Identification of relationships between demography and genetic variation is a crucial but difficult task in conservation and management of exploited or endangered organisms, especially those subject to hatchery-based supplementation, and/or those that experience highly variable population sizes and fluctuating environmental conditions. Here, we evaluated the ability of genetic monitoring to recover such potentially complex interactions in a species that has been subject to all three of these effects in recent times. Our study used long-term, empirical data from genetic (1987, 1999–2010) and population monitoring (1993–2010) in an attempt to synthesize this information with temporal records of environmental conditions, captive breeding, and wild population supplementation. A notable increase in mean values of key diversity metrics and a coincident decrease in inter-annual variability in these metrics indicated that population supplementation has been the single most important factor that has influenced trajectories of genetic diversity over the last decade in Rio Grande silvery minnow. Large-scale repatriation of hatchery-reared fishes into the Rio Grande has potentially obscured (and ameliorated) most genetic effects of density fluctuations and severe environmental conditions in the wild population. One important exception is that very low values of NeV are consistently observed in the wild population, suggesting that underlying causes of genetic decline continue to operate despite supplementation.
Population density, genetic diversity, and effective size
Extensive demographic surveys show that the wild population of Rio Grande silvery minnow has experienced multiple changes in density that exceed an order of magnitude over the past two decades (U.S. Fish and Wildlife Service 2010). From 2000 to 2004, densities of Rio Grande silvery minnow were less than one fish per 100 m2, and during this time, the threat of extinction in the wild was acute. A key premise of genetic monitoring is that population declines will be accompanied by erosion of genetic diversity and reduction in genetic effective size. However, in Rio Grande silvery minnow, strong positive relationships between population density, genetic metrics, and effective size were not observed over the 12-year time frame of the study.
It is apparent for both microsatellites and mtDNA that there is considerable inter-annual variability in gene diversity metrics and effective size estimates from 1987 and 1999–2004 (Fig. 1). However, following the onset of population supplementation with captively reared fishes, the general trend was toward stabilization and marginal increases in mtDNA and microsatellite diversity and number of alleles/haplotypes. Inter-annual variability in all of these measures decreased after 2005.
Temporal method estimates of genetic effective size remained very low over the entire study but increased slightly in more recent samples. Three estimates of NeV (derived from TempoFs) revealed an effective size of <100 (1999–2000, 2001–2002, 2002–2003), and five (TempoFs) and three (moments) comparisons were <200 during periods of low wild fish density (<1 to ∼6 fish per 100 m2) and no supplementation. From 2004 to 2010, estimates of NeV remained low [mean, 180 (moments)-422 (mlne)] but less variable (range, 115–307) than in the 1987–2003 period (range, 368–1186). For more recent comparisons (2008–2010), estimates of NeV from moments and TempoFs methods converged. Although mlne estimates were typically larger than those based on TempoFs or moments, they nevertheless indicate a decline in mean NeV from (1987–2004 = 842) comparisons to recent estimates (2004–2010 = 422). One estimate based on TempoFs is larger (NeV = 433) than other estimates obtained using this method and was recorded over a time frame (2003–2004) where some of the lowest densities of Rio Grande silvery minnow occurred. This result is somewhat counterintuitive, as this increase pre-dated a large boost in wild population densities in 2005 that resulted from favorable flow conditions in spring of that year and absence of river intermittency during the summer.
There are at least two reasons for the apparent disconnection of population density, genetic diversity, and effective size in Rio Grande silvery minnow. First, it may take several generations of very small population sizes for diversity to be depleted enough to be detected by genetic monitoring. It is well established that heterozygosity is a relatively insensitive indicator of population bottlenecks (Allendorf 1986), even ones of extremely small size. Secondly, it is an explicit goal of the supportive breeding program for Rio Grande silvery minnow to maintain genetic variability (USFWS 2009), so the lack of correlation between density and genetic diversity is likely due to supplementation practices and successful breeding of released fish. Supportive breeding may also explain lack of correlation of density and Ne estimates (discussed below).
