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Genetic Divergence Among Massachusetts Populations of the Vernal Pool Fairy Shrimp Eubranchipus vernalis (Crustacea: Anostraca)
S. Shawn McCafferty, Nicolas Warren, Christopher Wilbur, and Scott Shumway

Northeastern Naturalist, Volume 17, Issue 2 (2010): 285–304

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2010 NORTHEASTERN NATURALIST 17(2):285–304 Genetic Divergence Among Massachusetts Populations of the Vernal Pool Fairy Shrimp Eubranchipus vernalis (Crustacea: Anostraca) S. Shawn McCafferty1,*, Nicolas Warren1,2, Christopher Wilbur1,3, and Scott Shumway1 Abstract - The vernal pool fairy shrimp Eubranchipus vernalis (Eastern Fairy Shrimp) is an obligate freshwater invertebrate that is an important member of northeastern United States vernal pool ecosystems. The extent of gene flow among populations of vernal pool fairy shrimp in the Northeast is currently unknown, yet this information is important for understanding the ecology and evolution of this species. In order to infer the level of gene flow among E. vernalis populations, we used mtDNA sequence data to estimate the level of genetic divergence and the pattern of geographic variation among 22 vernal pool populations sampled across Massachusetts during spring 2003. We found significant differences among vernal pool populations in the frequency of unique mtDNA sequences, but no coherent geographic pattern in the distribution of these sequences, and there was no significant correlation between levels of genetic divergence and geographic distance. We argue that these results reflect priority effects rather than limited dispersal among these populations. Introduction Dispersal of aquatic organisms inhabiting isolated freshwaters has long been of interest to ecologists. Darwin pondered this question in On The Origin of Species and later in his last published work (Darwin 1882), inspired by a small freshwater bivalve attached to the leg of a water beetle sent to him by W.D. Crick, Francis Crick’s grandfather (Ridley 2004). Darwin reasoned, and many others have since demonstrated, that dispersal among isolated freshwater habitats readily occurs even in organisms that lack dispersing adult stages (reviewed in Bilton et al. 2001). Though understanding the pattern and rate of dispersal is important to understanding the evolutionary and ecological mechanisms driving populations of freshwater organisms, it is gene flow that provides the critical link between dispersal and evolution (Bohonak 1999). Clearly dispersal and gene flow are related to some degree, yet dispersal does not necessarily translate into gene flow (DeMeester et al. 2002). Therefore, in order to better understand the ecology and evolution of freshwater habitats and the species that live there, a key first step is to understand the extent of gene flow among populations. This understanding is particularly important in organisms that depend on small isolated freshwater habitats like vernal pools. 1Department of Biology, Wheaton College, 26 East Main Street, Norton, MA 02766. 2Current address - MRC Room 313, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543. 3Current address - Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064. *Corresponding author - smccaffe@wheatonma.edu. 286 Northeastern Naturalist Vol. 17, No. 2 Vernal pools are shallow, seasonal water bodies that provide critical habitat for many woodland amphibian and invertebrate species in New England (Colburn 2004). They vary in size, depth, hydrology, and degree of isolation, and characteristically experience a hydrologic cycle of seasonal flooding and drying that prevents fish populations from becoming established. Consequently, a variety of organisms lives and breeds only in these specialized environments. For this reason, vernal pools play an important role in regional ecosystem biodiversity (e.g., Colburn 2004, DeMeester et al. 2005, Zedler 2003), yet they are often threatened by man-made changes to the landscape (Belk 1998, Burne and Griffin 2005, Colburn 2004). Generally, a defining feature of vernal pools is the lack of any surface connection with other water bodies, making them, for all practical purposes, small isolated islands surrounded by inhospitable habitat (Holland and Jain 1981, Zedler 2003). This isolation is an important constraint on any obligate vernal pool organism; active dispersal of these organisms among vernal pools can only occur in adult stages adapted for such dispersal. For crustaceans lacking such an adult stage (such as fairy shrimp and many zooplankton species), dispersal can only occur via the passive transport of adults or diapausing eggs (Bilton et al. 2001, Havel and Shurin 2004, Panov et al. 2004). Though many authors consider passive dispersal mechanisms to be quite effective, recently this assumption has come under increasing scrutiny (e.g., Bohonak and Jenkins 2003, Bohonak et al. 2006, Cáceres