A DNA study of the Sheffield Peregrines

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 updates of the highlights are provided via the Sheffield Peregrines blog and the twitter account @SheffPeregrines 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:

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

To donate please visit our fundraising page:

Sheffield Peregrines Fundraising

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Summary of the work to date

No chicks fledged in 2019. However, feather samples have been collected from the St George’s peregrine chicks each year during 2015-2018 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. The 2019 chick with the orange ring 'PTA' is female. 

Secondly, we determined the DNA profile of each bird and compared individual DNA profiles at 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.

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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, 2018 and 2019 to look for signs of embryo development and 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

• Table 1: DNA profiling and genetic sexing-typing of a set of peregrine individuals which had been DNA-sexed independently by a commercial company (Biobest Labs Ltd)
• Table 2: UK Peregrine DNA Profile Bank - towards assigning individuals to nests
• Table 3: Samples of the peregrine's of St George's Church, Sheffield collected 2015-2019 and used for DNA profiling and genetic sex-typing of individuals

4. We are developing and validating a multiplex 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 and genotype embryos from addled eggs (2015-2019). 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

• Table 4: The Sheffield St George's Peregrine Family

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

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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. In 2019, four eggs were laid, one hatched but the chick died, three of the four eggs did not hatch and two of these addled eggs were collected.

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 the DNA team with samples from the egg membrane (chick's DNA) and the dead embryo for DNA sex typing and genetic profiling.

The DNA extraction and analyses were 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 2019, and also the shed feathers suspected to belong to the peregrines (species of origin to be checked). No DNA was obtained from the fresh faecal sample or the old pellets collected in 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 two 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 genetic 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 of feathers 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 chicks had just been fed and the DNA was a mixed with DNA from other bird species.  

Sexing of the 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.

Two of the four eggs laid in 2019 were collected and the unhatched chicks analysed.

Little development was observed in the brown egg and insufficient DNA was ontained to sex and DNA profile this individuals but the pale 2019 addled egg contained an embryo and this was found to be MALE.

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 fresh faecal sample obtained from the 2017 peregrine chick.

Table 1: DNA profiling and genetic sexing-typing of a set of peregrine individuals which had been 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 sizes of alleles amplified by the DNA sex marker and microsatellite markers	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 DNA Profiled.	The sexes of all 42 individuals matched those supplied by Biobest Labs.

Table 2: UK Peregrine DNA Profile Bank - towards assigning individuals to nests

Peregrine DNA profiles from Sheffield, Leicester, Nottingham, Derbyshire, Suffolk, Plymouth and Scotland have been added to the profile bank, more to be added soon. 