Population supplementation, diversity, and effective size
Supportive breeding has the potential to maintain diversity and to increase the effective population size either by increasing abundance or by reducing variance in reproductive success among parents (Ryman and Laikre 1991). In contrast, it may deplete genetic variation (Tessier et al. 1997) and depress the effective size (Ryman and Laikre 1991), depending on the genetic composition of fish repatriated from captivity. Ryman et al. (1995) found that risks to genetic diversity and effective size were greatest for species capable of producing large numbers of offspring in captivity (i.e., that exhibit type III survivorship); this characterizes the life history strategy of Rio Grande silvery minnow. We found that wild samples collected after the onset of population supplementation had significantly greater microsatellite diversity (Hec and Nac) and mitochondrial diversity (HR) than those prior to supplementation, which supports the prediction that supplementation buffers the population against loss of genetic diversity following bottlenecks in Rio Grande silvery minnow. This may be because the supportive breeding program for Rio Grande silvery minnow differs in some respects from traditional hatchery programs that spawn a small portion of the wild population in captivity. In Rio Grande silvery minnow, the preferred source of fish for population supplementation are eggs collected from natural spawning events. These egg collections should represent reproductive effort of a large segment of the wild population and therefore have the potential to capture a representative sample of its genetic variability. For example in 2002, more than 900 000 eggs were collected from the wild, and 230 000 of them were repatriated to the Rio Grande between 2003 and 2004. Some were retained by conservation hatcheries in New Mexico for use as the founding captive broodstock. Subsequent wild-breeding of captive-released fish likely made a significant genetic contribution to the wild population, and collection of eggs prior (2001–2003) to the population collapse that occurred from 2002 to 2004 may have helped to preserve diversity that would otherwise have been lost during these severe population contractions. Similarly, maintenance of diversity through periods of population supplementation has been demonstrated in both Chinook and Chum salmon (Eldridge and Killebrew 2008; Small et al. 2009).
Supportive breeding aims to reduce early-life mortality and associated variance in reproductive success, or the ‘sweepstakes mismatch’ process (Hedgecock 1994), that characterizes reproduction in Rio Grande silvery minnow in its currently fragmented habitat (Osborne et al. 2005; Turner et al. 2006). Although type III survivorship and a consequent small Ne:Nc ratio (Alò and Turner 2005) are typical for the species in the wild, supportive breeding likely ameliorated characteristically high variance in reproductive success by capturing a large portion of the species’ reproductive effort before it is transported to unsuitable habitat (Turner et al. 2006) and then rearing these progeny in protective custody. It is plausible that supplementation explains the observed increase in NeV for 2003–2004 in the wild. Wild population densities were sufficiently low in 2003 such that repatriated fish probably comprised a large portion of the population. Adult fish released to the wild from captivity in 2003 and 2004 were derived from wild-caught eggs collected from 2001 to 2003. Temporal comparison of allele frequencies between 2003 and 2004 was therefore based largely on progeny of repatriated fish and not wild fish, and thus yielded larger estimates of NeV.
Between 2005 and 2010, estimates of NeV were smaller than for the 2003–2004 comparison. During this time, densities of Rio Grande silvery minnow in the wild fluctuated greatly with densities ranging from a high of nearly 37 fish per 100 m2 (2005) to around one fish per 100 m2 (2006 and 2010). Over one million captive-spawned fish were released compared to only around 26 000 progeny of wild-caught and captive-raised eggs during the same time period. These fish were progeny of a captive broodstock derived from eggs collected in 2002. In years when recruitment of wild fish was poor (e.g., in 2006), progeny (produced in the wild) of captive-bred fish potentially comprised a disproportionate fraction of the subsequent generation. Despite efforts to maximize diversity of captive stocks, observed depression of Ne could reflect a Ryman–Laikre effect (Ryman and Laikre 1991), which occurs when variance in reproductive success of a population is increased owing to disproportionate contribution of offspring from relatively few captive breeders.
There are alternative hypotheses that could explain the apparent discrepancy between Ne and density in Rio Grande silvery minnow. For example, density-dependent effects such as genetic compensation can also cause Ne to be decoupled from Nc (Palstra et al. 2009; Saarinen et al. 2010). Compensation occurs when reduction in the effective number of breeders (Nb) is counterbalanced by reduced competition for mates or spawning sites when Nc is small, reducing among-spawner differences in offspring survivorship. It is not possible to distinguish between these two effects (compensation versus supplementation) with the current data set. However, stabilization of genetic diversity metrics and NeV coincides with the onset of supplementation and occurs despite dramatic fluctuations in the wild. Thus, supplementation appears to be the most plausible explanation for the results.