and Soluk 2002). High levels of dispersal can potentially lead to high levels of gene flow, which can result in low levels of genetic divergence among populations. Therefore, one expectation is that there should be little if any genetic divergence among vernal pool populations of obligate freshwater invertebrates with diapausing cysts. However, recent studies on various freshwater invertebrates tend to show just the opposite, i.e., a general pattern of high levels of genetic divergence among populations, suggesting limited gene flow (reviewed in DeMeester et al. 2002). These findings suggest an unexpected disconnect between dispersal and gene flow in some species. How common this disconnect is in obligate vernal pool species is currently unknown. Key members of North American vernal pool ecosystems are the diminutive crustaceans referred to as fairy shrimp (Class Branchiopoda, Order Anostraca). In fact, fairy shrimp are considered to be a key indicator group for identifying and certifying vernal pools (Colburn 2004, Kenney and Burne 2000). These small crustaceans swim ventral side up, are comparatively weak swimmers that are unable to escape fish and other fast visual predators, and therefore are restricted to temporary waters. Most fairy shrimp reproduce sexually, producing diapausing eggs that can withstand freezing and desiccation. In New England vernal pools, fairy shrimp hatch from diapausing cysts in the early spring, grow rapidly, mate, and deposit diapausing cysts in the sediment (where they contribute to the ongoing egg bank) before dying and completing their life cycle, typically by early May. In North America, there are seven families of Anostracans. The most common Anostracans in northeastern vernal pools are in the genus Eubranchipus 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 287 (Family Chirocephalidae; Smith 2001). In Massachusetts, there are two species of Eubranchipus, the common and widespread E. vernalis (Verrill) (Eastern Fairy Shrimp) and the more rare E. intricatus (Hartland-Rowe) (Smooth-Lipped Fairy Shrimp). Eubranchipus vernalis has a fairly wide range east of the Appalachians, from South Carolina north into Massachusetts and perhaps New Hampshire and Maine (Belk et al. 1998). Unfortunately very little is published on E. vernalis biology, though the species’ life history appears to be consistent with most other Anostracans (summarized in Colburn 2004). The purpose of this study was to determine the level and pattern of genetic divergence among Massachusetts populations of the fairy shrimp E. vernalis. We describe for the first time the levels and distribution of genetic variation within and among northeastern US vernal pool populations of an obligate freshwater invertebrate based on mitochondrial DNA (mtDNA) sequence data. The levels of genetic divergence among populations, the distribution of unique mtDNA sequences, and the relationship between levels of genetic divergence and geographic distance are used to infer the geographic structure of vernal populations in this recently deglaciated landscape. We interpret our results with respect to levels of dispersal and gene flow among vernal pools. Methods Study design and field collections To determine the level of genetic divergence among vernal populations, the authors and volunteers with the Vernal Pool Association (http://www.vernalpool. org, Reading, MA) used dipnets to collect samples of fairy shrimp from 22 vernal pools across Massachusetts during Spring 2003 (Fig. 1). Locales and sample sizes are presented in Table 1 (latitudes and longitudes are available from S.S. McCafferty upon request). Distances between the pools range from 0.1 km to more than 200 km. Individuals were identified to species according to Kenney and Burne (2000). All samples were preserved in 95% ethanol in the field. Laboratory methods The use of mtDNA in studies of geographic variation among populations has been an accepted approach for over 25 years and the methods for studying genetic variation in mtDNA are now fairly routine (Avise 2004). Genomic DNA is extracted from individual samples, and a region of the mitochondrial genome is sequenced using the polymerase chain reaction (PCR) coupled with direct sequencing. The resulting sequences can be used to estimate both the evolutionary relationship (phylogeny) among unique mtDNA sequences (haplotypes) and the frequency and occurrence of haplotypes within and among populations using routine methods. Genomic DNA was extracted from individual fairy shrimp using the Wizard Genomic DNA Extraction Kit (Promega Corp., Madison, WI) or by a standard chelex method (Walsh et al. 1991). Roughly 700 nucleotide 288 Northeastern Naturalist Vol. 17, No. 2 base pairs (bp) of the mitochonrial cytochrome oxidase I (COI) gene region were amplified in a standard PCR reaction using the primer pairs HCO2198 (Folmer et al. 1994) and FS-COIL (5’ CTGCTGGGTCACAGAATGAA 3’) under the following