# Testing the use of southern France and Cyprus mouflons as a reference to reconstruct birth season in sheep from oxygen isotope analysis in teeth

Eléa Gutierrez, Angelos Hadjikoumis, Eleftherios Hadjisterkotis, Jean-Denis Vigne, Fabien Dewilde, Denis Fiorillo, Lionel Gourichon, Marie Balasse

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## To cite this version:

Eléa Gutierrez, Angelos Hadjikoumis, Eleftherios Hadjisterkotis, Jean-Denis Vigne, Fabien Dewilde, et al.. Testing the use of southern France and Cyprus mouflons as a reference to reconstruct birth season in sheep from oxygen isotope analysis in teeth. Journal of Archaeological Science, 2025, 183, pp.106381. 10.1016/j.jas.2025.106381. halshs-05379241

HAL Id: halshs-05379241

https://shs.hal.science/halshs-05379241v1

Submitted on 24 Nov 2025

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# Testing the use of southern France and Cyprus mouflons as a reference to reconstruct birth season in sheep from oxygen isotope analysis in teeth

Eléa Gutierrez<sup>a,∗</sup>, Angelos Hadjikoumis<sup>b,c</sup>, Eleftherios Hadjisterkotis<sup>d</sup>, Jean-Denis Vigne<sup>a</sup>, Fabien Dewilde<sup>e</sup>, Denis Fiorillo<sup>a</sup>, Lionel Gourichon<sup>f</sup>, Marie Balasse<sup>a</sup>

## ARTICLE INFO

### Keywords:

Ovis gmelini

Enamel

Incremental sampling

Dental development

France

Cyprus

## ABSTRACT

Reconstructing past sheep birth seasons relies on oxygen isotope analysis in dental enamel, using modern reference datasets with known birth seasons for comparison. However, the comparability of dental growth calendar between modern and ancient sheep remains uncertain. The current mouflons in Europe, descended from Neolithic domestic sheep that later became feral in Corsica, Sardinia, and Cyprus, are amongst the closest living relatives of Neolithic sheep. Their suitability as references for the estimation of the birth season was explored by analysing the third molar of individuals from two populations giving birth in the spring: Cyprus mouflons (n = 7) and Mediterranean mouflons from southern France (n = 10). Results from oxygen isotope analysis of dental enamel reflect environmental and management differences (e.g., wild vs. captive conditions, local climate and seasonal temperature fluctuations). Isotopic sequence modelling indicated a good agreement between the Mediterranean mouflons from southern France and previously published western European sheep references. However, a radical shift in the record of the δ¹⁸O values of the seasonal cycle between the Cyprus mouflon and the sheep references has been observed.

This divergence suggests distinct dental growth calendars between the Cyprus mouflons and the remaining *Ovis* included in the study, which may be attributed to a different genetic lineage, reduced genetic diversity in Cyprus mouflons due to one or more founder events—potentially leading to the expression of traits not present or rarely present in the source population—and/or long-term isolation resulting in phylogenetic drift.

These findings suggest that Mediterranean mouflons from southern France are as suitable as modern sheep breeds as references for reconstructing past birth seasons in Western Europe. In contrast, the use of the Cyprus mouflons as a reference requires further investigation, both in the context of Cyprus and more broadly across the Near East.

## 1. Introduction

The socio-economic organisation of farming societies is intricately linked to the pastoral calendar, which is largely governed by the reproductive cycles of livestock. In particular, the birthing season requires increased labour from herders to ensure the highest possible rate in successful births, provide support during the initial stages of offspring development, and optimise dairying (Ferret, 2021). In temperate latitudes, sheep alternates between a period of sexual rest and a period of

fertility on an annual scale, tuned to the seasonal cycle by photoperiodic control, leading to seasonal lambing (Karsch et al., 1984; Malpaux et al., 1987). This seasonality imposes significant constraints on agropastoral activities, including milking, which was practised by the earliest Neolithic societies in Europe (Vigne and Helmer, 2007; Debono Spiteri et al., 2016), while also influencing the seasonal mobility of herds. The lambing season, therefore, constitutes a key element in attempts to reconstruct agropastoral calendars in past societies (Blaise and Balasse, 2011; Bréhard et al., 2014; Fabre et al., 2021; Manen et al., 2022;

<footnote><sup>a</sup> UMR 7209 BioArch: BioArchéologie, Interactions Sociétés Environnements, CNRS/MNHN, Paris, France</footnote>

<footnote><sup>b</sup> Science and Technology in Archaeology and Culture Research Center (STARC), The Cyprus Institute, Nicosia, Cyprus</footnote>

<footnote><sup>c</sup> School of Biosciences, The University of Sheffield, United Kingdom</footnote>

<footnote><sup>d</sup> Agricultural Research Institute, Ministry of Agriculture, Natural Resources and Environment, Cyprus</footnote>

<footnote><sup>e</sup> Université de Bretagne Occidentale (UBO, Brest), UMS 3113, Pole Spectrométrie Océan (PSO), Institut Universitaire Européen de la Mer, Plouzané, France</footnote>

<footnote><sup>f</sup> Université Côte d'Azur, CNRS, CEPAM, Nice, France</footnote>

<footnote>* Corresponding author. UMR 7209, BioArch, CP 56, 55 rue Buffon, 75005 Paris, France.</footnote>

<footnote>E-mail address: elea.gutierrez@mnhn.fr (E. Gutierrez).</footnote>
Balasse et al., 2017).

A significant dichotomy exists in sheep birthing season between the Mediterranean Basin and higher-latitude regions of Europe. In continental Europe, the lambing season is late winter and spring (Hafez, 1952). However, under specific conditions—including sheep genetics, zootechnics, and environmental factors defining the availability of food—the fertility period of sheep in the Mediterranean region can be extended, allowing for “out-of-season” births in autumn (Gómez-Brunet et al., 2012; Balasse et al., 2024). Today, autumn births can be achieved using hormonal treatments or exposure to artificial light regimens (Chemineau et al., 1992), but they also occur in non-industrialised husbandry as documented in Cyprus (Hadjikoumis et al., 2019), and they are evidenced during the 6th millennium in the northwestern Mediterranean (Tornero et al., 2020; Fabre, et al., 2021, 2023; Tejedor-Rodríguez et al., 2021). Autumn versus spring birthing would have shaped the pastoral calendar and the socioeconomic organisation of these societies differently.

