Phylogeography and cryptic introduction of the ragworm Hediste diversicolor (Annelida, Nereididae)...

Phylogeography and cryptic introduction of the ragworm Hediste diversicolor(Annelida, Nereididae) in the Northwest Atlantic

Anthony L. Einfeldt, Jeremy R. Doucet, and Jason A. Addisona

Department of Biology, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada

Abstract. Many benthic marine invertebrates show striking range disjunctions across broadspatial scales. Without direct evidence for endemism or introduction, these species remaincryptogenic. The common ragworm Hediste diversicolor plays a pivotal role in sedimentarylittoral ecosystems of the North Atlantic as an abundant prey item and ecosystem engineer,but exhibits a restricted dispersal capacity that may limit connectivity at both evolutionaryand ecological time scales. In Europe, H. diversicolor is subdivided into cryptic taxa andgenetic lineages whose distributions have been modified by recent invasions. Its origin inthe northwest Atlantic has not been adequately addressed. To trace the age and origin ofNorth American ragworm populations, we analyzed mtDNA sequence data (COI) from theGulf of Maine and Bay of Fundy (n=73 individuals) and compared our findings with pub-lished data from the northeast Atlantic. Our results together with previous data indicatethat two species of the H. diversicolor complex have independently colonized the northwestAtlantic at least three different times, resulting in two distinct conspecific assemblages in theBay of Fundy and Gulf of Maine (respectively) that are different from the species found inthe Gulf of St. Lawrence. North American populations had significantly lower geneticdiversity compared with populations in the northeast Atlantic, and based on patterns ofshared identity, populations in the Bay of Fundy originated from the Baltic Sea and NorthSea. Populations from the Gulf of Maine were phylogenetically distinct and most likelyoriginated from unsampled European populations. Analyses of the North American popula-tions revealed patterns of post-colonization gene flow among populations within the Gulf ofMaine and Bay of Fundy. However, we failed to detect shared haplotypes between the tworegions, and this pattern of complete isolation corroborates a strong phylogeographic breakobserved in other species.

Additional key words: founder event, cryptogenic, population divergence

The influence of human-mediated introductionson the global distribution of biodiversity is oftenunderestimated due to the common assumption thatspecies lacking documented histories of introductionare endemic (Carlton & Geller 1993). For soft-bod-ied animals that do not preserve well in the archaeo-logical record, direct evidence of endemism isdifficult to come by, as historical records for mostmarine taxa are poor and have been preceded byextensive shipping activity that facilitated introduc-tions during the Age of Exploration (15–17th centu-ries). Analyses of population genetic data provide a

means to demonstrate whether species with unusu-ally broad distributions are endemic, or the productof historic human-mediated dispersal (e.g., Carlton1996; Blakeslee et al. 2008; reviewed in Geller et al.2010 and Haydar 2012). Resolving such cryptogenicpopulation histories is important to our understand-ing of human impact on species distributions andbiogeography, as well as our interpretation of eco-logical and evolutionary processes in systems thatare not at equilibrium.

The polychaete Hediste diversicolor (O.F. M €ULLER

1776) (Polychaeta: Nereididae; previously known asNereis diversicolor) is an ecologically and economi-cally important littoral species complex with twomajor lineages (Gambi et al. 1994; Olive 1994).

aAuthor for correspondence.E-mail: [email protected]

Invertebrate Biology x(x): 1–10.© 2014, The American Microscopical Society, Inc.DOI: 10.1111/ivb.12060

Species A is common to the estuarine environmentsof the North Temperate Zone of the Atlantic andBaltic, and Species B is endemic to the Mediterra-nean, Black, and Caspian Seas (Scaps 2002). Theseomnivorous infaunal worms live in mud, sand,gravel, and turf, where they build U- or Y-shapedburrows at densities documented to exceed 3500 perm2, making them ecologically important as biotur-bators and prey items for many invertebrate andvertebrate (Scaps 2002; Cuny et al. 2007; Durouet al. 2007; Bowser et al. 2013). Larvae of H. diver-sicolor are incubated by the female and burrowimmediately following emergence, resulting in limi-ted dispersal that is expected to promote genetic iso-lation among populations separated by a lackof available habitat at a range of spatial scales(Bartels-Hardege & Zeeck 1990; Scaps 2002).