Year	Species	Location where sampled, wild/captive & supplier	Type of sample	Adult/Chick  and sex (as known)	Species based on size of alleles amplified by the DNA sex marker and microsatellite markers	Sex based on 5 DNA sexing markers
sent 2017	Peregrine	Gemany - captive (JW, CM)	DNA	Adult Male WF3 (assume sex based on body size, part of "WF" family mum, dad and 2 chicks)	Peregrine DNA Profiled. Father of WF1 and WF2 based on DNA profile.	Male
sent 2017	Peregrine	Germany - captive (JW, CM)	DNA	Adult Female WF4 (assume sex based on body size, part of "WF" family mum, dad and 2 chicks)	Peregrine DNA Profiled Mother of WF1 and WF2 based on DNA profile.	Female
sent 2017	Peregrine	German - captive (JW, CM)	DNA	"WF" chick 1, WF1 (unknown sex)	Peregrine DNA Profiled Offspring of WF3 and WF4 and sibling of WF2 based on DNA profile.	Male
sent 2017	Peregrine	Germany - captive (JW, CM)	DNA	"WF" chick 2, WF2 (female?, assume sex based on size)	Peregrine DNA Profiled. Offspring of WF3 & WF4 and sibling of WF1 based on DNA profile.	Female
sent 2017	Peregrine	Unknown origin - captive? (JW, CM)	DNA Sample names =  1.88, 2.88, 3.88, 4.88, 5.88, 6.88, 7.88	Seven individuals of unknown age and sex	All peregrine DNA Profiled DNA profiles reveal 6 individuals to be one family but 7.88 is unrelated.	4 Female, 3 Male
2015	Suspected Peregrine (feather)	Nottingham Trent University - wild (LG, EK)	Shed feather found in NTU nest on Newton Building. Same female (Mrs P., long beak) has been present since 2008. Adult male (Archie) first bred at NTU in 2017.	Adult feather Sample name = NTU_1_2015	Confirmed peregrine DNA Profile obtained. The female adult  feather may be from Mrs P. the Mother of NTU nest since 2008.	Female
2015	Suspected Peregrine (feather)	Nottingham Trent University - wild (LG, EK)	Shed feather found in NTU nest	Adult feather Sample name = NTU_2_2015	Confirmed peregrine  DNA profile obtained. Suspected adult NTU male breeding since 2012,  disappeared in 2016. Unrelated to NTU 2017_chick "Georgina" and unrelated to suspected NTU mother Mrs P. = NTU_1_2015 DNA profiles reveal feather DNA samples NTU_2_2015 and NTU_3_2015 are two feathers from same individual	Unknown sex. (Sex marker failed but DNA profile matches NTU_3_2015 so this is a DNA sample from a male
2015	Suspected Peregrine (feather)	Nottingham Trent University - wild (LG, EK)	Shed feather found in NTU nest	Adult feather Sample name = NTU_3_2015	Confirmed peregrine DNA profile obtained. Unrelated to NTU 2017_chick "Georgina" and supected NTU mother NTU_1_2015 DNA profiles reveal feather DNA samples NTU_2_2015 and NTU_3_2015 are two feathers from same individual	Male
2015	Peregrine	Belper, Derbyshire - wild (EW, LG, EK)	Feathers plucked from a single dead chick that fell from nest in 2015 NB. The Belper adult breeding male was shot in 2015.	Chick Three feathers provided = D1, D2, D3.	Peregrine DNA profile obtained (identical profile for D1 & D2 but D3 failed)	Male
sent pre 2010	Peregrine	UK - captive and wild (AD)	DNA	Samples from multiple individuals   provided	Peregrine  Still to be profiled.	Males and Females
sent 2016	Peregrine	Scotland - wild (LW)	DNA Sample name = V0048 from Southern Scotland	Adult of unknown sex	Peregrine DNA profile obtained.	Male
sent 2016	Peregrine	Scotland - wild (LW)	DNA Sample name = V0116 from Highland Scotland	Adult of unknown sex	Peregrine DNA profile obtained.	Female
2017	Peregrine	Nottingham Trent University - wild (LG, EK)	Tissue from dead fledgling ("George" / "Georgina", road accident)  Hatched =probably 23rd April 2017 (or 25th or 27th) Ringed =16th May 2017 (at 23 days old) Died = 6th June 2017 (approx. 64 days old)	Chick (suspected female based on size, fledged from NTU Newton Building nest and run over on road in city centre 6th June 2017)	DNA Profile obtained Possible daughter/ offspring of NTU_1_2015 (NTU_1_2015 = suspected mother at NTU nest)	to be tested
2017	Peregrine	Nottingham Trent University - wild (LG, EK)	Pellets (n=6, all old pellets)	Probably from chicks (but will also be genotyped to check species and individual identity)	No peregrine DNA amplified. Other bird species are amplifying (presumably prey species)	Mixed sample of bird species (will be useful for diet analysis). *
2017	Peregrine	Nottingham Trent University - wild (LG, EK)	Large shed feathers collected from NTU nest	Suspected to be peregrines and adults (these were genotyped to check species, individual identity and sex)	Not peregrine Sex marker allele sizes did not match sizes expected for a peregrines for several feathers.	Not peregrine
2017	Peregrine	Leicester Cathedral - wild (JG)	Broken thin eggshell provided (Sample name = Thin_Shell_2017)	Eggshell that includes shell membrane	Peregrine DNA amplified DNA profile obtained from Peregrine DNA.	Unable to sex due to being multiple prey bird species present. *
2018	Peregrine	Suffolk - wild (PM)	Unhatched egg	Embryo	to be tested	to be tested
2018	Peregrine	Ipswich - wild (PM)	Unhatched egg	Embryo	to be tested	to be tested
2018	Peregrine	Colchester - wild (PM)	Plucked feathers	Juvenille	to be tested	to be tested
2018	Peregrine	Colchester - wild (PM)	Pellet (Collected Fresh)	Juvenille	to be tested	to be tested
2019	Peregrine	Plymouth - wild (LS)	Unhatched egg (x2)	Embryo	to be tested	to be tested
2019	Suspected peregrine (feathers)	Lundy Island - wild (LS)	Shed Feathers (x5)	Adult feathers	to be tested	to be tested
2019	Peregrine	Cromer - wild (ZS)	Eggshell	Chick	to be tested	to be tested
2019	Peregrine	Captive (JMcK)	Accidental death, Heart muscle Sample name = DJR_Sept2019	Chick close to fledgling	to be tested	to be tested

