Invasive exotic species have caused significant problems, and the effects of extreme precipitation and drought, which might occur more frequently under the global climate change scenarios, on interspecific relationship between invasive and native species remain unclear.Rhus typhina and native Cotinus coggygria. Rhus typhina had lower height but bigger crown area than C. coggygria in the monoculture treatment. Rhus typhina had higher height, bigger crown area and total biomass than C. coggygria in the mixture treatment. Drought decreased the growth parameters, total chlorophyll concentration, and leaf biomass, but did not change gas exchange and other biomass parameters in R. typhina. The growth parameters, leaf area index, biomass parameters, total chlorophyll concentration, and net photosynthetic rate of C. coggygria decreased under drought conditions. The log response ratio (lnRR), calculated as ln , of R. typhina was lower than that of C. coggygria. The lnRR of R. typhina and C. coggygria decreased and increased with increase in soil water content, respectively.We conducted a greenhouse experiment with three soil water levels  and two cultivation treatments  to investigate soil water content effects on the relationship between invasive Rhus typhina has greater capacity to relatively stable growth to the drought condition than C. coggygria and has strong competition advantages in the mixture with C. coggygria, especially in the drought condition. Our study will help understand the causes of invasiveness and wide distribution of R. typhina under various moisture conditions and predict its expansion under climate change scenarios. The global climate change has resulted in an increasing trend in extreme precipitation in some areas, and severe drought is predicted to occur more frequently in the future in several regions of the world . Water lIdentifying the determinants of the invasiveness of alien plant species is essential in invasion ecology, and it has considerable implications . It has The competition for resources is a crucial process that influences plant competitive interactions and invasiveness , 9, 20. Lantana camara decreased under drought condition; thus, limiting its invasion into arid and sub-arid environments [Water plays an important role in the growth and physiology of plants and significantly affects the interaction between native and invasive species . Drier sronments . Furtherronments  or waterronments . ExperimRhus typhina) and a confamiliar native species (Cotinus coggygria). Rhus typhina, a large shrub or small tree native to eastern North America [Cotinus coggygria is widely distributed in the warm-temperate zone of China by local adaptation [R. typhina and C. coggygria exist in the temperate deciduous forest in the eastern Asia and have strong competition with each other, especially at the stage and have similar ecological niche at the stage of seedlings and mature trees. Besides, the two species are widely used for reforestation in North China [To examine the effects of invasive species on native species in response to various water conditions, we conducted a greenhouse experiment with an invasive species  and W2 (50–60% of field capacity) conditions, but lower than those of C. coggygria under W3 (70–80% of field capacity) condition. The root biomass of R. typhina was higher than that of C. coggygria in all the water treatments. In the mixture pots, the total biomass, leaf biomass, stem biomass, and root biomass of R. typhina were higher than those of C. coggygria in all the water treatments.In the monoculture pots, the total biomass and leaf biomass of R. typhina, neither cultivation nor interaction between cultivation and water affected any of the biomass and allocation parameters and water affected only the leaf biomass  to reach the upper space with higher maximum net photosynthetic rate (A) than that of R. typhina. Rhus typhina seedlings tend to grow horizontally (higher crown area than that of C. coggygria) to capture more light and deeply to obtain more water and nutrient (higher root biomass and RBR than those of C. coggygria). Rhus typhina maintained larger crown area, leaf biomass, and root biomass than those of C. coggygria. The upward growth of C. coggygria seedlings was strongly inhibited (indicated by distinctly lower shoot height than that of C. coggygria in monoculture) and its A was decreased most likely due to the shade environment caused by the exceedingly larger canopy of R. typhina seedlings in the upper layer in the mixture. Eventually the total biomass of C. coggygria seedlings was extremely lower than that of R. typhina seedlings in the mixture pots. Therefore, the invasive R. typhina and the native C. coggygria displayed differences in most crucial functional parameters related to growth when they were cultivated separately or together, indicating that our results support the phenotypic divergence hypothesis [This conferred  species , 36, 47 pothesis  rather tpothesis , 16.R. typhina possesses three advantages over the native C. coggygria in the mixed culture. First, with the stronger photosynthetic capacity than C. coggygria, R. typhina was able to achieve a higher growth rate and larger amount of carbohydrate production. Secondly, in the mixture pots, higher shoot height, crown area, RGR-H, and leaf biomass enabled R. typhina to exhibit dominance earlier by obtaining more light resource and shading the native C. coggygria. This asymmetric competition caused by the higher aboveground productivity of R. typhina over C. coggygria results in positive-feedback [R. typhina, leading to even higher competitive advantages of R. typhina. Finally, the higher root biomass and WUE of R. typhina than C. coggygria conferred R. typhina the ability to obtain water and nutrients, and utilize water more efficiently.Invasive alien feedback  to R. tyRhus typhina maintained its dominance under drought condition despite the fact that the competitive ability of R. typhina over C. coggygria decreased and concomitantly the competitive ability of C. coggygria over R. typhina increased to some extent. This observation generally supports the fluctuating resource availability theory [R. typhina over C. coggygria under drought conditions was probably related to the growth inhibition of R. typhina  under drought conditions. However, the shoot height, crown area, RGR-H, and leaf biomass of R. typhina were still substantially higher than those of C. coggygria under drought condition, consistently conferring R. typhina an aboveground competitive advantage. Therefore, the asymmetric competition of R. typhina over C. coggygria was alleviated to some extent, because the light-capturing ability of R. typhina decreased to some extent, although this ability was still conspicuously stronger than that of C. coggygria. Rhus typhina has been shown to outperform native Vitex negundo under variable levels of water supply frequency but with a constant level of total supplied water [y theory , which hed water .C. coggygria in the monoculture pots, whereas, those in the mixture pots was unaffected by drought, suggesting that the mixed culture alleviated the negative effects of drought on C. coggygria. This might be because R. typhina has a larger crown area than that of C. coggygria, which would intercept more light, reduce the temperature of the topsoil and evaporation of soil water, and eventually alleviate the negative effects of drought on C. coggygria when it is grown close to R. typhina. It has been reported that shade can alleviate the negative effects of drought on Acer buergerianum [C. coggygria under drought conditions provided further evidence to this view, because decreased chlorophyll a to chlorophyll b ratio enables the plant to obtain more light in shade [C. coggygria over R. typhina under drought condition was likely caused by weaker drought effects on C. coggygria in the mixture pots compared with that in the monoculture pots. In mixture pots, the root biomass and RBR of both species remained unchanged with the increasing drought stress, and R. typhina had higher root biomass, RBR, and WUE than those of C. coggygria, indicating that the belowground advantage of R. typhina to obtain water and nutrients, and utilize water more effectively than C. coggygria was independent of soil water content.Drought decreased all the biomass parameters of gerianum , which iin shade . TherefoC. coggygria simultaneously decreased by both drought and the competition with R. typhina, indicating the stomatal closure of C. coggygria in response to drought and competition. The responses of C. coggygria in the present study support the carbon-starvation hypothesis [Acer platanoides and Fagus sylvatica were found to decrease simultaneously under drought plus competition conditions [C. coggygria in the present study. In contrast to C. coggygria, R. typhina maintained constant A, and Gs in the mixture pots, regardless of the water conditions, indicating that R. typhina might have adopted another mechanism to cope with drought. Some species maintain positive carbon gain under drought conditions, allowing the midday leaf water potential to decline [R. typhina continued to accumulate carbohydrate under drought condition, maintaining the biomass constant compared with that under well-watered condition [The A and Gs  of pothesis , 58, in nditions , which i decline , 58 by kondition , and theR. typhina and the native C. coggygria displayed differences in many growth-related parameters when they were cultivated either separately or together. This is consistent with the phenotypic divergence hypothesis [Rhus typhina maintained its absolute dominance against C. coggygria in both well-watered and drought environments by possessing higher photosynthetic capacity, larger crown area, taller shoot height, and higher RGR-H, leaf and root biomass, and WUE, outcompeting C. coggygria vertically and horizontally. The growth of C. coggygria was strongly limited by the competition from R. typhina; however, this competition alleviated the negative effects of drought on C. coggygria mainly by providing shade environment. Drought alleviated the asymmetric competition of R. typhina over C. coggygria to some extent because drought had stronger negative effects on than on C. coggygria in the mixture pots and R. typhina is better capable of exploiting excess water resource. Our observation provides further evidence for the fluctuating resource availability theory [C. coggygria suffered from carbon starvation, whereas, R. typhina retained the normal carbohydrate synthesis, contributing to the dominance of R. typhina. However, both the species would experience the risk of hydraulic failure under severe drought conditions. Our observation confirmed the robustness of the comparative trait differences between invasive and non-invasive species across environmental gradients [The invasive pothesis  holding y theory . Under dradients .Rhus typhina grows slowly at the seedling stage, but grows fast once established [R. typhina