Past birth seasons can be estimated through analysis of the seasonal cycles recorded in dental enamel oxygen isotope ratios (δ¹⁸O). This approach involves comparison with modern reference sets from various European sheep breeds, spanning a wide latitudinal range in western Europe, from Corsica to Orkney (Balasse, et al., 2005, 2012, 2017, 2020, 2024; Blaise and Balasse, 2011; Tornero et al., 2018; Fabre et al., 2023). These reference sets have been applied to past sheep from contexts as chronologically and environmentally diverse as the Cypriot PPNB in the 8th millennium BCE (Hadjikoumis et al., 2019) and 3rd millennium BCE Orkney Islands (Balasse et al., 2017; Blanz et al., 2024).

Estimating the birth season in past populations using this approach relies on the assumption that the growth dental calendar of modern sheep breeds is comparable to that of ancient sheep. Although the consistency between observed modern and ancient patterns of sheep birth seasonality (Balasse et al., 2017) could support its validity, this initial assumption is nevertheless not directly verifiable. Modern sheep breeds exhibit morphological differences in bones (e.g., Van Vuren and Bakker, 2009; Jeanjean et al., 2023; Robin and Clavel, 2018), as well as in teeth (Jeanjean et al., 2025), although the latter is expected to be less phenotypically plastic than bones (Caumul and Polly, 2005). These differences reflect a long history of environmental adaptations (e.g., Sejian et al., 2017; Berihulay et al., 2019) and selection for production purposes (e.g., Pöllath et al., 2019, De Marinis and Asprea, 2006; Gaastra, 2023). Such pressures may also have influenced dental growth patterns over time. Hence, the use of modern domestic sheep as references should be scrutinised further.

One of the closest living relatives of Neolithic sheep in Europe is the European mouflon (Ovis gmelini), feralised from Neolithic sheep introduced by early agropastoralists (Poplin, 1979; Vigne, 1988) and which has survived to the present day in Cyprus, in Corsica, and in Sardinia (Chessa et al., 2009; Her et al., 2022). Domestic sheep were brought to Cyprus from the early 8th millennium cal BCE, possibly in successive waves, as suggested from sudden breaks in sheep size during the PPNB at Shillourokambos (Vigne et al., 2017). Sheep from the early and middle phases of Shillourokambos (8000-7400 BCE) do not seem to have feralised or, if so, they did not survive. From the morphology of horn cores, the Cyprus mouflon rather relates to the sheep found in the 7th millennium at Khirokitia (Vigne et al., 2014; Vigne, 2011). Chessa et al. (2009), then Her et al. (2022) and Atağ et al. (2024) showed that the Cyprus mouflon was an early feral lineage that split from the domesticated sheep gene pool.

Domestic sheep were transported to Corsica by the middle of the 6th millennium cal BCE (Vigne, 1984), while evidence for the presence of mouflon, also deriving from sheep feralisation, dates to the final Neolithic (2500-3000 BCE), with possible earlier origins (5th.4th millennium BCE) (Vigne, 1988, 1992). Subsequently, the Corsican mouflon was introduced and released to the wild in mainland France in the late 1940s, primarily for game hunting purposes. As a result, crossbreeding occurred with domestic or other wild mouflon species to enhance the

quality of hunting trophies. However, for the purpose of species preservation, the Caroux-Espinouse reserve was established mainly with individuals originating from Corsica and was primarily used to create new populations in various other French reserves (Cugnasse, 2001). The designation “Mediterranean mouflon” was proposed by the French administration to distinguish these potentially hybrid populations from the original source populations in Corsica and Sardinia (Cugnasse, 1994; Garel et al., 2022).

This study investigates whether mouflons are as suitable as domestic sheep as reference material for determining past birth seasons through sequential isotope analysis of tooth enamel. Teeth from modern mouflons with known lambing seasons, originating from populations in southern France and Cyprus, were analysed using the same protocol applied to archaeological specimens. The results are compared with previously obtained data from modern European sheep, which currently serve as reference sets. If found appropriate, mouflons could be incorporated into these reference sets.

## 2. Materials

### 2.1. Cyprus mouflons

A total of seven Cyprus mouflons were included in this study, consisting of seven lower third molars (M/3) selected for oxygen isotope analysis (δ¹⁸O). Considering the destructive nature of isotope analysis, the rarity of the Cyprus mouflons skeletons, and the greater availability of modern reference data for the lower third molar compared to the second molar; we targeted our analyses to the third molar. The Cyprian animals died from natural causes, with their carcasses collected between 1986 and 1987, except for one in 1984 and another in 2003 (Table 1) and then stored as a personal collection for scientific research. Of the seven mouflons, four were males, three were females, and they were aged between two and eight years old. Six of these mouflons were free-living individuals from the Paphos Forest in Cyprus, the natural habitat of the Cyprus mouflon. Amongst those, four originated from within the forest, with lambing grounds altitudes varying between 792 m and 880 m above sea level (asl). One mouflon had an uncertain provenance but was likely from within the forest. Another mouflon was from the forest-agricultural land (FAL) interface, with lambing grounds altitudes between 400 m and 510 m asl (Table 1). Interactions between individuals within the forest and those at its periphery are minimal or absent, as the distance between these areas exceeds their typical home range (Hadjisterkotis, 1992). Additionally, one mouflon was kept in a 40 × 50 m enclosure at an altitude of 900 m asl at the Stavros tis Psokas forest station (Fig. 1). It was fed a daily diet of domestic sheep pellets and had consistent access to water from a local reservoir throughout the year.