conditions: 94 °C for 2 minutes, followed by 35 cycles of 94 °C for 30 seconds, 52 °C for 30 seconds, and 72 °C for one minute, followed by a finishing step of 72 °C for five minutes. For each sample, the resulting PCR reactions produced a single fragment that was cleaned (Wizard Gel/PCR Cleanup Kit, Promega Corp., Madison, WI, or AmPure, Argencourt Bioscience Corp, Beverley, MA) and sequenced (BigDye 3.1, Applied Biosystems, Foster City, CA) in both directions. The resulting DNA sequences were analyzed on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) following the manufacturer’s recommendations. Statistical and phylogenetic analysis of mtDNA The analytical approach taken here is fairly routine when dealing with population samples of DNA sequence data (Avise 2004). First, the data are manually edited and aligned to detect ambiguities or problems in the sequences. Once aligned, haplotypes are identified, and their frequency estimated for each population. The phylogenetic relationship among haplotypes is inferred and subsequently used to determine if any pattern exists between the evolutionary relationship of the different haplotypes and their geographic origin, referred to as their phylogeographic relationship. Figure 1. Map of 22 sampling locations in Massachusetts. Locale ID numbers are by town alphabetically: (1) Belchertown, (2) Boxford, (3) Concord, (4) East Brookfield, (5) Groton, (6) Ipswich, (7) Leverett, (8) Nantucket, (9) Newburyport, (10) Norfolk, (11) North Hampton, (12) North Reading, (13–14) Norton, (15) Rowley, (16) Sturbridge, (17) Sudbury, (18) Wellfleet, and (19–22) Westborough. 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 289 Table 1. Frequency of mtDNA haplotypes from 22 vernal pool locations throughout Massachusetts. The order of haplotypes follows the evolutionary relationship among haplotypes found in Figure 2. The order of the first 14 locales (n > 4) follows the level of genetic similarity based on the frequency of haplotypes determined as in Figure 3. The remaining 8 locales (n < 4) are ordered alphabetically. Locale ID numbers were determined alphabetically. π is the level of nucleotide diversity within a population, estimated only for those populations with n > 4. ID Town Code O H10 H12 H13 H11 H4 H2 H3 H9 H8 H7 H1 H6 H5 n π 21 Westborough3 ANF 4 5 1 10 0.01011 22 Westborough4 ANN 1 8 4 13 0.00970 17 Sudbury SUD 3 2 1 6 0.00467 6 Ipswich IPS 2 2 1 5 0.00300 10 Norfolk NOR 4 1 5 0.00133 8 Nantucket NAN 6 1 1 8 0.00470 15 Rowley ROW 1 1 5 1 8 0.00315 7 Leverett LEV 1 4 5 0.00200 19 Westborough1 W5 7 3 10 0.00233 4 East Brookfield EBRK 5 5 0.00000 5 Groton GRO 5 5 0.00000 13 Norton NORT 7 7 0.00000 14 Norton Woods NORTW 1 5 6 0.00167 20 Westborough2 W9 10 10 0.00000 1 Belchertown BEL 1 1 2 - 2 Boxford BOX 4 4 - 3 Concord CON 1 1 - 9 Newburyport NEW 1 1 2 - 11 North Hampton NHAM 2 1 3 - 12 North Reading NRED 2 2 - 16 Sturbridge STU 1 1 2 - 18 Wellfleet CCNS 2 1 3 - Total 1 14 17 2 2 11 3 4 6 2 1 14 5 40 122 290 Northeastern Naturalist Vol. 17, No. 2 The resulting sequences were edited by eye and aligned using Sequencer 4.1 (Gene Codes Corp., Ann Arbor, MI). The aligned sequences were imported into MacClade 4.1 (Maddison and Maddison 2001), manually rechecked, and trimmed to 600 bp. An unweighted pair group method with arithmetic mean (UPGMA) tree was constructed based on the simple percent difference (with pair-wise deletion) among all mtDNA sequences using MEGA 3.1 (Kumar et al. 2004), and the resulting tree was used as a guide to determine the number of haplotypes. Representative haplotype sequences were constructed using majority rule consensus in MacClade when ambiguous base calls were present. The phylogenetic relationship among the resulting unique mtDNA haplotypes was inferred by neighbor joining (NJ) based on Kimura’s 2-parameter model, using the program Mega 3.1. In addition, a nonhierarchical phylogenetic view of the relationship among haplotypes was inferred by statistical parsimony using the program TCS (Clement et al. 2000). Nucleotide diversity (π) within vernal pools was estimated using the program Arlequin 3.11 (Excoffier et al. 2005). To test for spatial patterns in the data, an analysis of molecular variation (AMOVA) and estimates of pairwise Φst (analogous to Fst) were used to test for significant differences in mtDNA haplotype frequencies among populations (vernal pools) using Arlequin 3.11. Φst is a commonly used measure of genetic divergence among populations based on the variance in allele (or haplotype) frequencies. Values of Φst range from a low of 0.0 (no divergence) to a high of 1.0. AMOVA tests for significant differences in the frequency of genetic markers within and among hierarchical groups of populations and is analogous to an analysis of variance. The key difference is that AMOVA also factors in levels of genetic divergence. Because there was no a priori hierarchical structure to our sampling scheme, the