A method for estimating population sex ratio for sage‐grouse using noninvasive genetic samples

Population sex ratio is an important metric for wildlife management and conservation, but estimates can be difficult to obtain, particularly for sexually monomorphic species or for species that differ in detection probability between the sexes. Noninvasive genetic sampling (NGS) using polymerase chain reaction (PCR) has become a common method for identifying sex from sources such as hair, feathers or faeces, and is a potential source for estimating sex ratio. If, however, PCR success is sex‐biased, naively using NGS could lead to a biased sex ratio estimator. We measured PCR success rates and error rates for amplifying the W and Z chromosomes from greater sage‐grouse (Centrocercus urophasianus) faecal samples, examined how success and error rates for sex identification changed in response to faecal sample exposure time, and used simulation models to evaluate precision and bias of three sex assignment criteria for estimating population sex ratio with variable sample sizes and levels of PCR replication. We found PCR success rates were higher for females than males and that choice of sex assignment criteria influenced the bias and precision of corresponding sex ratio estimates. Our simulations demonstrate the importance of considering the interplay between the sex bias of PCR success, number of genotyping replicates, sample size, true population sex ratio and accuracy of assignment rules for designing future studies. Our results suggest that using faecal DNA for estimating the sex ratio of sage‐grouse populations has great potential and, with minor adaptations and similar marker evaluations, should be applicable to numerous species.     

Absence of repetitive DNA sequences associated with sex chromosomes in natural populations of the Trinidad guppy (Poecilia reticulata)

The genus Poecilia has been widely studied as a model for the evolution of sex chromosomes. In the course of molecular studies on population genetic structure and sexual selection in the Trinidad guppy, we examined our preparations for male-linked, repetitive DNA polymorphisms. We have not obtained any evidence of male-specific polymorphisms, in contrast to an earlier study. Our results have significant implications for theories on the evolution of sex chromosomes.Correspondence to: F. Breden     

Genetic detection of sex-biased dispersal

We investigated the application of a recently developed genetic test for sex bias in dispersal. This test determines an animal's 'assignment index' or the expected frequency of its genotype in the population in which it is captured. Low assignment indices indicate a low probability of being born locally. We investigated the use of this test with the white-footed mouse, Peromyscus leucopus, in which dispersal is predominantly male-biased, but not extreme. We found that male P. leucopus had significantly lower assignment indices than females. These data suggest that the genetic test for sex bias in dispersal has potential to be used with species that do not have extreme sex-biased dispersal tendencies.     

High error rates for avian molecular sex identification primer sets applied to molted feathers

ABSTRACT Molted feathers are becoming increasingly important as a source of DNA for identifying the sex of individuals, and accurate methods for molecular sex identification are needed. Three molecular sex identification primer sets have been developed for use in nearly all nonratite birds, but performance of these primer sets has not been evaluated for molted feathers. For two species of birds, the Ring‐necked Pheasant (Phasianus colchicus) and the Scarlet Macaw (Ara macao), we evaluated success and error rates among primer sets using DNA from molted feathers and assessed the percentage of times an incorrect sex would be assigned when analyses are completed in duplicate. Amplification success rates differed among the primer sets for both species, ranging from 67.5% to 89.2% (P= 0.0002 and 0.009), and error rates were high, ranging from 1.9% to 24.2%. Success rates and error rates were not consistent between species and among primer sets. To improve the accuracy of molecular sex identification tests when using molted feathers, we suggest determining acceptable confidence levels in the accuracy of sex assignment, conducting pilot tests to evaluate the performance of different primer sets, and using high‐resolution electrophoresis systems to increase detection of errors.     

A critique of avian CHD‐based molecular sexing protocols illustrated by a Z‐chromosome polymorphism detected in auklets

The sexes of non‐ratite birds can be determined routinely by PCR amplification of the CHD‐Z and CHD‐W genes. CHD‐based molecular sexing of four species of auklets revealed the presence of a polymorphism in the Z chromosome. No deviation from a 1:1 sex ratio was observed among the chicks, though the analyses were of limited power. Polymorphism in the CHD‐Z gene has not been reported previously in any bird, but if undetected it could lead to the incorrect assignment of sex. We discuss the potential difficulties caused by a polymorphism such as that identified in auklets and the merits of alternative CHD‐based sexing protocols and primers.     

Is variation in brood sex ratios adaptive in the great tit (Parus major)?