probably will continue its dominance when grown adjacent to C. coggygria in a long term. As the glasshouse study cannot simulate the complex natural environments, our observation should not be extrapolated arbitrarily to field conditions. Given the widely acknowledged invasiveness of R. typhina and the extensive use of the two species in reforestation and urban greening, field studies with closer attention on the interaction between R. typhina and C. coggygria in the future global change scenarios is necessary to better understand the invasion of R. typhina and to take appropriate precautionary and management measures. Our study contributes to a better understanding of invasive mechanisms of alien tree species such as R. typhina under various moisture conditions and predicts its future expansion under global climate change scenarios.ablished . TherefoThe experiment was conducted in the greenhouse of Fanggan Research Station of Shandong University , which is located in the central mountainous region of Shandong Province, China. The region has a typical temperate monsoon climate, with an average annual precipitation of approximately 600–800 mm, most of which occurs from June to August (60–70%), which was collected from National Climate Center of China in 2011. The predominant vegetation in this area is mixed forests of the warm temperature zone. The soil is a yellow cinnamon soil with limestone as the parent material [The seeds of both species were collected from the Loahu hill  near the research station during the October of 2011.The seeds were stored at 0–4 °C during winter in the fridge . During the late April of 2012, the seeds were soaked in distilled water for 24 h before they were allowed to germinate in a growth chamber . A voucher specimen of this material has been deposited in the herbarium of Shandong University. Healthy and uniformly germinated seedlings were transplanted into plastic pots (320 mm height × 290 mm diameter) with one or two seedling(s) per pot. Each pot was filled with 6 kg of loam and 2 kg of sand, which was carefully passed through a 2-mm sieve to remove debris, and thoroughly mixed.R. typhina or C. coggygria) and mixture pots that contained one seedling of both the species.Three water treatments, 30–40% (W1), 50–60% (W2), and 70–80% (W3) of field capacity (FC), were applied to three plant cultivation treatments. The plant cultivation treatments included monoculture pots that contained one seedling of either species  throughout the experiment. The pots received compensatory irrigation after weighing daily at 18:00 h to maintain constant soil moisture level. Insects and weeds were controlled manually. Each treatment contained eight pots. The study was carried out from July 6 to September 5 of 2012, and it lasted for 62 days.−2 s−1 (PAR) to ensure that all seedlings were light saturated. The airflow through the leaf chamber was set to 400 µmol−1 s −1, chamber temperature to ambient temperature, and CO2 concentration to 400 µmol mol−1 (Ca). The net photosynthetic rate (A), stomata conductance (Gs), transpiration rate (E), and intercellular CO2 concentration (Ci) were recorded. The water use efficiency (WUE) was calculated as A/E. Three healthy and fully expanded leaves on the upper shoot of three seedlings were used to measure the parameter in each treatment.The gas exchange parameters were measured on August 20 and 21, 2012, using a portable leaf gas exchange system . Light was supplied by a red-blue light-emitting diode set at an irradiance of 2000 µmol mThe concentration of chlorophyll was determined on August 24 and 25, 2012, according to a previous study , using aThe shoot height and crown area were measured at the end of the experiment. The crown area was calculated as: crown area = π a b . Five pots were selected to measure the shoot height and crown area. The relative growth rate of shoot height (RGR-H) was calculated as: RGR-H = (InH2 − InH1)/Δt; where, H2 and H1 are the shoot height values measured at the end and the beginning of the treatment, and Δt is the time duration.Five pots per treatment were harvested thereafter, washed thoroughly with tap water, and divided into stems, leaves, and roots. The leaf area of all the plants was measured using a scanner  and calculated using an image analyzer . The dry weight of various plant fractions was determined after drying in an oven for 48 h at 100 °C. The specific area , leaf area index , root biomass ratio , stem biomass ratio , and leaf biomass ratio  were calculated thereafter.To evaluate the major effects and interactions of water and cultivation treatments, the two-way analysis of variance (ANOVA) was performed using plant performance and functional traits of each species. The data were log-transformed when necessary to meet the normality and homogeneity of variance assumptions of ANOVA. However, for clarity the untransformed data are presented. The two-way ANOVA and Duncan’s multiple range test (DMRT) at 95% confidence level for all plant variables were conducted.The dominance of the target species in mixed cultures was estimated using the log response ratio (lnRR) , 62, 63,All the statistical analyses were performed using the IBM SPSS 21.0 software package . Bar charts were drawn using Origin 9.0 software .


Triticum aestivum L.), which not only significantly reduces grain yield, but also affects end-use quality. Breeding wheat cultivars with high FHB resistance is the most effective way to control the disease. The Chinese wheat cultivar Jingzhou 66 (JZ66) shows moderately high FHB resistance; however, the genetic basis of its resistance is unknown. A doubled haploid (DH) population consisting 209 lines was developed from a cross of JZ66 and Aikang 58 (AK58), a FHB susceptible wheat cultivar, to identify quantitative trait loci (QTL) that contribute to the FHB resistance. Five field experiments were established across two consecutive crop seasons (2018 and 2019) to evaluate the DH lines and parents for FHB response. The parents and DH population were genotyped with the wheat 55K single-nucleotide polymorphism (SNP) assay. Six QTLs associated with FHB resistance in JZ66 were mapped on chromosome 2DS, 3AS, 3AL, 3DL, 4DS, and 5DL, respectively. Four of the QTL  were detected in at least two environments, and the QTL on 3AL and 5DL might be new. The QTL with major effects, QFhb.hbaas-2DS and QFhb.hbaas-4DS, explained up to 36.2% and 17.6% of the phenotypic variance, and were co-localized with the plant semi-dwarfing loci Rht8 and Rht-D1. The dwarfing Rht8 allele significantly increased spike compactness (SC) and FHB susceptibility causing a larger effect on FHB response than Rht-D1 observed in this study. PCR–based SNP markers for QFhb.hbaas-2DS, QFhb.hbaas-3AL, QFhb.hbaas-4DS, and QFhb.hbaas-5DL, were developed to facilitate their use in breeding for FHB resistance by marker-assisted selection.Fusarium head blight (FHB) is a destructive disease of wheat ( Fusarium graminearum, is one of the most destructive diseases of bread wheat, especially in warm and humid regions [Fusarium mycotoxin contamination renders grain unsuitable for human and animal consumption [Fusarium head blight (FHB), mainly caused by  regions . In epid regions . Moreovesumption . The occsumption ,5. The dBreeding FHB-resistant cultivars depends on the use of resistant germplasm. Numerous elite wheat genotypes with high or moderate resistance have been identified worldwide, including Sumai 3 and Wangshuibai in China ,7, ChokwFhb1 on 3BS is the most reliable and widely used gene in FHB resistance breeding. Introgression of Fhb1 into locally adapted wheat cultivars in many breeding programs has demonstrated significant improvements in FHB resistance [Fhb1 was cloned as His, encoding a histidine-rich calcium-binding protein [Fhb1 largely facilitates the application of this gene [Fhb1, is an effective way to enhance FHB resistance in wheat [Fhb1), 3B-2, 4B (Fhb4), 5A (Fhb5), 6A (2), 6B (Fhb2), and 7B have been validated or used in MAS breeding [Genetic dissection of FHB resistance has identified hundreds of quantitative trait loci (QTL) distributed across all wheat chromosomes ,4,12. A sistance ,16,17. R protein ,19. The his gene . Pyramidin wheat . So far,3//”Mentana/Jingzhou Rye” F6) indicates a broad origin. However, the FHB resistance in JZ66 was unlikely to be Fhb1, based on pedigree and analysis with an Fhb1 diagnostic marker in our previous studies [Jingzhou 66 (JZ66) is a spring wheat cultivar, released in 1975 in Hubei province, which is located in the Middle and Lower Yangtze River Valley in China where FHB epidemics frequently occur. It combines moderately high FHB resistance with good agronomic traits. It was once widely planted in FHB-prevalent regions. The diverse parentage of JZ66  across all seasons and locations . JZ66 hap < 0.01) between 2018EZ and 2019EZ to 0.65 (p < 0.01) between 2019WH and 2019EZ  to QFhb.hbaas-2DS and QFhb.hbaas-4DS revealed that they were in the same region as the reduced height genes Rht8 and Rht-D1 (Rht2), respectively. QFhb.hbaas-3AL was detected in three environments, explaining 6.6–8.0% of the phenotypic variation. QFhb.hbaas-5DL was significant in 2019WH, 2019JZ, and the dataset of the mean of all the environments, explaining 7.0–9.1% of the phenotypic variation. The remaining two QTL on 3AS and 3DL were each detected in a single environment and the dataset of the mean of all the environments. QFhb.hbaas-3AS was detected in 2019JZ and the dataset of the mean value of the FHB index across the environments, explaining 2.9–6.6% of the phenotypic variation. QFhb.hbaas-3DL was significant in the 2018EZ environment and dataset of the mean value of all the environments, explaining 2.6–6.4% of the phenotypic variation. Therefore, even though there are phenotypic variation, large effect QTL is more stable than minor effect QTL, and is often detected in more environments.Six QTL for FHB resistance were detected on chromosomes 2DS, 3AS, 3AL, 3DL, 4DS, and 5DL , Table 5AX-111561744, AX-89398511, AX-110591324, and AX-109381281) of the four stable QTL  to evaluate the effects of single QTL and various combinations of the QTL on FHB response. DH lines carrying resistance alleles to all four QTL had much lower FHB indices (reduced by 78%) than lines with no favorable allele. Similarly, lines containing one, two or three favorable QTL had lower FHB indices  than those with no favorable allele  on plant height reduction than QPH.hbaas-2DS (PVE: 13.73%). These two QTL were coincident with QTL (QFhb.hbaas-2DS and QFhb.hbaas-4DS) for FHB resistance.The QTL for plant height and spike compactness were mapped, as they both had a significant correlation with FHB severity in this population. Eight QTLs for plant height, repeatedly detected across the environments, were located on chromosomes 2BL, 2DS, 3DL, 4DS, 5AL, 6BL, 7AL, and 7DL . AllelesQSC.hbaas.2DL and QSC.hbaas.5BL decreased spike compactness and all were derived from JZ66 .Due to high humidity and warm climate in conditions during the flowering stage, FHB