Biol Invasions (2010) 12:191–209 DOI 10.1007/s10530-009-9442-3

ORIGINAL PAPER

Reduction in post-invasion genetic diversity in pseudogracilis (: Crustacea): a genetic bottleneck or the work of hitchhiking vertically transmitted microparasites?

Johanna G. M. Slothouber Galbreath Æ Judith E. Smith Æ James J. Becnel Æ Roger K. Butlin Æ Alison M. Dunn

Received: 14 July 2008 / Accepted: 22 January 2009 / Published online: 7 February 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Parasites can strongly influence the suc- parasites may evade the stochastic processes and cess of biological invasions. However, as invading selective pressures leading to enemy release. As hosts and parasites may be derived from a small microsporidia may be vertically or horizontally subset of genotypes in the native range, it is important transmitted, we compared the diversity of these to examine the distribution and invasion of parasites microparasites in the native and invasive ranges of in the context of host population genetics. We the host. In contrast to the reduction in host genetic demonstrate that invasive European populations of diversity, we find no evidence for enemy release from the North American Crangonyx pseudogracilis have microsporidian parasites in the invasive populations. experienced a reduction in post-invasion genetic Indeed, a single, vertically transmitted, microsporid- diversity. We predict that vertically transmitted ian sex ratio distorter dominates the microsporidian parasite assemblage in the invasive range and appears to have invaded with the host. We propose that Electronic supplementary material The online version of overproduction of female offspring as a result of this article (doi:10.1007/s10530-009-9442-3) contains supplementary material, which is available to authorized users. parasitic sex ratio distortion may facilitate host invasion success. We also propose that a selective J. G. M. Slothouber Galbreath J. E. Smith sweep resulting from the increase in infected individ- & A. M. Dunn ( ) uals during the establishment may have contributed to Faculty of Biological Sciences, Institute of Integrative and Comparative Biology, University of Leeds, the reduction in genetic diversity in invasive Crang- Leeds LS2 9JT, UK onyx pseudogracilis populations. e-mail: [email protected] Keywords Biological invasions J. G. M. Slothouber Galbreath Institute of Biological and Environmental Sciences, Enemy release Emergent disease University of Aberdeen, Zoology Building, Microsporidia Sex ratio distortion Aberdeen AB24 2TZ, UK Vertical transmission

J. J. Becnel USDA/ARS, Center for Medical, Agricultural and Veterinary Entomology, P.O. Box 14565, Introduction Gainesville, FL 32604, USA Two important factors affecting the success of an R. K. Butlin Department of and Plant Sciences, University invasion and its impact on the native biota are the of Sheffield, Western Bank, Sheffield S10 2TN, UK changes in genetic diversity as a result of the invasion 123 192 J. G. M. Slothouber Galbreath et al.