Life-history theory predicts skewed offspring sex ratios ina range of situations in which the costs and benefits of producingthe two sexes differ. In recent years, many studies have demonstratedbiased sex ratios in a variety of bird species. However, manyof these investigations have been based on small sample sizes,on data from a single year, or both. Using a recently developedpolymerase chain reaction-based molecular DNA technique, 912great tit (Parus major) nestlings from 118 broods in 5 differentyears were sexed. As found in a number of previous studieson the same species, there were significant predictors of offspringsex ratio in individual years. However, there were no consistenttrends across years, and none of the measured variables significantlypredicted sex ratio over all years combined. Furthermore, broodsex ratio of the population did not depart from the expectedbinomial distribution. Although there are theoretical advantagesto manipulating the sex ratio in this and other species, thephysiological mechanism by which it is achieved in birds remainsobscure. We argue that data from several years are needed toconfirm whether facultative sex ratio manipulation is a consistentbreeding strategy used by birds.     

Pheromone production, male abundance, body size, and the evolution of elaborate antennae in moths

The males of some species of moths possess elaborate feathery antennae. It is widely assumed that these striking morphological features have evolved through selection for males with greater sensitivity to the female sex pheromone, which is typically released in minute quantities. Accordingly, females of species in which males have elaborate (i.e., pectinate, bipectinate, or quadripectinate) antennae should produce the smallest quantities of pheromone. Alternatively, antennal morphology may be associated with the chemical properties of the pheromone components, with elaborate antennae being associated with pheromones that diffuse more quickly (i.e., have lower molecular weights). Finally, antennal morphology may reflect population structure, with low population abundance selecting for higher sensitivity and hence more elaborate antennae. We conducted a phylogenetic comparative analysis to test these explanations using pheromone chemical data and trapping data for 152 moth species. Elaborate antennae are associated with larger body size (longer forewing length), which suggests a biological cost that smaller moth species cannot bear. Body size is also positively correlated with pheromone titre and negatively correlated with population abundance (estimated by male abundance). Removing the effects of body size revealed no association between the shape of antennae and either pheromone titre, male abundance, or mean molecular weight of the pheromone components. However, among species with elaborate antennae, longer antennae were typically associated with lower male abundances and pheromone compounds with lower molecular weight, suggesting that male distribution and a more rapidly diffusing female sex pheromone may influence the size but not the general shape of male antennae.     

SEXUAL COMPOSITION and ANALYSIS OF REPRODUCTIVE FEMALES IN THE NORTH ATLANTIC RIGHT WHALE, EUBALAENA GLACIALIS, POPULATION

To test hypotheses involving reproduction and demographics, the sex of individuals must be established, but many species of Cetacea are not obviously dimorphic. In the North Atlantic right whale, Eubalaena glacialis , population, the sex of 61 males and 55 females had been determined previously by observation of the urogenital region, and the sex of 43 more females had been inferred from repeated sightings with a calf. To confirm the sex of some of these animals and to identify the sex of mote animals, genomic DNA was isolated from skin samples of 95 individual right whales (54 from among those described above and 41 additional recognizable individuals). The DNA was surveyed using the human Y-chromosome probe pDP1007. With Eco RI-digested DNA, a clear, sex-discriminating banding pattern was apparent. This method verified the sex of all 54 animals whose sex was previously known or inferred and identified the sex of an additional 41 recognizable individuals. A total of 89 male and 111 female right whales was identified in the population. The most unbiased estimate of sex ratio available is the 36 male and 34 female calves identified by genital morphology and DNA techniques. The sex ratio of this sample does not differ significantly from unity (P = 0.811). Only 38% (58/152) of the females in the North Atlantic population are known to have been reproductively successful compared with 54% in the population of right whales in the western South Atlantic. The population growth rate reported for the North Atlantic population is only 33% of that reported for right whales in the South Atlantic. Thirteen adult North Atlantic females have been identified that have not been known to calve during the past 11 yr. These data suggest that the absence of measurable recovery may be due to a combination of fewer actively reproducing females and lower reproductive rates of some females.     