is a frequent occurrence in the Middle and Lower Yangtze River Valley region that includes Hubei province. Numerous wheat cultivars such as Wuhan 1, grown in the region have moderate FHB resistance . JZ66 waXgwm261 allele in the susceptible parent Gamenya. The same QTL was also detected in a RIL population developed from Ning 894,037 and Alondra, with resistance contributed by the moderately susceptible parent Alondra [Xgwm261, indicating that they are likely the same QTL. In all cases, the FHB resistance allele on 2DS was conferred by the susceptible parent, particularly in Sumai 3-derived populations. Interestingly, Basnet et al. (2012) [Xgwm261 in a Sumai 3 and Y119306 (highly susceptible Tibetan landrace) population. It is well-known that Xgwm261 is the diagnostic marker for the semi-dwarf gene Rht8; therefore, the QTL identified for FHB resistance on chromosome 2DS might be due to the effect of Rht8. Our laboratory data and previous studies [Xgwm261, indicating that it likely contains the dwarfing allele of Rht8 and probably inherited this gene from its Funo parent . However, in these abovementioned published studies, plant height data were not available for further comparisons. In our study, the closely linked marker (AX-111561744) to QFhb.hbaas-2DS is located at 23.4 Mb, close to Xgwm261 (19.6 Mb). Analysis of Xgwm261 indicated that JZ66 did not carry Rht8, whereas AK58 did (unpublished data). Therefore, we speculate that the taller Rht8 allele in JZ66 contributes its FHB resistance. Similar results were obtained in an YZ1 and NX188 RIL population. QFHB.caas-2D and QPH.caas-2D were simultaneously identified at the position of Rht8, the FHB-resistance allele contributed by YZ1 and the plant height reducing allele was from NX188 [The first stable QTL identified for FHB resistance was located on chromosome 2DS. Alleles from JZ66 were associated with enhanced FHB resistance. Several previous studies reported FHB resistance related QTL in the same chromosome region. In a DH mapping population derived from Sumai 3 and Gamenya, Xu et al. (2001)  reported Alondra . It is n Alondra . In the . (2012)  identifi studies ,30 showeom NX188 .QFhb.hbaas-4DS). The favorable allele of this QTL for FHB resistance was derived from JZ66 and co-localized with the plant height gene Rht-D1  by physical position comparison between the closely linked marker AX-89398511 of QFhb.hbaas-4DS (17.6 Mb) and Rht-D1 (19 Mb). This QTL was also detected for plant height, and the allele for reduced plant height was from AK58. It has long been known that the Rht-D1 locus is associated with FHB resistance. Draeger et al. (2007) [Rht–D1, and the Rht-D1a allele from the Swiss cultivar Arina contributed resistance. In a Spark and Rialto DH population, the reduced height allele Rht-D1b increased susceptibility to FHB [Rht-D1 on FHB rating in three segregating populations revealed that Rht-D1b reduced plant height by 7–18%, but increased FHB incidence by 22–53% [The second stable QTL, with a large effect on FHB response mapped in this study, was located on chromosome 4DS (. (2007)  detectedy to FHB . Investiy 22–53% . The autQFhb.hbaas-3AL was detected in three environments. The physical position of the closely linked marker AX-110591324 to QFhb.hbaas-3AL was around 530 Mb. In previous reports, a major effect QTL associated with FHB resistance on 3AL was identified in Brazilian cultivar Frontana, near marker Xdupw227 (598 Mb) [Xwmc264 and Xgwm155 (625–702 Mb). However, the physical positions of these markers were not close to QFhb.hbaas-3AL. We therefore speculated that the QFhb.hbaas-3AL identified in JZ66 was different from those reported in previous studies.(598 Mb) . In an AQFhb.hbaas-5DL, and was detected in two environments and combined mean, explaining 2.7–11.5% of the phenotypic variances. Lv et al. (2014) [QFHB.caas-5D), flanked by markers Xgwm292 and Vrn-D1, with a PVE of 12.8%. A 5DL QTL near marker Xgwm292 in the Wangshuibai and Wheaton population contributed three types (type I–III) of FHB resistance [Vrn-D1 [Xgwm292, which is adjacent to Vrn-D1. The physical position of the closely linked marker (AX-111212888) to QFhb.hbaas-5DL was 494 Mb, which is 26 Mb from marker Xgwm292 (468 Mb). Therefore, QFhb.hbaas-5DL is likely to be new.The fourth stable QTL was designated as . (2014)  reportedsistance . A minor [Vrn-D1 . The aboQFhb.hbaas-2DS and QFhb.hbaas-4DS) for FHB resistance coincided with reduced height QTL QPH.hbaas-2DS and QPH.hbaas-4DS. Both favorable alleles for FHB resistance were associated with tallness. The physical positions of these loci were most likely the same as the height genes Rht8 on 2DS and Rht-D1 (Rht2) on 4DS. QPH.hbaas-4DS had a much larger effect on reducing plant height than QPH.hbaas-2DS, as reported previously [QFhb.hbaas-2DS (mean PVE: 36.2%) on FHB resistance much larger than that of QFhb.hbaas-4DS (mean PVE: 14.5%). The favorable alleles from JZ66 decreased FHB severity by 69.9% for QFhb.hbaas-2DS, and 55.5% for QFhb.hbaas-4DS.Co-localization of QTL for FHB resistance and plant height has been found in numerous mapping populations ,32,33,38eviously . The effQSC.hbaas.2DS) was identical to the QTL for FHB resistance and plant height at the position of Rht8. Alleles from JZ66 reduced spike compactness by 16%, relative to alleles from AK58  from the Middle and Lower Yangtze River Valley compared to cultivars from Northern wheat regions (more FHB susceptible) [The QTL with the largest effect on spike compactness (rom AK58 . It was rom AK58 . A similrom JZ66 . These reptible) ,41.Fhb1 into local wheat cultivars through backcrossing will significantly improve FHB resistance in Hubei province. Recently, Zhu et al. [Fhb1 but with good agronomic traits, providing better parental material for incorporating Fhb1 in local breeding populations. Alternatively, FHB resistance of wheat cultivars can be improved by stacking resistant loci from local cultivars, such as those discovered in the present study. In particular, the PCR-based markers developed in this study will facilitate the use of FHB resistance in JZ66.Sumai 3 has been identified as the most FHB-resistant cultivar and is exploited worldwide in wheat FHB breeding. However, the poor agronomic performance of Sumai 3 and its progeny hampers its use in China. Moderately resistant cultivars often have multiple QTL with small or medium effects and tend to be more adapted. Therefore, a breeding strategy using cultivars with better yield and moderate FHB resistance was proposed by Cheng et al. (2003)  and was u et al.  proposedA doubled haploid population of 209 lines was developed from a cross between JZ66 and FHB-susceptible cultivar AK58. AK58 was a leading cultivar in the Yellow and Huai River Valley in China with semi-dwarf plant height. Wheat cultivars Sumai 3, E’en 1, and An’nong 8455 were used as resistant, moderately resistant, and susceptible checks in all the experiments.The DH lines and parents, along with the checks, were evaluated for FHB reaction in five environments across two cropping seasons (2017–2018 and 2018–2019) and three locations, Wuhan, E’zhou, and Jingzhou. The trials were abbreviated as 2018WH, 2018EZ, 2019WH, 2019EZ, and 2019JZ. All trials were arranged in randomized complete blocks with two replicates. Each plot consisted of single 1 m rows spaced 25 cm apart, and sown with approximately 60 seeds.F. graminearum collected from Huanggang and Wuhan, Hubei province. The suspension contained 1 × 105 spores mL−1 and was amended with 0.01% Tween 20 detergent. The FHB responses of the ten labeled spikes were scored by counting the number of infected and total spikelets at 21 d after inoculation. The FHB index (%) was calculated as FHB incidence times severity, where incidence was the average percentage of infected spikes, and severity was the average percentage of symptomatic spikelet [Experiments at Wuhan were in an FHB screening nursery equipped with an overhead misting system to facilitate disease development. Plots were misted hourly for 2 min from 9 am to 7 pm daily from inoculation to completion of disease assessment. At anthesis, ten flowering spikes were randomly selected in each plot, labeled with blue 3 M tape, and sprayed with ~30 mL a water suspension of conidiospores of two isolates spikelet .2 on the soil surface about three weeks before anthesis. The FHB incidence and severity were evaluated at 28 d post anthesis by counting infected spikes and infected spikelets per spike on 30 spikes in each plot.Inoculation was performed at E’Zhou and Jingzhou, by scattering 600 g scabby wheat grains per 100 mPlant height, days to flowering, spike length, and total spikelets per spike were also measured. Spike compactness was calculated by dividing the number of spikelets per spike by spike length. Days to flowering were recorded as the number of days from sowing to anthesis. Plant height was measured from the ground to top of the spike (awns excluded) at maturity. Spike length was measured from the base to the top of the spike, not including the awns.H2) was calculated from the ANOVA using the following formula: H2 = 1 − MS2/MS1, where MS1 and MS2 are the mean squares for genotype and genotype by environment interaction, respectively [The calculations for descriptive statistics, correlation analysis, and analysis of variance (ANOVA) were performed using SPSS . The frequency distribution for the FHB index at each site and BLUP value for all sites was produced using Origin programs . The BLUP value was calculated with the R package Lme4 (y = lmer (Trait ~ (1|Genetype) + (1|Year)). Broad-sense heritability (ectively . Mean vap < 0.05) were removed from the dataset.Genomic DNA was extracted from fresh leaves of DH lines and parents with a Plant Genomic DNA Kit . DNA integrity was checked on agarose gel, and the quantity was measured with a Nanodrop spectrometer. The population was genotyped using the wheat 55 K SNP array  that conThe filtered SNPs 4500) were analyzed to eliminate redundant markers using the BIN function in IciMapping 4.1 with the parameter “not considering the missing value” . One mar0 were anhttp://202.194.139.32/ to obtain the physical position.Estimates of QTL positions and their effect in each environment were determined by inclusive composite interval mapping of additive function (ICIM-ADD) in IciMapping V4.1 ,52. A LOhttp://www.gentides.com) for use in MAS breeding. Primers for PARMS markers were designed by PolyMarker (http://polymarker.tgac.ac.uk) according to sequences of flanking SNP markers. PCR were performed in 384-well PCR plates for PARMS genotyping. The 5 μL PCR reaction system contained 2 times PARMS PCR reaction mix, 150 nM of each allele-specific primer, 400 nM locus-specific primer, and 1.4 μL alkaline lysis DNA template; 5 μL mineral oil was added into each well to prevent evaporation of the PCR mix. The thermal cycler program of PARMS was denaturing at 95 °C for 15 min, followed by 10 cycles of denaturation at 95 °C for 20 s, annealing started at 65 °C for 1 min, decreasing 0.8 °C per cycle to 57 °C; followed by 32 cycles of denaturation at 95 °C for 20 s and annealing at 57 °C for 1 min. The well plate was read using a TECAN Infinite M1000 plate reader. SNP calling and plots were carried out by an online software snpdecoder (http://www.snpway.com/snpdecoder/) combining manual modification.SNPs significantly associated with FHB response were converted to PARMS markers  in WuhanRht8 and Rht-D1 (Rht2) loci. Co-localization of FHB resistance QTL with the Rht8 and Rht-D1 loci but not some other reduced plant height QTL suggested a pleiotropic effect of these two loci. FHB resistance in JZ66 was associated with decreased spike compactness, controlled by QSC.hbaas.2DS and QSC.hbaas.5DL. The moderate FHB resistance and better agronomic traits thus endowed JZ66 a good germplasm for FHB resistance breeding. The PARMS marker designed for the stable QTL identified in this study may facilitate the pyramiding of resistance gene/QTL.The results from the current study showed that the moderate FHB resistance in JZ66 is controlled by multiple QTL with additive effects. Two QTL on chromosomes 2DS and 4DS played dominant roles in FHB resistance, and co-localized with the reduced height