In this region of Cyprus, annual rainfall is usually around 800 mm and most precipitation occurs between November and March (IAEA/WMO (2024), DoM (2024); Fig. 1), while long drought periods are common from mid-May to mid-September. During dry periods, mouflons rely on spring water, though springs are scarce during summer. To mitigate water shortages, the Cypriot government established water reservoirs in 1997, which mouflons primarily use in autumn but abandon in winter when water stress decreases (Eliades et al., 2022).

The birthing period for both captive and free-living Cyprus mouflons is between late March and early May, with a peak in April (Hadjisterkotis, 1992). The onset of the lambing season tends to be earlier (10–17 days) amongst mouflons living in the FAL interface compared to those living in the forest. The lambing period is also longer (3–12 days) in the FAL interface (Hadjisterkotis, 1992), resulting in overlapping birthing peaks. Adding to this, a small sample set and the effect of interindividual variability in the timing of tooth growth on our time resolution (Balasse et al., 2024), make it unlikely that this difference will show in our results.
<caption>**Table 1**</caption>

List of the individuals used in this study with their respective sex, location, year of collection, and estimated age-at-death based on teeth (Payne, 1973, 1985; Deniz and Payne, 1982) or horns. FAL indicates forest-agricultural land; ND indicates Not Determined.

<table><thead><tr><th>Individual</th><th>Sex</th><th>Location</th><th>Year of collection</th><th>Age-at-death, dental row (in years)</th><th>Age-at-death, horns (in years)</th></tr></thead><tbody><tr><td colspan="6"><strong>Cyprus mouflons</strong></td></tr><tr><td>CYM36</td><td>Male</td><td>Inside the Paphos forest</td><td>1986</td><td>4-6</td><td>7-8</td></tr><tr><td>CYM77</td><td>Male</td><td>Inside the Paphos forest</td><td>1987</td><td>3-4</td><td>3-4</td></tr><tr><td>CYM80</td><td>Male</td><td>Inside the Paphos forest</td><td>1987</td><td>4-6</td><td>4</td></tr><tr><td>CYM82</td><td>Female</td><td>Inside the Paphos forest</td><td>1987</td><td>3-4</td><td>ND</td></tr><tr><td>CYM87</td><td>Female</td><td>FAL interface</td><td>1984</td><td>4-6</td><td>ND</td></tr><tr><td>CYM92</td><td>Female</td><td>Stavros tis Psokas enclosure</td><td>1987</td><td>2-3</td><td>ND</td></tr><tr><td>CYMN</td><td>Male</td><td>Unknown (Paphos forest?)</td><td>2003</td><td>3-4</td><td>4</td></tr><tr><td colspan="6"><strong>Southern France mouflons</strong></td></tr><tr><td>MD0646</td><td>Male</td><td>Alpes-de-Haute-Provence</td><td>ND</td><td>2-4</td><td>ND</td></tr><tr><td>MD1359</td><td>Male</td><td>Alpes-de-Haute-Provence</td><td>ND</td><td>3-5</td><td>ND</td></tr><tr><td>MD1502</td><td>Female</td><td>Alpes-de-Haute-Provence</td><td>ND</td><td>2-4</td><td>ND</td></tr><tr><td>MD1836</td><td>Male</td><td>Alpes-de-Haute-Provence</td><td>ND</td><td>3-5</td><td>~4</td></tr><tr><td>MD3031</td><td>Male</td><td>Alpes-de-Haute-Provence</td><td>ND</td><td>4-6</td><td>6-7</td></tr><tr><td>AR193</td><td>Male</td><td>Salon-de-Provence</td><td>ND</td><td>2-4</td><td>ND</td></tr><tr><td>AR232</td><td>Male</td><td>Saint-Paul-lez-Durance</td><td>ND</td><td>4-6</td><td>6-7</td></tr><tr><td>MD0102</td><td>Male</td><td>Salon-de-Provence</td><td>ND</td><td>4-5</td><td>5-6</td></tr><tr><td>MD0101</td><td>Male</td><td>Salon-de-Provence</td><td>ND</td><td>4-6</td><td>ND</td></tr><tr><td>MD1323</td><td>Male</td><td>Saint-Paul-lez-Durance</td><td>ND</td><td>4-6</td><td>ND</td></tr></tbody></table>

## 2.2. Mouflons from southern France

Ten mouflons from southern France were included in this study. The dental rows derive from hunting trophies, from which the bones were removed before taxidermy treatment. The dental rows were acquired to integrate the osteological research collections of the laboratory CEPAM (CNRS). They were all males, except one female, aged between two and six years old. They originated from two regions: (1) the Bouches-du-Rhône region, where three individuals were kept in a 400-ha enclosed area (140–220 m asl) at Roquerousse, Salon-de-Provence, and two other roamed the 800-ha Cadarache state forest near Saint-Paul-lez-Durance (250–430 m asl) as free-ranging animals, although occasionally provisioned with food and water; and (2) the Alpes-de-Haute-Provence, where all individuals were fully free-living. Regarding the habitat of this second group, due to the mountainous terrain, altitude can vary significantly over small areas, reaching up to 2600 m asl (Fig. 1 and Table 1). The observed birth period for the mouflons from southern France extends from the end of March to mid-June, with a peak in April–May (Catusse, 1996; Garel et al., 2005).