Genetics of sex determination in flowering plants

Most flowering plant species are hermaphroditic, but a small number of species in most plant families are unisexual (i.e., an individ-ual will produce only male or female gametes). Because species with unisexual flowers have evolved repeatedly from hermaphroditic progenitors, the mechanisms controlling sex determination in flowering plants are extremely diverse. Sex is most strongly determined by genotype in all species but the mechanisms range from a single controlling locus to sex chromosomes bearing several linked locirequired for sex determination. Plant hormones also influence sex expression with variable effects from species to species. Here, we review the genetic control of sex determination from a number of plant species to illustrate the variety of extant mechanisms. We emphasize species that are now used as models to investigate the molecular biology of sex determination. We also present our own investigations of the structure of plant sex chromosomes of white campion (Silene latifolia - Melan-drium album). The cytogenetic basis of sex determination in white campion is similar to mammals in that it has a male-specific Y-chromosome that carries dominant male determining genes. If one copy of this chromosome is in the genome, the plant is male. Otherwise it is female. Like mammalian Y-chromosomes, the white campion Y-chromosome is rich in repetitive DNA. We isolated repetitive sequences from microdissected Y-chromosomes of white campion to study the distribution of homologous repeated sequences on the Y-chromosome and the other chromosomes. We found the Y to be especially rich in repetitive sequences that were generally dispersed over all the white campion chromosomes. Despite its repetitive character, the Y-chromosome is mainly euchromatic. This may be due to the relatively recent evolution of the white campion sex chromosomes compared to the sex chromosomes of animals. © 1994 Wiley-Liss, Inc.     

Identification of Random Amplified Polymorphic DNA Markers Linked to Sex Determination in Calamus simplicifolius C. F. Wei

The random amplified polymorphic DNA (RAPD) molecular marker technique was used to determine the sex of Calamus simplicifolius C. F. Wei In the present study, DNA samples were extracted individually from 10 male and 10 female plants. After a total of 1 040 decamer primers had been tested, an approximate 500-bp male-specific DNA fragment was generated with the S1443 primer. It is feasible to identify sex at the early stages of plant life, which is beneficial for improving breeding programs of this dioecious species. In addition, we have obtained a proper RAPD protocol that is useful for other species of rattan.     

DNA‐based approaches for evaluating historical demography in terrestrial vertebrates

Contemporary DNA sequences can provide information about the historical demography of a species. However, different molecular markers are informative under different circumstances. In particular, mitochondrial (mt)DNA is uniparentally inherited and haploid in most vertebrates and thus has a smaller effective population size than diploid, biparentally inherited nuclear (n)DNA. Here, we review the characteristics of mtDNA and nDNA in the context of historical demography. In particular, we address how their contrasting rates of evolution and sex‐biased dispersal can lead to different demographic inferences. We do so in the context of an extensive review of the vertebrate literature that describes the use of mtDNA and nDNA sequence data in demographic reconstruction. We discuss the effects of coalescence, effective population size, substitution rates, and sex‐biased dispersal on informative timeframes and expected patterns of genetic differentiation. We argue that mtDNA variationin species with male‐biased dispersal can imply deviations from neutrality that do not reflect actual population expansion or selection. By contrast, mtDNA can be more informative when coalescence has occurred within the recent past, which appears to be the case with many vertebrates. We also compare the application and interpretation of demographic and neutrality test statistics in historical demography studies. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 367–386.     

Sex-ratio evolution in sex changing animals

Sex allocation theory is often able to make clear predictions about when individuals should facultatively adjust their offspring sex ratio (proportion male) in response to local conditions, but not the consequences for the overall population sex ratio. A notable exception to this is in sex changing organisms, where theory predicts that: (1) organisms should have a sex ratio biased toward the "first" sex: (2) the bias should be less extreme in partially sex changing organisms, where a proportion of the "second" sex matures directly from the juvenile stage; and (3) the sex ratio should be more biased in protogynous (female first) than in protandrous (male first) species. We tested these predictions with a comparative study using data from 121 sex changing animal species spanning five phyla, covering fish, arthropods, echinoderms, molluscs, and annelid worms. We found support for the first and third predictions across all species. The second prediction was supported within the protogynous species (mainly fish), but not the protandrous species (mainly invertebrates).     

Do operational sex ratios influence sex allocation in viviparous lizards with temperature‐dependent sex determination?

Under certain environmental situations, selection may favour the ability of females to adjust the sex ratio of their offspring. Two recent studies have suggested that viviparous scincid lizards can modify the sex ratio of the offspring they produce in response to the operational sex ratio (OSR). Both of the species in question belong to genera that have also recently been shown to exhibit temperature-dependent sex determination (TSD). Here we test whether pregnant montane water skinks (Eulamprus tympanum) utilise TSD to select offspring sex in response to population wide imbalances in the OSR, by means of active thermoregulation. We use a combination of laboratory and field-based experiments, and conduct the first field-based test of this hypothesis by maintaining females in outdoor enclosures of varying OSR treatments throughout pregnancy. Although maternal body temperature during pregnancy was influenced by OSR, the variation in temperature was not great enough to affect litter sex ratios or any other phenotypic traits of the offspring.     

Identification of Random Amplified Polymorphic DNA Markers Linked to Sex Determination in Calamus simplicifolius C. F. Wei.

