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Evidence for Population Differentiation in the Bog Buckmoth of New York State
Janet Buckner, Amy B. Welsh, and Karen R. Sime

Northeastern Naturalist, Volume 21, Issue 4 (2014): 506–514

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Northeastern Naturalist 506 J. Buckner, A.B. Welsh, and K.R. Sime 22001144 NORTHEASTERN NATURALIST 2V1(o4l). :2510,6 N–5o1. 44 Evidence for Population Differentiation in the Bog Buckmoth of New York State Janet Buckner1, Amy B. Welsh2, and Karen R. Sime3,* Abstract - Hemileuca maia (Bog Buckmoth; Saturniidae) is a rare, ecologically unique variant of the Hemileuca maia complex known only from ten wetlands in the Great Lakes region of North America. The Bog Buckmoth’s status as a threatened taxon meriting conservation has been subject to a debate largely centered on its degree of evolutionary isolation and species status. We studied the genetic variation of two New York Bog Buckmoth populations using amplified fragment-length polymorphisms (AFLP). Bayesian clustering analysis identified two genetically distinct population clusters, with membership that did not coincide consistently with the two sampled populations. There appears to be either historical or contemporary gene flow between Bog Buckmoth populations, with the results suggesting either dispersal between the two sampled populations or contributions from a third unsampled population. Genetic diversity levels were similar. These findings argue for the utility of population-level analyses of Bog Buckmoth as a tool in conservation practice as well as in understanding the taxon’s evolutionary history. Introduction Hemileuca maia (Drury) (Bog Buckmoth, Lepidoptera: Saturniidae), known also as Cryan’s Buckmoth, is a rare variant of the Hemileuca maia complex that was first discovered in 1977 and has been found in ten peatlands in the Great Lakes region of North America (Tuskes et al. 1996). Six Bog Buckmoth populations have been identified along the eastern lake plain of Lake Ontario in Oswego County, NY, and the remaining populations are in eastern Wisconsin and southern Ontario, Canada (Fig. 1). In 1999, Bog Buckmoth was listed as threatened under the New York Endangered Species Act largely because the few fens in which it occurs are disappearing or becoming unsuitable for its larval food-plant, the wetland herb Menyanthes trifoliata L. (Bog Buckbean, Menyanthaceae) (Stanton 2004). Habitat succession appears to be accelerating as a result of nutrient enrichment from runoff and hydrologic variation attributable to lake-level regulation and alteration of inflows (COSEWIC 2009, Stanton 2004). Also, invasive wetland plants such as Phragmites sp. (common reed), Lythrum salicaria L. (Purple Loosestrife), and Frangula alnus Mill. (Glossy Buckthorn) have displaced Bog Buckbean at some sites (Bonnano 2008, Gratton 2006). Furthermore, extant Bog Buckmoth population sizes are small and thus may be more vulnerable to extinction than larger 1Department of Ecology and Evolutionary Biology, University of California Los Angeles, 621 Charles E. Young Drive South, Los Angeles, CA 90095. 2Division of Forestry and Natural Resources, West Virginia University, PO Box 6125, Morgantown, WV 26506. 3Department of Biological Sciences, Shineman Hall, State University of New York at Oswego, Oswego, NY 13126. *Corresponding author - karen.sime@oswego.edu. Manuscript Editor: Adrienne Kovach Northeastern Naturalist Vol. 21, No. 4 J. Buckner, A.B. Welsh, and K.R. Sime 2014 507 populations. Most of the Oswego County populations have been monitored annually since the late 1990s, the results of which reveal patterns of population fluctuation that include dramatic declines and recoveries, and populations in at least three of the six known sites are apparently extinct (Bonanno 2013). The degree to which Bog Buckmoth should be prioritized for conservation efforts has been challenged by disputes over its species status. Hemileuca maia occurs across the eastern United States and Great Plains regions, and, with the exception of the Great Lakes Bog Buckmoth populations, feeds on Quercus spp. (oaks ) and Salix spp. (willows) in wooded habitats (Tuskes et al. 1996). Although H. maia exhibits clinal color variations across its broad range, Bog Buckmoth populations are not consistently distinguishable from other H. maia on the basis of any diagnostic set of color or other morphological characters (Tuskes et al. 1996). However, Legge et al. (1996) argued that behavioral and ecological differences between the Bog Buckmoth and other H. maia may warrant the delineation of the Bog Buckmoth as an evolutionarily significant unit, if not actually a separate species. These disparities include the occurrence of the Bog Buckmoth in peatland habitats and its use of Bog Buckbean as the larval food plant. Other populations of H. maia cannot develop on Bog Buckbean (Legge et al. 1996). Moreover, Bog Buckmoth exhibits numerous