## 3. Rationale and methods

Oxygen in the mineral fraction of teeth precipitates in isotopic equilibrium with body water (Longinelli, 1984), which primarily originates from ingested water, including drinking water and plant water, thus indirectly from precipitation (D'Angela and Longinelli, 1990). Seasonal variations in $\delta^{18}\text{O}_{\text{prec}}$ values at the Draix GNIP station in the South of France follow the temperature pattern with the highest values from June to August and the lowest between December and February (Fig. 1) as described for most continental Europe (Rozanski et al., 1993; Dansgaard, 1964). Cyprus receives precipitation from air masses of different origins and trajectories, whose relative influence changes in the course of the year: Mediterranean westerly air masses prevail in summer, while northern European air masses prevail in winter. The lowest $\delta^{18}\text{O}_{\text{prec}}$ values are recorded in December and the highest in July (Christofi et al., 2020; IAEA/WMO, 2024, Prodromos station, 2018–2021, Fig. 1). Stable oxygen isotopes in surface water in the central part of the Troodos range follow similar seasonal trends: the Karkotis River surface water has higher $\delta^{18}\text{O}$ values in spring and summer and lower during winter, at a time when snowmelt can also contribute to the river water although to a varying degree according to the year. Weak fluctuations in $\delta^{18}\text{O}$ values (<0.5 ‰) are seasonally observed in spring water (Christofi et al., 2020). An additional important factor influencing body water balance and $\delta^{18}\text{O}$ is the animal's drinking behaviour and physiology (e.g., water excretion), which are partly shaped by environmental conditions such as air temperature and moisture (Chen et al., 2017).

These factors combine and result in temporal annual variations in body water $\delta^{18}\text{O}$, recorded in tooth bioapatite as a seasonal signal, capturing approximately one year's worth of data in the third molar of sheep (Balasse et al., 2003). This recording is influenced by the timing of enamel formation and the season of birth (Bryant et al., 1996; Fricke and O'Neil, 1996). The latter can be determined by comparing the specimens to modern reference sets for which the birth dates are known (Balasse et al., 2012).

Residual soft tissues were removed from the molars by gently brushing with warm water. After removing the cementum and dental calculus using a tungsten carbide burr, enamel was sampled from the buccal surface of the medial lobe. Sampling was conducted by drilling with a diamond burr from the apex to the enamel-root junction (ERJ) at regular intervals, perpendicular to the tooth's growth axis. Between 2.0 and 14.9 mg of enamel powder was collected per sample, with an average of 7.8 mg. No pre-treatment was applied. The samples from the Cyprus mouflons were analysed at the SSMIM (MNHN Paris, France). Approximately 600 µg of the collected enamel powder was placed into individual vessels to react under vacuum with phosphoric acid at 70 °C in a Kiel IV automated carbonate device interfaced to Delta V Advantage isotope ratio mass spectrometer (Thermo Scientific). The IAEA NBS-19 reference material was used to normalise $\delta^{18}\text{O}$ values for CYM77, CYM80, and CYM82, while a two-point calibration (NBS-18 and NBS-19) was used for CYM36, CYM87, and CYM92. The SSMIM in-house check standard is Marbre LM ($\delta^{18}\text{O} = -1.88$ ‰ normalised to NBS-18 and NBS-19). The analytical precision, estimated from the analysis of 5–8 Marbre LM per analytical session, was lower than 0.06 ‰.

The enamel samples from the mouflons from southern France were analysed at the Stable Isotope Platform of the European Institute for Marine Science (IUEM, Université de Bretagne Occidentale, France) on a Kiel IV Carbonate Device interfaced to a MAT-253 (Thermo Scientific). The standard deviation was calculated using the SSMIM in-house standard (Marbre LM). It was $\pm 0.03$ ‰ for this range of masses (20–60 µg). To formulate the values in the V-PDB scale, all samples were calibrated using two international carbonate standards: NBS-19 ($\delta^{18}\text{O} = -2.20$ ‰) and NBS-18 ($\delta^{18}\text{O} = -23.01$ ‰).

Measurements of the distance between the ERJ and various samples were taken using digital callipers. The $\delta^{18}\text{O}$ sequences were modelled with an equation derived from a cosine function (Balasse et al., 2012,
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Distribution area of the Cyprus mouflons in (1) the Paphos forest and in (2) the Stavros tis Psokas forest station; distribution of the southern France mouflons in the South of France in (3) Salon-de-Provence, (4) Saint-Paul-lez-Durance, and (5) Alpes-de-Haute-Provence. Location of Global Network of Isotopes in Precipitation (GNIP) stations in (6) Prodromos and (7) Draix. Data of the GNIP stations for precipitation monthly amount (purple), ambient temperature (green), and stable oxygen isotope ratios in rain (black) are from IAEA/WMO (2024). Air temperature and precipitation data from Prodromos station are from 2021, while δ¹⁸Oprec values represent an average from 2018 to 2021. Monthly precipitation amounts at Stavros tis Psokas forest station are from 1976 to 2013 (Department of Meteorology, 2024). No δ¹⁸Oprec data for this station were available. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

Supplementary Mat 1C) to determine the position (distance to the ERJ) of the maximum δ¹⁸O value (x₀). The four-parameter model (the period X, the amplitude A, the delay x₀, and the mean M) was applied, with the exception of one tooth (CYM92), for which the slope (parameter p) was added (Balasse et al., 2012). The cycle period (X), representing the distance over which the isotopic record spans one annual cycle, was used to normalise the distances (x₀/X), eliminating inter-individual variability in tooth size. Each modelled δ¹⁸O sequence produces an (x₀/X) ratio that allows direct comparison between individuals.

# 4. Results

## 4.1. Mouflons from Cyprus

The results, provided in detail in Supplementary 1A and 1B, are summarised in Table 2 and shown in Fig. 2. Amplitude of intra-tooth variation was observed between individuals, with CYM92, the captive individual, having the lowest amplitude (2.2 ‰), while all others ranged between 3.5 ‰ and 4.3 ‰. Additionally, CYM92 showed the lowest mid-range (δ¹⁸Omin + δ¹⁸Omax)/2 of δ¹⁸O value (−8.6 ‰), while free-living individuals had mid-range values between −5.7 ‰ and −2.7 ‰ (Fig. 2 and Table 2). Excluding CYM92, which did not share the same water source as the other individuals due to its captivity, no significant differences were found between male and female δ¹⁸O values (Wilcoxon test, W = 8, *p*-value = 0.13; test conducted on individual mid-range values). Individual CYMN, the most recently collected individual (in

<caption>**Table 2**</caption>

Number of samples per tooth, minimum and maximum δ¹⁸O values (‰) measured in each intra-tooth sequence, amplitude of intra-tooth variation, mid-range values (δ¹⁸Omin + δ¹⁸Omax)/2, and (x₀/X) ratios.