The random amplified polymorphic DNA （RAPD） molecular marker technique was used to determine the sex of Calamus simplicifolius C. F. Wei In the present study, DNA samples were extracted individually from 10 male and 10 female plants. After a total of 1 040 decamer primers had been tested, an approximate 500-bp male-specific DNA fragment was generated with the S 1443 primer. It is feasible to identify sex at the early stages of plant life, which is beneficial for improving breeding programs of this dioecious species. In addition, we have obtained a proper RAPD protocol that is useful for other species of rattan.     

Co-occurrence of multiple, supposedly incompatible modes of sex determination in a lizard population

Sex is determined genetically in some species (genotypic sex determination, or GSD) and by the environment (environmental sex determination, or ESD) in others. The two systems are generally viewed as incompatible alternatives, but we have found that sex determination in a species of montane lizard ( Bassiana duperreyi , Scincidae) in south-eastern Australia is simultaneously affected by sex chromosomes and incubation temperatures, as well as being related to egg size. This species has strongly heteromorphic sex chromosomes, and yet incubation at thermal regimes characteristic of cool natural nests generates primarily male offspring. We infer that incubation temperatures can over-ride genetically determined sex in this species, providing a unique opportunity to explore these alternative sex-determining systems within a single population.     

The neurogenetics of sexually dimorphic behaviors from a postdevelopmental perspective

Most sexually reproducing animal species are characterized by two morphologically and behaviorally distinct sexes. The genetic, molecular and cellular processes that produce sexual dimorphisms are phylogenetically diverse, though in most cases they are thought to occur early in development. In some species, however, sexual dimorphisms are manifested after development is complete, suggesting the intriguing hypothesis that sex, more generally, might be considered a continuous trait that is influenced by both developmental and postdevelopmental processes. Here, we explore how biological sex is defined at the genetic, neuronal and behavioral levels, its effects on neuronal development and function, and how it might lead to sexually dimorphic behavioral traits in health and disease. We also propose a unifying framework for understanding neuronal and behavioral sexual dimorphisms in the context of both developmental and postdevelopmental, physiological timescales. Together, these two temporally separate processes might drive sex‐specific neuronal functions in sexually mature adults, particularly as it pertains to behavior in health and disease.     

Can the meiotic sex ratio explain the sex ratio bias in adult populations in the dioicous moss Drepanocladus lycopodioides?

Sex ratio variation is commonly observed in natural populations of many organisms with separate sexes and genetic sex determination, including bryophytes. Most bryophyte populations exhibit female-skewed expressed adult sex ratios, generally inferred from counts of sexually mature plants. For the rarely sexually reproducing perennial dioicous moss Drepanocladus lycopodioides, we showed that a female bias also exists in the genetic adult sex ratio, using a specifically designed molecular sex-associated marker. Here, we investigated whether the meiotic spore sex ratio contributes to the observed bias in genetic adult sex ratio in natural populations. Earlier attempts to study meiotic sex ratios have involved commonly cultivated ruderals that rapidly express sex in the laboratory. We established single-spore cultures from field-collected sporophytes from these populations and used the marker to assess the sex of individual sporelings. Spore germinability was (near) complete, and mortality among sporelings was virtually absent. The true meiotic sex ratio did not differ from equality, but strongly differed both from the observed genetic sex ratios in the natural adult populations, and from the European scale genetic sex ratio. We conclude that the biased population sex ratios in this species arise at life cycle stages after spore germination. Sexual dimorphism may selectively favour female proliferation during some phase of gametophyte development. Based on methodological progress, we successfully used a perennial study species with rare sexual reproduction, which significantly broadens the life history spectrum investigated in bryophyte sex ratio studies.     

Genetic evidence for co-occurrence of chromosomal and thermal sex-determining systems in a lizard

An individual's sex depends upon its genes (genotypic sex determination or GSD) in birds and mammals, but reptiles are more complex: some species have GSD whereas in others, nest temperatures determine offspring sex (temperature-dependent sex determination). Previous studies suggested that montane scincid lizards (Bassiana duperreyi, Scincidae) possess both of these systems simultaneously: offspring sex is determined by heteromorphic sex chromosomes (XX-XY system) in most natural nests, but sex ratio shifts suggest that temperatures override chromosomal sex in cool nests to generate phenotypically male offspring even from XX eggs. We now provide direct evidence that incubation temperatures can sex-reverse genotypically female offspring, using a DNA sex marker. Application of exogenous hormone to eggs also can sex-reverse offspring (oestradiol application produces XY as well as XX females). In conjunction with recent work on a distantly related lizard taxon, our study challenges the notion of a fundamental dichotomy between genetic and thermally determined sex determination, and hence the validity of current classification schemes for sex-determining systems in reptiles.