Genetic Variability and Family Relationships in a Reintroduced Osprey (Pandion haliaetus) Population: A Field-Lab Integrated Approach

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Reviewer 1

I would like to thank the authors for all their work and decades long continuous effort in creating this manuscript describing the population genetics of a small newly established osprey population in Tuscany partly aided by translocation programs. The conservation monitoring combined field observations with genetic analysis to assess the genetic health of this population and provide a baseline data, which is crucial for future long-term monitoring.

It was my absolute pleasure to read this paper and I would most definitely advocate for its publication. However, I have highlighted some minor issues that need to be addressed before it being published and I hope that the authors will find these beneficial.

Firstly, this paper would benefit from editing the English grammar. I have tried to correct some of the most unclear parts, but there are other minor mistakes throughout the text too frequent to correct them all. If no suitable editor can be found, I am happy to do so prior the publication for the revised final version.

I have split my comments into broader more conceptual issues and grammar-related problems for each of the sections. The original text is highlighted in Italic font with my comments in normal font. Please see below.

 

Answer from the authors: we want to thank the reviewer for the positive feedback and for appreciating this study; we are thankful mainly for the strong effort in reviewing the draft. We agree with all the observations that will improve and ameliorate the text.

 

INTRODUCTION

Overall/Conceptual items.

1) Line 92 Native birds from Corsica, Balearics, Northern Africa, and Canary Islands are genetically different from ospreys breeding in Central and Northern Europe.

 

Could you please add some information about whether this difference is purely genetic or are Mediterranean basin ospreys also distinct morphologically at any extent (e.g. colouration/ size) and behaviour wise (e.g. are they migratory or sedentary; are they philopatric like N. and W. EU populations).

 

Answer from the authors: Individuals are not morphologically differentiated but show different migratory behaviour. We added some details about it in the text. More specifically, we focused on the fact that, in parallel to genetic differences, northern and southern ospreys populations also show distinct migratory strategies with respect to both temporal and spatial components of migration, with the former being long-distance migrants and the latter mostly resident or short-distance migrants.

 

2) Could you add some information of typical generation intervals for the ospreys.

Adding this information in the introduction will help to improve the discussion by providing stronger link between observed genetic patterns, life-history of the species and adaptive aspects.

 

Answer from the authors: we added this information in the text. In particular, we stressed the fact that although the species can reach sexual maturity already at the age of 2 years, the recruitment in the breeding population can require more time, sometimes with first successful breeding attempts occurring after 3-5 years (e.g., Englund and Greene 2008) and with an estimated mean generation time of 9.6 years (Wahl and Barbraud, 2019). For this reason, the settlement of the first breeding pair in the framework of a reintroduction project is an important milestone that can be interpreted as a chance for the success of the project itself.

 

3) Could you clarify if the familial relationship between 33 translocated chicks from Corsica are known (via previous either genetic or ecological monitoring) or completely random?

 

Answer from the authors: we added this information as a Supplementary file. Please refer to the new Table S1.

 

Technical/language/clarity issues.

Lines 72 - 74 For example, the trend of several reintroduction projects has been documented by using Single nucleotide polymorphisms (SPNs) or microsatellite loci (STRs), allowing the description and the recording of the genetic variability trend in several taxa (e.g. [18; 20-21]).

Delete the word “trend” in the latter part of the sentence to read: “For example, the trend of several reintroduction projects has been documented by using Single nucleotide polymorphisms (SPNs) or microsatellite loci (STRs), allowing the description and the recording of the genetic variability in several taxa (e.g. [18; 20-21]).

 

Answer from the authors: we deleted the word as suggested.

 

Lines 121 - 123 We genotyped 16 loci from individuals both translocated (2006-2010) and belonging to the new population (2011-2021) to: i) assess the genetic variability and ii) verify any modification of the genetic composition in the long term, from the early stages of the re- introduction program to 2021.

Not entirely clear sentence. I suggest rewording to read:

“We genotyped 16 loci from individuals belonging to both the source population of the translocated birds (2006-2010) and those belonging to the newly established population (2011-2021) to: i) assess the genetic variability and ii) verify any modification of the genetic composition in the long term compared to the early stages of the re-introduction program to 2021.”

