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.

An evaluation of three field techniques for sexing live Gould's petrels (Pterodroma leucoptera) (Procellariidae)

Many petrels show no obvious sex-linked dimorphism in plumage or size and consequently many researchers fail to sex the living individuals they study. Several methods of sex discrimination that do not rely on plumage- or obvious size-dimorphism can be used to sex live petrels. The effectiveness of three such techniques was evaluated: body condition at the time of laying, cloacal inspection, and discriminant function analysis (DFA) of external morphometrics. Gould’s Petrel (Pterodroma leucoptera leucoptera) was used as the subject species. Sexing of breeding adults on the basis of body condition at laying proved to be highly accurate (100% of birds sexed correctly) but required detailed knowledge of the breeding biology. Following training, cloacal inspection proved to be an accurate (96%) method of determining the sex of breeding adults, but not of chicks. Unlike molecular sexing, the latter two methods of sex discrimination provide immediate knowledge of the sex of individuals in the field. DFA of external morphometrics predicted the sex of adults with an accuracy of 73% and the sex of near-fledged chicks with an accuracy of 66%. However, the probability of correct assignment of sex was low in most cases and, therefore, this is the least useful of the three techniques assessed here.

Molecular sex-typing in shorebirds: a review of an essential method for research in evolution, ecology and conservation

Knowing the correct sex of individuals is essential both for research in evolutionary ecology and for practical conservation. Recent molecular advances have produced cheap, quick and reliable methods for sexing birds including chicks, juveniles, immatures and adults. Shorebird researchers have not yet fully utilised these advances. Here we provide an overview of work in this area to date with two objectives: (i) to review the major applications of molecular sexing and findings of shorebird research so far, and (ii) to provide an essential guide on how to carry out molecular sexing using current methods whilst avoiding methodological pitfalls. We encourage shorebird researchers to make better use of molecular sex-typing techniques in studies of conservation, migration, foraging ecology and breeding behaviour.

Molecular determination of sex-ratio in Yellowhammer Emberiza citrinella offspring

Theory predicts skewed offspring sex-ratios in a range of situations in which the economics of producing the two sexes differ. Offspring sex-ratio skews in birds are relatively scarcely observed compared to other taxa. This could be because avian molecular sexing techniques, which allow young birds to be sexed, have only recently become available. Alternatively, birds may be largely constrained from adaptively manipulating the sex-ratio of their offspring. We used a recently-developed molecular sexing technique for birds to sex 420 Yellowhammer Emberiza citrinella offspring from 168 clutches found in Oxfordshire. Clutch sex-ratio of the population did not depart from the expected binomial distribution, and there was no variation in clutch sex-ratio with laying date, breeding attempt, or a variety of habitat variables which were predicted to differentially affect the survival and future reproductive success of offspring of the two sexes. There was no difference in size or growth rate of the sexes and nestling mortality was not sex-biased. Hence, although we can identify possible advantages of manipulating the sex-ratio in this species, it seems not to be used as a breeding strategy. Given the lack of consistent evidence for skewed avian offspring sex-ratios, more experimental work is required to determine whether, and how, birds may adaptively manipulate their offspring sex-ratio.

Biometrics and sexing criteria of the yellow-faced honeyeater lichenostomus chrysops

Morphometric data on 99 adult and 13 juvenile Yellow-faced Honeyeaters Lichenostomus chrysops that were independently sexed using molecular techniques were analysed to investigate size dimorphism between the sexes. Our results support previous studies that have demonstrated Yellow-faced Honeyeaters are sexually dimorphic in size, with males being the larger sex. Discriminant analyses of morphometric data were used to develop a simple method for sexing adult Yellow-faced Honeyeaters in the hand. As five observers collected the measurements our sexing criteria are conservative and should have wide application for field ornithologists working on the species.

Methods for aging and sexing the Black-eared Miner, Manorina melanotis, and Yellow-throated Miner, M. flavigula

Morphometric data on 92 Black-eared Miners and 47 Yellow-throated Miners that had been independently sexed using molecular techniques were analysed to investigate size dimorphism between the sexes. We found that both species are sexually dimorphic in size, with males being the larger sex. Discriminant analyses of morphometric data were used to develop a simple method for sexing both species in the hand. Additionally, alula shape was consistent with other methods that we applied for ageing individuals. Sex-specific size differences between Black-eared and Yellow-throated Miners detected here add further support to the contention that they represent different taxa. The application of these sexing and ageing techniques for both species of mallee miner will improve ongoing field management of the endangered Black-eared Miner.

Sexing little penguins eudyptula minor using bill measurements

In Little Penguins Eudyptula minor there are no reliable plumage or body size differences that can be used visually to distinguish the sex of individuals. However, sexual dimorphism of morphometric measures has been noted, with males always being a little larger than females. In this study, differences between E. minor sexes at eight colonies in south-eastern Australia were determined statistically via discriminant function analysis (DFA) and through the utilization of DNA-based techniques developed for non-ratite birds. The DFA correctly determined gender in 91.1% of cases and molecular methods were 100% accurate. Our DFA success rate of classification is similar to that previously published for Little Penguins in Victoria. In this study statistically significant differences in mean bill depths and lengths were found between Little Penguin colonies at St Kilda, Phillip Island and Gabo Island, compared to colonies at Kangaroo Island, Granite Island, Middle Island and London Bridge. As birds in eastern populations (St Kilda, Phillip Island, Gabo Island) exhibit statistically significantly smaller beaks (bill depth and bill length), separate discriminant functions were investigated for each phenotypically distinct geo-spatial cohort. Interestingly, cluster analysis for bill length identified three groups: western (Kangaroo Island and Granite Island), eastern (St Kilda, Phillip Island and Middle Island) and the London Bridge Little Penguin colony, which constituted a separate group. We conclude that while there is a slight increase in DF power for colonies west of Cape Otway and for some specific colonies, colony-specific DFA is not required to identify the sex of Little Penguins in south-eastern Australia.