Modeling Wolf, Canis lupus, Recolonization Dynamics to Plan Conservation Actions Ahead: Will the “Big Bad Wolves” Howl Again in Slavonia, Croatia?

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article is devoted to a relevant topic, however, in similar works based on the analysis of data on the number of large predators, including the wolf, whose populations have recovered in Croatia. On the other hand, for such an object as the wolf, which affects agriculture, the preservation of the population and has commercial value, data on the number is important, including for making a decision on regulating the number and harvesting of wolf food resources, such as wild boar. In general, the presented study is relevant, as is the presentation of scenarios for the development of the situation. In Croatia, the harvesting of animals - wolf food - is considered excessive, and they also require linking the wolf population with the number of sheep, which indicates a tense situation, additional research and the adoption of biotechnical measures, for example, to support the food supply and limit the area of ​​grazing livestock, the use of fences and paddocks. Comments:
1. Lines 73-75: "By the early 1990s, however, wolves were present only in Gorski kotar and Lika [26] (Frković et al. 1992), which represent only 11% of Croatia (Figure 1)." Please clarify the numbering of the figures in the text and the figures themselves.
2. Lines 416-420: "The analyses revealed that corridor construction and translocation supported optimal population viability, with conservation effectiveness improving over time, while habitat fragmentation significantly affected recolonization success, and adverse events (disease, illegal hunting, and human-wolf conflicts) posed the greatest risks of extinction." Additional data are needed to analyze the causes of population decline, as well as the characteristics of recolonization processes. Additional data are needed to analyze the causes of population decline, as well as the characteristics of recolonization processes.

3. Lines 470-472: “These highways have cut off the Slavonia region (the modelled area) from Banovina, where a stable wolf population exists. Other reasons could be illegal hunting, competition with other predators (probably with jackal – [47], scarcity of prey, and other conflicts with humans. Initially, it is vital to modify the existing highway structures into wildlife passages.» The authors point out that highways are the main factor of isolation, which fragments habitats. It seems to me that it is necessary to study and indicate the most favorable areas for the construction of crossings for animals. Or propose a new study. It is recommended to indicate these areas on the map.

4. Since in practice, dead animals are usually examined, is there any data on their condition (fatness, presence of injuries, helminth infestation). Analysis of such information will help to adequately assess the state of wolf populations during recolonization. One of the factors in the decline of wolf populations in Italy is considered to be poisoning of animals by rodents after field treatment «First evidence of widespread positivity to anticoagulant rodenticides in grey wolves (Canis lupus)» (Musto et al., 2024, doi 10.1016/j.scitotenv.2024.169990)

5. Information is needed on the facts of the appearance of dog-wolf hybrids. It is necessary to take into account the facts of genetic pollution of populations with the formation of wolf-dog hybrids. For example, the manual "Keeping Wolf Hybrids in the UK" (2000) provides a large amount of information on the origin of hybrids and their identification. It is noted that hybrids are more synanthropic and more often attack farm animals.

6. A factor in the decline in numbers, such as parasitic invasion, in neighboring countries (France, Italy) is associated with environmental pollution after dog walking, the composition of the diet and the population density of wolves (Molnar et al., 2019, doi: 10.1016/j.ijppaw.2019.09.002).

References:
Musto C, Cerri J, Capizzi D, Fontana MC, Rubini S, Merialdi G, Berzi D, Ciuti F, Santi A, Rossi A, Barsi F, Gelmini L, Fiorentini L, Pupillo G, Torreggiani C, Bianchi A, Gazzola A, Prati P, Sala G, Apollonio M, Delogu M, Biancardi A, Uboldi L, Moretti A, Garbarino C. First evidence of widespread positivity to anticoagulant rodenticides in gray wolves (Canis lupus). Sci Total Environ. 2024.10;915:169990. doi: 10.1016/j.scitotenv.2024.169990
Keeping hybrid wolves in the UK // Royal Society for the Prevention of Cruelty to Animals. June 2000
Molnar B., Ciucci P., Mastrantonio G., Betschart B.Correlates of parasites and pseudoparasites in wolves (Canis lupus) across continents: A comparison among Yellowstone (USA), Abruzzo (IT) and Mercantour (FR) national parks. International Journal for Parasitology: Parasites and Wildlife, 2019. 10, 196-206. doi: 10.1016/j.ijppaw.2019.09.002.

