Genetic purging in captive endangered ungulates with extremely low effective population sizes

We have analyzed the inbreeding-purging process in four captive populations of different ungulate species with effective sizes ranging 4–40 and with available pedigrees as well as survival and productivity records. This allows us to explore the role of inbreeding and purging in determining the evolution of fitness traits in a range of scenarios relevant in the context of conservation.

In A. lervia (Ne ≈ 4), purging is expected only for the most severely deleterious alleles (those giving dNe > 1, which implies d > 0.25 as, for example, in completely recessive alleles with deleterious homozygous disadvantage s > 0.5). Thus, it could be that purging has not been detected for this species because such severely deleterious alleles had been purged during the demographic decline in the wild, before the foundation of the captive population. This would be consistent with the low and non-significant inbreeding load estimated in this species. It is also possible that these estimates are non-significant due to the relatively small number of individuals available.

G. cuvieri and N. dama have significant initial inbreeding loads that, adding up the direct and maternal components, is about 1.25 in both cases, which is on the order of other estimates published for captive populations (Ralls et al. 1988). Since in both species Ne > 10, purging should be efficient against less severely deleterious alleles than in A. lervia (d > 0.1). Purging is detected for both species with very low P values. This result is in agreement with Moreno et al. (2015), who suggested that purging had occurred in G. cuvieri as they found an increased juvenile survival parallel to an increased inbreeding coefficient. The relative contribution of severe and mild deleterious effects to the inbreeding load of populations is under a scientific debate with direct implications in conservation biology (Ralls et al. 2020, Kyriazis et al. 2021, Pérez-Pereira et al. 2021). The large d estimates obtained in our analysis indicate that a substantial fraction of the initial inbreeding load is being purged under modest effective population sizes, implying that such substantial fraction is due to relatively severe deleterious mutations in these two populations. As far as we are aware, these are the first estimates of this purging parameter obtained in managed, non-experimental populations. Previous estimates of d were obtained in D. melanogaster bottlenecked populations, first for egg-to-pupae viability in lines with Ne = 6 or 12 under noncompetitive conditions (d = 0.09, Bersabé and García-Dorado 2013), and second in lines with higher Ne ≈ 40–50 under more competitive conditions, giving a larger estimate of d, of the order of that estimated in these two ungulate endangered species (d ≈ 0.3, López-Cortegano et al. 2016).

Regarding G. dorcas, given its larger population size, purging is expected even against alleles with mild recessive component of the deleterious effect (d > 0.025). However, although a significant (if modest) inbreeding load was estimated, no significant purging was detected. Nevertheless, the number of equivalent complete generations by the end of the pedigree (EqG = 7) was smaller than our proposed minimum number of generations required to detect purging (tm = 10). This suggests that, due to the large size of this population, more generations are needed to detect purging.

The results above support the use of tm to get an approximate idea about when a pedigree is too shallow for purging to be detected. Should the number of generations available be larger than tm, IP predictions could additionally be computed to search the d values that can be expected to produce detectable purging. Supplementary Fig. S3 shows that the true number of generations required to detect purging becomes increasingly larger than tm for alleles with smaller d values, as they suffer weaker purging each time they are exposed in homozygosis. The tm approach helps to understand the failure of many studies to detect purging. Such is the case of the extensive meta-analyses on 119 zoo populations by Boakes et al. (2007), where the median Ne value was 22.6 while the median number of generations was t = 3 meaning that, for most species, at least 5 more generations were needed before purging could be detectable. On the contrary, and in agreement with this tm approach, purging was experimentally detected in lines of D. melanogaster with Ne = 43 (i.e., tm ≈ 10) where, after an initial period of inbreeding depression, fitness experienced a substantial recovery beginning between generations 10 and 20 (López-Cortegano et al. 2016).

A reason why detecting purging in captive populations is challenging is that a fitness rebound can also be due to adaptation to captive conditions or to environmental effects, such as those derived from improved husbandry (Clifford et al. 2007). In fact, this might have been the case in Speke’s gazelle breeding program, where the observed rebound of fitness was first ascribed to purging (Templeton and Read 1984, 1998), while Kalinowski et al. (2000) suggested that husbandry improvements could also be responsible for these findings. Our estimates of d and δ, however, are based on the association between the fitness trait and purged inbreeding at the individual level (Wi, gi) which, in our data, is mainly expressed within cohorts while average survival showed little variation through time. In addition, the analyses included temporal factors (YOB or POM) that should have removed confounding effects from adaptation to captivity or improved husbandry. Therefore, adaptive processes or time-dependent environmental factors are not expected to have biased our IP estimates.

