A DNA study of the Sheffield Peregrines

     

A breeding pair of peregrines has nested on St George's Church near the University of Sheffield since 2012.  A 24 hour live stream web camera allows viewers around the world to observe the life of the Sheffield peregrines as they raise their chicks. The webcam has had over a million views and weekly updates of the highlights are provided via the Sheffield Peregrines blog and the twitter account @peregrines2018 throughout the breeding season (February-July).

   

In collaboration with the Sheffield Bird Study Group,
we are appealing for donations to support a DNA study of the Sheffield Peregrines.

    

If sufficient funds are raised we intend to:

I. Determine the sexes of the peregrine chicks.
II. Obtain a DNA profile for each peregrine chick.
III. Investigate if the same pair of adult birds are raising chicks across different years.
IV. Archive the peregrine samples, the DNA extractions and the DNA profiles for all individuals to assist future investigations and research.
V. Assist in DNA-based investigations of wildlife crime.

    

To donate please visit our fundraising page -

Make a donation

Sheffield Peregrines Fundraising webpage
St Georges Peregrine

Summary of the work to date

Feather samples have been collected from the St George’s peregrine chicks each year from 2015 and used for DNA analyses. After extracting the DNA from a single feather, we amplify and analyse target regions of the genome with two aims:

Firstly, we determine whether each chick is male or female. As adults, the plumage patterns of both sexes look very similar, but adult female peregrines are about a third larger than adult males. However, as chicks, peregrines are more difficult to tell apart. We used five DNA sex markers, which identified that all of the eight St George’s chicks that hatched between 2015-2017 were male. It was therefore great news when we discovered one of the three new 2018 chicks to be female (with the orange ring 'PTA').

Secondly, we determine the genetic profile of each bird based on 23 highly-variable regions of the genome known as microsatellites. These microsatellite profiles can be used to identify each individual peregrine, to match them with their biological parents, and also to match them with their local population of origin.

    

The work flow:

1. Sample collection.
Various sample types have been collected:
Feathers were collected from each peregrine chick during ringing in 2015 to 2018.
Mouth swabs were taken from the chicks in 2017 and 2018. 
Shed feathers that were suspected to be from the peregrines were collected from the nest box in 2017 and from the graveyard in 2017 and 2018.
Unhatched eggs were taken in 2016, 2017 and 2018 to investigate why they have failed to hatch (performed by Dr Nicola Hemmings).
Bird pellets were collected from the church roof in 2017 and from the graveyard in 2018.
A fresh faecal sample was obtained during ringing in 2017.
(The pellets and the faecal sample will be used to test their potential for use in DNA-based diet analyses.)

2. Genomic DNA was extracted from each sample.
3. Genetic bird sexing markers were used to sex the peregrines.
4. We are developing and validating a mutliplex set of peregrine microsatellite markers suitable for use to obtain a DNA profile for any peregrine individual.
5. We will use this validated marker set to genotype the peregrine chicks (n=11) that fledged from St George's in 2015-2018. The DNA profiles of these chicks will then be compared across years to see if the same pair of adult individuals are raising chicks each year. 
6. Tissue and DNA samples from all peregrines included in the study and the DNA profiling data obtained have been archived, for future investigations and research.
7. We also intend to test genetic and sex markers in other bird of prey species, and develop genetic tools to assist DNA-based investigations of wildlife crime.

       

The progress so far (steps 1-3):

  

1. Sample collection

Collection of samples from Sheffield Peregrines:
Samples from chicks: Feathers were collected from the peregrine chicks in 2015, 2016 and 2017 whilst ringing the birds. The three 2017 chicks were ringed at 21 days old on the 18th May 2017.
In 2017 and 2018, mouth swabs (as well as feathers) were collected from each chick.
In 2017, one chick kindly donated a fresh faecal sample on one of the ringers and this was collected by the team.
Samples from the adults: Shed feathers suspected to be from the adult birds were collected from the churchyard in 2015/2016 (donated by the public) and were collected from the nest in 2017. Pellets were found on the roof of the church were also collected in 2017 and in the churchyard in 2018.
Unhatched eggs: In 2016, 2017 and 2018 a single egg did not hatch. These unhatched eggs were also collected and delivered to Dr Nicola Hemmings, who has expertise to investigate why the eggs failed to hatch. Nicola provided samples from the egg membrane (chick's DNA) and the dead embryo for DNA sex typing and genetic profiling.