Despite the expected isolation between disjunctpopulations across the Atlantic and the importanceof this species across its range, the phylogeographicorigin of H. diversicolor has not been adequatelyaddressed. Genetic data for northwest Atlanticpopulations of H. diversicolor are limited to onepopulation in Chaleur Bay, Quebec (Breton et al.2003), which has been suggested as a potentialsource for the cryptic H. diversicolor Species B intro-duced to the Baltic (Audzijonyte et al. 2008). How-ever, genetic data collected by Virgilio et al. (2009)suggest that Species B colonized the Baltic from theWestern Mediterranean and Black/Caspian Seas,alongside another lineage originating from the NorthSea. The cryptogenic status of North American popu-lations thus stands unresolved, despite the dramaticimplications that a recent invasion of this speciesacross the Atlantic would have for the assemblageand dynamics of local community structure.

The population genetic structure of H. diversicolorhas been well studied in Europe, where there is sig-nificant population heterogeneity within estuaries(R€ohner et al. 1997; Virgilio & Abbiati 2004, 2006),phylogeographic breaks between the Black Sea/Cas-pian Sea, Mediterranean, and Atlantic Ocean (Virgi-lio et al. 2009), and the presence of cryptic specieswithin the Baltic (Fong & Garthwaite 1994;Audzijonyte et al. 2008). While these studies confirmthat barriers to dispersal can promote localizedintraspecific genetic differentiation, populationgenetic patterns among northwest Atlantic popula-tions of H. diversicolor have not been characterized.Aside from a strong biogeographic break that hasbeen identified between populations of the amphi-pod Corophium volutator (PALLAS 1776) sampledfrom mudflats in the Gulf of Maine and the Bay ofFundy (Einfeldt & Addison 2013), there are few

comparisons of population genetic patterns of othermarine invertebrates across this region. Evidencefrom additional species to test the generalization ofthis phylogeographic break at the community levelis necessary, and as H. diversicolor inhabits the samesoft-sediment habitat as C. volutator and has a limi-ted benthic dispersal ability, it is an ideal specieswith which to further investigate this divide.

In this study, we use patterns of mitochondrialDNA (mtDNA) sequence variation to test thehypothesis that populations of H. diversicolor areinvasive across the north Atlantic (suggested byAudzijonyte et al. 2008). We compare our newsequence data with published data to test the predic-tions that populations established through a recentfounder event (i) will have a high frequency of haplo-types identical to those in the founding populationsand (ii) exhibit relatively low genetic diversity com-pared with source populations, and that (iii) diver-sity in introduced populations will be nested withinthat of endemic lineages (Corre & Kremer 1998;Dlugosch & Parker 2007). Alternatively, if popula-tions of H. diversicolor have long been establishedon both coasts of the North Atlantic (i.e., they areendemic to both regions), we expect to recover highgenetic diversity in all populations and observe deepphylogenetic divergence between both coasts (Avise2000). Our study also characterizes the patterns ofgenetic structure among NW Atlantic populationsand assesses the potential phylogeographic breakbetween the Bay of Fundy and the Gulf of Maine.As H. diversicolor has a life history that lacks apelagic dispersal stage, we expect the strong currentsisolating the Bay of Fundy from the Gulf of Maineto limit post-colonization gene flow via demersalswimming, and result in significant populationgenetic subdivision between the two regions.

Methods

Sampling and species identification

Specimens of Hediste diversicolor were collectedfrom nine mudflats within the inner Bay of Fundyand Gulf of Maine (Table 1, Fig. 1) between May2012 and December 2012. To avoid the possibility ofsampling kin groups, individual polychaetes werecollected haphazardly from each mudflat across spa-tial scales of 500–1000 m with a minimum separationof 10 m between samples. Specimens were preservedin the field using 95% ethanol, and identified asH. diversicolor with a compound microscope usingmorphological features of the proboscis and anteriorcirri (Smith 1964; Appy et al. 1980; Weiss 1995).