AMOVA analysis simply tested for significance among vernal pools. The relationship among vernal pool populations based on the population pairwise estimates of Φst (negative values were converted to 0.000) was visualized using hierarchical clustering (UPGMA) and the nonhierarchical ordination method nonmetric multidimensional scaling (NMDS) coupled with a minimum spanning tree using the program NTSYSpc (Rohlf 2002). Used together, these methods provide a powerful multivariate approach for detecting groups of populations based on overall levels of genetic similarity (Allendorf and Luikart 2007). Isolation by distance (IBD), the relationship between geographic distance and genetic divergence (Φst), was tested using the program IBDWS (Bohonak 2002, Jensen et al. 2005) with 1000 permutations of the Mantel (1967) test. Geographic distance among vernal pools was calculated from latitude and longitude using the geographic distance matrix generator (Estes 2007). If the latitude and longitude of a particular vernal pool was unknown, we used the latitude and longitude of the town center where the vernal pool was located as a proxy estimate. 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 291 Results Thirteen unique mtDNA sequences, arbitrarily labeled haplotypes H1– H13, were found in 121 E. vernalis from 22 vernal pools distributed across Massachusetts (GENBANK accession numbers EU169880-EU169893). A single individual E. intricatus was also sequenced and used as an outgroup in the phylogenetic analyses. There were 18 variable nucleotide positions found in the aligned sequences, 16 of which allowed us to determine the relationship among haplotypes (parsimony informative). No insertions or deletions were found, and within E. vernalis, all substitutions were transitions (C/T or G/C) located at the third codon position, meaning that none of these substitutions leads to an amino acid substitution. The levels of nucleotide diversity within populations were relatively low, ranging from 0.00000 to 0.01010 with an average of 0.00775 ± 0.00213 (Table 1). The relationship among the 13 haplotypes can be found in the NJ tree and as an unrooted parsimony network in Figure 2. The two trees are very similar in terms of the evolutionary relationship or branching order among haplotypes. The 13 haplotypes are closely related and generally fall into two primary groups (clades), one consisting of haplotypes H10–H13, the other of haplotypes H1–H9. There is a tendency for subgroups to form within these two main clades (H11–H13; H1–H6, H7–H9), though these are not well supported by bootstrap analysis, a measure of confidence in the resulting branching pattern (results not presented). The frequency of the 13 haplotypes can be found in Table 1. The most common haplotype is H5, which occurs in a little over a third of the pools and Figure 2. The phylogenetic relationship among haplotypes as determined by (a) the neighbor joining (NJ) method and (b) statistical parsimony (see text for details). 292 Northeastern Naturalist Vol. 17, No. 2 represents 10–100% (when n > 4) of the populations where it occurs. Three other haplotypes (H1, H4, and H12) are less common, occurring in 5–6 of the pools at frequencies of 12–80%. The remaining eight haplotypes (H2, H3, H6, H7, H8, H9, H10, H11, and H13) are relatively rare, occurring in only 1–3 of the sampled pools, although at within-pool frequencies of up to 62%. Several haplotypes are confined to one (H6, H7, and H13) or two (H7, H9, and H11) populations. No single haplotype is found in all populations. There is a highly significant difference among populations based on the frequency of mtDNA haplotypes, with a little over half the variation accounted for among populations (Table 2). We include only those populations with a sample size of n > 4, which results in a final data set containing 104 of the 121 original sequences from 14 populations and all 13 haplotypes. The pairwise estimates of Φst are highly variable (ranging from 0.000 to 0.961) but on the average quite high (mean Φst of 0.490 ± 0.038; Table 3). We find no geographically coherent pattern in the relationship among vernal pools based on the Φst values. The results of the UPGMA and NMDS (Fig. 3) are very similar; those populations forming clusters in the UPGMA are also closely associated in the ordination. There is a tendency for the vernal pool populations to fall into three groups, one consisting of vernal pools from East Brookfield (EBRK), Groton (GRO), Westborough (W9), and Norton (NORT and NORTW), a second group consisting of pools from Nantucket (NAN), Sudbury (SUD), and Westborough (ANF and ANN), and a third group consisting of pools from Ipswich (IPS), Leverette (LEV), Norwood (NOR), Rowley (ROW), and Westborough (W5). However, these groups do not appear to have any coherent geographic basis. There is extensive geographic overlap in the distribution of the three groups (Fig. 4), clearly