Inter-relationships of effective size estimates
NeV estimates obtained using different methods should be positively inter-correlated, but this was not observed for values obtained over the entire 12-year time series. However, after the commencement of supplementation, all three commonly used estimators of NeV produced positively correlated values. Lack of correlation overall may be due to known biases of methods used to estimate NeV (e.g., Waples 1989; Turner et al. 2001; Wang 2001; Jorde and Ryman 2007). Likewise, NeV and NeD estimates were not correlated for any period; this result is not unexpected, as these estimates use different aspects of the data to estimate variance and inbreeding effective size.
Estimators of NeV used in this study are subject to specific biases that influence accuracy and precision in different ways. For example, mlne tends to overestimate Ne when calculated from loci with highly skewed allele frequencies (Jorde and Ryman 2007) and can provide imprecise estimates in nonequilibrium populations (Wang 2001). Both Waples (1989) and Turner et al. (2001) noted that moments estimates obtained using the most commonly employed measures of allele frequency change (Nei and Tajima 1981; Pollak 1983) tended to be downward biased (resulting in overestimates of Ne) when allele frequencies are close to zero or one. Jorde and Ryman (2007) and Antao et al. (2010) also noted that unbiased estimates of effective size (TempoFs) may come at the cost of precision, with wider confidence intervals than moments estimates. Regardless, in all except two cases (2003, 2004) for which sufficient samples were available, noninfinite upper-bound CIs were obtained.
Several benchmarks for ascertaining extinction risk have been suggested for interpreting effective size in threatened species. The most conservative targets deemed necessary to maintain long-term genetic security of a species range from an effective size of 500–5000 (Franklin and Frankham 1998; Lynch and Lande 1998). Regardless of potential biases, all NeV estimates suggest values of Ne that are smaller than the minimum benchmark of Ne = 500 in seven of eight (mlne) and all (moments) of the most recent temporal comparisons.
Values of NeD, which provide a measure of the inbreeding effective size, were uniformly higher than and not positively correlated with estimates of NeV. The underlying principle of the LD method is that as Ne decreases, genetic drift increases nonrandom association among alleles at different loci (Hill 1981). As erosion of linkage disequilibrium can take several generations, NeD may also contain information on the effective size from several generations that precede a population decline. In addition to this upward bias, single-sample Ne estimators including NeD provide an estimate of the effective number of parents that produced the progeny from which the sample is drawn (Waples 2005). Estimates of NeV and NeD were paired accordingly (refer to Methods for details) to account for this potential source of bias, so this is unlikely to explain the lack of correlation between NeV and NeD estimates.
Using computer simulations, Antao et al. (2010) evaluated the ability of NeD and NeV estimators to (i) detect a population decline, (ii) correctly identify a bottleneck with low bias and high precision, and (iii) evaluate whether the methods were subject to a high rate of false positives (i.e., indicate a bottleneck when none had occurred). They found that temporal-method estimates of NeV were very close to the bottlenecked population size in the first generation, whereas NeD always overestimated Ne with relatively low precision. Moreover, in the first generation following a decline, values of NeD were much closer to the prebottleneck population size. This result is consistent with the idea that estimates of NeD include information on the effective size of previous generations (Waples 2005).
From a management perspective, there are a number of theoretical and practical distinctions between NeI (to which NeD estimates are most closely associated) and NeV. These two measures should be similar in stable populations but show predictable differences in declining (or growing) populations. Waples (2002) demonstrated that in declining populations, NeV is reduced more rapidly than NeI, and, as a consequence, NeV will be smaller than NeI until equilibrium is reached. Conversely, Waples (2002) found the opposite for increasing populations, as increasing population size rapidly attenuates the magnitude of genetic drift, but inbreeding effects persist longer because of inherent relatedness among individuals derived from a bottlenecked (or reduced) population. Thus, the observed discrepancy between NeV and NeI in Rio Grande silvery minnow is precisely the signature expected for a declining population. In fact, the only significant correlation we found between NeV (mlne) and NeD was negative (r = −0.69, P = 0.047). At present, Rio Grande silvery minnow are subject to source–sink dynamics. Specifically, captive stocks (source) contribute breeders to the wild (sink) each year, where reproductive success and recruitment are highly variable. Under these circumstances, we would expect discrepancy between NeV and NeD. Such dynamics are also likely to occur in other endangered but highly fecund species, especially fishes.