For productivity, the estimates of inbreeding load were high (overall inbreeding load ~5, P value < 10−8), but no significant purging was detected in any species (Supplementary Table S7). Note, however, this trait was assayed only in females, and only in those that had completed their reproductive life by the end of the pedigree (see Methods). This implies less statistical power than for survival and, more importantly, fewer generations for purging to occur (only about six generations in the case of G. cuvieri and N. dama, for which tm ≈ 6). Thus, detecting purging for productivity was in fact hardly expected.

Genomic data are useful to measure inbreeding and, therefore, to estimate inbreeding depression (Kardos et al. 2018). The footprint of slow purging has been detected as a reduction of the genomic burden of putatively deleterious alleles in populations that suffered historical bottlenecks (Xue et al. 2015; Grossen et al. 2020). In line with these advances, inferred functional genetic variation observed at the genomic level has been recently proposed as a tool to select individuals in conservation biology (Kyriazis et al. 2021; Teixeira and Huber 2021, but see Ralls et al. 2020; García-Dorado and Caballero 2021). However, the magnitude of the deleterious effect or of the potentially adaptive effects of most genomic variants cannot at present be inferred with any certainty, and there is yet no way to infer from genomic information the amount of purging accumulated in the ancestors of an individual. Therefore, the analysis of pedigreed fitness data is an essential tool to evaluate the fitness impact of purging during ex situ breeding and its conservation impact, although additional genomic analysis can be helpful as, for example, to infer previous demographic and selective processes.

The evaluation of the efficiency of purging may help to determine the minimum viable population size (MVP) that has been proposed as a rule of thumb in conservation guidelines. Classically, genetic considerations lead to a 50/500 MVP rule being proposed for the effective population size, where the lower figure was aimed to prevent excessive inbreeding depression in the short to medium term, while the larger one was intended for the long-term preservation of adaptive potential (Franklin 1980; Jamieson and Allendorf 2012). There has been some debate on the appropriateness of updating the rule to 100/1000 which, regarding the first figure, was based on the high estimates of the inbreeding load reported in the wild (δ ≈ 6, O’Grady et al. 2006; Frankham et al. 2014a, b; Franklin et al. 2014). However, our results illustrate that purging can be relevant even in populations with effective size scarcely over 10, where no evidence of purging during the first generations after a bottleneck is not indicative that purging will not be able to induce substantial fitness recovery later on. Thus, although increasing the size of endangered populations should always be a major aim, considering purging can lead to a more flexible value for the lower MVP figure regarding inbreeding depression. Although attention needs to be paid to the threat derived from the loss of genetic diversity and adaptive potential, these results encourage conservation efforts even on populations that seem stalled in a too small census (García-Dorado 2015; Caballero et al. 2017).

Due to the nature of the data, our results are particularly relevant to ex situ conservation. Since the facility at La Hoya allowed most surviving females to breed every year, our populations were maintained with the main priority of increasing population size. This strategy contrasts with the Minimum Kinship protocol (MK), where breeding adults are chosen to minimize the average coancestry of the progeny in order to maximize genetic diversity (Ballou and Lacy 1995; Fernández and Toro 1999). MK is recommended to preserve adaptive potential and to slow adaptation to captive conditions, but implies setting the number of breeding offspring contributed by each parent, which becomes independent of the parents’ reproductive fitness. This leads to some relaxation of natural selection with different consequences for survival and reproductive fitness: MK is favorable regarding survival, since both inbreeding and purging (which occurs just within families) are slowed; MK relaxes purging for reproductive fitness, so that it could be risky in the medium to long term (García-Dorado 2012). On the contrary, the managing protocol followed at La Hoya should have allowed genetic purging for both survival and reproductive fitness. The detection of purging when the data were appropriate (survival in Cuvier´s and Dama gazelles), together with recent evidence that productivity has not declined in Cuvier’s gazelle despite its high initial inbreeding load (Moreno et al. 2020), suggests that purging may have also occurred for productivity but was not detected due to the limited sample size and pedigree depth.

Our results illustrate that, when it is necessary to decide which individuals are going to breed, some compromise may be necessary between two extreme options: (i) allowing breeders to contribute breeding offspring proportionally to their observed reproductive fitness in order to favor purging; (ii) choosing breeders according to MK to maximize genetic diversity and slow inbreeding. Such compromise should be dependent upon the population size, the inbreeding load (particularly that for reproductive traits), the average reproductive potential, and the time horizon of the captive breeding program. If the population size is so small that inbreeding depression and loss of adaptive potential are an immediate concern, MK is to be advised. If the effective population size is on the order of several tens, it could be advisable to assign some weight to productivity when choosing breeding individuals instead of relying exclusively on minimizing average kinship or even, in some cases, to move toward a breeding system with no genetic management, as under reintroduction. This could prevent the medium to long-term consequences of the relaxation of purging for reproductive traits.