<table><thead><tr><th>Individuals</th><th>N</th><th>δ¹⁸Omin<br>(‰)</th><th>δ¹⁸Omax<br>(‰)</th><th>Amplitude<br>(‰)</th><th>Mid-<br>range<br>(‰)</th><th>x₀/<br>X</th></tr></thead><tbody><tr><td colspan="7"><strong>Cyprus mouflons</strong></td></tr><tr><td>CYM36</td><td>13</td><td>-6.8</td><td>-2.8</td><td>4.0</td><td>-4.8</td><td>0.80</td></tr><tr><td>CYM77</td><td>17</td><td>-6.7</td><td>-2.4</td><td>4.3</td><td>-4.5</td><td>0.86</td></tr><tr><td>CYM80</td><td>15</td><td>-5.7</td><td>-1.6</td><td>4.1</td><td>-3.7</td><td>0.90</td></tr><tr><td>CYM82</td><td>19</td><td>-7.5</td><td>-3.2</td><td>4.3</td><td>-5.7</td><td>0.88</td></tr><tr><td>CYM87</td><td>12</td><td>-7.5</td><td>-3.3</td><td>4.2</td><td>-5.2</td><td>0.95</td></tr><tr><td>CYM92</td><td>18</td><td>-10.1</td><td>-7.9</td><td>2.2</td><td>-8.6</td><td>0.93</td></tr><tr><td>CYMN</td><td>14</td><td>-4.6</td><td>-1.1</td><td>3.5</td><td>-2.7</td><td>0.73</td></tr><tr><td colspan="7"><strong>Southern France mouflons</strong></td></tr><tr><td colspan="7">Alpes-de-Haute-Provence</td></tr><tr><td>MD0646</td><td>18</td><td>-5.7</td><td>-2.9</td><td>2.8</td><td>-4.3</td><td>0.30</td></tr><tr><td>MD1359</td><td>18</td><td>-4.4</td><td>-2.1</td><td>2.3</td><td>-3.2</td><td></td></tr><tr><td>MD1502</td><td>17</td><td>-7.4</td><td>-2.9</td><td>4.5</td><td>-5.2</td><td></td></tr><tr><td>MD1836</td><td>18</td><td>-7.3</td><td>-3.8</td><td>3.4</td><td>-5.6</td><td>0.37</td></tr><tr><td>MD3031</td><td>14</td><td>-5.3</td><td>-2.3</td><td>2.9</td><td>-3.8</td><td></td></tr><tr><td colspan="7">Bouches-du-Rhône</td></tr><tr><td>AR193</td><td>20</td><td>-3.9</td><td>-1.2</td><td>2.6</td><td>-2.6</td><td></td></tr><tr><td>AR232</td><td>18</td><td>-5.5</td><td>-1.5</td><td>4.0</td><td>-3.5</td><td>0.21</td></tr><tr><td>MD0102</td><td>21</td><td>-4.8</td><td>-1.4</td><td>3.4</td><td>-3.1</td><td></td></tr><tr><td>MD0101</td><td>17</td><td>-4.1</td><td>0.4</td><td>4.5</td><td>-1.8</td><td>0.22</td></tr><tr><td>MD1323</td><td>18</td><td>-4.3</td><td>-0.6</td><td>3.7</td><td>-2.4</td><td>0.35</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Results from the sequential stable oxygen isotope analysis in third molar (M/3) enamel bioapatite of the Cyprus and southern France mouflons. F: female; M: male; FAL: forest-agricultural land.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

2003), exhibited the highest mid-range δ¹⁸O values (−2.7 ‰).

All individuals showed a sinusoidal pattern consistent with the seasonal cycle, considerably attenuated in the captive mouflon (CYM92) compared to the free-living mouflons (Fig. 2). Results from the modeling of the δ¹⁸O sequences are reported extensively in Supplementary Mat 1C. The (x₀/X) ratios range from 0.73 to 0.95 (Table 2 and Fig. 3), which fall within an interval defined by other current reference datasets

corresponding to autumn. Furthermore, these ratios indicate a birth period of approximately 2.6 months. No difference was observed between males and females in their (x₀/X) ratios.

## 4.2. Mouflons from southern France

The mouflons from southern France exhibited an amplitude of intra-tooth variation in δ¹⁸O values ranging from 2.3 ‰ to 4.5 ‰. The sole female (MD1502) displayed a mid-range value of −5.2 ‰, while the remaining individuals (n = 9) had mid-range values ranging from −5.6 ‰ to −1.8 ‰ (Table 2). Individuals from Alpes-de-Haute-Provence had significantly lower δ¹⁸O values compared to those from Bouches-du-Rhône (Wilcoxon test, W = 24, *p*-value = 0.016; test conducted on individual mid-range values) (Fig. 2).

The (x₀/X) ratios ranged from 0.21 to 0.37 (Table 2), indicating a birth period of approximately 1.9 months (Fig. 3). Five teeth amongst the southern France mouflons (MD0102, MD1359, MD1502, MD3031, and AR193) could not be modelled because of a non-sinusoidal signal or the absence of a clear maximum. Visual comparisons suggest they do not differ greatly from the other individuals (Fig. 2).

# 5. Discussion

## 5.1. Environmental and behavioural factors driving interindividual variability in δ¹⁸O values

The dataset from Cyprus exhibits inter-individual variability, likely linked to differences in the mouflons living conditions. First, CYM92, kept in an enclosure, shows the lowest δ¹⁸O values compared to the other individuals, as well as a highly attenuated seasonal variation. In the free-living individuals, the use of different water sources through the year (including streams, springs, or plants water), as well as seasonal variations in the δ¹⁸O values of streams and plants would lead to a marked amplitude of seasonal variation. In comparison, the water consumed by CYM92 comes from a reservoir, resulting in water mixing and a dampened seasonal signal. The difference in annual (mid-range)

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Distribution of mouflon births in Cyprus (CYM) and southern France (MD or AR), as reflected by the position of the maximum δ¹⁸O value in tooth crown (x₀) normalised to the period of the cycle (X). The birth season is compared with preexisting modern reference sheep sets (Balasse, et al., 2005, 2012, 2017, 2020, 2024; Blaise and Balasse, 2011; Tornero et al., 2018; Fabre et al., 2023).</figcaption></figure>
values may also be explained by the consumption of reservoir water.