Invertebrate Biologyvol. x, no. x, xxx 2014

2 Einfeldt, Doucet, & Addison

DNA extraction, PCR, and sequencing

Tissue was dissected from the anterior region toavoid contamination by intestinal content. GenomicDNA was extracted using a cetyltrimethylammoni-um bromide (CTAB) protocol (Grosberg et al.1996). A fragment of the cytochrome c oxidasesubunit I (COI) mitochondrial gene was amplifiedusing the polymerase chain reaction (PCR) primersLCO1490 and HCO2198 (Folmer et al. 1994).Amplification was performed in a 20 lL volume

consisting of 10 ng DNA, 1X ThermoPol reactionbuffer (New England Biolabs, NEB), 0.2 mmol L�1

dNTPs (NEB), 2.5 mmol L�1 MgSO4, 0.5 lmolL�1 forward and reverse primers, and 0.8 U Taqpolymerase (NEB). Thermal cycling conditions were95°C for 3 min, followed by 34 cycles of 95°C(30 s), 45°C (45 s), 72°C (60 s), and a final exten-sion at 72°C for 60 s. Amplicons were separatedusing agarose gel electrophoresis and visualizedunder UV light prior to sequencing using for-ward, reverse, or both PCR primers at the

Table 1. Geographic region, population grouping, number of individuals (n), number of segregation sites (S), numberof haplotypes (H), haplotype diversity (h), and mean nucleotide diversity (p) for the 271 bp COI alignment of Hedistediversicolor species A sequences from this study, Virgilio et al. (2009), and Audzijonyte et al. (2008).

Region Location n S H h p

Northwest Atlantic Bay of Fundy 29 17 6 0.663 0.015Northwest Atlantic Gulf of Maine 44 26 4 0.720 0.037NW Atlantic Total 73 37 10 0.847 0.033Northeast Atlantic Baltic 64 42 28 0.949 0.021Northeast Atlantic Northwest European Coast 33 51 27 0.989 0.029Northeast Atlantic Portugal 28 15 7 0.820 0.011NE Atlantic Total 125 68 56 0.975 0.029

All samples 198 76 61 0.966 0.033

Fig. 1. Locations of sampled populations of Hediste diversicolor in the northwest Atlantic Ocean. Refer to Table 1 forabbreviations. Arrows depict circulation patterns for the Bay of Fundy (adapted from Aretxabaleta et al. 2008) andthe Gulf of Maine (adapted from Pettigrew et al. 2005).


Phylogeography of Hediste diversicolor in the NW Atlantic 3

Genome Quebec Innovation Centre (Montreal,Quebec).

Sequence alignment and analysis

Sequences were aligned, edited, and trimmed to alength of 580 base pairs using Sequencher 5.0 (GeneCodes) (GenBank accession no. KF958754-826). Wecomputed measurements of nucleotide diversity (p),number of segregating sites (S), number of haplo-types (H), and haplotype diversity (h) using ARLE-QUIN v3.5 (Excoffier et al. 2005) for each region(Baltic, Northwest European Coast, Portugal, Bayof Fundy and Gulf of Maine). To test for a signa-ture of a founder event consistent with a recentinvasion across the north Atlantic, we aligned oursequences with European COI data for H. diversicol-or species A and B (sensu Audzijonyte et al. 2008;species A synonymous with Clade 1, species B syn-onymous with Clades 2+3 from Virgilio et al. 2009)presented by Audzijonyte et al. (2008; FJ030956-994) and Virgilio et al. (2009; EU300637-786). Thisnew 271 bp alignment indicated that all newsequences grouped with H. diversicolor species A,whereas the Canadian Chaleur Bay populationbelonged to the cryptic species B. Further analyseswere therefore restricted to comparisons withinH. diversicolor species A. To evaluate the efficacy ofsampling effort, a rarefaction analysis wasperformed using the Chao2 indicator in ESTI-MATES 8.0 to estimate total diversity in eachregion (Colwell et al. 2012). The genealogical rela-tionship of haplotypes was estimated using bothBayesian and maximum-likelihood methods. Genetrees and the posterior probability of the nodes wereestimated with MrBayes3 (Ronquist & Huelsenbeck2003) using the HKY model of substitution selectedby the Bayesian Implementation Criterion imple-mented in the program jMODELTEST 2.1.3 (Darribaet al. 2012). Independent analyses of 10 chains wererun (temp=0.1) for 3 million generations, samplingtrees every 1000 steps. Convergence was achieved byrunning the analysis long enough to ensure that thestandard deviation of the split frequencies of bothindependent runs was less than 0.01. A phylogenetictree was also inferred by maximum likelihood usingPhyML 3.1 (Guindon et al. 2010), with an HKY85substitution model, gamma distributed rate hetero-geneity at sites, and an SPR tree search. Nodal sup-port was estimated using nonparametric bootstrapanalysis with 1000 replicates.