demonstrating a lack of any coherent geographic pattern in the data. In addition, there is no significant relationship between geographic proximity and cluster membership (tested by a Mantel test, P = 0.85), and there is no obvious geographic feature separating the three groups. The clustering of vernal pools into these three groups appears to be due to the frequency of haplotype H5 and of clades 1 and 2 (Fig 2). Group 1 (EBRK, GRO, NORT, NORTW, and W9) is characterized by either fixation for or a high frequency of haplotype H5. Group 2 is distinguished from Group 3 by having a high frequency of haplotypes from clade 2, while Group 3 is dominated by haplotypes from clade 1. These results are clearly seen in Figure 3 where the frequency of the two clades is plotted on the results from the NMDS. Table 2. Results of the AMOVA testing for significant differences among vernal pool populations of E. vernalis (***P < 0.001). Variance Percentage Source of variation d.f. Sum of squares components of variation Among populations 13 136.976 1.30157 53.59*** Within populations 88 99.200 1.12727 46.41 Total 101 236.176 2.42884 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 293 Table 3. Pairwise estimates of Φst and geographic distance among pools. The Φst values are below the diagonal, the estimates of geographic distance (km) above. ANF ANN EBRK GRO IPS LEV NORT NW NAN NOR ROW SUD W5 W9 ANF 0.000 0.16 34.23 39.46 80.34 74.23 52.20 49.40 167.67 30.12 74.53 22.82 2.97 2.98 ANN 0.047 0.0000 0.16 34.23 39.46 80.34 74.23 52.20 49.40 167.67 30.12 74.53 2.82 2.97 EBRK 0.316 0.526 0.000 57.97 111.36 44.80 79.45 78.18 193.05 61.01 104.35 55.05 36.34 36.36 GRO 0.316 0.526 0.000 0.000 60.52 78.08 81.13 76.66 191.72 58.45 51.75 28.49 41.16 41.15 IPS 0.249 0.432 0.654 0.654 0.000 138.45 85.61 80.36 167.06 73.92 9.56 57.53 79.70 79.67 LEV 0.288 0.494 0.813 0.813 0.440 0.000 123.66 121.96 237.83 103.71 129.81 89.52 76.97 76.98 NORT 0.369 0.566 0.000 0.000 0.709 0.846 0.000 5.34 115.50 23.20 85.53 52.68 49.23 49.22 NW 0.280 0.507 0.000 0.000 0.479 0.605 0.028 0.000 118.41 19.59 80.20 48.16 46.44 46.24 NAN 0.305 0.325 0.773 0.773 0.586 0.658 0.803 0.728 0.000 137.98 172.91 164.07 164.7 164.69 NOR 0.232 0.449 0.867 0.867 0.145 0.359 0.891 0.672 0.600 0.000 71.53 30.32 27.19 27.18 ROW 0.348 0.494 0.571 0.571 0.228 0.537 0.620 0.458 0.651 0.514 0.000 51.80 74.18 74.16 SUD 0.278 0.313 0.791 0.791 0.596 0.651 0.824 0.733 0.000 0.618 0.652 0.000 22.38 22.36 W5 0.331 0.545 0.577 0.577 0.433 0.049 0.620 0.353 0.705 0.422 0.483 0.696 0.000 0.02 W9 0.428 0.612 0.000 0.000 0.764 0.878 0.000 0.091 0.835 0.915 0.673 0.858 0.667 0.000 294 Northeastern Naturalist Vol. 17, No. 2 Furthermore, there is no obvious coherent spatial pattern based on the frequency of the two clades when mapped onto sampling locales (Fig. 4), reinforcing our conclusion that the three groups are not defined by geographic proximity. This overall lack of coherent geographic structure is further seen when testing for isolation by distance. No significant relationship is found between geographic distance and genetic divergence Φst (P = 0.557; P = 0.727 for the log-log analysis; Fig. 5). Discussion Our results clearly demonstrate significant differences among fairy shrimp populations in the frequency and occurrence of mtDNA haplotypes. However, even though we find a significantly high level of spatial variation among populations (Φst values and the AMOVA results), no coherent geographic pattern can be detected. There are no distinctive geographic boundaries that would suggest population breaks, and there is no relationship between Figure 3. The relationship among sites (n > 4) as determined by (a) unweighted pair group method with arithmetic mean (UPGMA) and (b) nonmetric multidimensional scaling (NMDS) of Φst values (see text for details). A minimum spanning tree connecting similar populations is overlain on the plot. Abbreviation for locations can be found in Table 1. Also shown are the frequencies of clade 1 (black) and 2 (white) found at each site. 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 295 geographic distance and levels of genetic divergence. Populations that are geographically close to each other are no more genetically similar to each other than they are to populations that are more geographically distant. There are two important aspects of our results. The first is finding significant levels of genetic divergence among vernal pool populations of Figure 4. (a) Plot of the three groups of populations identified by UPGMA and NMDS (Fig. 3) onto their geographic location. Group 1 is black, Group2 is grey, and Group 3 is white. (b) Plot of the frequency of mtDNA clades 1 (black) and 2 (white) onto their geographic locations. All locations sampled are plotted in (b), whereas only locations with n > 4 are plotted in (a). 