 

Answer from the authors: we modified the sentence, as suggested.

 

Lines 124 -127 Moreover, notwithstanding the monogamy of the species, we obtained evidence from video camera recordings of cases of extra-copulation, a behavior that has been already described in the species [39-40] but never recorded in the early stage of a population settlement [39-40] but never recorded in the early stage of a population settlement.

Delete the second “never recorded in the early stage of a population settlement”.

 

Answer from the authors: we deleted the duplicated sentence.

 

Lines 128 - 130 As we could not assess if this tendency behavior resulted in diverse brood composition, we carried out parentage tests iii) for the identification of direct relationships among individuals and full relationships among individuals to verify any occurrence of chicks associated with non-mate individuals.

Delete the “tendency” to read: “130 As we could not assess if this behavior resulted in [genetically?] diverse brood composition, we carried out parentage tests iii) to infer direct relationships among individuals as well as full relationships among individuals and enable the verification of any occurrence of chicks associated with non-mate individuals.”

 

Answer from the authors: we deleted the word and modified the sentence, as suggested.

 

MATERIALS AND METHODS

Technical/language/clarity issues.

Line 138 Two of them successively settled in the study area in two …

It would be good to mention here if they were male or female and provide the tag number and nest site number/name.

 

Answer from the authors: we provided the information.

 

Line 144 - 145 Remarkably, this sample includes two individuals born respectively in 2011 and 2016 in the framework of the reintroduction project, settled and bred in two new and different nests in 2014 and 2019.

Replace “born” with “hatched” throughout the text and also in the Supplement.

born respectively in 2011 and 2016: please provide the nest sites where they were hatched (if known)

settled and bred in two new and different nests in 2014 and 2019: please provide the name/number of these nest sites.

 

Answer from the authors: we substituted born with hatched as suggested and provided the information regarding the two settled individuals.

 

Line 159 following the prescriptions …

Please replace “prescriptions” to “in accordance with Law 157/1992 [Art.4(1) and Art 7(5)]…”

 

Answer from the authors: we replaced, as suggested.

 

To make it more clear and brief, the figure 2 legend could be re-written as follows:

Figure 2. Graphic scheme of nests and reproductive events between 2011-2021. BELLOQ is the un- ringed male that in 2020 replaced I1-E4647 in NEST_1 and is the father of solely the two chicks born in 2020 in NEST_1 (1-IBM-E2427 and 1-IBS-E2428). Two individuals born in 2011 and in 2016 in the NEST_1 and NEST_3 (indicated by *), upon reaching sexual maturity, reproduced in the NEST_2 and NEST_5, respectively (see notes below). In black the ospreys sampled, while in red are reported other individuals of the population that have not been sampled for this study. Blue and pink squares indicate male and female birds, respectively. **= individuals translocated during 2006-2010 that became successful breeders; °= non-translocated Corsican adult female (ringed in Corsica as a pullus) that naturally settled in NEST_4 in 2018. n.a. = two individuals born in NEST_3 in 2021 but not ringed/sampled. \ = indicates unsuccessful breeding events (e.g., eggs did not hatch or pullus died before fledging).

 

Answer from the authors: we modified the sentence, as suggested.

 

Overall/Conceptual items.

• It would be great if a table with technical information on multiplexes for the primers during the amplifications and dyes used for each locus could be added (in Supplemental Information).

 

Answer from the authors: We added a table in the supplementary. Please refer to the new Table S2.

 

2) Lines 206 – 209 According to the manufacturer's instructions, DNA was isolated from feather or blood samples using the DNeasy Blood & Tissue Kit (Qiagen) and amplified at 16 nuclear microsatellite loci, Balbu17, Balbu18, Balbu23, Balbu28, Balbu40 [19], PHA04, PHA11, PHA13, PHA14, PHA16, PHA27, PHA28, PHA29, PHA35, PHA36, PHA37 [42] in an 8μl final volume reaction.

 

The correct publication (reference No 42 here) of the PHA microsatellite markers is:

Dawson DA, Kleven O, Dos Remedios N, Horsburgh GJ, Kroglund RT, Santos T, Hewitt CR. A multiplex microsatellite set for non-invasive genotyping and sexing of the osprey (Pandion haliaetus). Conservation genetics resources. 2015 Dec;7:887-94.