Carum carvi) is a widespread and frequently used spice and medicinal plant with a long history of cultivation. However, due to ongoing climatic changes, the cultivation is becoming increasingly risky. To secure caraway cultivation in future, timely breeding efforts to develop adapted material are necessary. Analysis of genetic diversity can accompany this process, for instance, by revealing untapped gene pools. Here, we analyzed 137 accessions using genotyping by sequencing (GBS). Hence, we can report a broad overview of population structure and genetic diversity of caraway. Population structure was determined using a principal coordinate analysis, a Bayesian clustering analysis, phylogenetic trees and a neighbor network based on 13,155 SNPs. Genotypic data indicate a clear separation of accessions into two subpopulations, which correlates with the flowering type . Four winter-annual accessions were closer related to biennial accessions. In an analysis of molecular variance, genetic variation between the two subpopulations was 7.84%. In addition, we estimated the genome size for 35 accessions by flow cytometry. An average genome size of 4.282 pg/2C (± 0.0096 S.E.) was estimated. Therefore, we suggest a significantly smaller genome size than stated in literature.Caraway ( Carum carvi L., 2n = 2x = 20 [Caraway  2x = 20 ) is a hex = 20  i 2x = 20 . There i 2x = 20 . Besides 2x = 20  and prov 2x = 20 ,6. ThereSince the medieval period, biennial ecotypes of caraway have been cultivated from Italy to Scandinavia . Until tet al. [et al. [Until now, only a few studies dealt with the molecular genetics of caraway. These studies used randomly amplified polymorphic DNA (RAPD) markers and treated small sets from 5 to 24 accessions –9. Basedet al.  conducte [et al. . Disadva [et al. ,11.The primary goal of this study was to detect population structure and genetic diversity within a large set of caraway accessions. This set should represent a majority of available caraway germplasm and not a small selection of genotypes as in prior studies. In addition, we asked whether population structure could be correlated with available data on the accessions like flowering type, classification, geographic origin, national breeding programs or germplasm provider. Furthermore, we wanted to know whether domestication and breeding reduced the genetic diversity in caraway cultivars. For this purpose, we wanted to implement a genome-wide and cost-effective method, which requires no reference genome and allows simultaneous genotyping of a vast number of genotypes. Due to protandry, caraway is expected to be predominantly an outcrossing species ,12. HencGenotyping by sequencing (GBS) fulfills all these requirements. This next generation sequencing (NGS) method has already been implemented for a variety of crops, including horticultural species, with the aim of investigating population structure and genetic diversity –16. GBS Knowledge of genome size is advantageous before conducting a GBS. In particular, genome size plays a role for the choice of the restriction enzyme. Das  publisheTable 1 provides a summary assigning accessions to classification and flowering type. Detailed information on accessions is provided in S1 Table.A set of 137 caraway accessions was assembled from gene banks, breeding companies and institutes: The Leibniz Institute of Plant Genetics and Crop Plant Research  and the United States Department of Agriculture  each provided all their available caraway accessions. Accessions from the Crop Research Institute , the Centre for Genetic Resources, the Netherlands  and the Nordic Genetic Resource Center  were added to cover additional countries or cultivars. Further breeding material and cultivars were provided by the Julius Kuehn-Institute , by Dr. Junghanns GmbH , by Agritec Ltd. , by the National Research Council Canada , by Chrestensen GmbH  and by Pharmasaat GmbH . In total, the accessions originated from 27 countries (collecting or breeding site). Due to the assumed outcrossing nature of caraway, we expected most biennial varieties and wild material to be highly heterozygous populations. This should also be true for annual varieties. In contrast, annual breeding material of the JKI and Dr. Junghanns GmbH was produced by inbreeding. It mainly originated from crossings between annual and biennial material and was selected for annual flowering. The breeding lines are in the fourth or fifth generation of inbreeding . Two annual breeding lines are classified as doubled haploid (DH) lines. These lines were produced by the NRC using microspore culture [S1 Table.In May 2018, pre-grown plants  were pl. Three individual plants per accession were grown in greenhouse and were sampled at medium leaf-stage with six or more developed leaves  cultivar 'Stupické' was used as reference standard of known genome size (2C = 1.96 pg) [A set of 35 accessions was selected for genome size estimation. This set includes accessions out of all classification groups and flowering types . The sa1.96 pg) . Leaf fr1.96 pg) . Tests o1.96 pg) .Plant material for DNA extraction was grown under greenhouse conditions in two replicates for each accession. Young leaf material (BBCH 13–16 ) of fiveet al. [GBS was carried out by Elshire Group Ltd. . For GBS analysis, the method published by Elshire et al.  was adapThe platform Galaxy  was emplS1 Table.In total, the number of reads per genotype after trimming ranged between 1,722,352 and 14,361,058 with an average number of 5,621,037 reads. The clustering resulted in 1,196,473 contigs with an average length of 145.56 bp. The SNP calling generated 13,155 SNPs after filtering. On average, this set contained 4.42% missing values per accession and at maximum 16.19%. Detailed values per accession are provided in Bayesian clustering analysis was performed using STRUCTURE (v2.3.4) –40. A puO) and expected heterozygosity (HE) were calculated using R software [Allele frequencies and observed . Annual flowering types (n = 54) had a genome size of 4.285 pg/2C (± 0.013 S.E.) and biennial flowering types (n = 51) of 4.279 pg/2C (± 0.014 S.E.). This small difference was not significant . An estimated mean value for the genome size of each measured accession is given in S1 Table.Across all measured samples, an average genome size of 4.282 pg/2C (± 0.0096 S.E.) was estimated within a range from 4.051 to 4.482 pg/2C . P1 (n = 62) merely contains annual accessions and P2 (n = 75) contains all biennial accessions and a group of five annual accessions. Based on this result, we conducted separate PCoA for P1 and P2 to get deeper insights into the substructure of these subpopulations.–PCo2 shows that one of those five annual accessions is closely related to some annual accessions of P1 . Therefore, this accession was analyzed with P1. –PCo1 and PCo2 explained 10.96% and 2.66% of the variance and PCo1 divided the accessions into two subpopulations defined as P1 and P2 , these accessions tend to split into two subgroups (SG1 and SG2), which is clearer on PCo4 (not shown). SG1 contains German cultivars (n = 4), including several breeding lines of the JKI, whereas SG2 includes all accessions of P1 originating from other countries (n = 8).P1 predominantly contains breeding material (n = 53). Only a few accessions are cultivars, landraces, wild material or are unclassified (n = 10). On PCo3 Fig 2E and 2F), further on defined as SG3. The interesting subgroup SG4 of four annual accessions, including the Czech cultivars 'Aprim' and 'Ol-Alfa', is separated from the larger cluster of P2 accessions. This is also true for the subgroup SG5, which consists of biennial accessions originating from Russia, India and China.The PCoA for P2 shows an accumulation of most cultivars in the left end on PCo1 . For K = 2, 62 accessions were assigned to subpopulation (or cluster) C1 and 75 accessions to C2 (membership coefficient > 0.5) . This assignment is congruent with the assignment based on PCoA. For K = 6, twelve, sixty-one, six, nine, six and eight accessions were assigned to subpopulation C1-C6 (membership coefficient > 0.5), respectively , including SG3, were assigned to C2 and all other biennial accessions (n = 9), including SG5, to C4. The assignment to C1 contains the annual accessions of SG1. Accessions assigned to C3, C5 or C6 form groups of related annual inbred lines. However, the majority of annual accessions could not be assigned to a subpopulation and were assigned to the admixed group. No assignment contains annual as well as biennial flowering types. Therefore, STRUCTURE results for K = 6 basically subdivide the subpopulations retrieved from K = 2. These subdivisions can be observed by PCoA as well .STRUCTURE results revealed two equally high ΔK-value peaks, for K = 2 and for K = 6 . For K = 6, we analyzed the admixed group as a seventh subpopulation. In both cases (K = 2 and K = 6), genetic variation between subpopulations was low .Fig 4; r = 0.94; for comparison: simple NJ, r = 0.86 and UPGMA, r = 0.79). The BIONJ tree also displays a clear partition between annual and biennial flowering types although the annual subpopulation emerges as a subgroup of the biennial subpopulation. SG4 appears as the nearest subgroup of annual flowering types to the biennial subgroup SG5. We also recover the biennial subgroup SG3, which contains most biennial cultivars. The BIONJ tree clearly separates German annual cultivars (SG1) from all annual accessions from other countries (SG2).For a better visualization of the genetic relations between single accessions, we built phylogenetic trees. A hierarchical clustering (UPGMA), a simple NJ and an improved NJ algorithm (BIONJ) were tested to build phylogenetic trees. The best correlation between the Euclidian distance matrix and a phylogenetic tree was found for the BIONJ tree  supports the partition