The free-living Cyprus mouflons show a striking similarity in the range of variation of their enamel $\delta^{18}$O values and in intra-tooth amplitude of variation (4.0 ‰to 4.3 ‰), except in CYMN (3.5 ‰). The establishment of water troughs since 1997 to mitigate water stress in mouflons within the forest may account for the lower amplitude observed in CYMN. These water troughs are sourced from a larger reservoir (holding between 700 and 1000 L; Eliades et al., 2022). A reservoir effect could attenuate the record of seasonality in animals drinking from them, although to a lower extent than year-round drinking from a reservoir, as the troughs are used primarily in summer and autumn, before being deserted by the mouflons from December onwards (Eliades et al., 2022). The individual CYMN, collected in 2003, would likely have undergone third molar mineralisation around 2001, given its age at death, whereas the other individuals would have all experienced dental mineralisation at least before 1985. Moreover, CYMN stands out from the other individuals by its higher mean annual values (–2.7 ‰ versus –5.7 ‰ to –3.7 ‰ in others). This difference may be explained by an increase in annual temperature over time. CYMN was collected more than 15 years after the majority of the group and a temperature rise of more than 1 °C per decade between 1975 and 2021 has been recorded in north Cyprus (Bey et al., 2024).

In southern France, warmer winter and summer temperatures are recorded in Bouches-du-Rhône under a typical Mediterranean climate, compared to a more continental climate at higher altitudes in the mountainous region of the Alpes-de-Haute-Provence (Joly et al., 2010). This could explain that individuals from Bouches-du-Rhône exhibit higher annual $\delta^{18}$O values compared to those from Alpes-de-Haute-Provence.

## 5.2. Length of the birthing season and difference between sexes

Although obtained from a limited number of individuals, the results are in agreement with a constrained period of birth for the Cyprus and southern France mouflons, which aligns with observations made on these mouflon populations (Hadjisterkotis and Bider, 1993; Catusse, 1996). Furthermore, no differences in the ($x_0$/X) ratios were observed between sexes. The reference materials used until now to determine the season of birth have consisted exclusively of females. Although these references have been applied to archaeological sheep regardless of sex, the question could arise as to whether a difference in dental development between males and females could limit the applicability of female references. No significant difference in the timing of second and third molar eruption has been noted between sexes in Shetland sheep (Worley et al., 2016). Based on our results, a similar conclusion is supported for the third molar of mouflons.

## 5.3. Agreement between the southern France mouflon and existing European sheep references

The mouflons from southern France align with existing reference data on Western European sheep breeds giving birth in winter/spring (Shetland cross, North-Ronaldsay, Préalpes du Sud, Ouessant x Lande de Bretagne, Arles Merino, and Lacaze). A significant part of genetic diversity in modern European sheep breeds can be traced back to a wave of diffusion subsequent to the first wave of neolithisation in Europe (Chessa et al., 2009), likely occurred during the Late Neolithic. Due to their confined geographical positions at the periphery of north-western Europe, the Corsican mouflon, as well as the Orkney and Soay sheep, were not affected by this later migration (Chessa et al., 2009). Neither the specificity of their long-term history, nor the hardiness of the Orkney sheep breeds seem to have induced a significant difference in the timing of molar eruption in comparison to more improved breeds, although this is based on a limited number of individuals and as long as our resolution allows to say (Balasse et al., 2024). Similarly, the results of the present study do not point to a significant difference in the timing of the third

molar development, between the southern France mouflon and current sheep breeds from western Europe.

The question could still arise, whether the mouflon introduced to southern France from Corsica is a good representative of the Corsican mouflon in this regard. Indeed, selective breeding efforts to enhance hunting trophies, a key reason for their introduction to the continent (Bon et al., 1991; Luddeni, 1993), may have further influenced their genetic makeup. Additionally, hybridisation between domestic sheep and mouflons is well-documented (Pfeffer and Genest, 1969). Sheep have contributed to the genetic heritage of mouflons in continental Europe (Schröder et al., 2016), whereas these introgressions have remained limited in the mouflon's population of Corsica (Barbato et al., 2017).

## 5.4. Difference in the recording of the birth season between the Cyprus and southern France mouflons

Cyprus and southern France mouflons share a similar birth season, spanning from March to May/June, with a peak in April/May (Hadjisterkotis, 1992; Catusse, 1996). Therefore, we would expect the $\delta^{18}$O sequence recorded in the M/3 to be similar between both populations. Instead, the ($x_0$/X) values from both groups exhibit diametrically opposite positions in the annual cycle, with those obtained from the Cyprus mouflons coinciding with autumn births observed in other reference sheep breeds (Fig. 3). The seasonal isotopic sequence recorded in a given tooth is determined by three key factors: (1) the seasonal variation of the input signal, (2) the birth season, and (3) the dental growth calendar.