Although supplementation could confound relationships between NeV and NeI in a declining population, theoretical and empirical treatments of this issue are too limited to provide firm guidance with respect to Rio Grande silvery minnow. For example, a theoretical evaluation by Ryman et al. (1995) was limited to cases of critically low census sizes (Nc < 50); therefore, it is unclear how effects on severely bottlenecked populations relate to supplemented populations in which thousands to millions of individuals potentially contribute to annual reproduction, which is the case for Rio Grande silvery minnow. However, Waples and Do (1994), based on empirical evaluation of supplemented Pacific salmon, found that the most important factor that determines effects on NeI after supplementation is whether the population maintains a large size; this finding is consistent with observations of larger NeI than NeV in the hatchery supplemented Rio Grande silvery minnow. A simulation study is currently underway (by EWC, MJO and TFT) to determine relationships between NeI, NeV, and supplementation in this species.
The value of NeD dropped substantially in 2004 (NeD = 595) from estimates of ∼2000 in prior years. This coincided with very poor wild recruitment into the 2002 and 2003 year-classes. Subsequent increases in NeD ranged from 2000 to 4400 and coincided with an increase in wild fish densities (2005), the input of large numbers of captive-spawned fish (∼106 between 2005 and 2010), and somewhat more favorable environmental conditions (e.g., less extensive channel drying).
Estimates of Ne varied across methods, but all estimators indicated a genetic effective size that is one or more orders of magnitude smaller than the census size (estimated between ∼24 000 and 3.5 million, Dudley et al. 2011). In contrast to the NeD estimates (which were generally in the 1000’s), all but three NeV estimates (across all estimators) were lower than 500 and three of the unbiased estimates from TempoFs were <100. We can conclude, therefore, that the genetic effective size of the wild Rio Grande silvery minnow population is smaller than expected from census size. Relatively large values of NeD reflect a population in decline rather than evidence of robust genetic resistance to extinction.
The results are consistent with our previous studies that have shown that NeV in wild Rio Grande silvery minnow is up to three orders of magnitude lower than adult census size (Alò and Turner 2005; Turner et al. 2006; Osborne et al. 2005). To explain this result, we proposed a model whereby the vast majority of reproductive output from spatially discrete spawning aggregations, comprised of semi-buoyant eggs and larvae, move passively downstream, past dams to relatively poor nursery habitat (such as reservoirs) or areas with a higher propensity for channel drying (such as the San Acacia reach). Once displaced, progeny either fail to recruit or cannot migrate back upstream to the natal reach. The model predicts that negative effects of downstream transport of reproductive output on NeV are largely density independent. In other words, loss of productivity and variance among spawning aggregates in the wild should persist despite enormous supplementation from captive sources. Low values of NeV observed in the wild prior to and after the onset of supplementation support this idea. In the absence of supplementation, we would also expect substantial losses of genetic diversity and values of NeD to converge with those obtained from NeV estimators if this model is correct.
The downstream distance travelled by Rio Grande silvery minnow eggs and larvae is determined, in part, by development time required for hatching and transition from a yolk-sac larva to a free-swimming stage. Time required usually exceeds 4 days, and downstream drift distances can exceed 100 kms (Dudley and Platania 2007b). Passively drifting propagules are swept past diversion dams that occur roughly every 60–90 kms in the current range of the species. In other species of pelagophiles, and likely in Rio Grande silvery minnow prior to supplementation, diversion dams are highly likely to be responsible for population declines in upstream reaches because these structures prevent upstream movement of any spawned fish displaced over dams as eggs or larvae (Dudley and Platania 2007b). Hence, genetic diversity in subpopulations in upstream reaches should be eroded in the absence of inputs from the hatchery or downstream sources and will eventually impact the entire population if upstream subpopulations represent a source and the downstream subpopulations act as a sink. In the Rio Grande, there are also reach-specific environmental effects such that flow conditions are more reliable in the Angostura reach, but drying is more likely in the San Acacia reach. These dynamics have predictable effects on genetic diversity. For example, mtDNA diversity in Rio Grande silvery minnow is highly variable across the time series in the San Acacia reach, whilst there appears to be more stability in the Isleta reach. The Isleta reach is less subject (compared to the San Acacia reach) to severe drying events.
Broodstock effective size and NeD
As predicted, a significant and positive correlation was observed between NeD estimates and the number of broodstock used for matings in captive brood lots. Likewise, stocks reared from wild-caught eggs tended to have larger effective sizes than those produced through captive spawning. This is not surprising as wild-caught eggs should reflect a large number of wild parents. Interestingly, many NeD estimates were less than the broodstock census size. A number of these instances involved paired matings, which suggests that the linkage disequilibrium method may underestimate the true effective size or alternatively, and perhaps more likely, that there is some variance in reproductive success (i.e., not all breeding pairs contribute equally) among captive spawners.