*Those samples for which DNA was detected from multiple bird species (ie contaminated by prey species) can still be used in comparing peregrine individuals because the genetic markers for individual profiling are peregrine specific so do not amplify other non-falcon bird species.

Table 3: Samples of the peregrine's of St George's Church, Sheffield collected 2015-2019 and used for DNA profiling and genetic sex-typing of individuals

Year	Species	Location where sampled	Type of sample	Status	Species based on allele sizes of the DNA sex marker and microsatellite markers	Sex based on 5 DNA sexing markers
2015	Peregrine	Nest box	Plucked Feather	Chick 1	Peregrine	Male
2015	Peregrine	Nest box	Plucked 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	Plucked Feather	Chick 1	Peregrine	Male
2016	Peregrine	Nest box	Plucked Feather	Chick 2	Peregrine	Male
2016	Peregrine	Nest box	Plucked 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	Plucked Feather (sexed twice) and mouth swab	Chick 1 (Colour ring = PRF)	Peregrine	Male (found dead in Sheffield May 2019)
2017	Peregrine	Nest box	Plucked Feather (sexed twice) and mouth swab	Chick 2 (Colour ring = PSF)	Peregrine	Male
2017	Peregrine	Nest box	Plucked Feather (sexed twice) and mouth swab	Chick 3 (Colour ring = PTF)	Peregrine	Male
2017	Peregrine	Nest box	Tissue from dead chick (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	Plucked Feather	Chick 1 (Colour ring = PRA)	Peregrine	Male
2018	Peregrine	Nest box	Plucked Feather	Chick 2 (Colour ring = PSA)	Peregrine	Male
2018	Peregrine	Nest box	Plucked 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 (old)	No peregrine DNA amplification	No bird DNA amplified	Sex would have been contaminated by bird prey. (No bird DNA amplification.)
2019	Peregrine	Nest box	Dead embryo's tissue (embryo's DNA from pale egg with speckles)	Embryo died approx. 2.5 days after lay date (N. Hemmings pers. comm.)	Peregrine DNA Profiled.	Male (2 sex  markers)
2019	Peregrine	Nest box	Dead embryo's tissue (embryo's DNA from brown egg)	Zero or minimal (<24 h) development (N. Hemmings pers. comm.)	No DNA amplification	Unknown sex. (No DNA amplification.)

*Those samples for which DNA was detected from multiple bird species (ie contaminated by prey species) can still be used in comparing peregrine individuals because the genetic markers for individual profiling are peregrine specific so do not amplify other non-falcon bird species.  However the DNA markers used for sexing will sex all birds so mouth swabs and pellets cant be used for sexing peregrines (plucked breast feathers are used).   

4. Creation of a peregrine multiplex microsatellite marker set

We have created a powerful multiplex microsatellite marker set to DNA profile peregrines. This allowed us to quickly and efficiently create DNA profiles and has enough discriminating power to identify individuals. This has been successfully used with peregrine mouth swabs, roadkill tissue and embryo samples. We will test the set in various other types of samples (egg membrane, shed feathers, fresh faecal samples and bird pellets).