NOT

Currently Ref 42: Dawson, D. A.; Bird, S.; Horsburgh, G. J.; Ball, A. D., Autosomal and Z-linked microsatellite markers enhanced for cross-species utility and assessed in a range of birds, including species of conservation concern. Conservation Genet Resour 2015, 7 (4), 881-886.

 

Answer from the authors: Thanks for pointing it out. We replaced the citation.

 

RESULTS

Technical/language/clarity issues.

Line 306. All 81 individuals were fully genotyped. Biomolecular sexing evidenced that 12 and 33 specimens respectively out of 33 translocated individuals and 46 newborn chicks were males with an evidently different sex ratio between the two project sampling phases.

Consider rewording for clarity. “All 81 individuals were fully genotyped. Biomolecular sexing determined that 12 out of 33 translocated individuals and 33 out of the 46 post-translocation chicks were males, which is evidently a different sex ratio between the two project sampling phases.”

 

Answer from the authors: we modified the sentence, as suggested.

 

Line 308. A strong departure from Hardy-Weinberg equilibrium was found either in WSS and in POP_2 while no disequilibrium was recorded in POP_1. One (Balbu23) and three loci…

Delete “either” in this sentence.

 

Answer from the authors: we deleted the word, as suggested.

 

Line 321. Detailed results are shown in Table 1.

Please could you also add a table of allele numbers & richness for each of the 16 loci (in Supplementary Data), would allow for a good indication to the best marker choice in future osprey studies in different regions.

 

Answer from the authors: we added the table in the supplementary files.

 

Line 350 … the full-sibling analysis, conducted by considering only the relationships between newborn individuals…

Consider replacing “newborn” to “chicks hatched post translocation”

Replace “born” by “hatch” throughout the text.

 

Answer from the authors: we replaced “newborn” to “chicks hatched post translocation” in this sentence and substituted “born” with “hatched” throughout the text.

 

Line 354 However, when removing the individuals born in the NEST_1 and NEST_3 and that reproduced in NEST_2 and NEST_5, respectively, all the individuals were correctly…

Rewrite to: “However, when removing the individuals born in the NEST_1 and NEST_3 that subsequently reproduced in NEST_2 and NEST_5, respectively, all the individuals were correctly…”

 

Answer from the authors: we modified the sentence, as suggested.

 

Lines 358 – 360. Two individuals (1-IBM-E2427 and 1-IBS-E2428) that were born in NEST_1 after the male of the breeding pair has been replaced by another individual, were assigned from the software to different cluster families (Table 2), thus explaining the presence of six clusters with respect to the five nests.

 

Consider rewording to for clarity: “Two individuals (1-IBM-E2427 and 1-IBS-E2428) that hatched in NEST_1 in 2020 after the male of the breeding pair was replaced by another individual, were assigned to different genetic cluster than their half-siblings from the previous years (Table 2), thus explaining the presence of six clusters with respect to the five nests.”

 

Answer from the authors: we modified the sentence, as suggested.

 

Figure 3

3a. I would ask to consider to remake the structure cluster figure based on individual breeding pairs and indicating the nest number they bred at– it would make it easier to infer the genetic relations for reader.

3b & 3c – need to be produced in a better resolution as they are currently unreadable.

 

Answer from the authors: it was quite difficult to write in such a small space breeding pair IDs, so we reported the nesting sites at the top and the nest at the bottom. We provided a better resolution for Figures 3b and 3 c, and also for Figure S3 in the Supplementary file

 

DISCUSSION

Overall/Conceptual items.

1)The title of the paper states “Genetic variability and family relationship in a reintroduced osprey (Pandion haliaetus) population: an integrated approach.” However, the importance of the field monitoring needs to be emphasised more. For example, Figure 2 – is the only figure presumably based on primarily ringing and video monitoring, so that should be stated. It would also be beneficial to include a more comprehensive table of the field records of key life history events in the Supplement if possible.

 

Answer from the authors: we added further field information in the text and the known relationships in a Supplementary file (Table S1). According to this suggestion, we also decided to slightly change the title in “Genetic variability and family relationship in a reintroduced osprey (Pandion haliaetus) population: a field-lab integrated approach”. Thanks.