Author Response

Comments 1:

1. Lines 73-75: "By the early 1990s, however, wolves were present only in Gorski kotar and Lika [26] (Frković et al. 1992), which represent only 11% of Croatia (Figure 1)." Please clarify the numbering of the figures in the text and the figures themselves.

Response 1: Thank you for pointing this out. We have carefully reviewed and revised the numbering of all figures in the manuscript to ensure consistency between the in-text citations and the figure captions. The reference to Figure 1 on lines 79 and 88 has been updated accordingly, and all figures are now correctly numbered throughout the text.

Comments 2:

2. Lines 416-420: "The analyses revealed that corridor construction and translocation supported optimal population viability, with conservation effectiveness improving over time, while habitat fragmentation significantly affected recolonization success, and adverse events (disease, illegal hunting, and human-wolf conflicts) posed the greatest risks of extinction." Additional data are needed to analyze the causes of population decline, as well as the characteristics of recolonization processes. Additional data are needed to analyze the causes of population decline, as well as the characteristics of recolonization processes.

Response 2: Thank you for this valuable observation. In response, we have revised the relevant passage (now L417–434) to include more specific results from the scenario analyses, particularly quantitative data on extinction risk. We clarified that scenarios involving corridor construction and translocation resulted in zero recorded extinctions across all time frames (10, 30, and 100 years), while all other scenarios averaged extinction risks exceeding 27%. This revision better supports the claim that habitat fragmentation and insufficient intervention significantly hinder recolonization success and magnify the impact of adverse events such as disease, illegal hunting, and human–wolf conflict.

Additionally, we acknowledged the need for further ecological, demographic, and health-related data to fully understand the underlying drivers of population decline and recolonization processes, in line with your comment. We also indicate that these issues will be further explored in the subsequent discussion section through a detailed comparison of scenario groups.

Comments 3:

3. Lines 470-472: “These highways have cut off the Slavonia region (the modelled area) from Banovina, where a stable wolf population exists. Other reasons could be illegal hunting, competition with other predators (probably with jackal – [47], scarcity of prey, and other conflicts with humans. Initially, it is vital to modify the existing highway structures into wildlife passages.» The authors point out that highways are the main factor of isolation, which fragments habitats. It seems to me that it is necessary to study and indicate the most favorable areas for the construction of crossings for animals. Or propose a new study. It is recommended to indicate these areas on the map.

Response 3: Thank you for your constructive suggestion. We fully agree with the importance of identifying and mapping the most favourable areas for wildlife crossings to mitigate the barrier effect of highways. In response, we revised the relevant paragraph (now L499-511) to explicitly recommend a detailed spatial analysis focused on locating optimal sites for the construction or adaptation of wildlife passages. This includes proposing the development of a corridor suitability model based on landscape connectivity, proximity to core habitats, and minimization of human disturbance. We would like to note that Figure 5, which was already included in the original manuscript, illustrates 27 existing highway structures within the modelled area, eight of which have been preliminarily identified as potentially suitable for minor modifications. We apologize if this was unclear and have now ensured that the figure is explicitly referenced and better integrated within the main text. We agree that further spatial modelling and visualization are essential for effective conservation planning and policy implementation, and we propose that such work be addressed in future studies.

 

Comments 4:

4. Since in practice, dead animals are usually examined, is there any data on their condition (fatness, presence of injuries, helminth infestation). Analysis of such information will help to adequately assess the state of wolf populations during recolonization. One of the factors in the decline of wolf populations in Italy is considered to be poisoning of animals by rodents after field treatment «First evidence of widespread positivity to anticoagulant rodenticides in grey wolves (Canis lupus)» (Musto et al., 2024, doi 10.1016/j.scitotenv.2024.169990)

Response 4: Thank you for this important observation. In response, we have expanded the manuscript (now L544-560) to include relevant necropsy data from Croatia and Bosnia & Herzegovina, noting that 96.5% of wolf deaths between 1986 and 2001 were caused by humans, primarily through shooting and traffic incidents, and that the average age at death was only 1.9 years. This highlights that anthropogenic mortality disproportionately affects young individuals and deviates from natural mortality patterns observed in undisturbed populations, where intraspecific aggression accounts for the majority of deaths.