5. Comparing individuals and family relationships of Sheffield's Peregrines from chick DNA samples collected 2015-2019 

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. comm.). 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 (St Georges has had peregrines since 2012).

We used microsatellite DNA profiling to investigate if a new female joined the original male in 2016, and checked for evidence of a change in either parent between 2015-2018. In 2019, a second unringed female appeared and DNA investigation of the chicks DNA would be needed to identify which of the two females was the mother of each of th eggs laid was unknown

Table 4. The Sheffield St George's Peregrine Family

DNA sampling began in 2015 and all surviving chicks and two embryos from addled eggs from 2015 onwards were sexed with genetic markers and DNA profiled to study parentage.

Year	Male parent	Female parent	Eggs	Number of chicks hatched (sex)	Unhatched eggs (sex)
2012	Metal BTO ring Not sampled Suspected to be young (D. Wood pers. comm.)	?    (No webcam) Not sampled	4?	2 (No samples taken)  First year the peregrines successfully bred at St Georges Church, Sheffield	2 ? (No samples taken)
2013	Metal BTO ring Not sampled	?    (No webcam) Not sampled	4	3 (No samples taken) Hatched after 32 days	1 (No samples taken)
2014	Metal BTO ring Not sampled	?    (No webcam) Not sampled	4	4 (No samples taken) Hatched after 32 days First year the peregrines chicks at St Georges Church were ringed 2014_GC20731 2014_GC20732 2014_GC20733 2014_GC20734 2014_GC20732 - is male and has bred on Wakefield Cathedral since 2015	0
2015	Metal BTO ring,   Possibly "G/Jxxxx" Not sampled "George"	Web camera set up. Unringed (not sampled) "Mildred"	4	2 (both male)  Hatched after 32 days   2015_GC20794 2015_GC20795	2 (not sampled)
2016	Metal BTO ring Not sampled "George"	Unringed (not sampled) "Mildred"	4	3 (all male)  Hatched after 31 days    2016_GV0084 2016_GV0085 2016_GV0086 GV00085 – found dead on 12 May 2018 near Bradford (had died within a week).	1 (male)   2016_Embryo
2017	Metal BTO ring Not sampled "George"	Unringed (not sampled) "Mildred"	4	3 (all male)    2017_GV0090_PRF  2017_GV0091_PSF 2017_GV0092_PTF Mummified corpse of GV0090_PRF was found in May 2019. Probably died in 2018.	1 (no DNA)
2018	Metal BTO ring Not sampled "George"	Unringed (not sampled) "Mildred"	4	3 (one female=PTA, and two males=PRA and PSA)    2018_GV00097_PRA 2018_GC00098_PSA 2018_GV00099_PTA	1 (no DNA)
2019	Metal BTO ring Not sampled  "George"	2 adult females present (one unringed and one with a metal ring) Metal ringed female laid the first (single) white egg. A week later an unringed females laid 3 brown eggs, one of these 3 was paler than the other two. DNA testing of unhatched embryo from the pale egg (laid in second batch of 3 eggs) reveals its mother is the unringed "Mildred"	1 & 3	1 Only one chick hatched, on 7th May 2019, from the first (white) egg laid on 23rd March 2019 (incubation began after the last egg was laid (4th April) =33 days incubation). The chick died same day it hatched (not sampled). (Chick was removed from nest by father.)	3 Laid 30/03/19, 02/04/19, 04/04/19. A brown egg was damaged/ smashed and disappeared from nest. The two remaining addled eggs were recovered and sampled = a brown and a pale egg. No DNA amplified from brown egg The embryo in the pale egg displayed some development. This "pale" embryo was successfully DNA profiled.   Sample name = 2019_Pale_egg_embryo DNA results suggest it's parents = George and Mildred
2020	Metal BTO ring Not sampled "George"	2 adult females present. One unringed female - seen rarely (could this be Mildred?), and a large female with a metal BTO ring who mated with George frequently - "Bertha".	2	2 First egg laid 5.52am 29th March 2020 by metal ringed female "Big Bertha" Second egg laid 1st April 2020. Laying not seen. Both eggs hatched on 7th May 2020 between 4-7 am. = 37 days incubation. First shell eaten at 4.38 am, chick seen, second shell eaten 5.25 am.	0 Incubation takes approx. 32-38 days and starts from the date the last egg was laid (Newton 1979), which was 1st April 2020, so hatching was due 2nd - 8th May. Newton I (1979) Population Ecology of Raptors. T & AD Poyser Ltd. Table 18, page 343.