   

All of the lab work and DNA analyses will be performed in the Molecular Ecology Laboratory, Department of Animal and Plant Sciences at the University of Sheffield.

     

2. DNA extraction

DNA was successfully extracted from the feathers and mouth swabs obtained from the chicks, the unhatched embryo from 2016, and also the shed feathers suspected to belong to the peregrines (species of origin to be checked). No DNA was obtained from the faecal sample or the pellets from 2017.

      

3. DNA sex-typing

METHODS

Controls: Peregrine samples were kindly donated by various people and included samples from adults of known sex (22 females and 22 males; see Table 1). Adult female peregrines are 40% bigger than males and this allows us to distinguish between adult males and females. A single adult female and one male peregrine whose sex was based on size were provided, these were samples from a family of peregrines including mother, father and chicks. Additionally a batch of samples was also provided which had been sexed using a CHD-based marker by a commercial company (Biobest Labs Ltd). These known-sex DNA samples were PCR amplified with five sex typing marker sets (CHD and non-CHD based) and the products visualised on an ABI DNA analyser. All females (ZW) produced 2 products and males (ZZ) produced only one product for all five markers, and this confirmed that these markers can be used to accurately assign sex in peregrines.

The Sheffield chick samples were PCR amplified with the five sex-typing markers, the products were compared against the results obtained from the 44 peregrine individuals of known sex.

   

RESULTS

SEXING OF THE CONTROL (KNOWN SEX) PEREGRINE SAMPLES
The sexes obtained with the 5 markers matched the sex based on size or based on previous CHD sex-typing by Biobest for all of the 42 known-sex peregrine individuals. This confirmed the 5 sex markers were correctly identifying the sex of the peregrines.

SEXING OF THE ST GEORGE'S CHICKS FROM FEATHERS
The genetic sexing revealed that ALL eight chicks that hatched at St Georges Church, Sheffield between 2015-2017 are MALE (see Table 1).

The probability of the chicks being the same sex 8 times in a row is 1 in 128.

The likelihood of eight male chicks in a row is 0.5^8 = 0.00390625 or 1 in 256.
The probability of a male chick 8 times in a row is 1 in 128, because the probability of 8 females in a row is also 1 in 256). [Many thanks to Prof. Jon Slate for advice on calculating probabilities.]

The sexing of the 2018 chicks revealed that one chick was a FEMALE (orange ring 'PTA') and two were male.

SEXING OF THE ST GEORGE'S CHICKS FROM MOUTH SWABS
Swabs were only taken in 2017 and 2018. The results from the 2017 chicks matched the results from the feather samples (ie all male). The swabs taken in 2018 could not be sexed because the checks had just been fed and the DNA was a mixed with DNA from other bird species.  

SEXING OF TISSUE FROM THE UNHATCHED EGGS 
Samples were not taken from the two unhatched eggs laid in 2015.
The unhatched chick from 2016 was found to be MALE.
The unhatched chick from 2017 and 2018 could not be sexed because the embryo was too small and too degraded. The unhatched embryo was extremely small (estimated to be 3 days development by Dr Nicola Hemmings) and DNA could not be extracted from the egg membrane nor the embryo tissue.

SPECIES IDENTIFICATION OF THE SHED FEATHERS
The allele sizes obtained from PCR amplification of the peregrine DNA with the sex markers are of a specific size in peregrines and this allowed us to reveal that none of the shed feathers collected in 2016 and 2017 (and suspected to be peregrine) belonged to a peregrine. These feathers probably belong to various prey species brought into the nest. The feather suspected to peregrine and found in the churchyard in 2018 will be tested soon.  

SPECIES IDENTIFICATION OF THE PELLETS AND THE FAECAL SAMPLE

The bird pellets found in 2017 on the roof were very old and mossy and it was not possible to extract DNA from these. DNA could not be extracted from the faecal sample obtained from the 2017 chick.