To explore patterns of genetic structure within thenorthwest Atlantic populations, we computed mea-sures of diversity (as above) and visualized a 95%

parsimony network using the computer programTCS 1.21 (Clement et al. 2000). We represented thephylogeography by overlaying the geographic distri-bution of samples on a neighbor-joining tree con-structed using Kimura 2 parameter (K2P) distancesimplemented by MEGA (Tamura et al. 2011). Popu-lation subdivision was evaluated by computing Weir& Cockerham’s (1984) global FST using the K2Pmodel implemented in ARLEQUIN (Excoffier et al.2005). To test for a biogeographic break at themouth of the Bay of Fundy as observed in C. volu-tator (Einfeldt & Addison 2013), a hierarchicalanalysis of molecular variance (AMOVA) in ARLE-QUIN (Excoffier et al. 2005) was conducted bygrouping populations in the Bay of Fundy or theGulf of Maine (Table 1). The significance of boththe FST and AMOVA tests was assessed by 10,000permutations of the data.

Results

Comparison with European populations

Our 580 bp COI alignment from 73 NW Atlanticindividuals was added to 125 published Hediste di-versicolor species A sequences from the NE Atlanticto create a final trimmed alignment of 271 bp(Table 1). The NW Atlantic showed a significantreduction in genetic diversity compared with the NEAtlantic (chi-square test statistic X2=30.2, d.f.=1,p<0.001). Of the 59 unique H. diversicolor species Ahaplotypes detected overall, 10 were found in theNW Atlantic, and 56 were found in the NE Atlan-tic. Consistent with the hypothesis that H. diversicol-or was introduced to the NW from the NE Atlantic,five of the six haplotypes recovered in the Bay ofFundy were identical to those unique to the Baltic(four haplotypes) or shared with the Baltic andNorth Sea (one haplotype) (Fig. 2). None of thefour haplotypes recovered in the Gulf of Mainewere shared with NE Atlantic populations, andalthough two of these haplotypes are highly diver-gent, they did not cluster with the cryptic species B(Fig. 2), which included all samples from the Cana-dian Chaleur Bay population studied by Bretonet al. (2003). Using haplotype estimation (Chao2)analyses, the estimated total maximum number ofhaplotypes in the NW Atlantic was 10.7 (95% CI10.1–17.8), compared to 123.0 (95% CI 54.3–365.0)in the North Sea and 60.4 (95% CI 37.8–135.4) inthe Baltic (Table 2). These analyses suggest that thenumber of unique H. diversicolor species A haplo-types in the NE Atlantic is at least an order of mag-nitude greater than in North America, and that



while sampling in North America was sufficient tocapture total diversity, increased sampling in Europewould continue to reveal additional uniquehaplotypes.

Population structure

In the 580 bp sequence alignment of COI for 73individuals of H. diversicolor from the NW Atlantic,

Table 2. Observed number of 271-bp COI haplotypes in the samples, and expected number in the total population forHediste diversicolor species A in the NW Atlantic, Northwest European Coast (Portugal excluded), and Baltic. Maxi-mum expected number of haplotypes (Hmax-exp) and 95% confidence intervals were calculated using the Chao2 estima-tor. Non-overlapping confidence intervals indicate much higher genetic diversity in Europe than in North America,suggesting that unique North American haplotypes are likely among the many unsampled haplotypes in Europe.