We also added a reference to recent findings from Italy (Musto et al., 2024), where widespread exposure of wolves to anticoagulant rodenticides has been documented. While toxicological and parasitological data from Croatia are still lacking, we emphasize in the revised text the need to include assessments of body condition, presence of injuries, parasitism, and toxicological exposure in future necropsy protocols. Such integrative data will be crucial for evaluating the health and viability of recolonizing wolf populations, as you rightly pointed out.

Comments 5:

5. Information is needed on the facts of the appearance of dog-wolf hybrids. It is necessary to take into account the facts of genetic pollution of populations with the formation of wolf-dog hybrids. For example, the manual "Keeping Wolf Hybrids in the UK" (2000) provides a large amount of information on the origin of hybrids and their identification. It is noted that hybrids are more synanthropic and more often attack farm animals.

Response 5: Thank you for highlighting the importance of addressing wolf–dog hybridization. In the revised manuscript (now L561-583), we have incorporated current knowledge on this issue, including a recent Croatian study that used genetic markers and phenotypic data to confirm that 2.8% of tested wild canids were wolf-dog hybrids. All hybrids were found in Dalmatia, a region characterized by high human disturbance and recent wolf recolonization. These hybrids originated from matings between female wolves and male dogs. Consistent with the literature, we note that hybrids tend to be more synanthropic and more frequently attack farm animals, which could exacerbate human–wildlife conflicts.

We also emphasize that although comprehensive toxicological, parasitological, and genetic data are still lacking for Croatia (including Slavonia), these aspects should be integrated into future necropsy and monitoring protocols. Given the ecological similarities with neighbouring regions, ongoing genetic monitoring is crucial to prevent hybrid establishment, maintain genetic purity, and guide effective conservation and recolonization strategies. Our revision underscores the necessity of including hybridization assessments alongside spatial and demographic studies to better evaluate population viability.

 

Comments 6:

6. A factor in the decline in numbers, such as parasitic invasion, in neighboring countries (France, Italy) is associated with environmental pollution after dog walking, the composition of the diet and the population density of wolves (Molnar et al., 2019, doi: 10.1016/j.ijppaw.2019.09.002).

Response 6: Thank you for your insightful comment regarding parasitic invasion as a factor influencing wolf population declines in neighbouring countries. We have addressed this in the revised manuscript (now L553-583) by including recent findings from France and Italy that link parasite invasions to environmental contamination from domestic dog feces, diet composition, and wolf population density (Molnár et al., 2019). We highlight that these factors can facilitate the transmission of pathogens such as Echinococcus and Toxocara, especially in areas with high densities of free-ranging dogs and significant human disturbance.

Although specific toxicological, parasitological, and genetic data for Croatia, including the Slavonian region, are currently lacking, we emphasize the need to incorporate these aspects into future necropsy and monitoring protocols. Given the ecological similarities with neighboring countries, future research should integrate assessments of body condition, cause of death, parasite load, toxicological exposure, and signs of hybridization in recolonizing wolves. We stress the importance of ongoing genetic monitoring to prevent hybrid establishment and to support conservation strategies, thereby enhancing the accuracy of population viability assessments.

 

Additional references included into revised version of MS

63. Mech, L. D.; Adams, L. G.; Meier, T. J.; Burch, J. W.; Dale, B. W. The Wolves of Denali; University of Minnesota Press: Minne-apolis, MN, 1998; 227 pp.
64. Peterson, R. Wolves as Interspecific Competitors in Canid Ecology. In Ecology and Conservation of Wolves in a Changing World; Carbyn, L. N.; Frits, S. H.; Seip, D. R., Eds.; Canadian Circumpolar Institute, Occasional Publication No. 35; Canadian Circumpolar Institute: Edmonton, Canada, 1995; pp 315–323.
65. Musto, C.; Gazzola, A.; Milanesi, P.; Ghidini, A.; Pizzi, R.; Apollonio, M.; Bassi, E. First Evidence of Widespread Positivity to Anticoagulant Rodenticides in Grey Wolves (Canis lupus). Sci. Total Environ. 2024, 915, 169990.
66. Molnar, B.; Ciucci, P.; Mastrantonio, G.; Betschart, B. Correlates of parasites and pseudoparasites in wolves (Canis lupus) across continents: A comparison among Yellowstone (USA), Abruzzo (IT) and Mercantour (FR) national parks. Int. J. Parasitol. Par-asites Wildl. 2019, 10, 196–206.
67. Kusak, J.; Fabbri, E.; Galov, A.; Gomerčić, T.; Arbanasić, H.; Caniglia, R.; Galaverni, M.; Reljić, S.; Huber, Đ.; Randi, E. Wolf-dog hybridization in Croatia. Vet. arhiv 2018, 88 (3), 375–395.