Selection of breeding individuals and the management of their mating have different consequences and should better be established through a two-step protocol (Fernández and Caballero 2001). Mating management is usually intended to avoid inbred mating, which is expected to reduce homozygosis. This implies less inbreeding depression in the first generations, but also less-efficient purging and no additional long-term protection of adaptive potential, as well as some increase in genetic drift (Caballero and Toro 2000). This mating management should in principle be applied as far as population growth is compromised by immediate fitness inbreeding depression, as in a very small population with high inbreeding load. In the case of our populations, from the mid-1980s onwards, matings at La Hoya have been managed to minimize coancestry between pairing individuals in order to reduce inbreeding in the offspring, a strategy that should reduce the efficiency of purging. However, based on the trend of more-inbred mothers to produce more surviving daughters than sons (Moreno et al. 2011) and in agreement with other authors (Tella 2001), beginning in 2006 more-inbred females were promoted to be the ones mating males with lower coancestry in G. cuvieri (Moreno et al. 2015). This implies that: (i) they contribute offspring with lower inbreeding and, therefore, higher survival; (ii) due to their mothers’ high inbreeding, these offspring are more purged and, those surviving, also show a higher female/male ratio. Thus, very likely, this method, besides producing a more convenient sex ratio for the management of polygynous species, has improved purging opportunities. The high purging detected in this population suggests that the reduction of purging from minimum coancestry mating has been mitigated by favoring the breeding contribution of more-inbred females, which should have also contributed to the maintenance of genetic diversity (Caballero and Toro 2000). In ex situ conservation programs, alternative mating strategies have been proposed to improve purging while controlling inbreeding depression, as in the case of circular mating (Theodoru and Couvet 2015). However, when considering such strategies, the extinction risk from short-term inbreeding depression should be carefully evaluated considering the population reproductive potential (Caballero et al. 2017).

As Tudge (1991) asserts, the proper end point of captive breeding is reintroduction. So, the success of the above recommendations in captive breeding programs depends on how far purging occurring in captive conditions will reduce the inbreeding depression expressed in the wild. Obviously, purging during ex situ conservation is not expected to act upon traits that are not expressed in captivity but that could be fitness components in the wild. For example, the inbreeding load for heat shock resistance in Drosophila was not purged in lines that had been maintained in stable lab conditions (Bundgaard et al. 2021). However, mutation is expected to produce unconditionally deleterious alleles much more often than alleles that are deleterious in some conditions but advantageous in others. This is not in contradiction with the notion that adaptation to captive conditions should entail some maladaptation in the wild, but implies that the genetic basis of adaptive tradeoffs may contribute a small fraction of the inbreeding load. Thus, the purging of such unconditional deleterious alleles occurred ex situ should, to some extent, be expressed in the wild. Furthermore, it is usually considered that the deleterious effects of mutations tend to be larger when expressed in harsh environments (Halligan and Keightley 2009), accounting for a larger inbreeding load when measured in wild conditions (Keller and Waller 2002; Armbruster and Reed 2005; Fox and Reed 2011). For example, López-Cortegano et al. (2016) found that purging under competitive conditions was efficient against inbreeding depression expressed for both competitive and noncompetitive fitness, and their results suggested that the larger inbreeding load estimated in competitive conditions could be mainly ascribed to the same deleterious alleles as in noncompetitive ones but with larger effects. In such situation, although purging is expected to be more efficient if it occurs in the wild, even purging in captivity could remove a substantial fraction of the inbreeding load to be expressed in wild conditions (Swindell and Bouzat 2006b). In our case, the successful recent reintroduction of G. cuvieri in Tunisia (Moreno et al. 2020) has produced a healthy population with vigorous productivity since 2016 and that shows no apparent sign of inbreeding depression, suggesting that purging in captivity has reduced the inbreeding depression expressed in wild conditions Nevertheless, additional research is required regarding the consequences of purging in captive conditions on population survival after reintroduction.

Overall, the large d estimates obtained for survival in G. cuvieri and N. dama, the two species where Ne and the time scale provided opportunity for purging detection, illustrate that a large fraction of the inbreeding load can be purged when breeding contributions are governed by natural selection. Our results suggest that, during ex situ conservation, it may be appropriate to progressively move to a breeding strategy allowing for purging on productivity based either on management protocols that take this trait into account or on reduced breeding intervention allowing selection to act. The latter option encourages early reintroduction efforts, with the additional advantage of allowing purging to occur under wild conditions. However, this should be considered only after an initial ex situ phase of demographic recovery (say, Ne above a few tens), so that the loss of adaptive potential and the early inbreeding depression are not threatening and purging is reasonably efficient.

Source: Ecology -

Macroecological distributions of gene variants highlight the functional organization of soil microbial systems

Making roadway spending more sustainable