Region Populations sampled Hobs Hmax-exp Lower 95% Upper 95%

Bay of Fundy 5 6 7.0 6.1 17.1Gulf of Maine 4 4 4.0 4.0 4.8NW Atlantic 9 10 10.7 10.1 17.8Northwest European Coast 3 27 123.0 54.3 365.0Baltic 12 28 60.4 37.8 135.4NEC+Baltic 15 49 171.5 92.5 394.3

Fig. 2. Bayesian tree of the unique 271 bp COI haplotypes collected in this study and sequences published in Gen-bank. Haplotypes of Hediste diversicolor are identified as species A and B or Clade 1, 2, and 3 (following Audzijonyteet al. 2008 and Virgilio et al. 2009, repectively). Haplotypes from the northeast Atlantic are indicated with circles andhaplotypes from the Mediterranean/Black/Caspian Seas are indicated with triangles. Haplotypes from the northwestAtlantic (Fundy/Maine) are identified with arrows, and those shared with the northeast Atlantic are indicated with abox. Nodal support is presented for nonparametric bootstrap (1,000 replicates) of the maximum-likelihood tree (above)and Bayesian posterior probability (below). Only values greater than 60% are reported.



there were 12 highly divergent and unique haplo-types distinguished by a total of 65 segregating sites(Table 3). The NW Atlantic haplotypes clusteredinto six distinct haplotype groups, which were sepa-rated by mean distances ranging from 12 to 28.5nucleotide differences (Fig. 3). Two lineages in theGulf of Maine (clusters V and VI) diverged fromthe other H. diversicolor species A sequences by 3.4–5%, suggesting the possibility that they may repre-sent a cryptic species in the H. diversicolor complex.The geographic distribution of the 12 unique haplo-

types demonstrated strong differentiation among thenine sites, and clusters of haplotypes did not repre-sent reciprocal clades between the Gulf of Maineand the Bay of Fundy (Fig. 3b). Eight of the 12haplotypes were found exclusively in the Bay ofFundy, and the remaining four were restricted tothe Gulf of Maine (Fig. 3).

The four Gulf of Maine populations generallyhad more segregating sites (S=32–40) and highernucleotide diversity (p=0.021–0.031) than popula-tions within the Bay of Fundy (Table 3). Within the

Table 3. Hediste diversicolor. Sampling sites, geographic region (BF=Bay of Fundy, GM=Gulf of Maine), number ofindividuals sequenced (n), number of segregation sites (S), number of haplotypes (H), haplotype diversity (h), andmean nucleotide diversity (p) for the 580-bp COI alignment.

Site Population Abbr. Region n S H h p

1 Avonport AV BF 5 19 3 0.800 0.0172 Daniels Flat DF BF 5 11 2 0.400 0.0083 Grande Anse GA BF 6 19 4 0.867 0.0164 Minudie MN BF 7 16 3 0.524 0.0105 Saint John SJS BF 6 21 4 0.867 0.0176 Falmouth FMS GM 11 40 4 0.764 0.0317 Machiasport MAC GM 12 40 4 0.636 0.0218 Milbridge MIL GM 11 32 3 0.636 0.0269 Waldoboro WAL GM 10 32 3 0.711 0.028

Total 73 65 12 0.854 0.026

Fig. 3. A. A 95% parsimony network for Hediste diversicolor COI (580 bp) sequences from North America. Each sym-bol represents a unique sequence (12 total). Circle area is proportional to haplotype frequency (n=1–18). Solid linesconnect haplotypes differing by one mutation; small open circles indicate missing intermediate haplotypes. Color-coding indicates the origin of each private haplotype (solid circles) or frequency of shared haplotypes (pie diagrams):blue=Bay of Fundy, and green=Gulf of Maine. Dotted lines connect networks with 90% confidence. B. Neighbor-join-ing tree (NJ) constructed using Kimura 2-parameter distances with clusters of haplotypes and their geographic originsindicated as in (A).



Bay of Fundy, one high frequency haplotype(shared by 72% of the individuals within the region)was common to all five mudflats, while four of themudflats each had their own unique haplotype. Incontrast, three of the four haplotypes detected inthe Gulf of Maine were common (20–41% totalfrequency per haplotype) and shared by all fourpopulations, while the haplotype with the lowest fre-quency in the Gulf of Maine (11%) was restrictedto two of the four populations (Fig. 3). These largedifferences in haplotype frequencies among popula-tions resulted in a highly significant global fixationindex (FST=0.198; p<0.001), indicating strong geneticstructure throughout the region.

Results of the AMOVA support the strong diver-gence between the Bay of Fundy and the Gulf ofMaine (Table 4). Grouping of populations based onthe previously described biogeographic breakbetween the Bay of Fundy and Gulf of Maineexplained 26.2% of the variation in nucleotide diver-sity (ΦCT=0.262, p<0.001). Including Saint John inthe Gulf of Maine grouping (ΦCT=0.191, p=0.032),or as a distinct third region (ΦCT=0.224, p=0.011) assuggested for Corophium volutator by Einfeldt &Addison (2013), did not improve the geographicresolution of the genetic break.