68.         Cusdin, P. A.; Greenwood, A. G. The Keeping of Wolf-Hybrids in Great Britain; Department of the Environment, Transport and the Regions and Royal Society for the Prevention of Cruelty to Animals: Keighley, UK, 2000.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presents a robust model-based approach to predicting and planning for the recolonization of wolves Canis lupus in Slavonia, Croatia, which is a region historically inhabited by the species but currently without a permanent population. Using the VORTEX simulation framework, the Authors model eleven scenarios that span adverse events, habitat improvements, and population supplementation. The study’s aim is to inform conservation strategy by projeciting extinction risk and population viability under these varied future conditions.

The study's strengths include the use of a methodologically sound, stochastic, and spatially explicit population viability model. The model’s parameters are derived from well-established literature values and are transparently documented in robust supplementary materials. Additionally, the simulations explore a baseline scenario and ten alternative scenarios incorporating various threats (e.g., disease outbreaks, illegal hunting), management interventions (e.g., habitat improvements, translocation), and combinations of these factors. Results are analysed over short (10-year), medium (30-year), and long-term (100-year) horizons, providing insights into how different interventions affect extinction risk over time and underscoring the importance of sustained conservation investment. Notably, corridor construction and population translocation emerge as the most effective interventions, yielding zero extinction probability in the simulations, whereas habitat improvements alone prove insufficient. This finding highlights the critical need for landscape connectivity and direct population support.

Despite these strengths, there are some important limitations. First, the modelling does not incorporate genetic variability or inbreeding dynamics, which could lead to underestimating long-term viability risks. Second, socio-political factors are not integrated into the model (for example, while the discussion acknowledges human–wolf conflict and public attitudes, these aspects are not explicitly included in the simulations). Third, there is uncertainty in several model assumptions, as some inputs (e.g., carrying capacity adjustments and translocation success rates) rely on expert judgment rather than empirical data. These uncertainties should be acknowledged, as they may affect the reliability of the long-term projections.

In conclusion, this study makes a significant contribution to applied conservation biology, particularly in the Central European context. It offers a proactive blueprint for wolf recolonization planning and underscores the importance of landscape connectivity and targeted interventions for species recovery. The modelling approach and insights could be readily adopted to other large-predatory species and regions facing similar recolonization or reintroduction challenges.

Comments on the Quality of English Language

The manuscript is generally understandable and well-organized; however, the quality of English could be improved to enhance clarity and precision. Several sentences are syntactically awkward or overly long (particularly in the methodology and discussion sections), and minor grammatical issues occasionally obscure meaning. A careful language edit by a native English speaker or professional scientific editor is recommended to improve readability and ensure that the research is communicated as clearly as possible.

Author Response

Comments:

The paper presents a robust model-based approach to predicting and planning for the recolonization of wolves Canis lupus in Slavonia, Croatia, which is a region historically inhabited by the species but currently without a permanent population. Using the VORTEX simulation framework, the Authors model eleven scenarios that span adverse events, habitat improvements, and population supplementation. The study’s aim is to inform conservation strategy by projeciting extinction risk and population viability under these varied future conditions.

The study's strengths include the use of a methodologically sound, stochastic, and spatially explicit population viability model. The model’s parameters are derived from well-established literature values and are transparently documented in robust supplementary materials. Additionally, the simulations explore a baseline scenario and ten alternative scenarios incorporating various threats (e.g., disease outbreaks, illegal hunting), management interventions (e.g., habitat improvements, translocation), and combinations of these factors. Results are analysed over short (10-year), medium (30-year), and long-term (100-year) horizons, providing insights into how different interventions affect extinction risk over time and underscoring the importance of sustained conservation investment. Notably, corridor construction and population translocation emerge as the most effective interventions, yielding zero extinction probability in the simulations, whereas habitat improvements alone prove insufficient. This finding highlights the critical need for landscape connectivity and direct population support.