Based on DNA profiling, we found no evidence of a switch in either parent between 2015-2018. 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 initial proposed parent switch in 2016.

However the likelihood of detecting a switch in parents was high because we used a large number of powerful highly variable autosomal genetic markers (n=23) to DNA profile the birds and compare individual profiles.

We genotyped 11 chicks (sampled 2015-2018) 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 between 2015-2018.

In 2019, a second female appeared at the nest box. This resulted in disputes between the three birds. Four eggs were laid but it was not clear which female laid which egg. One egg did hatch but died after a few days and was removed from the nest by one of the adults.

The remaining 3 eggs did not hatch. One of the eggs was destoyed or lost from the nest. The remaining two eggs were collected and checked for any signs of development.

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 Dr 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 will also compare the St Georges individuals against peregrine individuals from other nests across the UK and abroad to investigate genetic diversity and population structure.

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

We have identified genetic markers suitable for sexing peregrines and other species of bird of prey, including eagles and falcons.

see the Bird Sex-typing Database

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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 2019 or previous years).

We are looking to obtain samples from more peregrines or addled eggs 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 addled eggs, 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).  

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Personnel

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

Laboratory work and DNA analyses - DNA extraction, genetic sex-typing, microsatellite genotyping, data analyses

• Lucy Knowles - Research Technician 2019-2020
• Dr Natalie dos Remedios - Research Technician 2017-2018
• Gracie Adams - Undergraduate Summer placement volunteer 2017
• Dr Paul Parsons - Research Technician 2020
• Reviewer of camera observation data recorded during 2019 by SBRG and public observations on blog and twitter
• Jennifer Sealy - Undergraduate Summer placement volunteer 2019

Sample collection teams

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

• Steve Samworth, Sorby Beck Ringing Group, (2015-2018)
• Dr Simon Mills, University of Sheffield, (2015-2018)
• Dr Dean Rea, Sorby Beck Ringing Group, (2017)
• Luke Nelson, Sorby Beck Ringing Group, (2018 & 2019)
• Emma Hughes, Sorby Breck Ringing Group, (2018)
• John Rochester, Climber (2019)
• Andy Thomson, Climber (2019)

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.
   
Peregrine samples, including DNA controls of known sex, were kindly provided by the following:

• AD - Dr Andrew Dixon (International Wildlife Consultants Ltd, Wales, UK),
• BTO - British Trust for Ornithology,
• CM - Dr Celia May (University of Leicester, UK),
• DJR - Dr Douglas Ross (Bakewell Vets)
• EK - Esther Kettel (Nottingham Trent University, UK),
• EW - Elizabeth Woodward (Derbyshire Wildlife Trust, UK),
• JG - Jim Graham (Leicester Cathedral Peregrines, UK),
• JMcK - James McKay (Honeybank Falcon Displays)
• JW - Dr Jon Wetton (University of Leicester, UK),
• LG - Dr Louise Gentle (Nottingham Trent University, UK),
• LS - Luke Sutton (The Peregrine Fund & Global Raptor Impact Network),
• LW - Dr Lucy Webster (SASA Scottish Government Wildlife DNA Forensic Unit, Edinburgh, Scotland),
• NB - Nick Brown (Derby Cathedral Peregrine Project, UK),
• PM - Peter Merchant (DEFRA/Natural England Licenced rehabilitation keeper and BTO monitor for Suffolk area),
• SMcC - Sarah McCallum (Biobest Laboratories Limited, Edinburgh, Scotland),
• ZS - Zoe Smith (Peregrine Network, Hawk and Owl Trust).

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