(1) Amongst the parameters potentially influencing the pattern of seasonal variations in the $\delta^{18}$O values of the input signal (ingested water), we investigated (1a) the pattern of seasonal variations in $\delta^{18}$Oprec.; (1b) the seasonal contribution of melting snow to river waters, and (1c) the effect of altitudinal mobility in the Cyprus mouflon. (1a) The $\delta^{18}$Oprec. values in the Troodos region follow the same seasonal pattern as observed in southern France (Fig. 1), with lower values during winter (December to February) and higher values in summer (June to August). Although no $\delta^{18}$Oprec. and temperature data were available at the Stavros tis Psokas station, precipitation amounts follow the same pattern of seasonal variation as in Prodromos (Fig. 1), and the same is expected for temperature. Therefore, we also expect the $\delta^{18}$Oprec. values to follow a similar pattern of seasonal variation. (1b) In the mountainous context of the Troodos, snow meltwater may seasonally contribute to the isotopic signal of river waters but only over a brief period of the year, during the spring (Christofi et al., 2020). Moreover, the Paphos forest remains free of snow for the majority of the year. (1c) Finally, altitudinal mobility, combined with the altitude effect in $\delta^{18}$Oprec. values—as described by Rozanski et al. (1993), whereby lower temperatures at higher elevations lead to a decrease in $\delta^{18}$Oprec. values—has been pointed out as potentially influencing the recording of the seasonal cycle in intra-tooth $\delta^{18}$O sequences (Fisher and Valentine, 2013; Tornero et al., 2016). Cyprus mouflons undertake altitudinal migrations as part of their seasonal movements, such as to reach their lambing grounds. Some individuals have been observed at elevations exceeding 1200 m in late spring and summer (Hadjisterkotis, 1992). Mobility over an altitudinal gradient of over 560 m for the mouflons inside the forest and assuming a decrease of approximately 0.2 ‰ per 100 m of elevation in $\delta^{18}$Oprec values (Christofi et al., 2020), would result in a lowering in the summer $\delta^{18}$O signal of about 1.1 ‰. Considering that the amplitude of annual variation in $\delta^{18}$Oprec values is around 5 ‰ (Fig. 1), the altitudinal effect alone cannot account for a reversal in the input signal. Furthermore, the captive mouflon (CYM92)—without the possibility of altitudinal movements—exhibits oxygen isotope sequences in
phase with those of free-living individuals, with their maximum and minimum values occurring at the same positions on the tooth. This suggests that there is no reversal of winter and summer values between mobile free-living and sedentary individuals. This result supports the conclusions of *Knockaert et al. (2018)* and *Tornero et al. (2018)*, who, based on $\delta^{18}O$ sequences measured in modern sheep teeth, found no difference in the recording of the seasonal cycle between sedentary and trans-humant sheep in the Pyrenees.

(2) The birth season is known to be the same in the Cyprian and southern France populations as well as between captive and free-living Cyprian individuals. Moreover, in a harsh environment such as that of Cyprus, the lambing season is very short in order to ensure better survival of the offspring. Between 1955 and 1969, the latest recorded births occurred in June, although such cases remained rare (Hadjisterkotis and Bider, 1993). Therefore, it is unlikely that our sample included only off-season births.

(3) This leaves the dental growth calendar as the primary remaining factor to explain the observed differences. Given that both populations originate from the feralisation of different populations of domestic sheep, which occurred at different time periods, a divergence in tooth development may have emerged due to different origins and evolutionary history.

### 5.5. Evolutionary pathways of southern France and Cyprus mouflons

Although the Cyprus and Corsican mouflons originate from the same ancestral mouflons domesticated in Eastern Anatolia (Kaptan et al., 2024), some morphological differences suggest that they evolved from different lineages. The Cyprus mouflon has a characteristic conformation of its horn cores showing a flattened basal section, similar to the early domestic sheep found at Khirokitia (phases III to I) in Cyprus and in the Jordan valley (Munhatta, Ducos, 1968), from which they possibly derive. By contrast, in this character they do not resemble the early Neolithic Anatolian sheep first introduced to Cyprus at Shillourokambos, nor any of the later Neolithic sheep from the Central and Western Mediterranean area and therefore also diverge from the Corsican mouflon (Vigne, 2011; Vigne et al., 2017). These morphologically different sheep from the Jordan valley and the Southern Levant may have evolved into a dead-end lineage. These distinctions are supported by genetic data, which show a clear genetic separation between Cyprus and Corsican mouflons (Guerrini et al., 2015; Sanna et al., 2015; Garel et al., 2022; Her et al., 2022). However, these different evolutionary histories do not necessarily lead to a difference in dental growth timing.

Nearly 2500 years passed between the introduction of sheep to Cyprus and to Corsica. Thus, even though the feralisation occurred during the Khirokitian period (7th millennium BCE), that is to say 1500 years after this first introduction (Vigne, 2011), the insular history of the Cyprus mouflon should be significantly longer than that of the Corsican mouflon, which feralised between the late 6th and the 3rd millennium BCE (Vigne, 1988). The effect of insularity on tooth development has been investigated in taxa such as black-tailed deer, where a delay in tooth eruption was observed in insular compared to mainland individuals (Long et al., 2019). However, the study by Geiger et al. (2020) on Soay sheep from St Kilda Island (with a considerably shorter history of insularity than the Cyprus mouflon) did not confirm a significant delay in cheek tooth formation previously reported for this population (Clutton-Brock et al., 1990). They show instead that Soay sheep molar eruption timing falls within the range of other domestic breeds (Karakul and Swiss sheep), although it tends to be on the later side. The authors also conclude that phylogenetics rather than insularity may explain this delay. However, considering that the history of insularity is much longer in Cyprus mouflons than in Soay sheep, we cannot dismiss the possibility of a phylogenetic drift due either to a founder effect and/or to a long isolation leading to a difference in dental growth timing.

Whether insular colonisation results from natural immigration or

anthropogenic introduction, its success depends on two factors. First, one or several founding events, which entail a reduction in the genetic diversity of the resulting insular lineages compared to that of the source population; this selection of a reduced genome may confer genetic traits to the insular lineage that are rarely or not at all expressed in the source population. Second, a phylogenetic drift resulting from changes in selective pressures experienced by the insular lineage in its new environment. In wild populations, this includes the release from predation and interspecific competition (due to the typically low biodiversity of islands), the intensification of intraspecific competition (linked to higher population densities on islands), and access to ecological niches that were unavailable on the mainland because they were occupied by species with greater fitness. In domestic populations, these evolutionary shifts are likely less pronounced, but not absent, since the husbandry systems themselves are adapted to the lack of predators and reduced interspecific competition. Under such conditions, it is conceivable that certain traits rare on the mainland (e.g., flattened basal section of horn cores observed at Munhatta—or possibly a different dental growth schedule) might become predominant in insular lineages such as those of Cyprus.