into two subpopulations correlating with flowering type. SG4 and SG5 appear as rather intermediate subgroups between P1 and P2. However, net structures towards biennial accessions seem to be stronger. In general, net structures are prominent at the basis of subpopulations but are low between individuals within subpopulations. Branches of individual accessions are long, indicating that a lot of genetic diversity is stored on individual level.The neighbor network . The HO of the annual breeding material, consisting of 27 S4 lines, 24 S5 lines and two DH lines, (26.34%) was significantly lower than the HO of annual cultivars (30.75%) . In contrast, the HE was significantly higher (2.11%) in annual breeding material . The highest HO was found in the annual subgroup SG4 with 35.03%. No larger differences in HO or HE were found between biennial cultivars, biennial landraces and biennial wild material. In general, the HO of biennial material is on the same level as the HO of annual cultivars. The lowest HO (20.93%) among all accessions was found in a Canadian DH line (ID: 127) and the highest HO (39.91%) in a wild Russian accession (ID: 002). Single values for heterozygosity per accession are listed in S1 Table.HWe investigated a broad collection of caraway by GBS to reveal population structure and genetic diversity in caraway germplasm. We applied distance-based and model-based methods, which mutually supported our findings. Different types of visualization may enable the reader to retrieve general and detailed information on the relations between the 137 analyzed caraway accessions. We found a strong correlation between the upper most level of population structure and the flowering type. Therefore, we focused on this phenomenon for further analysis and in the following discussion. Nonetheless, we will also discuss some interesting findings within the substructures of the subpopulations. In addition, we were interested in the genetic diversity of cultivars compared to present breeding material and wild accessions.S1 Table).The flowering type was assessed by a field trial sown in spring. We defined accessions that flowered in the year of sowing as annual and in the year after sowing as biennial. Biennial flowering types need a cold stimulus  for flowering induction ,4. ThereArabidopsis thaliana and cereals [In crossing experiments between annual and biennial material, annual flowering was found to be dominant . Since m cereals –53. ThesSolanum lycopersicum cultivar 'Stupické' as well accepted reference standard [C. carvi var. KBI 1434 used by Das [Genome size was investigated by flow cytometry with standard ,54,55, wstandard ). In constandard  publishestandard  pointed standard . The larstandard  used an standard , but no d by Das  is descrd by Das  reports Daucus carota, Coriandrum sativum and Apium graveolens are the nearest relatives of C. carvi with an available reference genome  [Daucus genus, Eryngium duriaei subspecies and diploid Seseli libanotis reported genome sizes from 0.92 pg/2C to 3.019 pg/2C [et al. [For a set of 35 caraway accessions, no considerable intraspecific variation was found. To the best of our knowledge, ctively) –60. Furt19 pg/2C ; 5.52 pg19 pg/2C  and 2.5919 pg/2C , respect19 pg/2C ,62. IndeAs expected, GBS enabled the discovery of a large number of SNPs, suitable for a variety of downstream applications. For species for which a reference genome is available imputation of missing values can be a valuable method to improve the SNP data quality . HoweverO. However, Ho of 26.34% is much higher than theoretically expected for lines of the 4th or 5th inbreeding generation. Checking the frequency of heterozygous SNP calls per genotype in the raw SNP data reveals that it is much lower and that it is increased by the MAF filter applied. Nevertheless, the frequency of heterozygous SNPs per genotype is yet higher than expected for these advanced generation breeding lines. Selection mechanisms and/or unintended outcrossing could be reasons for this. Partially, the existence of multiple copies of highly similar but non-allelic sequences might lead to heterozygous SNPs [As expected, annual breeding material, which mainly consists of advanced generation breeding lines, showed the lowest Hous SNPs ,63,64. WFigs 3A). This indicates that the flowering type causes the main structure in the population of 137 caraway accessions under investigation. In addition, STRUCTURE results also support the presence of six subpopulations, which show differentiation within the annual (P1) and biennial (P2) subpopulation . In general, occurrence of two peaks of K = 2 and K = 6 is not contradictory. K = 2 describes the upper most level of population structure caused by the flowering type, whereas K = 6 describes a lower level of population structure, which is also indicated by PCoA.Bayesian clustering analysis and PCoA revealed an optimal number of two subpopulations, which differ in flowering type of accessions . We hypothesize that both subpopulations mainly differ in the genomic region of the locus controlling vernalization, whereas in most other genomic regions, both subpopulations could be similarly variable. This might be attributable to the fact that most annual breeding lines originate from crossings between annual and biennial breeding material.Vice versa, alleles of the biennial parents, which promote high essential oil content, were not strongly selected. We assume that alleles for higher essential oil content were already present in parental annual material used for crossings. Indeed, breeding history at the JKI shows that recurrent selection within original annual breeding material could increase the essential oil content in the same way as crossings between annual and biennial breeding material [Those crossings, mentioned above, aimed to bring a higher essential oil content of biennial breeding material into the annual breeding pool. During multiple steps of inbreeding, annual flowering and high essential oil lines were selected. Hence, we expected a large intermediate group, but instead most breeding lines cluster nearby original annual accessions . Therefore, the selection towards annual flowering might have had the largest impact on the genetic constitution of the breeding material. As discussed above, this selection might only affect a small genomic region because little variation is explained by the separation into subpopulations in accordance with flowering type. material .et al. [Figs 4). This subgroup contains 'SZK-1' and 'Karzo' as well as Tunisian, German, French, Canadian and North American accessions.–The German accession, which is classified as “wild”, has to be escaped from cultivation or is wrongly classified.–The close relationship indicates an identical origin and an active exchange of annual material between breeders, farmers and gene banks. German cultivars seem to belong to a separate but related subgroup (SG1). Thus, there might have been a slightly different origin or strong selection.According to Pank , annual et al.  found thet al. . Toxopeuet al.  reports E was significantly higher for annual breeding material than for annual cultivars . However, due to different numbers (n = 53 to n = 6), both groups are scarcely comparable. To the best of our knowledge, only four annual cultivars . Literature provides no more information to reconstruct the breeding history, but breeders state that some annual breeding material was crossed and backcrossed for several times with biennial material . 'Aprim' most likely originates from these breeding schemes. Several backcrossings would perfectly explain the assignment to the biennial subpopulation. The appearance of an annual Russian accession classified as “wild” in this subgroup is enigmatic . It might be the most parsimonious way to assume a wrong classification. Alternatively, we could assume a distinct (Russian) origin of 'Alpha' and 'Aprim', but there are no breeding reports that support this thesis. Although ‘Aprim’ was bred for winter-annual growing season with sowing in August, as mentioned above, it was sown in spring and no vernalization requirement was observed. This subgroup is a valuable gene pool for breeding of winter-annual caraway showing an outstanding frost-resistance. In summary, we can report that winter-annual ecotypes  are more closely related to biennial ecotypes than to summer-annual ecotypes.Pank  reports Fig 4). Having a closer look at the STRUCTURE results for K = 2 or K = 6, SG5 contains "annual clusters" to a larger proportion  and appears in all investigations as the closest subgroup to annual accessions . In nature, these accessions might overlap geographically with or be related to an annual population in the Middle East, whose extensions to the north and west are unknown. Accessions from Japan and North Korea do not belong to that subgroup, so that we should not define SG5 as an Asian subgroup . In addition, we found some wild (or unclassified) accessions of nearby geographic origin that cluster in one subgroup, for instance, five Scandinavian accessions . However, some wild accessions seem to be randomly distributed regarding their geographic origin . We can only speculate about reasons. In some cases, passport data might be incorrect. Alternatively, wild accessions might originate from escaped plants, which were not indigenous in a country.The biennial subpopulation P2 contains many more accessions classified as wild than P1 and contains geographically diverse caraway accessions. The subgroup SG5 contains three Russian, one Indian and one Chinese accession  indicates that a certain gene pool was preferred in caraway breeding. This subgroup contains cultivars from the Netherlands, Czech Republic, Germany, Austria, Poland and Denmark, indicating an active exchange between national breeding programs. It can be suspected that this subgroup keeps many advantageous alleles for agronomic traits, but untapped valuable alleles might exist in other subgroups as well. Wild accessions located in SG3 originate from Sweden, Denmark and Great Britain. This might lead to the assumption that the majority of improved biennial cultivars originated from North-West European wild material. However, North European wild material  appears outside SG3 as well. A more expanded sampling in wild caraway populations would be necessary to get reliable insights into the domestication history of biennial caraway. However, there probably is more than one domestication event.The accumulation of biennial cultivars in SG3 . For this purpose, SNP loci from GBS can be easily converted into diagnostic markers. Further investigations are necessary to identify SNP loci associated with flowering type and other traits of interest.S1 FigFor one to ten assumed subpopulations based on STRUCTURE results processed by STRUCTURE harvester.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 TableThis table provides detailed information on passport data and gathered data of all 137 accessions.(XLSX)Click here for additional data file.