(Miura 2007) and the impact of parasitism (Hatcher ranges (Mitchell and Power 2003; Torchin et al. et al. 2006). Invasion success is dependent on the size 2003) as well as individual empirical studies demon- and origin of introduced populations and on the strating a reduction in parasitism in invasive range frequency of introductions (Kolar and Lodge 2001; compared with the native range. This reduced impact Suarez et al. 2005) and invasion filters are predicted on host fitness may be realized as a reduction in to cause a reduction in post-invasion genetic diversity parasite diversity (Marr et al. 2007) or a reduction in with genetic bottlenecks historically considered a parasite prevalence and intensity in the invasive host general characteristic of invasion events (Cristescu (Torchin et al. 2001). However, invading hosts and et al. 2004; Muller et al. 2002). For example, the parasites are often derived from a small subset of a Ponto-Caspian freshwater amphipod Echinogamma- generally much larger pool of candidate genotypes in rus ischnus has experienced a severe reduction in the native range (Colautti et al. 2004). Hence, tests post-invasion genetic diversity throughout its Euro- for enemy release that do not restrict comparison pean and North American invaded range (Cristescu between the invasive population and the source et al. 2004). However, several recent studies find no population from which it was founded may lead to reduction in genetic diversity (e.g. Astenei et al. exaggerated estimates of enemy release (Colautti 2005; Wattier et al. 2007) or even an increase in et al. 2004). diversity. A study of invasive populations of the North American amphipod Gammarus tigrinus Study system revealed decreased genetic diversity in some invasive European populations, whilst others had increased Amphipod Crustacea are successful invaders globally genetic diversity reflecting multiple sources of intro- (Cristescu et al. 2004; Dick and Platvoet 2000; duction (Kelly et al. 2006). Hence it is important to Jazdzewski et al. 2004; Devin and Beisel 2008) and consider the effect of the source and the history of an many species are successful intercontinental invaders invasion on genetic diversity. (Colautti et al. 2005; Holeck et al. 2004; Bij de Vaate Parasites have been shown to be important in et al. 2002). Amphipod invasions have led to determining the success and impact of biological dramatic changes in community structure (Krisp invasions. Parasites may directly influence the suc- and Maier 2005; Van Riel et al. 2006) including cess of the invading host (Prenter et al. 2004) as well extinction of native species, reduced species diversity as mediate the outcome of interactions between and richness (Dick and Platvoet 2000), and changes native and invasive species during invasion events in fish productivity (Kelly and Dick 2005). Some (MacNeil et al. 2003a, b; Prenter et al. 2004). invasive amphipods, including Echinogammarus is- Invasive species may introduce novel pathogens to chnus (Cristescu et al. 2004), have experienced a naı¨ve hosts (Daszak et al. 2000; Lips et al. 2006; sharp decline in post-invasion genetic diversity. For Tompkins et al. 2003), they may themselves acquire others, the invasion process appears to have had little new parasites from the invasive range (Krakau et al. discernable effect on post-invasion genetic diversity. 2006), or they may benefit from the loss of parasites One example is the freshwater amphipod Dikero- (Enemy release) during an invasion event (Mitchell gammarus villosus which has successfully invaded and Power 2003; Torchin et al. 2003). Release from the major river systems of Western Europe from a natural enemies has been proposed as a major factor Ponto-Caspian origin without any discernable loss of in the success of biological invasions. Subsampling, genetic diversity or loss of microparasites (Wattier stochastic factors, and selective pressures (Drake et al. 2007). There have also been several instances of 2003; Mitchell and Power 2003; Prenter et al. 2004) cryptic invasions by amphipod which during the invasion event may all contribute to the were only detected through the use of molecular loss of hosts with parasite-impaired fitness and markers (Muller 2001; Muller et al. 2002). consequently of parasites and susceptible host geno- Amphipods are host to a diverse range of parasites types (Mitchell and Power 2003; Torchin et al. 2003). (Dunn and Dick 1998) several of which play a role in Support for the enemy release hypothesis has native invader interactions (MacNeil et al. 2003a, b; come from both meta-analysis of the natural enemies Rigaud and Moret 2003; Bauer et al. 2005). For of non-indigenous species in their native and invasive example, the microsporidian Pleistophora mulleri 123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 193 reduces the predatory impact of the native G. duebeni Furthermore, there has been relatively little charac- on two invasive amphipods, thereby facilitating the terisation of the invasion history of C. pseudogracilis invasion process (MacNeil et al. 2003a, b; MacNeil compared to other gammarid amphipods invasive in et al. 2004). Acanthocephalan infections do not Europe. This amphipod is found mainly in ponds, manipulate the behaviour of the invasive amphipod lakes and slow moving streams (Holland 1976; Zhang Gammarus roeseli (Bauer et al. 2005; Tain et al. and Holsinger 2003; Josens et al. 2005) and is 1 of 42 2006), but do alter the phototactic response of the species of Crangonyx described from North America native amphipod G. pulex in France, making it (Zhang and Holsinger 2003). It is a primarily fresh- vulnerable to predation by the definitive fish host water species that will tolerate some salinity (Chlo- (Cezilly et al. 2000), and so may facilitate invasion. ride: 250–350 mg/l) but does not become established Furthermore, when the invasive D. villosus is present in organically polluted sites or sites experiencing in the system, the resulting manipulation of host fluvatile conditions (Holland 1976). Crangonyx has behaviour by the acanthocephalan Polymorhpus often been found in seemingly isolated sites and this minutus is stronger to ensure predation by the appro- has been attributed to introduction by anglers (acci- priate ultimate host rather than this predatory amphi- dental or deliberate; Holland 1976) and movement pod (Medoc et al. 2006). between sites by means of damp interstitial spaces as Microsporidia, a diverse phylum of eukaryotic well as the water table below the surface habitat parasites that infect all animal phyla (Wittner 1999), (Harris et al. 2002; Jass and Klausmeier 2003). are particularly widespread and diverse in amphipods Identification to species level within the Crangonyx (Terry et al. 2004) and so are of great use in studying has been difficult and, because the proposed native the impact of the invasion process on these hosts and range is so large (Fig. 1) and potentially overlaps with their parasites. In addition, microsporidia may use so many other species of Crangonyx, misidentification vertical and horizontal transmission routes (Dunn has occurred repeatedly in previous studies (Hynes et al. 2001). Studies of parasitism in native and 1955; Zhang and Holsinger 2003). In the native range, invasive populations have generally focused on sympatric species include Gammarus fasciatus, horizontally transmitted parasites, which are typically G. pseudolimnaeus, C. gracilis, C. minor, C. floridanus virulent and dependent on host density for transmis- and Hyalella azteca. A particular cause for concern is sion. In contrast with horizontally transmitted the sister species C. floridanus, which may be parasites, vertically transmitted parasites are typically confused with C. pseudogracilis, and which has avirulent and transmission is independent of host successfully invaded Japan and areas of the North density (Bandi et al. 2001; Dunn et al. 2001; Dunn American Pacific Seaboard (Zhang and Holsinger and Smith 2001). Therefore, these parasites may 2003; Kanada et al. 2007). As cryptic invasions may evade the stochastic processes and selective pressures be most robustly identified by molecular genetic leading to enemy release and may have an increased approaches (Miura 2007) and as there have been likelihood of invasion with the host (Slothouber several cases of cryptic amphipod invaders (Muller Galbreath et al. 2004). Furthermore, microsporidia 2001; Muller et al. 2002), confirming the identity of the are of interest as several species of vertically invasive Crangonyx in Europe is of some importance. transmitted microsporidia that infect dipteran and Species of the genus Crangonyx are morphologically amphipod hosts cause host sex ratio distortion (Terry similar, there is a need to combine morphological and et al. 2004) and thus have the potential to affect host molecular genetic data in order to confirm species population size and stability (Hatcher et al. 1999). It identity in native and invasive populations. It has been is therefore of particular interest to look for evidence proposed that European populations of C. pseudograc- of vertical transmission and sex ratio distorting ilis may have originated either from Canada by means microsporidia in invasive species. of the timber trade (Maitland and Adams 2001)or The North American amphipod Crangonyx elsewhere from the native range by means of ballast pseudogracilis was chosen for this study as it has water transport (Holmes 1975; Zhang and Holsinger spread rapidly across Western Europe since it was 2003). Both proposals are feasible as the native range first detected in England in 1936 (Crawford 1937) of C. pseudogracilis is enveloped by international and Holland in 1979 (Zhang and Holsinger 2003). shipping routes and transected by internal transport 123 194 J. G. M. Slothouber Galbreath et al.