296 Northeastern Naturalist Vol. 17, No. 2 E. vernalis. This result is fairly consistent with previous studies of other fairy shrimp species based on protein electrophoresis (Bohonak 1998, Boileau et al. 1992, Davies et al. 1997, Hulsmans et al. 2007, Ketamaier et al. 2003, Meglecz and Theiry 2005). Those few examples of low levels of genetic divergence tended to be among populations that were geographically very close (e.g,. Bohonak 1998, Riddoch et al. 1994). In a general review of the literature on Anostracans, Hulsmans et al. (2007) suggest an overall trend for high levels of genetic differentiation on geographic scales of greater than 0.1 km, with low levels of differentiation at geographic scales of less than 0.1 km due to the effects of dispersal and gene flow. Similar patterns of high levels of divergence among populations are also reported for a variety of other diapausing freshwater invertebrates (e.g., DeMeester et al. 2002, Marten et al. 2006, Zeller et al. 2006, and references therein), suggesting this may in fact be a general phenomenon in organisms with this particular reproductive strategy. The second aspect of our results that is worth discussing is that even though we find significant levels of divergence among populations, there is no coherent geographic pattern to this variation. How do we explain this seemingly paradoxical result? Though phylogeographic boundaries (defined here as large breaks in the distribution of haplotypes that correspond to some geographic feature) have been reported in other studies of diapausing freshwater invertebrates, these were invariably found among populations separated by very large geographic distances (hundreds to thousands of kilometers, e.g., De Gelas and DeMeester 2005, Dooh et al. 2006, and references therein). The general tendency is for studies at medium or smaller geographic scales to show limited if any evidence of phylogeographic structure (see references above). This can be attributed to the effects of ongoing dispersal and gene flow coupled with a general absence of any major geologic features that would interfere with historical or contemporary dispersal. Our data would certainly be consistent with these findings. Considering the Figure 5. Plot of untransformed pairwise genetic distance (Φst) versus geographic distance (km). 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 297 geographic range of our sampling sites, the topography of eastern and central Massachusetts, and the potential for high rates of dispersal among fairy shrimp populations, there may be no compelling reason to expect substantial phylogeographic structure among vernal pool populations in this region. Though dispersal and gene flow tend to erase phylogeographic signals, they can, however, lead to a correlation between levels of genetic divergence and geographic distance among populations. Termed isolation by distance (IBD), this relationship is expected to occur among populations if levels of gene flow are dependent on geographic distance (e.g., Hutchison and Templeton 1999, Slatkin 1993). As geographic distance among populations increases, levels of gene flow tend to decrease. This trend leads to increasing levels of divergence among populations that are geographically distant, which translates into a positive relationship between levels of genetic divergence and geographic distance. Bohonak (1998) interpreted the results from previous studies on various species of fairy shrimp as demonstrating clear evidence for IBD. In fact, a general consensus seems to be emerging from the literature that IBD may be common in diapausing freshwater species. However, this relationship may also depend on geographic scale. IBD has been demonstrated among populations that are geographically close, but the relationship appears to break down at larger geographic distances (Haag et al. 2006, Hulsman et al. 2007, Zeller et al. 2006). At what point IBD breaks down tends to be species specific, ranging from ca. 0.1 km in Branchipodopsis wolfiDaday and in two species of Daphnia (Haag et al. 2006, Hulsman et al. 2007), to between 0.28 km and 50 km in Branchinecta sandiegonensis Fugate (San Diego Fairy Shrimp) (Davies et al. 1997), to less than 100 km in a species of diapausing copepod (Zeller et al. 2006). In E. vernalis, we found no evidence for IBD. However, this may simply be a reflection of the geographic scale of our sampling, since only 3 of the 91 possible pairwise comparisons were less than 1 km distant (mean distance of 71.5 km ± 5.27 km). However, it is interesting to note that the only comparison less than 0.1 km in our data has a Φst value of 0.667, which suggests a high level of divergence even in geographically close populations and an overall lack of IBD in E. vernalis. Dispersal, gene flow, and priority effects Our results strongly suggest that there is limited gene flow among Massachusetts vernal pool populations of E. vernalis, resulting in a pattern of high levels of divergence among vernal pool populations with little coherent geographic pattern. How do