The success and viability of an ex-situ conservation program lie in the establishment and potential maintenance of a demographically and genetically viable insurance population. Such population reserve may support reintroduction and reinforcement activities of wild populations. White-naped mangabeys are endangered restricted-range African primates which have experienced a dramatic population decrease in their natural habitats over the last few decades. Since 2001, some European zoos singularly monitor an ex-situ population aiming to seek the recovery of the current wild population. The aim of the present paper is to evaluate the genetic status and population demographics of European zoo-captive white-naped mangabeys based on pedigree data. The captive population is gradually growing and preserves specific reproductive and demographic parameters linked to the species. The intensive management program that is implemented has brought about the minimization of inbreeding and average relatedness levels, thus maintaining high levels of genetic diversity despite the existence of fragmented populations. This finding suggests white-naped mangabey ex-situ preservation actions may be a good example of multifaceted conservation throughout studbook management which could be used as a model for other ex-situ live-animal populations. To counteract this global extinction crisis, ex-situ conservation programmes have been increasingly implemented, seeking the sustainable maintenance and breeding of threatened species under controlled conditions outside their natural habitat.The Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) warned about the high risk of extinction of over one million animal and plant species in 20194. Integral management of captive-bred populations implies genetic and demographic routine monitoring tasks must be performed to reach conservation objectives in these captive populations4.Strategically combined with reintroduction activities, ex-situ techniques have become effective measures to preserve endangered species, when the efficient preservation of wild populations is compromised6. High levels of genetic diversity do not necessarily imply a high heritability of features that may be desirable from a conservation perspective. Evolutionary potential depends on external, and often complex, components for example, the variability that is due to environment. Provided evolutionary potential7 is thought to be partially driven by genetic diversity8, it is the effectiveness of genetic management policies , which may determine whether long-term population viability and reintroductions into the wild are successfully maximized.Monitoring tasks seek to ensure that genetic diversity and effective population sizes are maintained within acceptable levels, while inbreeding and average relatedness between mating animals are reduced to a minimum9. Under the scope of the Zoos European Directive10, EAZA member institutions are instructed to maintain standardized individualized records to make the interpretation and utilization of animal databases easily accessible for all the members involved in cooperative management plans and formal research projects11. Internally, these databases allow animal management staff to plan and monitor population conservation and care programmes for long-term survival and potential in-situ conservation assistance12.The European Association of Zoos and Aquaria (EAZA) gathers together the leading zoos and aquariums in Europe and the Middle East and regularly designs standards for the conservation of nature and wildlife both at its member institutions (400 member zoos and aquariums across 48 countries) and outside the zoo premises13. To ensure these goals are accomplished, each individual animal record should comprise all the relevant data concerning its current status, origin, genealogy, possible transactions, health and other practical advice 14.Updated records of zoo collections are a valuable source of information for scientific researchers pursuing to reach the key mission conservation goals of zoos and aquaria as stated in the first set of guidelines of the World Association of Zoos and Aquariums15. This information may not only enhance the ability to adapt housing and living conditions to approximate captivity to wild environmental conditions, but also to replicate the genetic interaction of the individuals in this recreated environment16.The documentation of the complete historical record of every individual lays the basis for better coordinated genetic and demographic population management practices in threatened taxa17 with reliable reproduction and increasing group growth rates18. Such recommendations may involve translocations among zoological nuclei, and the objective selection of most appropriate individuals for reproduction and/or identification of related animals19.Pedigree information analysis constitutes an invaluable tool for genetic diversity quantification and demographic structure evaluation in captive populations. This information can be used to formulate recommendations to maintain a genetically-healthy population20. The effectiveness of pedigree analyses relies on the strict control of genealogical information carried in endangered populations, from the moment when the base captive-founder populations were established. The integrity of the genealogical information present in pedigrees may be compromised by problems associated to the veracity and effectivity of the tools used23, pedigree completeness levels 25 and of the thoroughness of the operators participating in the process of data collection and registration21, among others. The estimates derived from the analyses of non-robust pedigrees  can be favoured if empirical estimates of relatedness via genetic markers (microsatellites or SNPs) are determined26. Hence, endeavouring to improve pedigree robustness may always be sought to improve the accuracy of genetic parameters27.Pedigree-based strategies are cost-effective alternatives to perform routine genetic diversity evaluations, population demographics and viability and to track the improvement of genetic diversity29 may frequently compromise the routinely application of genomic tools and restrict their utility to small-sized threatened populations, with limited or missing genealogical background, in which the proportion of polymorphic loci is commonly small31. Otherwise the use of large numbers of genomic markers may be needed32. These limitations may result in allele frequencies of the historical population being unknown, which may bias the inference of inbreeding as a direct consequence of potential changes occurring due to genetic drift33. As a result, molecular techniques, which may not distinguish between identity by descent (IBD) and identity by state (IBS) probabilities underlying genetically mediated similarities among relatives35, may compliment the information comprised in pedigrees.In this context, budget limitationsex-situ management programmes using pedigrees are routinely carried out by EAZA in a wide range of animal species. These species mainly consist of terrestrial and marine mammals 15. In the case of non-human primates, nearly 60% of the species are endangered and 75% account for wild reduced populations as a result of human-induced disturbs during the last three decades36.Based on the aforementioned comparison, Cercocebus atys lunulatus), a West African endemic non-human primate from 37 currently classified as ‘Endangered’ by The Red List of IUCN due to habitat fragmentation and bushmeat38. The genealogical information  of captive white-naped mangabeys19 has been monitored in different European zoos since 1994.Among other genus and families, EAZA institutions host the largest captive population of white-naped mangabeys (19. Supported by the West African Primate Conservation Action (WAPCA) in Ghana and Côte d’Ivoire since 2010, the first research outcomes obtained consisted of a population viability analysis (PVA) simulating different scenarios combining deterministic and stochastic factors potentially affecting white-naped mangabey wild populations’ dynamics. The study concluded genetic diversity may remain high under all assumptions (> 90%)19, which suggested European captive mangabeys may act as an insurance population to accomplish in-situ conservation goals.Since 2000, a European Endangered Species Programme (EEP) was implemented to respond to the classification of the species as ‘Critically Endangered’ by IUCNThe present study evaluates the effectiveness of conservation activities along the history of the captive population of white-naped mangabeys. The genetic and demographic structure of the captive population, genetic parameters and the trends described by them were quantified. Afterwards, a breeding strategy is proposed to recommend the most appropriate animal matings among the individuals present in the current population. To conclude, the genetic distances among hosting institutions were quantified and traced. The present set of analyses may guide future conservation aimed breeding strategies and act as a model for other species under the similar circumstances.The historical and current distribution of individuals across institutions is shown in Fig. Pedigree completeness indexes (PCIs) for one, two, three, four, five and six generations, the maximum number of traced generations, maximum number of complete generations and number of equivalent generations in the two population sets, are shown in Table The maximum progeny per male and female decreased almost linearly in the current population. However, the mean (± SD) progeny per male was 1.57 ± 4.17 in the historic population and 2.07 ± 4.19 in the current population; 1.68 ± 3.02 and 1.92 ± 2.78 in the historic and current population, respectively, for females. The female/male ratio is increased in the current population (1.22/1) in respect of the historic population (0.90/1). The percentage of males with progeny selected for breeding, that is all males whose offspring has acted as a breeding male or female, was 45.62% and 84.10% in the historic and current population, respectively; 41.34% and 75.73% for females, all females whose offspring has acted as a breeding female or male (Table Although mean (± SD) inbreeding is low (3.19% ± 0.07% in the historic population and 1.64% ± 0.05%) in the current population), highly inbred animals are present in each population set .The percentage of inbred animals was 23.15% and 17.5%; the average (± SD) coancestry was 4.21% ± 2.00% and 4.18% ± 2.00%; and the degree of non-random mating presented mean values of − 0.02 ± 0.07 and − 0.03 ± 0.05, for the two population sets, respectively. The highest values for these three parameters were 0.25 (25%) for different ages between 1985 and 2018, 0.0914 (9.14%) for coancestry in 1997 and 0.225 for non-random mating degree in 1999.Matings resulting in highly inbred animals have occurred in the population: 2 (0.67%) mating between full sibs, 18 (6.04%) mating between half-sibs y 18 (6.04%) mating between parent-offspring.Registered values for mean (± SD) Genetic Conservation Index (GCI) were of 2.84 ± 1.48 and 3.52 ± 1.52 for the historic and current population, respectively. The summary of identity by descent estimators, non-random mating degree and genetic conservation index parameters is presented in Table α), inbreeding rate (F), average relatedness (ΔR), and Genetic Conservation Index (GCI) of the European captive white-naped mangabey from 1951 to 2019 is represented in Fig. F) and average relatedness (ΔR) up to 15 generations are shown in Fig. 2)39.The evolution of the non-random mating degree  explained 16.99% of the total genetic pool within the population (91.62%), 1.69% of the total inbreeding coefficient (3.09%) and 1.68% of the total coancestry (3.93%). The 10 ancestors with higher marginal genetic contributions were responsible for the total inbreeding and 3.72% of the total coancestry in the population.NeCi) was 17.76 ± 1.59. The number of equivalent subpopulations (± SD) was 0.37 ± 0.17.The mean (± SD) effective population size calculated by the individual inbreeding rate was 47.33 ± 21.04 in the reference population, whereas the mean (± SD) effective population size based on the individual coancestry rate  was 0.13 (Table The mean (± SD) number of animals per zoo was 8.51 ± 10.26, ranging from 1 to 40. Related to Wright’s F statistics, the