 

409 -412 The values of allele number, allele richness and observed heterozygosity were in line with a recent investigation carried out by Skujina and colleagues [17] that used a combination of the panel markers from two former studies [42] and [19]. This approach returned values of genetic variability higher than the two sets of STRs of the latter studies analyzed alone.

I am not sure I fully agree/follow this statement. In the study of newly establishing Welsh osprey population [17] authors used the following loci with the ones in bold overlapping with the markers used in this study (n=11).

Pha02, Pha04, Pha13, Pha16, Pha27, Pha28, Pha29, Pha30, Pha33, Pha35, Pha36, Pha37, Balbu-05, Balbu-12; Balbu-18, Balbu-23, Balbu-26; Balbu-30, Balbu-37.

As you can see, the marker panel is comparable. Study by Skujina et al 2021 indeed found higher diversity levels in the Welsh population, but that could be attributed to a wider pool of mixed founding populations of most likely Scottish and Northern European ancestry. The other crucial finding was that the marker set had a definite effect on genetic variability determination: Dawson et al. (2015) loci (PHA) were considerably more polymorphic than the Balbu loci used by Monti et al. (2018) loci and the higher levels of variability in PHA loci almost certainly are linked to the smaller repeat motifs (mostly dinucleotides) than those used by Monti et al. (2018) - trinucleotide up to hexanucleotide, and the greater repeat number for the former. But since the study by Skujina et al and this study here are both using a comparable combination of these two marker sets, the lower diversity levels in Italian populations can be considered a real biological difference and excludes this being merely a consequence of different marker choice.

 

Answer from the authors: the significance of the sentence was not clear and we used part of your comment to rewrite the paragraph and make it clearer.

 

Line 445 AMOVA identified that 98% of the variance was distributed among individuals.

This is in stark contrast to the statement in the results: Line 335 - Fst recorded a significant difference between POP_1 and POP_2 (Fst = 0.02102; P = 0.000) although AMOVA computation confirmed that almost the whole recorded variance (98%) was within individuals.

To distinguish between intra- and inter- individual variation has very important conservation implications especially for a small recovering populations. If almost all the genetic variance occurs WITHIN the individuals, it means that every single nest is very important contributor to overall population success and could nearly be viewed as evolutionary significant unit (ESU) in its own right. This to me is one of the most important genetic findings from this study and should be expanded in the discussion.

 

Answer from the authors: we apologize because there was a mistake in the text: 98% of the variance was within individuals. Among individuals, the variance was = 0%, thus confirming the data from other analyses. Data were correctly reported in the figure. We added the information on variance among individuals

 

Line 495 A possible explanation could be that attempts of extra-pair mating potentially inform about the good quality of an individual as a partner, but not necessarily result in a change of the partner.

Could this be because of sperm competition?

 

Answer from the authors: we added some information in the text

 

Technical/language/clarity issues.

Line 422. This mitigated effect might be explained by considering the recorded natural integration...

Delete “considering”.

 

Answer from the authors: we deleted the word, as suggested.

 

Line 429. However, respecting the genetic origin and evolutionary history of the reestablished population it’s also extremely..

Avoid abbreviations: it’s – it is

Line 435. …observed a re-established sex ratio with respect to POP_1, we recorded a departure from…

 

Answer from the authors: we changed, as suggested.

 

Please clarify what do you mean by “re-established sex ratio”

 

Answer from the authors: Since it was not clear, we preferred to modify and delete part of the sequence

 

Line 448. This piece of evidence could be explained by considering that only two out of the translocated individuals were able to successively reproduce in the study area and contribute to the actual genetic composition. Anyway, it is not possible to affirm whether causality or any better adaptative genomic variants of breeding individuals have driven the successful nesting.

Correct English:

“This result could be explained by the fact that only two of the translocated individuals were able to successfully reproduce in the study area and contribute to the genetic composition. At this point, it is not possible to affirm whether this success is attributed to a random chance or better adaptative genomic variants of these breeding individuals.

 

Answer from the authors: we substituted the sentence, as suggested.