behavioral adaptations to its wetland habitat, including oviposition on plants other than the larval food plant and resulting peculiarities of the larval foraging strategies (Pryor 1998, Tuskes et al. 1996). Genetic studies have attempted to address the question of whether the Bog Buckmoth merits classification as a species separate from H. maia. To date, neither allozymes (Legge et al. 1996) nor mitochondrial DNA (mtDNA) (Rubinoff and Sperling 2004) have differentiated Bog Buckmoth from other H. maia. Analysis of the mitochondrial COI gene in specimens from Wisconsin and New York showed that the Bog Buckmoth populations are paraphyletic with respect to other H. maia, and thus do not comprise a separate species (Rubinoff and Sperling 2004). More likely, H. maia, like other widespread Saturniidae, exhibits a variety of rapidly evolved adaptations to local habitats and food plants (Tuskes et al. 1996). However, we suggest that small sample sizes, limited sampling across the Great Lakes region, and reliance on a single gene marker render these findings somew hat inconclusive. Rubinoff and Sperling (2004) recommended analysis at the population level to better understand the population genetic structure within H. maia, and our study was directed towards that end. Our specific objective was to conduct a comparative genetic study of Bog Buckmoth to assess genetic variation between two Oswego County populations and the levels of genetic diversity within each population. Although this approach does not resolve the question of species status, knowledge of population structure can help delineate management units, gauge evolutionary potential, and assess the extinction risk of local populations in the absence of recolonization (Crandall et al. 2000, Frankham et al. 2002, Gompe rt et al. 2006). Like other Bog Buckmoth populations, the study populations occurred in concentrated areas within fens that span no more than a few hundred square meters. The two fens we studied are separated by 30 km of wooded or developed habitat unsuitable for Northeastern Naturalist 508 J. Buckner, A.B. Welsh, and K.R. Sime 2014 Vol. 21, No. 4 the Bog Buckmoth (Fig. 1; Bonnano 2008, Olivero 2001). The topography of the area includes gently rolling hills and drumlins up to a few hundred meters high. Although males fly well, female H. maia are weak fliers, do not feed as adults, are short-lived, and thus not likely to make long-distance dispersive flights (Collins and Tuskes 1979). Females tend to perch on plants where they release pheromones and wait for males to arrive, then after mating make short, clumsy flights to deposit eggs. Males make longer flights, but limit their movements to the natural borders of the fen, turning back when approaching open water or the forest edge. For both sexes, maximum flying height is about 2 m above the surface of the fen (Pryor 1998; K. Sime, pers. observ.). With no obvious corridor for migration, travel between the two sampled sites is probably very infrequent. Our aim was to test the hypothesis that Bog Buckmoth populations are differentiated, because its low dispersal capability is among the justifications for listing the Bog Buckmoth as threatened (Stanton 2004). We hypothesized that we would observe genetic differentiation between the two Oswego County populations we studied. Methods We sampled two Bog Buckmoth populations in Oswego County—Selkirk Fen and Silver Lake Fen (hereafter, Selkirk and Silver Lake; Fig. 1). Selkirk is on the eastern shore of Lake Ontario near Port Ontario, NY, approximately 30 km northeast of Silver Lake, which is an inland site about 10 km south of the lakeshore, near Minetto, NY. We collected adult males (22 from Silver Lake and 14 from Selkirk) in late September 2009. Males fly rapidly across wide swaths of the fens in search of emerging females (Pryor 1998). To ensure a random sample representative of each population, volunteers scattered throughout both sites took the moths in flight. Within hours of collection, we killed the insects by freezing, and dissected the thorax away from the rest of the body. We preserved the samples in 100% ethanol and stored them at -40 °C until we conducted DNA analysis. We extracted DNA from the thoracic muscles using the Gentra PureGene Tissue Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Extracts were quantified using a Thermo Scientific NanoDrop 2000 spectrophotometer (Wilmington, DE) and amplified fragment-length polymorphisms (AFLPs; Vos et al. 1995) were targeted according to the protocol provided by Beckman Coulter, Inc. (Pasadena, CA). We used three selective PCR-primer combinations: EcoACT-MseCAA, EcoAGC-MseCTC, and EcoACT-MseCAT. Fragments were visualized by capillary electrophoresis on a Figure 1 (following page). Map of Bog Buckmoth populations in the vicinity of Lake Ontario. Two localities in Wisconsin approximately 900 km due east of Oswego County, NY (highlighted area) are not shown. Two Oswego County sites, Selkirk Fen