inbreeding coefficient relative to the total populationZoo structure assessment revealed none of the zoos could be considered the population nucleus neither totally isolated. The number of zoos that used foreign father was 19, whereas 17 used own fathers and 14 used both foreign and own fathers. In total, 28 pairs of zoos showed the greatest Nei’s genetic distance 50%) among them. The minimum Nei’s genetic distance was 0.0619 (6.19%) and was shared between one pair of zoos  describes a cyclical trend which lasts approximately 20 years, a period after which white-naped mangabeys naturally display signs of reproductive senescence42.Conservation measurements implemented in captive European white-naped mangabeys have focused on maintaining a healthy ex-situ population mimicking the natural framework of the species. The captive population has maintained high genetic diversity and minimized inbreeding levels since 1951 see Fig. . Two of ex-situ population since 201219. The progressive increase in pedigree knowledge up to 90% at first generation occurred in the context of the scarcely available data from other threatened species held at offsite emplacements for similar conservation purposes .The demographic evolution of captive population is parallel to the improvement of the risk situation faced by white-naped mangabeys wild populations, which promoted the reclassification of the species by The Red List of IUCN to the lower threat category of “Endangered” in 2015. The inclusion of twenty-one new institutions reinforced EAZA’s network and brought about the addition of new founders  and genealogy-known individuals to the 23, low generation depth24, unknown founder relationships25, and human error21, among others45.This lack of information occurs even if institutions make every effort to implement the most efficient standardized methods. Pedigrees can remain problematic due to multiple reasons, including difficulties associated with discerning parentage with herd or flock breeding46. For instance, 78% of bird and 52% of mammal captive populations registered in EAZA’s studbooks47, have pedigree completeness index levels below 85% and 58% fail to achieve the target conditions for sustainability 46.As a result, the analysis of incomplete pedigree records may lead to biased calculations of demographic and genetic parameters, even if self-sustainability could be expected from most managed populations19.The pedigree completeness levels in our study provide the first evidence of the success of white-naped mangabey ex-situ programme, which in turn enhances the possibilities for protection and recovery at medium and long-term, as long as genealogical recording and intensive management husbandry practices continue48.The captive population constitutes itself a short-term backup reserve if the imminent extinction of wild populations occurred. In fact, it is the intensive management implemented, which aims at preserving the demographic and biologic structure of white-naped mangabeys wild counterparts, which potentializes the breeding capacities of the individuals to effectively retain high levels of genetic diversity. Maximum progeny per male and female were higher in the historic population. However, this could be ascribed to the fact that in the origin of the captive population, the main objective was to ensure a number of animals which may permit the captive population’s long-term viability49. This was supported by the negative values of FIS  earlier, with the consequent increase in birth number and population growth rate.Prolonging generational intervals can effectively increase the number of animals selected for breeding, progressively increasing effective population sizes and, therefore, generating a proportional reduction in inbreedingIS, which may be indicative of the promotion of breeding policies that consider unrelated animals at a rate at which the population does not excessively depart from Hardy–Weinberg equilibrium.Additionally, the balance between such strategies may lead to the success of the breeding programme, as suggested by the relatively low negative values of FCercocebus54 which could be ascribed to the philopatric nature of primate females.Mean generation intervals were higher for the current sire-daughter and dam-daughter pathways, which could be explained by a sex-ratio which favours females Table , a commo55. Additionally, in Old World monkeys (Family Cercopithecidae), milk amount and quality may be influenced by offspring sex, to the detriment of baby females57, which determines a lower growth rate in their early stages and a delay in the mean age of prepuberal females in reproduction.This biological condition provides primates with an important functional role for ecosystem health and wellbeing which simultaneously enables the survival and success of offspring to breed58) and maximum periods of fertility  of captive individuals.Parents’ mean age at their offspring’s birth was slightly lower than generation intervals Table , suggest58), functionally (reproductive effectiveness) or conservationally (higher levels of genetic diversity and reduced inbreeding)] as suggested for other species59. This policies simulate the sexual dispersion of this species, in which the males constitute the migratory sex60.Historical and current percentages of females with progeny selected for breeding were lower than those of males . This suggests breeding selection policies pay a greater attention to males [either phenotypically  and FIS values, which suggested higher rates of random matings among closely-related individuals may occur, which is common in small-sized populations which develop in limited spaces.Inbreeding has remained below coancestry levels, suggesting matings among closely-related individuals were unintentionally performedF slightly exceeds the recommended maximum of 1%, level below which the fitness of a population steadily decreases65. Hence, effective population size may still not reach the recommended threshold to maintain genetic variability . However, the trends described for the historical evolution of ΔF report promising outcomes, as there has been a decrease of around 4 points, which may imply genetic variability may be recovering acceptable levels. As Lee and Wilcken66 stated, a population of any size can be sustainable if a supplementing source population can effectively suit the required harvest of new individuals and cooperation across institutions is well-established. The incorporation of Accra’s Zoo  in 2010, meant an invaluable source for genetically unrelated wild-born animals which may prevent genetic erosion.Current Δ67, population subdivision may be beneficial, provided the extinction risk derived from compromising events such as accidents or health-related factors, may only cause the disappearance of population sections. Furthermore, genetic diversity may reach its highest levels when populations subdivide into as many separate groups as possible. Still, caution should be taken, provided the benefits of subdivision may be counteracted by the negative effects derived from the reduction in effective size and increase in inbreeding.The number of equivalent subpopulations below 1 revealed a high level of population structuration. According to Fernández, et al.F, it hardly affects coancestry increase, hence NeCi may more accurately estimate effective population size than NeFi68. Hence, progressively adding individuals through the participation of new institutions may be beneficial to maintain a high degree of genetic diversity for as long as possible19. Simultaneously, the proportion of translocated males is higher than that of females. This practice may be an additional attempt to simulate the male sexual dispersion of the species60, an implicit evolutionary strategy for the prevention of inbreeding increase71.Although population structure can greatly affect Δfe (15.41) and fa/fe (0.71) may suggest the frequent use of a small number of animals for breeding may lead to the loss of genetic variability, which may be supported by the low number of ancestors (5) which explains 50% of population's gene pool. Still, founders’ genotypes are represented in the current population. The unequal contribution of founders may be confirmed by the values of fg (11.85) and fa/fe (0.71) as one of the main causes for the current genetic diversity loss. The difference between fe and fa suggests bottlenecks, although not sharply, may have reduced population’s genetic variability. The lower the fe/fa is, the greater impact bottlenecks have on the population.Values of 72. Mutations that are only mildly deleterious are difficult to eliminate and are the principle cause of inbreeding depression73. Furthermore, even if lethal and semi-lethal mutations disappear rapidly due to inbreeding, the large costs of this process may affect population viability73.These bottlenecks may be associated to a progressive increase in the occurrence of abnormalities and susceptibility to disease or stressful environmental situations. Such an increased susceptibility may derive from the increase in the incidence of deleterious recessive mutations, which may potentially lead populations to extinction58, could potentially be ascribed to increased inbreeding levels (around 25%) in primate captive populations among other factors74. However, theoretically, species that are naturally inbred to some degree in the wild, should potentially show less of a deleterious effect when subjected to inbreeding in captivity, which may somehow explain the low representativity of the loss of genetic diversity derived from the occurrence of bottlenecks and genetic drift in the population under study (0.97%)74.For instance, infant mortality levels of in white-naped mangabeys in captivity of 37.2% with most deaths occurring within the first two months of lifefg and fa (11) would suggest the effects of genetic drift on genetic diversity may have been compensated by the higher value of fg. The difference between fg and the number of founders (f) (29) may be indicative of the loss of founder’s offspring, inbreeding increase at founder stages, or a combination of both causes75. This could be justified by the absence of an intensive population breeding programme until 2000 when the EEP76 of this species was set19. Nevertheless, according to genetic theory, twenty unrelated individuals may be enough to retain 97.5% of the wild gene diversity within the founder population72.Additionally, the difference between 77, with levels of genetic diversity (95.78%) in the captive population progressively increasing since 2012 (93%)19. Considering that intensive management for this captive population is relatively recent, the maintenance of such high genetic diversity levels may depend on multiple simultaneous factors. For instance, not enough generations may have passed since the decrease in the effectives comprising the wild population pushed the species to its current endangered situation. Hence, generation number in captivity may not be enough to verify the magnitude of genetic variability reduction78.Long generation intervals found in primates may permit genetically self-sustainability with few founders. In fact, this specific ex-situ programme may have effectively captured at least 90% of wild gene diversity for 100 years since the captive population was established, as most of the EEPs and ESBs within EAZA institutions19, the number of founder genome equivalents was half the number of founders in captive white-naped mangabeys in 2016, which was indicative of either lost descendants of the original founders or that the original founders were inbred, or a combination of these factors.However, in the absence of conservation efforts, a substantial loss could be confirmed in the near future for these primates. For instance, as reported by Jara et al.79 as described for other captive populations of critically endangered species80.In this context, the lower difference between founder genome equivalent and number of founders in the present study confirms special efforts may be being made on the preservation of descendants from the founder population, as if the cause for such differences may have been the fact that founder animals were inbred, these values may have remained somehow stable. This supports the fact that the founder population of captive white-naped mangabeys may have been highly genetically diverse