The intentional and unintentional spread of non-native species is contributing to the global reduction of plant diversity and the homogenization of plant communities [1
]. Non-native insect pests and pathogens represent major destructive forces in forests, impacting productivity, biogeochemical cycling, hydrology and forest successional dynamics [3
]. Conserving the genetic diversity of imperiled forest tree species is essential for maintaining the long-term sustainability and resilience of forest ecosystems. Genetic diversity provides the basis for adaptation to environmental and anthropogenic change [6
], increases ecosystem stability and resilience [7
], and promotes species diversity [9
]. Particularly influential is the genetic diversity of foundational species, such as large trees. Although it is well established that conserving genetic diversity is crucial in preventing the extinction of populations and species, very few management plans incorporate genetic monitoring in their action plans [10
Conserving genetic diversity typically relies on both an ex situ approach, in which a species is preserved outside of its natural habitat, and an in situ approach, in which viable, reproducing populations are maintained within the species’ natural habitat. Ex situ conservation can involve either maintenance of living trees, or storing seeds in long term storage such as a seed bank. Although ex situ approaches have the potential to save a majority of a species’ diversity, they also have their limitations. Seed banks only work for species with orthodox seeds, and accessions may be compromised either during the collection (selection bias) [11
] or storage (e.g., seed death, mutation accumulation). Likewise, trees maintained in living collections require large planting areas, diversity can be lost through cycles of regeneration, and there is potential for adaptation to cultivated conditions [12
]. The value of ex situ collections may also be limited if it is not associated with any in situ strategies. Reintroductions, with ex situ material, may be hampered by the degradation of underlying ecosystem properties associated with the initial disturbance and extirpation and loss of in situ mutualists, like pollinators, seed dispersers, and fungi. Recent evidence suggests that Fraxinus
are associated with unique soil microbial assemblages which may be altered if ash were to be lost from a forest ecosystem [14
]. Additionally, black ash (Fraxinus nigra
Marshall) has been shown to help to maintain the hydraulic balance of sensitive wetland forests, and its loss from these systems because of the invasive emerald ash borer (Agrilus planipennis
Fairmaire, EAB) may prevent the future establishment of black ash and other tree species [15
]. Therefore, in situ conservation is often the preferred means of conserving a species, especially when used in conjunction with ex situ conservation.
Refinement of in situ approaches for maintaining imperiled species is required if managers are to maximize the genetic diversity conserved while minimizing costs. Developing such tools is increasingly necessary as outbreaks of forest pest and pathogen become more prevalent and result in widespread disturbances [16
]. EAB, a non-native forest pest accidentally introduced into North America in the 1990’s, has spread from south eastern Michigan to over 31 states, resulting in a dramatic decline in numbers of ash trees [17
]. There are 16 native species of ash trees (Fraxinus
spp.) across North America [18
], which collectively represent ~2.5% of the trees in the United States, with the greatest density and abundance in the Great Lakes Region [19
]. EAB larvae, which feed predominately on phloem and cambial tissue, create serpentine galleries that effectively girdle host trees [20
] and result in >99% tree mortality (for black, white and green ash see [22
], relative to ~71% survival for blue ash see [24
]). Due to this infestation, five ash species are now listed as critically endangered on the International Union for Conservation of Nature red list (F. pennsylvanica
Marshall, F. americana, F. nigra, F. profunda
(Bush) Bush, and F. quadrangulata
Michx.) and one is listed as endangered (F. caroliniana
]. The loss of ash trees has been estimated to cost upwards of $
60 billion USD, not including replacement costs [26
], which will be significant in cities like Chicago, where there were significant numbers of ash trees (>600,000). Because of the lethal effects and speed with which EAB is spreading, managers need new tools to mitigate the impact.
The threat to ash trees has led to the development of a combined ex situ and in situ conservation approach. Ex situ methods include seed collection and storage, which have yielded collections from 1982 ash trees from five species (US National Plant Germplasm System). Meanwhile, the in situ methods include insecticide treatment of standing trees to protect them from future EAB attacks. In situ preservation will help maintain breeding populations of large trees in the landscape, thereby protecting ecosystems services, helping to ensure future ash reintroduction, and allowing continued natural selection and adaptation. Evidence from early in situ Fraxinus
conservation efforts suggest a degree of associational protection, in which insecticide treatment of a small number of ash trees in a stand can promote the health of untreated trees [27
]. However, the optimal treatment densities to achieve maximal associational protection remain unknown and are under investigation [28
]. While insecticides have been used frequently for protection of urban street trees and yard trees, these cultivated ash are not representative of, and do not help to preserve, natural levels of genetic diversity. To address this concern, insecticide treatment has been expanded to multiple, naturally-occurring populations, managed by a diverse assemblage of landowners. Multiple insecticide formulations are available, but all require repeated treatments (yearly or every 3 years, depending on the insecticide used). The cost of emamectin benzoate injections, which have been demonstrated to be most effective at controlling EAB [29
], range from $
50 to $
150 USD per tree, depending on tree diameter. Multiple entities have expressed interest in expanding these efforts; however, practical information is needed to help managers maximize the benefits of insecticide treatments while minimizing resource use.