and Silver Lake Fen, were sampled for the current study. Selkirk Fen is the southernmost of a complex of five fens (not distinguishable at this resolution) along the eastern shore of the lake, separated from each other by a few km of forested or developed land, that historically have been known to harbor Bog Buckmoth. By 2009, when we took our samples, only one fen other than Selkirk had a significant Bog Buckmoth population, and in 2013 all but the Selkirk population seemed to have disappeared (Bonnano 2013). Northeastern Naturalist Vol. 21, No. 4 J. Buckner, A.B. Welsh, and K.R. Sime 2014 509 Beckman Coulter CEQ8000 and scored with the CEQTM 8000 genetic analysis system software. A single individual evaluated all genotypes. We used AFLP data to characterize genetic variation between and within the two populations. Expected heterozygosity (assuming Hardy-Weinberg equilibrium), the percentage of polymorphic loci, and population differentiation (FST) were calculated Northeastern Naturalist 510 J. Buckner, A.B. Welsh, and K.R. Sime 2014 Vol. 21, No. 4 with the program AFLP-SURV (Vekemans et al. 2002) using the method described by Lynch and Milligan (1994). Significance of FST was based on 1000 permutations. We employed a bayesian approach to determine the most likely number of populations (K) given the genetic data, using the software STRUCTURE (Pritchard et al. 2000). We used the admixture model (allowing for mixed ancestry of individuals) and assumed correlated allele frequencies between populations, as recommended by Falush et al. (2003), to detect subtle differences in population structure. Sampling location was not used as a prior in the analysis. Values of K ranging from 1 to 4 were tested using 5 replicates for each value of K, a burn-in period of 100,000 iterations, and 100,000 Markov-chain Monte Carlo iterations following the burnin period. Values of K exceeding the number of sampled locations were tested to allow for the possibility of multiple populations existing at a single location. Loglikelihood (LnP[D]) values for the tested K-values were plotted, and the most likely number of populations was determined based on the K-value with the highest likelihood and greatest change in K (ΔK), based on the method described by Evanno et al. (2005), as implemented in the software STRUCTURE HARVESTER (Earl and von Holdt 2012). Membership coefficients (Q) were calculated for each individual to estimate the fraction of its genome with ancestry in each of the K clusters. Results and Discussion LnP(D) and ΔK both peaked at K = 2, indicating that the most likely number of populations represented in our sample collection was two (Fig. 2). However, the Figure 2. Determination of most the likely number of Bog Buckmoth population clusters (K) in Oswego County, NY, given the genetic data from five replicate runs for each value of K in STRUCTURE. The most likely number of populations is two, based on the highest probability (LnP [D]) with lowest variability between runs and greatest change in K (ΔK). Northeastern Naturalist Vol. 21, No. 4 J. Buckner, A.B. Welsh, and K.R. Sime 2014 511 two populations did not separate based on sample location (Fig. 3). Instead, a single cluster was predominant in the two locations. At Silver Lake, 82% of the sampled individuals had a high proportion of membership (Q > 0.70) in this dominant cluster. At Selkirk, 57% of the sampled individuals belonged to the dominant cluster (Q > 0.70). One possible explanation is that Silver Lake and Selkirk represent a single population, with contributions from a third population, i.e., the second cluster identified in the samples could represent individuals from another population that we did not sample. These potential migrants (with Q > 0.7 in cluster 2) account for 9% of the sampled individuals at Silver Lake and 21% of the sampled individuals at Selkirk. The most likely unsampled source of migrants is the population nearest Selkirk in South Pond Fen (hereafter South Pond), located approximately 3 km north of Selkirk. Since 2001, the South Pond population has been very small compared to the Selkirk population, and no Bog Buckmoths have been observed there since 2012, but historically the South Pond population had probably been larger (Bonnano 2013). An alternative explanation is that Silver Lake and Selkirk are genetically distinct, with gene flow between the two populations. Migration seems to be asymmetrical, with a higher proportion of migrants going to Selkirk than to Silver Lake, which may in turn reflect the fact that prevailing winds would tend to move moths in that direction. Based on the FST value, the Silver Lake and Selkirk populations were genetically distinct (FST = 0.13, P < 0.001). Some individuals appear to have mixed ancestry (9% at Silver Lake and 21% at Selkirk; Q < 0.70 in either cluster), which could have resulted from mating between individuals from the two locations. These two explanations are not mutually exclusive; a combination