Discussion

Trans-Atlantic introduction

The genetic patterns found for Hediste diversicolorbetween Europe and North America resemble thosefound for marine species that have had multipleintroductions to the NW Atlantic: the green crabCarcinus maenas (LINNAEUS 1758) (Roman 2006),the snail Littorina littorea (LINNAEUS 1758) (Blakes-lee et al. 2008), and the brown algae Fucus serratus(LINNAEUS 1753) (Brawley et al. 2009). The nearly20-fold reduction in genetic diversity in the NWAtlantic and widespread sharing of identical haplo-types nested within the diverse NE Atlantic lineagesare indicative of a founder event, strongly suggestingthat the disjunct distribution of H. diversicolor inthe North Atlantic is the result of human-mediated

introduction. Based on patterns of shared identity,our results suggest that the sources for the Bay ofFundy populations were located in the Baltic Seaand North Sea (The Netherlands and Germany). Incontrast, all the genetic variants detected in the Gulfof Maine are unique to the NW Atlantic, which sug-gests either a second independent introduction froman unsampled European population, or a historicaldistribution in the NW Atlantic that predates therecent invasion. We can at least in part reject thelatter hypothesis based on the lack of reciprocalmonophyly of haplotypes from both the Bay ofFundy and Gulf of Maine, which would be expectedunder a model of allopatric divergence (Avise 2000).The nesting of the unique Gulf of Maine haplotypesin the phylogeny of European lineages (Fig. 2)strongly suggests that these populations were inde-pendently introduced from an unknown NE Atlanticpopulation, similar to the nesting of the ChaleurBay population from Breton et al. (2003) within theH. diversicolor species B phylogeny, as outlined byVirgilio et al. (2009). However, two highly divergentlineages in the Gulf of Maine are monophyletic withrespect to the other species A haplotypes, whichsuggests that the alternative explanation of an ende-mic cryptic species in the Gulf of Maine may beplausible. More intensive sampling of the Europeancoastline will help in identifying potential sourcepopulations, clarifying the degree of cryptic diversityin the Gulf of Maine, and resolving cryptogenicpopulation history.

Previous studies have documented the introduc-tion of several marine species across the Atlantic viaballast water, which enables trans-oceanic dispersalof species with pelagic larvae or adult stages smallenough to pass exclusion filters (e.g., Zmudzinski1996; Bastrop & Blank 2006; reviewed in Ruiz et al.2000). While whole benthic invertebrates of largersize than H. diversicolor, such as C. maenas andRhithropanopeus harrisii (GOULD 1841), have beendocumented in ballast tanks arriving in Canada(Briski et al. 2012), H. diversicolor itself has notbeen documented in ballast tanks to date. Prior to~1880, when ballast tanks came into widespread use,material stored and collected from the intertidal

Table 4. Hediste diversicolor population structure from analysis of molecular variance (AMOVA) in mtDNA sequences(580-bp COI) sampled from the NW Atlantic (see Table 3).

Source of the Variation d.f. Sum of squares % of variance Fixation index p-value

Among groups 1 92.5 26.2 ΦCT=0.262 <0.001Among populations|groups 7 61.4 3.2 ΦSC=0.043 0.094Within populations 64 413.5 70.6 ΦST=0.294 <0.001



(dry ballast) was loaded onto boats to adjust buoy-ancy (Galil et al. 2011). As H. diversicolor lacks apelagic phase and inhabits sediment in intertidalestuaries associated with historic shipping ports, dryballast is the most probable introduction vector forthis species. This dates its introduction to NorthAmerica prior to 1880, earlier than the first biologi-cal surveys of the sedimentary intertidal in the NWAtlantic.