 

Line 478. Some factors seem to influence a new settlement more than genetic composition, firstly, the presence of other individuals of the same species that produces a conspecific attraction [30]. The sentence is not quite clear. Please reword.

 

Answer from the authors: we removed the sentence to avoid any misunderstanding.

 

Line 483. …the protocol was able to identify the correct full sibling through the years 483 without using preliminary information…

I think what you mean here is: “ the genetic protocol was able to identify the correct full sibships through the years without using any prior information.”

 

Answer from the authors: we added “genetic”, as suggested.

 

Line 492: …recorded several extra-pair copulation attempts of at least a male and a female, but the genetic analysis excluded the occurrence of a successful extra-copulation event and confirmed the full-sibling relationships, as indicated by field observations…

What is meant by “of at least a male and a female”?

 

Answer from the authors: The video-surveillance system allowed us to record this behaviour in only a male and a female, but we cannot ensure that it was carried out by other individuals in the area (when out of view).

 

Line 508. These times are in lineage with those of other projects: in the Twin Cities project (Minnesota, U.S.A.).

English: replace “lineage” with “line”

 

Answer from the authors: we replaced “lineage” with “line”.

 

Line 507. The first successful breeding event was recorded 5 years after release started, mainly due to the delayed maturity of the species.

I would not consider this “delayed maturity” but a typical “generation interval” for ospreys: reaching sexual maturity at age of 3 followed by first breeding attempts within 3-7 years.

 

Answer from the authors: we modified the text as follows: “The first successful breeding event was recorded 5 years after release started, mostly due to the average age at first known successful nest which, in this species, is ca 4.39 years for males and 3.64 years for females.”

 

Table S1. Distribution of private alleles. a) Private alleles distributed in the translocated (POP_1) and lacking in the POP_2; b) alleles found in the injured ospreys and not found either in POP_1 or in POP2.

Ospreys: was there more than one injured osprey?

Answer from the authors: we corrected in osprey. There was a unique injured osprey

 

Reviewer 2 Report

Genetic variability and family relationship in a reintroduced osprey (Pandion haliaetus) population: an integrated approach

 

The manuscript is interesting and overall well-written. Standard laboratory and analyses methods have been employed. The MS would benefit from some minor corrections before publication.

Remove full stop sign from the title.

I felt major improvement needed in the abstract. Major part of the abstract (Line 17-26) is on the background. It could be shortened and the major results could be presented. Please consider rewriting it making it more interesting to the readers.

Line 29: We did not observe a reduction in genetic…………. Reduction to which reference?

Line 30: … a slight sign of ……………. What does the slight sign mean? How was the sign confirmed? I think it would be better to mention here.

Line 73: SPNs – SNPs

Line 101: 42°39’ N, 11°05’ E; - please give the geographic range of the park rather than a point

Lines 126-127: repetition of the texts.

Line 134: A total of 81 samples………… what were sampled? How many bloods and how many feather samples? Line 162 says both types of samples were collected.

There are some other areas where improvement in write up are essential. Authors could improve it if revised carefully.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Genetic variability and family relationship in a reintroduced osprey (Pandion haliaetus) population: an integrated approach

 

The manuscript is interesting and overall well-written. Standard laboratory and analyses methods have been employed. The MS would benefit from some minor corrections before publication.

Answer from the authors: we thank the reviewer for the positive feedback and for the comments that allowed us to ameliorate the draft. We tried to address all of them.

 

Find below the comments from review 2 and relative answers from the authors:

 

Reviewer 2: Remove full stop sign from the title.

Answer from the authors: we removed the full stop sign.

 

Reviewer 2: I felt major improvement needed in the abstract. Major part of the abstract (Line 17-26) is on the background. It could be shortened and the major results could be presented. Please consider rewriting it making it more interesting to the readers.

Answer from the authors: We removed a sentence dealing with the starting phase of the project and added further information on the data obtained in the study.

 

Reviewer 2: Line 29: We did not observe a reduction in genetic…………. Reduction to which reference?

Answer from the authors: we specified that the comparison was between the two sampling periods.

 

Reviewer 2: Line 30: … a slight sign of ……………. What does the slight sign mean? How was the sign confirmed? I think it would be better to mention here.

Answer from the authors: we specified that a sign of the founder effect was found in the reestablished population.