we account for this limited gene flow? Either (a) there is limited dispersal among populations, which translates into limited gene flow, or (b) dispersal is occurring but it does not lead to gene flow among populations; in other words, there is a decoupling between dispersal and gene flow. Limited dispersal means limited gene flow, which results in increasing levels of genetic divergence among populations over time. It is generally assumed that diapausing cysts are an effective mechanism of dispersal in 298 Northeastern Naturalist Vol. 17, No. 2 obligate freshwater invertebrates. However, Bohonak and Jenkins (2003) convincingly argue that passive dispersal via diapausing cysts may not be as frequent as commonly assumed. Without clear direct estimates, the assumption of passive dispersal should be carefully evaluated on a species by species basis. However, though Bohonak and Jenkins’ (2003) argument is compelling and would certainly explain our results, there are aspects of the life history of E. vernalis that suggest an alternative explanation. Many early studies on the population genetics of freshwater organisms with passive dispersal revealed unanticipated high levels of genetic divergence among populations (Boileau et al. 1992, DeMeester et al. 2002). This result was considered a paradox; the life-history strategy clearly suggests substantial levels of dispersal, yet the genetic data point otherwise. Boileau et al. (1992) first proposed a solution to this apparent paradox, which they termed persistent founder effects. In this model, colonization of new habitats by a limited number of individuals followed by rapid population expansions lead to the preservation of founder effects (stochastic differences among populations) over substantial periods of time (Boileau et al. 1992). This long-term lack of equilibrium between gene flow and drift can result in a random spatial pattern of genetic divergence that can persist even when levels of dispersal are high. Because the populations are at or near carrying capacity, there is little opportunity for immigrant genotypes to contribute to the population, dampening the homogenizing effects of gene flow. In effect, dispersal occurs, but it is effectively decoupled from gene flow. The presence of a large cyst bank would further reinforce the long-term persistence of founder effects by swamping out any immigrant genotypes entering the population (Hairston 1996). In a further expansion of this model, DeMeester et al. (2002) proposed that rapid local adaptation tended to even further accentuate persistent founder effects by locking out immigrant genotypes with lower fitness, a model they referred to as the monopolization hypothesis. There are various aspects of the life history of E. vernalis and the nature of vernal pools that makes persistent founder effects and/or the monopolization hypothesis (here we combine the two models under the general category of priority effects) compelling explanations for our results. First, populations of E. vernalis in New England tend to be rather young, reflecting colonization events that can be no more than 11,000–14,000 years old, based on the glacial history of eastern North America (Ruddiman 1987). Some populations may be even younger, considering the agricultural history of this region may have led to extensive habitat destruction prior to recent reforestation (Gerhardt and Foster 2002, Hall et al. 2002). Because of their relatively young ages, these populations may not have had sufficient time to reach an equilibrium with respect to gene flow and drift (e.g., Nurnberger and Harrison 1995), a situation that can lead to persistent founder effects. Second, population sizes of E. vernalis vary greatly by location and year, though they can be quite high in some instances (M. Burne, Vernal Pool Association, MA, pers. observ.). Rapidly fluctuating populations can also lead to a lack of equilibrium between gene 2010 S.S. McCafferty, N. Warren, C. Wilbur, and S. Shumway 299 flow and drift. Third, though no formal estimates on cyst-bank size has been reported in the literature for E. vernalis, it is reasonable to assume they tend to be rather high. For example, estimates of cyst-bank density from other Anostraca tend to be around 103 to 105 per square meter (Brendonck and DeMeester 2003, Hulsman et al. 2006). In addition, hatching success from cysts may also be quite variable. Hatching rates appear to be highly dependent on the environmental conditions, ranging from around 3% to 85%, depending on the species and environment (Belk 1998, Hulsman et al. 2006). A large cyst bank coupled with variable hatching rates would act to limit the contribution of immigrant genotypes to a population. The physical and chemical nature of vernal pools also suggest that priority effects may play an important role in determining the pattern of genetic divergence we find in E. vernalis. Vernal pools tend to be relatively limited