Ecological impact

The short evolutionary history of H. diversicolorin North America revealed by our data has strongimplications for habitat structure, intertidal ecology,and community evolution. While the impact of non-indigenous species is often evaluated according todirect effects on biodiversity, species that physicallyalter their environments (ecosystem engineers) canalter the rules of existence for the entire marinecommunity (Vitousek et al. 1997; Wallentinus &Nyberg 2007). By constructing burrows and creatingcurrents through them while feeding, H. diversicolorcauses bioturbation that decreases phosphate efflux-es, enhances ammonia release and nitrogen uptake(Mortimer et al. 1999), redistributes green algae intothe sediment (Nordstrom et al. 2006), and is amajor cause of saltmarsh loss and coastal erosion(Paramor & Hughes 2004). As a high impact ecosys-tem engineer, the introduction of H. diversicolor tothe Gulf of Maine and Bay of Fundy may have fun-damentally changed both the habitat and structureof the soft-sediment intertidal. By examining therole of ecosystem engineers like H. diversicolor inboth introduced and native habitat, we will gainvaluable insights into the ecological principles thatgovern the assembly of marine intertidal communi-ties, enhancing our ability to predict ecosystem-leveleffects of introductions. Our results highlight theimportance of genetic studies for both understand-ing and reconstructing historical and contemporarycommunity assemblages, and play an essential rolein improving our understanding of the causes andconsequences of marine invasions.

Phylogeography in the northwest Atlantic

We detected strong patterns of genetic subdivisionand deep divergence among lineages of H. diversicol-or sampled in the Bay of Fundy and Gulf of Maine.Our data reveal a striking pattern of independentcolonization of mudflats in the Bay of Fundy andGulf of Maine, with each region being populated bya different subset of European haplotypes. Genetic

similarity among populations within regions reflectsefficient post-colonization gene flow at smaller spa-tial scales, while restricted gene flow between regionsis consistent with the hypothesis of a phylogeo-graphic break between the Bay of Fundy and theGulf of Maine. The strong gyre at the mouth of theBay of Fundy appears to provide an efficient barrierto gene flow in H. diversicolor; this pattern is identi-cal to that observed in Corophium volutator, inwhich no shared mitochondrial haplotypes betweenthe Bay of Fundy and Gulf of Maine were identified(Einfeldt & Addison 2013). This concordant phylo-geographic break suggests that for species with simi-lar dispersal ability, the Bay of Fundy is a distinctbiogeographic region isolated from the Gulf ofMaine and Atlantic Ocean.

Understanding the position and strengths ofgenetic breaks such as that identified in this study isan important component to understanding thedynamics driving complex ecosystems. Our resultsshow that the strong hydrological features separat-ing the Bay of Fundy from the Gulf of Maineseverely limit gene exchange between the tworegions following multiple introduction events. Thefactors driving recruitment of individuals within andbetween the biogeographic regions, both pre- andpost-introduction, are also consequential to identify-ing the roles that natural selection and stochasticprocesses play in shaping patterns of genetic diver-sity in marine populations (Grosberg & Cunning-ham 2001). Hediste diversicolor displays a widetolerance for salinity, thriving in habitats rangingfrom freshwater to two times the normal salinity ofseawater (Oglesby 1969), but is susceptible toanthropogenic stress; when exposed to elevated lev-els of toxic trace metals, the species exhibits reducedfecundity and fitness (Scaps 2002; Durou et al.2005; Moreira et al. 2006). While little is known ofthe range of physical and chemical stresses that popu-lations of H. diversicolor experience in the Bay ofFundy and Gulf of Maine, it is possible that naturalselection may contribute to the success of differentlineages in each of the two regions, or to the main-tenance of the strong genetic break between them.

The striking differentiation we report in this studysuggests that this biogeographic break currently rep-resents a significant barrier to dispersal for H. diversi-color. Given its importance as a bio-indicator inmany marine and brackish water habitats, limiteddispersal ability, and wide tolerance to stressors,more detailed genetic studies of H. diversicolorwill deepen our understanding of the mechanismsdriving genetic change in marine systems. Additionalstudies on other members of the shallow water com-



munity with a variety of dispersal abilities will pro-vide a more detailed understanding of the influenceof self-recruitment, biogeographic barriers, geneticdrift, and natural selection on the genetic structure ofmarine species in the Bay of Fundy and elsewhere.

Acknowledgments. We thank J Starkey-Seto and MABarbeau for help with field collections, and G Rouse, RV€ain€ol€a, and an anonymous reviewer for constructive crit-icism. Funding was provided by NSERC Discovery, CFILeaders Opportunity FUND, and NBIF grants to JAA.

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