### 5.6. Implications of reference selection for reconstructing birth season in the past

The dispersal of early domestic lineages across Europe, Africa, and Asia into a wide variety of regions, climates, and landscapes led to the emergence of numerous lineages that underwent phylogenetic divergence, resulting in a broad range of phenotypic traits. This divergence may include differences in dental growth rates, as suggested by the case of the Cyprus mouflon. Although most European lineages appear to exhibit a consistent pattern of dental growth, it is reasonable to expect that some divergent lineages may display different growth patterns. This possibility warrants careful consideration.

In particular, questions remain concerning Neolithic sheep from Cyprus. The birth season of sheep from the Late PPNB in Cyprus, at Ais Yorkis (AY) and Shillourokambos (SHI), has previously been investigated (Hadjikoumis et al., 2019). Using the only modern reference available at the time—a third molar from an ewe born in winter (KMZ; Balasse et al., 2017)—Hadjikoumis et al. (2019) concluded that part of the births at AY and SHI occurred in early winter, while another group was possibly born in late summer or autumn, similarly to traditional herding practices documented through ethnoarchaeology. The use of a broader set of modern domestic sheep reference specimens now available confirms births in autumn and winter (and even early spring) at AY, and, predominantly in winter, and in summer-autumn at SHI (Fig. 4A).

In contrast, using the Cyprus mouflon as a reference would shift the birth season to spring, summer, and autumn at AY, and to winter, spring, and predominantly summer at SHI (Fig. 4B). A major difference, therefore, when using the Cyprus mouflon reference, would be the emergence of a predominantly summer birthing group across both sites—an unlikely scenario under a Mediterranean climate. As we have no information about when the distinct dental growth pattern observed in the Cyprian lineage first emerged and it is unclear whether this unique dental growth pattern results from a founder effect or from a later phylogenetic drift, further investigation into the use of the Cyprus mouflon is warranted.

## 6. Conclusion

Estimating birth season using sequential oxygen analysis of two mouflon populations—from Cyprus and southern France—yielded markedly different results, despite both populations being known to give birth in spring. The southern France mouflons produced results comparable to existing sheep reference datasets in terms of the position of their $\delta^{18}O$ maximum in the tooth ($x_0$/X), whereas the Cyprus mouflons were radically shifted. Given that environmental factors do not explain
<figure><img src="image_7.png" /><figcaption>Fig. 4. Distribution of late PPNB sheep births in Ais-Yorkis and Shillourokambos (Cyprus) from Hadjikoumis et al. (2019), as reflected by the position of the maximum δ¹⁸O value in tooth crown (x₀) normalised to the period of the cycle (X). The birth season is compared with (A) preexisting modern reference sheep sets (Balasse, et al., 2005, 2012, 2017, 2020, 2024; Blaise and Balasse, 2011; Tornero et al., 2018; Fabre et al., 2023) and (B) Cyprus mouflon reference.</figcaption></figure>

this discrepancy between the two mouflon populations, it may be linked
to differences in dental growth schedules. However, the underlying
cause of this divergence remains unclear. It may reflect a distinct genetic
lineage, reduced genetic diversity in Cyprus mouflons due to one or
more founding events—potentially conferring traits rarely or not
expressed in the source population—and/or the long-term isolation that
led to phylogenetic drift. Further investigation is needed to clarify these
possibilities. Based on these results, we conclude that Mediterranean
mouflons from southern France are as reliable as current sheep reference
datasets for determining past birth seasonality. As such, they can be
incorporated into established reference sets. In contrast, the applica-
bility of the Cyprus mouflon as a reference for reconstructing birth
seasonality should be further assessed, both within the context of Cyprus
and more broadly across the Near East. Furthermore, no differences in
the (x₀/X) ratios were observed between sexes in either mouflon pop-
ulations, confirming that the exclusive use of females so far in the
established reference sets remains valid. Despite the applicability of the
reference sets being appropriate, it is essential to continue expanding
those sets to explore variability amongst different domestic sheep
breeds, mouflon subspecies (in particular the Corsican mouflon), and
different teeth (M/₂ and M/₃), as well as to further test the applicability
of these references across populations differing in space and time.

CRediT authorship contribution statement

**Eléa Gutierrez:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis, Data curation. **Angelos Hadjikoumis:** Writing – review & editing, Resources, Funding acquisition, Formal analysis, Conceptualization. **Eleftherios Hadjisterkotis:** Writing – review & editing, Resources. **Jean-Denis Vigne:** Writing – review & editing. **Fabien Dewilde:** Formal analysis. **Denis Fiorillo:** Formal analysis. **Lionel Gourichon:** Writing – review & editing, Resources, Funding acquisition, Conceptualization. **Marie Balasse:** Writing – review & editing, Writing – original draft, Resources, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declarations

The authors have no competing interests to declare that are relevant
to the content of this article.

Reproductibility Results

The Associate Editor for Reproductibility could download all mate-
rials and reproduce all results and figures presented by the authors.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The Agence Nationale de la Recherche (ANR), project AUTUMN LAMBS (ANR-22-CE27-0011-01) funded a post-doctoral fellowship to EG and the stable isotope analyses of the mouflons from southern France. AH has received support from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Actions [Program/Call: H2020-MSCA-IF-2020, Grant Agreement: 101029092-SACHROFICS]. The mouflon mandibles from southern France were either generously donated or purchased by the CEPAM from La Maison de la Taxidermie (Salernes, Var), operated by the Fanucchi family, master craftsmen in taxidermy. Special thanks to their works and donations.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jas.2025.106381.

Data availability

The data that support the findings of this study are included as part of the paper and supplementary materials.

The code generated during this study is available 10.5281/zenodo.16813755 as the date of the publication.

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