but variable in size. Persistent founder effects are thought to be more important in species occupying small habitats than in those occupying larger, more stable environments (Boileau and Taylor 1994, DeMeester et al. 2002, Vanoverbeke et al. 2007). In addition, vernal pools can vary extensively in their hydrology, water and sediment chemistry, and other variables such as dissolved oxygen and temperature (Colburn 2004). Recent studies have shown that variation in physical and chemical variables play an important role in determining the community composition of vernal pools (E.A. Colburn, Harvard Forest, Petersham, MA, unpubl. data). This variation could lead to adaptive differences among vernal fairy shrimp populations as has been shown for other freshwater crustaceans (De- Meester et al. 2002). Though we have no evidence for the role of natural selection in driving levels of genetic divergence among vernal pool fairy shrimp populations, and we certainly are not implying selection acting on the mitochondrial gene region studied here, adaptive differences among populations could further reduce levels of gene flow among populations, as proposed in the monopolization hypothesis (DeMeester et al 2002). Observed geographic patterns of genetic divergence among populations would then result from an interaction of persistent founder effects and natural selection with the end result being a complex spatial pattern among vernal pools, with no clear phylogeographic signal. Priority effects versus lack of dispersal Which explanation bests accounts for our results, a lack of dispersal among vernal pools or priority effects? Resolving this issue clearly depends on direct estimates of dispersal among vernal pools. However, though passive dispersal may not be as prevalent as previously thought (Bohonak and Jenkins 2003), it is clear that some level of dispersal occurs in diapausing invertebrates, as demonstrated in recent studies using experimental pools (Cáceres and Soluk 2002, Frish and Green 2007, Louette et al. 2007). Extrapolating from these results should proceed cautiously, however, since we should expect dispersal rates to vary greatly among species regardless of the fact that they share the common character of having a diapausing cyst. 300 Northeastern Naturalist Vol. 17, No. 2 Finding strong evidence for dispersal in one species having a diapausing egg stage need not imply extensive dispersal occurs in all such species. One point does seem to be generally agreed upon: dispersal does occur at least on a microgeographic scale. Geographically proximate populations may experience a fairly high level of dispersal among populations due to wind, waterfowl, and other animal vectors (Bilton et al. 2001, Bohonak and Jenkins 2003, DeMeester et al. 2002, Figuerola et al. 2005, Green and Figuerola 2005). Therefore, the primary mechanism driving patterns of divergence in species with passive dispersal may vary according to geographic scale. Priority effects may explain high levels of population differentiation in geographically close populations, while a lack of dispersal leading to genetic drift (stochastic lineage sorting) predominates at greater geographic distances. That transition zone where we see a shift from the lack of gene flow being driven by priority effects versus lack of dispersal should vary by species according to species specific patterns of dispersal and should be reflected in the observed patterns of genetic divergence among populations. In our particular example, the extent of contemporary dispersal among vernal pool populations of E. vernalis is unknown at any scale; we are not aware of any direct estimates of dispersal in this species. It is difficult to imagine a lack of contemporary dispersal of fairy shrimp among Massachusetts vernal pools when clearly it had to have occurred in the recent past, but this is of course no evidence that dispersal continues today. Regardless, the high levels of genetic divergence among E. vernalis populations, a lack of any coherent phylogeographic association among haplotypes, and no isolation by distance all suggest limited gene flow among Massachusetts’ populations of the vernal pool fairy shrimp. Whether this is due to priority effects or simply limited dispersal remains to be determined. Be that as it may, we have clearly demonstrated that vernal pool fairy shrimp populations tend to be genetically distinct entities with limited gene flow among them. In light of the growing awareness of the critical link between community ecology and evolutionary processes (e.g., DeMeester et al. 2007, Emmerson and Gillespie 2008, Stockwell et al. 2003, Vellend and Geber 2005), these results lend valuable insights to our overall understanding of the ecology and evolution of vernal pool organisms. Acknowledgments The majority of the laboratory research reported here was performed by C. Wilbur and N. 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