of the two scenarios could be that Silver Lake and Selkirk are genetically distinct, with genetic contributions from a third, unsampled population. The use of the three selective PCR-primer pairs resulted in the generation of 203 fragments. The percentage of polymorphic loci (Selkirk: 63%, Silver Lake: Figure 3. Membership coefficients from program STRUCTURE (y-axis) for each individual (represented by vertical bars) for Bog Buckmoth individuals sampled from Silver Lake (n = 22) and Selkirk (n = 14) when K = 2. Clusters are represented by light vs. dark gray shading. The black line separates the two sampling locations. Northeastern Naturalist 512 J. Buckner, A.B. Welsh, and K.R. Sime 2014 Vol. 21, No. 4 67%) and expected heterozygosity (Selkirk: 0.233, Silver Lake: 0.227) were similar for the two populations. The heterozygosity values are within the range expected for Lepidoptera, according to genetic studies that used allozyme data to produce a range of values from about 0.04 to 0.32 in various taxa (Packer et al. 1998). The similar results are not surprising because population densities at the two sites have been similar since monitoring at Selkirk began in 2004 and Selkirk and Silver Lake have consistently been two of the most populous Bog Buckmoth sites (Bonnano 2013). Additionally, gene flow between the two populations, or into these populations from a third source, could result in similar levels of genetic diversity. Our results suggest that either explanation is plausible. Rubinoff and Sperling (2004) argued that their mtDNA results, which showed the Bog Buckmoth as a paraphyletic assemblage within H. maia, indicated that the Bog Buckmoth is not isolated from surrounding populations of H. maia. They suggested however that AFLP data might shed light on recent isolation events within the Bog Buckmoth / H. maia complex. Although population differentiation is apparent among Bog Buckmoth populations, our results indicate that some between-population gene flow has occurred. Our data are consistent with two possible scenarios: gene flow between the two sampled fens, or from an additional unsampled population. Thus, the 30 km of unsuitable habitat between Selkirk and Silver Lake may not be enough to isolate these populations. It seems most likely that moths are occasionally delivered by winds from one to the other, with prevailing winds tending to carry them toward Selkirk. Migration between Selkirk and South Pond as well as the nearby lakeshore fens is likely as well. These explanations are not mutually exclusive, and neither can be eliminated by analysis of our data. Our conclusions are limited by having sampled only two populations, and further sampling and analysis that includes additional Bog Buckmoth populations as well as other H. maia would provide a clearer picture of gene-flow patterns and colonization and isolation events. It would be particularly instructive to obtain comparable AFLP data from the Wisconsin and Ontario populations, as well as from non-Bog Buckmoth members of the H. maia complex. Many Saturniidae are reported to persist at low densities compared to other Lepidoptera, and cyclic population explosions and crashes appear to be regular demographic features of the family (Tuskes et al. 1996). Such events can be a cause for conservation concern when drastic population reductions result in a reduced effective population size and lowered genetic diversity (Frankham et al. 2002). Bog Buckmoths may be susceptible to such challenges because recolonization of the fen by nearby populations may not occur rapidly after a population goes extinct. However, our finding that there may be some gene flow between populations separated by as much as 30 km indicates that recolonization is possible. Genetic studies that focus on the relationships within Bog Buckmoth, rather than considering the H. maia complex as a whole, may aid management efforts directed at the insect’s recovery. Studies comparing the genetic structure between all Oswego County localities as well as more distant populations would help delineate management units and inform conservation priorities accordingly. Northeastern Naturalist Vol. 21, No. 4 J. Buckner, A.B. Welsh, and K.R. Sime 2014 513 Acknowledgments We thank the State University of New York at Oswego for support through Scholarly and Creative Activities Grants awarded to K.R. Sime and to J. Buckner, and the McNair Scholars Program for supporting J. Buckner’s undergraduate research. The New York State Department of Environmental Conservation granted us access to Selkirk Fen and an Endangered Species License to collect moths, and the Central New York Land Trust gave us access to Silver Lake. Peter A. Rosenbaum, Eric Hellquist, Sandy Bonnano, John Laundré, Andy Nelson, and two anonymous reviewers provided helpful guidance at various stages of this project and in preparing the manuscript. Literature Cited Bonanno, S.E. 2008. 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