As treating all trees in an area is usually not an option, a subset of trees will need to be chosen for treatment. If the objective is to maintain local standing genetic diversity, population models suggest that treating more trees will maximize the chance of conserving a substantial portion of the local alleles [31
]. However, the relationship between allelic diversity and sample size is non-linear, hence there is a point at which additional individuals provide minimal gains in diversity. Ex situ seed collections protocols suggest that in order to maximize the local diversity collected, you need to sample a minimum of 50 unrelated maternal trees per population, and repeat for as many populations as possible [33
]. However, the appropriate design of a conservation collection depends on various aspects of the target species, including population size, reproductive biology, recent population history, and connectivity among populations [34
]. Given that it is nearly impossible to preserve all genetic material, determining the optimum number and spatial arrangement of treated trees will be important in efforts to conserve the majority of local genetic variation.
Here we investigated the genetic diversity of white ash trees (F. americana) across the Allegheny National Forest (ANF), and utilized the results to optimize a regional in situ conservation plan. Our goals were specifically to: (1) determine the genetic diversity and structure of white ash populations across the ANF; (2) quantify how varying levels of treatment (number of trees and populations) will affect the preservation of ash genetic diversity; and (3) formulate optimal strategies, including identifying which populations preserve the greatest proportion of genetic diversity. To achieve this, we conducted a genetic survey of white ash trees, then used computer algorithms to subsample the dataset with different possible sampling strategies (100 plants, 20 plants, etc.). Finally, we discuss implications for white ash, as well as caveats to this approach.
In this study, we have found that the optimal tree sampling strategy identified here, i.e., treating more populations, rather than more trees per population, maximizes conservation of genetic diversity of ash while reducing management costs by >20%. The F. americana
populations of the Allegheny National Forest showed minimal genetic structure (in terms of low Fst), suggesting that genetic diversity within white ash is evenly distributed across the region. Efficiencies may be even higher in forests with more structured populations. The cost savings associated with reduced treatment expenses can easily offset the costs of genetic analyses. The low genetic structure and high genetic diversity observed herein compliments findings in other ash species (i.e., F. excelsior
]), and is consistent with other common, wind-pollinated tree species (oaks [58
] such as pine [60
] and beech [62
]). This likely reflects adequate gene flow between populations, common in species with wind-dispersed pollen and seeds. Hence, we suggest that our findings on landscape population patterns and management may be transferable to conservation efforts of other wind dispersed tree species. However, in complex landscapes with high levels of environmental variability, a high degree of adaptation to local environments may occur.
A number of studies have examined how many trees and populations to sample for ex situ conservation, both empirically [34
], and theoretically [33
]. These studies have shown that appropriate sampling strategies depend on the species and the situation. They have also provided several consistent messages: (1) a relatively small number of well-chosen samples can preserve most of the genetic diversity; (2) many samples are needed to preserve all of the genetic diversity; and (3) diminishing returns are observed as more samples are added. More importantly, protecting multiple populations is almost always necessary to capture most of the genetic diversity. These results are consistent with these commonalities from previous studies. We found that even for a species with moderately low genetic differentiation across populations, protecting more populations is more valuable for maintaining a maximum level of genetic diversity. Our study helps address the paucity of specific information aimed at designing an optimal in situ or ex situ strategy for conserving the genetic variation in plants (though see [66
]), and provides the first gene conservation strategy for designing in situ pest management approaches.