and may have included individuals from a wide range of geographic origins hence, the variability to be expected from wild populations’ sub-structuration may be well represented81. In this context, research seeking to understand viability and resilience mechanisms in captive populations may bring about the development of tools which may enable to evaluate genetic diversity levels indirectly. This in turn may help to fulfill conservation purposes more efficiently and practically, as the more diverse populations are, the more capable to adapt to captivity environments they will be as well.Genetic diversity may be the basis for individuals’ resilience to the factors that extensively threat their wild populations and the adverse effects of adaptation to captive breeding82. Hence, the subdivided populations could contribute to the conservation of genetic diversity, as shown in wild fragmented populations of other nonhuman primates85.In this sense, a recent population viability analysis simulating different scenarios combining deterministic and stochastic factors and their potential impact on the viability of wild isolated populations of white-naped mangabey has suggested high levels of genetic diversity may be generally maintained under all assumptions (> 90%)The minimum Nei’s genetic distance between institution pairs, effective population size and Wright’s F statistics confirmed a certain subdivision degree. A single institution (26) is at the top of the relationship cladogram Fig. . This ma86. Table 87.Reproductive policies normally consider a small number of ancestors as the basis for subsequent generations, which indirectly replicates the natural isolation patterns found for fragmented wild animal populations88 measures the proportion of genes of founder animal i in the pedigree of each particular individual in the population. GCI is a measure of the representativity of founding population in the individuals, and acts as a measure of genetic diversity in the range of the genetic pool of the base population. The highest score in the rank was given to the model obtaining the most desirable value for each particular criterion. For instance, those individuals presenting the lowest inbreeding coefficients may be ranked higher, while those animals presenting the highest genetic conservation indexes will be ranked higher as well. Then, the rest of positions in the rank were determined in ascending or ascending order from the most desirable values to the lowest desirable ones, which are ranked with the value of 1.Bearing this in mind, current pairing/transfer criteria focuses on ranking animals in the population considering their individual inbreeding coefficients (F) and genetic conservation index (GCI). GCI89 to summarize the position in the rank for each of the two parameters. The combined index used (ICO) was as follows;1 is the weight for inbreeding coefficient, W2 for GCI rank position. All criteria are given the same relevance in the ICO, hence, no coefficient was used, that is the proportion of 1:1 is followed. As a result, the animals presenting greater ICO values are the ones presenting the highest levels of genetic diversity from the pool of the founding population and having those founding genes from the least related animals. Conclusively, the individual values for ICO and mean kinship rankings between pairs of individuals are considered to determine which the most appropriate pairing/transfer candidates are.Afterwards, as aforementioned inbreeding and GCI differ in terms of which their most desirable values are and what their magnitude is, a combined selection index (ICO) is developed following the premises in Van Vleck4. Such condition may translate into a more successful genetic and demographic intensive management of biodiversity conservation43. Comparing genetic diversity and structure between captive and wild populations using genomic markers would help to determine the magnitude of the potentially occurring bias when using information from pedigrees and which of these two alternatives may eventually more effective90.This use of mean kinship, inbreeding and GCI values for best guiding of animal pairing is proposed to be more attainable in zoo-kept intensive-managed populations than in other large housing facilities where social structure and therefore mate choices cannot be accurately handledBarcelona Zoo coordinates the European Studbook (ESB) for the white-naped mangabey. This population breeding/management programme registers the information in respect birth place, birth and death dates, average kinship  and transfers of the individuals that are housed in EAZA-member institutions.19.Through the compilation of this information, a demographic and genetic assessment is regularly performed to effectively manage the general status of the population. If derived results indicate a non-self-sustaining population at a given time, more intensive management  are proposed for the ongoing population viabilityTransfer decisions are based on internal criteria such as lack of space for more individuals in a particular location for welfare issues, existing heavy disputes among congeners sharing resources, desired phenotypic traits and/or low reproduction performance within a captive herd.Concerning exchanging rates, sixty-eight males and fifty-seven females have been subjected to translocation activities for improved pairing since 1994. In fifty-five cases, this genetic exchange has been made through assisted reproduction techniques by expert veterinarians instead of removing the animals from their living emplacement for mating attending to animal welfare-related logistic and biological constraints .The historical population comprises 298 animals  born between January 1951 and January 2019. The current population comprised 120 white-naped mangabeys  which were born between September 1987 and January 2019 and are alive. Only thirty-three animals (11%) were wild-born.19 62 computing the maximum number of traced generations; the number of complete traced generations; the number of complete equivalent generations ; and the quality of the genealogical information of the pedigree were determined after the calculation of the proportion of known parents through to great-great-great-great-grandparents (first to fifth generation inclusive).New-born annual median number, maximum and mean number of offspring per sire and dam were calculated. Pedigree completeness index (PCI), which summarizes the percentage of known ancestors of each ascending generation, was evaluated as in Navas et al.94 and the mean age of parents at offspring’s birth (selected for breeding or not), were calculated for each of the four gametic pathways: sire to son, sire to daughter, dam to son and dam to daughter. These parameters were obtained from the birthdate for every animal together with those of its parents. Female/male ratio was considered the relationship between total number of females and males in historical and current populations.Generation intervals were computed as the mean age of parents at the birthdate of their offspring selected for breedingF) was computed according to Luo95. The average relatedness (ΔR) of each individual or the probability that an allele randomly selected within the population belongs to a given animal, was obtained as proposed by Gutiérrez et al.91. The individual rate of inbreeding  per generation and average relatedness (ΔR) were used to issue regression equations fitting lineal, logarithmic and polynomic functions to predict for the evolution of inbreeding and relatedness up to fifteen generations onwards. Non-random mating (α) was calculated as described by Caballero and Toro98. Genetic Conservation Index (GCI) or the effective number of founder ancestors of each pedigree, was estimated as proposed by Alderson88.Individual inbreeding (fe) or founders equally contributing that are expected to generate the same genetic diversity that in the studied population, was computed as;qk is the probability of gene origin of the founder and f the real number of founders75.The effective number of founders (fa), founders or not, necessary to explain the entire genetic constitution of the population, was determined as;pk is the marginal contribution of an ancestor k, which means the contribution not explained yet by the rest of ancestors99. Both parameters (fe and fa) can be used to summarise the loss of genetic variability because of the non-proportional breeding animals’ contribution100.The minimum number of ancestors (fg) or the number of equally contributing founders without founder alleles loss that are expected to generate the same genetic diversity than in reference population (both parents known), was obtained by calculating the inverse of twice the average coancestry98.The effective number of founder genomes (j (the largest genetic contributing founders or not) was computed as the expected genetic contribution independently of the rest of ancestors’ contribution99.The expected marginal contribution of each major ancestor 101. An inbreeding loop exists when the ancestor of an individual is that by both maternal and paternal pathway. Mean effective population sizes  that form inbreeding loops, were obtained according to Colleau and Sargolzaei103 was assessed as the relationship between 104;The number of equivalent subpopulations98,The GD loss (GDL) in the population since the founder generation was estimated as 75.The difference between GD and GD* is referred to genetic drift accumulated since the foundation of the populationNef) was calculated as proposed by Caballero and Toro98 to describe the relationship between the effective number of founders and the number of equivalent genomes of founders.The effective number of non-founders (105 for each subpopulation (35) were calculated according to Caballero and Toro106. Wright’s F statistics allow pairwise comparisons among subpopulations or populations but those pairwise "distances" take account only of the data for the two populations concerned, not all the data simultaneously. Still this provides relevant information in the context of pedigree evaluation as the differences between both parameters may account for the estimation bias that may occur. For this reason and to quantify the degree to which populations differs from the entire pool of data using distance measures that make biological assumptions, Nei’s distances were used as well. Nei’s genetic distance69 between subpopulations i and j was computed as;ij is the average pairwise coancestry between individuals of the subpopulations i and j, including all Ni × Nj pairs. Cii and Cij are the average pairwise within subpopulations i and j, to assess interzoo relationships.The relationships between zoos were evaluated using Wright’s F statistics and Nei’s genetic distance. The Wright’s F statisticsAfterward, a simulation was made to determine the maximum limit of relatedness coefficient existing in the population between mated animals to determine which matings maintained (Ne = 50. Below these levels fitness of a population noteworthily decreases107. Relatedness coefficient (ΔR) can be defined as the probability that two individuals share an allele because of common ancestry. Relatedness coefficient (ΔR) of a pair of mating animals is the potential inbreeding coefficient of their potential offspring. This parameter ranges from 0 (unrelated) to 1 . This definition excludes alleles that are shared because of belonging to the same species or population.These levels of individual increase in inbreeding correspond to 96 was calculated by averaging the individual inbreeding increase through;ti is the number of complete equivalent generations108 and Fi the inbreeding coefficient of the individual i.Five mating groups were considered for the simulation. The average relatedness coefficient between mated animals was kept below 0.00%, 5.00%, 10.00%, 15.00% and 20.00% . The inbreeding coefficient of the offspring for each mating was estimated as one-half of the parental relationship coefficient. The inbreeding rate42 using SPSS Inc.109. Thirty replicates were evaluated within each group to calculate the average effective population size (Ne) as described by Gutiérrez et al.96.For each group, 17 random matings were selected, basing on the number of births in the last natural complete year (2018: 17 births) and on the assumption of one baby per femaleSupplementary Information.