        

Table 1. DNA Sexing-typing of control Peregrine individuals DNA-sexed independently by a commercial company (Biobest Labs Ltd)

 

Year
Species Location where sampled (& supplier) Type of sample Adult/Chick and sex (as known) Species based on size of allele amplified by the DNA sex marker Sex based on 5 DNA sexing markers
sent 2017 Peregrine Unknown (SMcC) DNA 21 male and 21 female samples. Sexes based on DNA sex-typing by Biobest Labs Ltd* Peregrine The sexes of all 42 individuals matched those supplied by Biobest Labs.


     

Table 2. DNA Sexing-typing of Peregrine samples from Leicester, Nottingham, Derbyshire, Suffolk and other (non-Sheffield) sites (some of known sex based on body size)

Year Species Location where sampled (& supplier) Type of sample Adult/Chick  and sex (as known)
Species based on size of allele amplified by the DNA sex marker
Sex based on 5 DNA sexing markers
sent 2017 Peregrine UK, possibly Leicester (JW/CM) DNA Adult Male WF3 (assume sex based on body size, part of "Leicester" family mum, dad and 2 chicks) Peregrine Male
sent 2017 Peregrine UK, possibly Leicester (JW/CM) DNA Adult Female WF4 (assume sex based on body size, part of "Leicester" family mum, dad and 2 chicks) Peregrine Female
sent 2017 Peregrine UK, possibly Leicester (JW/CM) DNA "Leicester" chick 1, WF1 (unknown sex) Peregrine Male
sent 2017 Peregrine UK, possibly Leicester (JW/CM) DNA "Leicester" chick 2, WF2 (assume sex based on size) Peregrine Female
sent 2017 Peregrine UK (JW/CM) DNA Seven individuals of unknown age and sex All peregrine 4 Female, 3 Male
2015 Suspected Peregrine Nottingham Trent University (LG/EK)  Shed feather found in NTU nest Adult 1? Confirmed peregrine Female
2015 Suspected Peregrine Nottingham Trent University (LG/EK) Shed feather found in NTU nest Adult 2? Unknown species.
(No amplification.)
Unknown sex.
(No amplification.)
2015 Suspected Peregrine Nottingham Trent University (LG/EK) Shed feather found in NTU nest Adult 3? Confirmed peregrine Male
2015 Peregrine Derbyshire (LG/EK) Tissue from dead Derbyshire chick (died when fell from nest in 2015) Chick Peregrine Male
sent pre 2010 Peregrine UK (AD) DNA Samples from multiple individuals   provided Peregrine Males and Females
sent 2016 Peregrine UK (LW) DNA Samples from two individuals of unknown sex Peregrine One male and one female.
2017 Peregrine Nottingham Trent University (LG/EK) Tissue from dead chick (road accident) Chick (suspected female based on size, fell from nest and run over on road in 2017) Lab work due to start July 2017 Lab work due to start July 2017
2017 Peregrine Nottingham Trent University (LG/EK) Pellets Probably from chicks (but will also be genotyped to check identity) Not peregrine (other birds amplifying) Mixed sample of birds (will be useful for diet analysis). *
2017 Peregrine Nottingham Trent University (LG/EK) Large shed feathers collected from NTU nest Suspected to be peregrines and the adults (but will be genotyped to check species, identity and sex) Not peregrine Not peregrine
2017 Peregrine Leicester Cathedral (JG) Broken eggshell provided Eggshell used that includes shell membrane Multiple bird species present due to debris on shell. Unable to sex due to being multiple species present. *
2018 Peregrine Suffolk (PW, BTO) Unhatched egg Embryo to be tested to be tested

 *Those samples for which DNA was detected from multiple birds can still be used in comparing peregrine individuals because the genetic markers for individual profiling are peregrine specific so do not amplify other species.

