes, was examined using the polymerase chain reaction (PCR) and a nonradioactive dot blot procedure. DNA was analyzed from 20 modern individuals of known sex and 20 skeletons from an archaeological site in central Illinois dating to A.D. 1300. An independent assessment of the sex of each skeleton was made according to standard osteological methods. The sex of 19 ancient and 20 modern individuals was accurately determined using this molecular genetic technique. Molecular sex determination will be especially useful for juvenile and fragmentary remains when it is difficult, or impossible, to establish an individual's sex from morphological features. © 1996 Wiley-Liss, Inc.