Despite these strengths, there are some important limitations. First, the modelling does not incorporate genetic variability or inbreeding dynamics, which could lead to underestimating long-term viability risks. Second, socio-political factors are not integrated into the model (for example, while the discussion acknowledges human–wolf conflict and public attitudes, these aspects are not explicitly included in the simulations). Third, there is uncertainty in several model assumptions, as some inputs (e.g., carrying capacity adjustments and translocation success rates) rely on expert judgment rather than empirical data. These uncertainties should be acknowledged, as they may affect the reliability of the long-term projections.

Response: We thank the reviewer for highlighting these important limitations. We have acknowledged these points explicitly in the revised manuscript:

Genetic variability and inbreeding (now L552-582):

Genetic variability and inbreeding dynamics were not incorporated into our model due to limited availability of empirical data, as now noted in the manuscript (L560-561). Nevertheless, we discuss genetic threats such as wolf–dog hybridization in the region, supported by recent genetic studies identifying hybrids in Croatia, particularly in Dalmatia. We emphasize the need for continued genetic monitoring and integrative health assessments to safeguard genetic integrity and improve long-term viability evaluations.

Socio-political factors (now L613-627):

While socio-political factors—including human–wolf conflict, public attitudes, and policy interventions—are recognized as critical to successful recolonization, these aspects could not be explicitly modelled here, as now noted in the manuscript. We stress the importance of managing public perceptions and adaptive policy measures in the discussion as indispensable complements to ecological strategies for wolf conservation.

Model uncertainties (now L428-434):

We now noted in the manuscript that some model inputs, such as carrying capacity adjustments and translocation success rates, were based on expert judgment due to a lack of comprehensive empirical data, which introduces uncertainty in long-term projections. We underline the importance of future empirical research to validate and refine these parameters, thereby enhancing model reliability and conservation planning.

In conclusion, this study makes a significant contribution to applied conservation biology, particularly in the Central European context. It offers a proactive blueprint for wolf recolonization planning and underscores the importance of landscape connectivity and targeted interventions for species recovery. The modelling approach and insights could be readily adopted to other large-predatory species and regions facing similar recolonization or reintroduction challenges.

Comments on the Quality of English Language:

The manuscript is generally understandable and well-organized; however, the quality of English could be improved to enhance clarity and precision. Several sentences are syntactically awkward or overly long (particularly in the methodology and discussion sections), and minor grammatical issues occasionally obscure meaning. A careful language edit by a native English speaker or professional scientific editor is recommended to improve readability and ensure that the research is communicated as clearly as possible.

Response: We sincerely thank the reviewer for the valuable feedback regarding the language quality of our manuscript. We acknowledge that clearer and more concise writing is essential for effective communication of our research. To address this, we have carefully revised the manuscript to improve clarity and readability, simplifying complex sentences and correcting grammatical issues. Additionally, we have arranged for a thorough language edit by a native English speaker, Adam Peter Maguire, to ensure the highest standard of language quality throughout the text. We appreciate the reviewer’s suggestion, which has helped us enhance the manuscript significantly.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The article has been significantly improved and revised. Most of the comments have been taken into account. The reviewer thanks the authors for their corrections, additions and comments.
Note:
Lines: 154-155. "Figure 2."
The figure legend is difficult to read, I recommend increasing the font or sharpening the image.
Lines: 542-543. "Figure 4." I recommend increasing the font or sharpening the image.

Author Response

Comment 1:

The article has been significantly improved and revised. Most of the comments have been taken into account. The reviewer thanks the authors for their corrections, additions and comments.

Note:

Lines: 154-155. "Figure 2."

The figure legend is difficult to read, I recommend increasing the font or sharpening the image.

Lines: 542-543. "Figure 4." I recommend increasing the font or sharpening the image.

Response 1: We thank the reviewer for their positive feedback and constructive comments.

Regarding the specific suggestions:

Lines 154-155, Figure 2: We have increased the font size in the figure legend and enhanced the overall image quality to improve readability.

Lines 542-543, Figure 4: We have improved the image quality of Figure 4 as recommended.

We appreciate the reviewer’s careful review and helpful suggestions, which have contributed to improving the manuscript.

Author Response File: Author Response.pdf