   

Table 3. DNA Sex-typing of the Sheffield Peregrine's of St George's Church, from samples taken 2015-2018

       

Year Species Location where sampled Type of sample Status Species based on DNA sex marker allele sizes Sex based on 5 DNA sexing markers
2015 Peregrine Nest box Feather Chick 1 Peregrine Male
2015 Peregrine Nest box Feather Chick 2 Peregrine Male
2015 Peregrine Nest box Unhatched egg 1 - No samples taken -
2015 Peregrine Nest box Unhatched egg 2 - No samples taken -
2015/ 2016 Suspected Peregrine St George's Church yard Shed feather (n=1) Suspected Adult Not peregrine -
2016 Peregrine Nest box Feather Chick 1 Peregrine Male
2016 Peregrine Nest box Feather Chick 2 Peregrine Male
2016 Peregrine Nest box Feather Chick 3 Peregrine Male
2016 Peregrine Nest box Dead chick's tissue (chick's DNA) Dead chick in unhatched egg Peregrine Male (unhatched chick tissue)
2017 Suspected Peregrine St George's Roof Pellets (n=4, old and mossy) Suspected Adult Unknown species.
(No DNA amplification.)
Unknown sex.
(No DNA amplification.)
2017 Suspected Peregrine Nest box Shed feathers (n=2) Suspected Adult Not peregrine (n=2) -
2017 Peregrine Nest box Feather (sexed twice) and mouth swab Chick 1 (Colour ring = PRF) Peregrine Male
2017 Peregrine Nest box Feather (sexed twice) and mouth swab Chick 2 (Colour ring = PSF) Peregrine Male
2017 Peregrine Nest box Feather (sexed twice) and mouth swab Chick 3 (Colour ring = PTF) Peregrine Male
2017 Peregrine Nest box

Tissue from dead (unhatched chick's DNA)

Dead chick in unhatched egg

Chick died approx. 3 days after lay date (N. Hemmings pers. comm.)

No DNA amplification

Unknown sex.
(No DNA amplification.)

2018 Peregrine Nest box Feather Chick 1 (Colour ring = PRA) Peregrine Male
2018 Peregrine Nest box Feather Chick 2 (Colour ring = PSA) Peregrine Male
2018 Peregrine Nest box Feather Chick 3 (Colour ring = PTA) Peregrine FEMALE
2018 Peregrine Nest box Mouth swabs Swab samples taken from all 3 chicks Multiple bird species present (ie includes food species. Chicks had just been fed.) Unable to sex due to being a mixed sample of birds (DNA will be useful for diet analysis) *
2018 Peregrine Nest box Tissue from dead (unhatched) chick's DNA)  Dead chick in unhatched egg No DNA amplification Unknown sex.
(No DNA amplification.)
2018 Peregrine St George's Church yard Small shed feather to be tested to be tested to be tested
2018 Peregrine St George's Church yard Pellet to be tested to be tested to be tested

*Those samples for which DNA was detected from multiple birds can still be used in comparing peregrine individuals because the genetic markers for individual profiling are peregrine specific so do not amplify other species.    

                        

4. Creation of a peregrine multiplex microsatellite marker set

We are currently validating a peregrine multiplex microsatellite marker set. This will allow us to quickly and efficiently create DNA profiles and identify individuals from various types of samples (shed feathers, mouth swabs, fresh faecal samples and bird pellets).

   

5. Comparing individuals and family relationships of Sheffield's Peregrines from chick DNA samples collected 2015-2018 

It has been suggested that the adult female appeared to be in poor condition in 2015 and she may have died resulting in a new adult female in 2016 (David Wood pers. com.). The adult male has a metal ring and although the number can not be read from a distance, it is believed that the same male has occupied the nest since 2015 and before this date. We used microsatellite DNA profiling to investigate if a new female has joined the original male in 2016, and checked for evidence of a change in either parent between 2015-2018.

       

Table 4. St George's Peregrine Chick DNA Samples Available

Year Male parent Female parent Number of chicks Unhatched eggs
2015 Metal BTO ring (not sampled) Unringed (not sampled) 2 (all male) 2 (not sampled)
2016 Metal BTO ring (not sampled) Unringed (not sampled) 3 (all male) 1 (male)
2017 Metal BTO ring (not sampled) Unringed (not sampled) 3 (all male) 1 (no DNA)
2018 Metal BTO ring (not sampled) Unringed (not sampled) 3 (one female=PTA, and two males) 1 (no DNA)

     

We found no evidence of a switch in either parent. It is not possible to be 100 percent confident of this result because no DNA is available from any of the parents across the different years and because only two chicks were sampled before the proposed parent switch. However the likelihood of detecting a switch in parents was high because we used a large set of 23 highly variable autosomal genetic markers 


We genotyped the 11 chicks with a set of 23 peregrine microsatellite DNA markers and compared the profiles of all the chicks to check if the chicks within clutches and between years are full siblings or if they have different parents. We constructed all of the potential parental DNA profiles and compared these against all the chick DNA profiles to see if any new alleles or mismatching profiles appeared in any year. We found no chick-parent mismatches, ie no evidence of a new adult female (or male) parent.