 

Reviewer 2: Line 73: SPNs – SNPs

Answer from the authors: we corrected the text.

 

Reviewer 2: Line 101: 42°39’ N, 11°05’ E; - please give the geographic range of the park rather than a point

Answer from the authors: we removed it from the text and provided a detailed description in MM.

 

Reviewer 2: Lines 126-127: repetition of the texts.

Answer from the authors: we removed the text.

 

Reviewer 2: Line 134: A total of 81 samples………… what were sampled? How many bloods and how many feather samples? Line 162 says both types of samples were collected.

Answer from the authors: We added this information.

 

Reviewer 2: There are some other areas where improvement in write up are essential. Authors could improve it if revised carefully.

Answer from the authors: we agree with you. We carefully read the draft and modified several sentences.

Reviewer 3 Report

Dear authors,

Please, make additions to the manuscript.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

Comments from the authors: we thank the reviewer for the suggestions. We carefully attempted to address them correctly.

 

L37-38 It is necessary to characterize the problem comprehensively. Provide alternative existing methods for the conservation of endangered birds. Focus on the advantages and disadvantages of different methods. Smoothly leading the reader to the reintroduction.

 

Answer from the authors: we integrated the text, providing a description of alternative methods for the conservation of endangered birds and referred to some of the following suggested references. Thanks.

 

L39 Endangered bird species are usually restored through the creation of artificial nests [Ivanovski, 2000; Lapshin et al., 2022], the implementing artificial insemination [Blanco et al., 2009], the use of artificial incubators [Kuehler et al., 2001] and the fight against pathogens of these birds [Jones, 2004].

Ivanovski, V. V., Construction of artificial nests as conservation measure for rare birds of prey. Buteo 2000. 11, 131-138.

Lapshin, A. S.; Andreychev, A. V.; Alpeev, M. A.; Kuznetsov, V. A., Methods and Techniques for Making Artificial Nests for Eagle-Owls (Bubo bubo, Strigiformes, Strigidae) to Increase Their Population. Biology Bulletin 2022. 49 (9), 1503-1507.

Blanco, J. M.; Wildt, D. E.; Höfle, U.; Voelker, W.; Donoghue, A. M. Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species. Theriogenology 2009. 71 (1), 200-213.

Kuehler, C.; Lieberman, A.; Harrity, P.; Kuhn, M.; Kuhn, J.; McIlraith, B.; Turner, J., Restoration techniques for Hawaiian forest birds: collection of eggs, artificial incubation and hand-rearing of chicks, and release to the wild. Studies in Avian Biology 2001. 22, 354-358.

Jones, C. G., Conservation management of endangered birds. Bird ecology and conservation: a handbook of techniques

Answer from the authors: we added a short reference dealing with the restoration of endangered birds and included suggested references.

 

L134 It is necessary to describe in detail the method of collecting the material. Here it is necessary to indicate from what area these samples were collected. What is the distance between sample collection points. Preferably in a table. Separately indicate the distance between the nearest samples from nesting pairs of birds.

 

Answer from the authors: we integrated several pieces of information in MM and in a supplementary file.

L306 Give the results of genetic studies based on the spatial structure of the studied osprey population.

 

Answer from the authors: we apologize but we cannot understand the request. We are available to address any further suggestions if clearer explained

L398 What video-cameras were used. Give their characteristics.

 

Answer from the authors: Thanks for pointing it out. we provided them in the text

 

L399 Likewise. Give characteristics in methods.

 

Answer from the authors: Thanks for pointing it out. we provided them in the text when referring to the injured osprey originating from Northern Europe and that was equipped with gps/gsm transmitter.

 

L518 The text does not contain the total hours of video recordings made for the entire time, as well as separately for nesting pairs of birds.

 

Answer from the authors: The hours of video recordings made over the entire time are quite difficult to provide because the video-surveillance monitoring program has been carried out in several stages. We provided info about the system used and additional information is reported in the literature. In any case, all nests were equipped with video-surveillance systems thus providing precious information on osprey activity at nests all year round (of course with a particular focus during the breeding period).

 

L576 Add sources of literature listed above in the introduction

Answer from the authors: we integrated them in the Reference section.