We show that for F. americana
in the Allegheny region, the current strategy of treating 536 trees from 27 plots (of which 10 plots were in our analyzed data) will likely protect the majority of the known genetic variation, based on our samples. The alleles that are not protected are typically rare (frequency less than 0.01), or are restricted to, but common in, one population (locally common). We demonstrate that protection of a larger number of populations with insecticide treatment is more useful and cost effective than a treatment applied to more trees per population. Protecting numerous populations has the additional benefit of ensuring against disturbances caused by fire, windstorms, or other local environmental change. However, the number of trees protected per population should still be substantial (20 or more if possible) so that future seed production will remain highly heterozygous. To test whether it is possible to reduce costs without sacrificing genetic diversity, we examined alternative strategies for treating fewer trees (thus reducing the amount of insecticide needed), or fewer populations (reducing travel time and transportation costs for treatment). Our results show that protecting the same number of trees but in fewer populations would result in a small loss (2%) of diversity, but a larger loss (13%) in locally common diversity. An attempt to reduce cost expenditures further might consider reducing the number of individuals treated by half; this would result in a larger loss (16%) of diversity (primarily rare alleles). Lastly, we demonstrate that an optimal set of populations can be identified that could result in greater genetic preservation and cost reductions. This is likely due to the principle of complementarity; an optimal set of populations has minimal overlap in alleles, and thus each population complements the others [34
]. Considering that it was possible to identify optimal strategies for F. americana
, a common and wind-pollinated species with low genetic structure, this method should provide even greater benefit for species with higher genetic structure.
While we consider our results to be robust in these main findings, we need to be cautious as we used a small number of neutral microsatellite markers and a small sample size of untreated trees. While neutral markers are good at detecting effective population sizes and migration rates, their connection to fitness and “adaptive potential” is more tenuous. A number of studies have demonstrated that with low differentiation there can still be extensive between-population differences in adaptive traits [60
]. Thus, there may be adaptive genetic differences that were not captured in our study, nor might they be captured in a treatment program guided by neutral markers; indeed, capturing them is a difficult task, regardless of methods used [69
]. Despite this limitation, neutral markers are the most cost-effective tools available, and alleles are the only “currency” we have for measuring the gain in genetic diversity from a conservation action. In addition, sparse densities of ash as well as EAB induced mortality in the region limited the number of untreated trees available for the study. The exact estimates of the ‘proportion of genetic variation’ preserved for a given number of trees would decrease as more of the untreated trees were genotyped, and future studies may benefit from collecting additional samples before EAB expands to those areas (Figure 6
). An accumulation curve of resampling our datasets, adding individuals one by one, supports this supposition; there are likely many more rare alleles in existence, but the other categories have likely reached an asymptote (Figure 6
). It is important to note that the broad conclusions about sampling more trees or more populations derived from our approach is unlikely to be changed by using other marker approaches or increasing sampling size.
Natural demographic loss is an important consideration when determining an optimal number of populations and trees for treatment. With ex situ seed collections, the minimum number of seeds needed to preserve genetic variation is increased to account for germination and recruitment failures [70
]. For our in situ treatment, the main dangers include the loss of populations due to abiotic disturbances (e.g., fire or windstorms), or the lack of treatment efficacy [30
]. A recent estimate of disturbance levels gleaned from Forest Inventory and Analysis Data suggests that in the eastern US, approximately 3% of all trees are lost across a 5-year period [71
]. Although these disturbance levels are relatively low, we suggest factoring disturbance into treatment plans, in order to help ensure that the 27 treated populations persist long term (i.e., 100 years). This may be achieved through a moderate increase in the number of populations treated (5 to 10 additional populations), and an increase in individuals per population (to perhaps 25), as well as the wide spatial distribution of populations. A more detailed regional analysis of mortality frequency from past catastrophic events may be useful to determine the likelihood that sufficient treated populations and trees will survive over the long term. Insecticide treatment efficacy should also be considered when deciding how many individuals to treat within a population. Because treatment efficacy decreases in less healthy trees [30
], more trees must be treated in strains that are already in decline, to ensure the survival of the target number of trees for genetic conservation. In sum, our analysis represents a minimum number of white ash trees and populations to treat based on standing genetic diversity; as some trees will be lost to disturbance or insecticide failure, this minimum number could be increased to ensure that a minimum persists into the future.
A more complete genotyping of the populations containing fewer samples would help fully verify these results. Sequencing a large number of genes with a known function would provide an important complement to this work, by providing information on functional genetic variation [72
]. Additionally, layering in a biological control strategy may help mitigate EAB pressures while contributing to the maintenance of ecosystem stability [73
]. As such, efforts should be made to incorporate insecticide treatments into broader in situ or combined in situ and ex situ conservation program(s) [74
]. Lastly, including information on the soil, vegetation, slope and other environmental variables may be useful to ensure the preservation of ecologically relevant adaptations, by demonstrating that treated trees are found through the full environmental range of the species.