To be confident if a parent switch has occurred, we require DNA from the parent birds. If anyone finds any peregrine feathers suspected to be from the St George's adult peregrines, which they would be willing to donate for us to use in DNA analyses, please get in touch with Deborah Dawson d.a.dawson@sheffield.ac.uk

   

6. Archives for Future DNA Investigations

The peregrine DNA and the DNA profiles obtained have been stored as a reference to allow future DNA investigations and research.
This will, for example, allow all of the Sheffield peregrines to be identified from any samples found in the future (eg shed feathers, fresh faecal samples, mouth swabs, tissue and bird pellets). It will allow pedigree investigations, such as a comparison of individuals DNA profiles in the database to the DNA profiles of future chicks that hatch at St Georges (eg to investigate if chicks have the same parents across different years). We can also compare the St Georges individuals against peregrine individuals from other nests across the UK and abroad to invesitigate genetic diversity and population structure.

   

7. Genetic tools to assist DNA-based investigations of wildlife crime.

We have tested sex markers in other species of bird of prey, including eagles and falcons.

see the Bird Sex-typing Database

    

Ways to support this work:

We have set up a Virgin Money Giving Page to raise funds to support this work.

We are also keen to obtain any shed peregrine feathers, especially if believed to be from either of the adult Sheffield peregrines (from 2018 or previous years).

We are looking to obtain samples from more peregrines at other locations in the UK. If you can help with this please get in touch with Dr Deborah Dawson (d.a.dawson@sheffield.ac.uk). We can extract DNA from most types of samples, including feathers, pellets, faecal samples and mouth swabs (we can supply the swabs).

        

If you are involved in a UK Peregrine project and would like us to sex your chicks from mouth swabs or feathers please get in touch (Dr Deborah Dawson d.a.dawson@sheffield.ac.uk).  

 

Personnel

Dr Deborah Dawson - Leader of the DNA study
(d.a.dawson@sheffield.ac.uk, @ddawson777)

     

Dr Natalie dos Remedios - Research Technician
DNA extraction, genetic sex-typing and microsatellite genotyping, 2017-2018

         

Gracie Adams - Undergraduate Summer placement volunteer 2017
Laboratory assistance

  

Sample Collection Teams

        

An expert team of qualified bird ringers collected the peregrine samples whilst ringing the St George's chicks:

Steve Samworth, Sorby Beck Ringing Group, (2015-2018)
Simon Mills, University of Sheffield, (2015-2018)
Dean Rea, Sorby Beck Ringing Group, (2017)

Luke Nelson, Sorby Beck Ringing Group, (2018)
Emma Hughes, Sorby Breck Ringing Group, (2018)


Peregrines and their nests are protected under the Wildlife and Countryside Act 1981 and are a Schedule 1 Protected Species so this work was done under a special licence issued by Natural England.

   
Control peregrine DNA samples, including those of known sex, were kindly provided by the following:


Dr Andrew Dixon (International Wildlife Consultants Ltd),
Dr Louise Gentle and Esther Kettel (Nottingham Trent University), 
Nick Brown (Derby Cathedral Peregrine Project),  
Elizabeth Woodward (Derbyshire Wildlife Trust),
Jim Graham (Leicester Cathedral Peregrines),
Dr Celia May and Dr Jon Wetton (University of Leicester),

Sarah McCallum (Biobest Laboratories Limited, Edinburgh),
Dr Lucy Webster (Wildlife DNA Forensic Unit, Science and Advice of Scottish Agriculture (SASA)).

 

Peregrine Chick

Images supplied by David Wood, Chairman of the Sheffield Bird Study Group and taken from the Sheffield Peregrines Blog.

                                  

           Thankyou for your support !!

                                                           Thankyou