Fig. 1 Map showing North American sampling sites (numbers locations within a seaboard, there is also a high volume of sea correspond to sites listed in Table 1. For both Crangonyx traffic on long-distance routes between seaboards. Further- pseudogracilis and C. floridanus, the lightly shaded areas more, shipping traffic from ports along the seaboards and the encompass all reported populations and the darker shaded areas Great Lakes region may reach distant seaboards via the indicate the core areas of the range. Lines indicate International Panama Canal (after US Army Corps of Engineers Waterway shipping routes and internal transport waterways from US Network (2004)) seaports. In addition to a high volume of sea traffic between waterways connecting to freshwater ports along the common to native amphipods and C. pseudogracilis seaboards (Fig. 1). While historical records cannot in Europe. We look for evidence that the invader has provide evidence in support for either of these introduced novel parasites to European amphipods in hypotheses, molecular genetic data may illuminate invaded habitats. Finally we test for sex host ratio the origin of C. pseudogracilis and the historical distortion for the different microsporidia detected. invasion process (Miura 2007). Using molecular and morphological characteris- tics, we confirm the identity of the Crangonyx species Materials and methods in native and invasive populations. We compare the genetic diversity and parasite diversity in native and Screening invading populations of C. pseudogracilis. We use nuclear (18S rDNA) and mitochondrial (COI) mark- We collected 441 individuals from 21 sites (Figs. 1, 2; ers to investigate whether invasive populations have Table 1) in the native and invasive range of C. pseudo- undergone a reduction in genetic diversity and to gracilis using a standard kick-sample method at each investigate the possible geographical origin of inva- site (Slothouber Galbreath et al. 2004). Using a sive C. pseudogracilis. We compare the diversity of standard net (230 mm 9 250 mm opening, 1 mm microsporidian parasites in native and invasive mesh size), the littoral zone of each site was sampled habitats using 16S rDNA markers specific to micro- for a period equivalent to a 2 h effort (i.e. 1 worker for sporidia. We test for enemy release by comparing 2 h, 2 workers for 1 h, etc.) or until at least 60 microsporidian parasite diversity in C. pseudogracilis amphipods had been collected. This method is pref- from native and invasive habitats and determine erable over quantitative methods because of the habitat whether these parasites are vertically or horizontally preferences (hiding in leaf litter, among roots, under transmitted. We test whether the invader has acquired stones, roots, and submerged wood as well as clinging microsporidian parasites from sympatric amphipods to floating macrophytes and dense vegetation) of the in the new range by looking for evidence of parasites amphipods sampled (Jazdzewski et al. 2004). The 123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 195

Table 1 List of sampling locations in Europe and North America Number Location Site

1 United Kingdom Middleton Park Pond, Leeds 2 The Netherlands Boerakker, Matsloot 3 The Netherlands De Eem, Eembrugge 4 The Netherlands De Aa, Rijsbergen 5 France Canal de Bourgogne, Dijon 6 France Le Loing, Souppes-s.-Loing 7 France Le Bouvron, Cellettes 8 France La Loire, Lignieres-de-Touraine 9 US: Louisiana Calcasieu River, Lake Charles 10 US: Louisiana Calcasieu River, Lake Charles 11 US: Louisiana Ira Breaux Rd. Ditch, Lake Charles 12 US: Louisiana Hwy 3095 Ditch, Lake Charles 13 US: Mississippi Lake Grada, Blue Mountain 14 US: Tennessee Barnishee Slough, Memphis 15 US: Wisconsin Lake Beulah, Walworth Co. 16 US: Wisconsin Green Lake, Walworth Co. 17 US: Florida Cross Creek, Gainesville 18 US: Florida Lab Ditch, Gainesville 19 US: Florida River Styx, Gainesville Fig. 2 Map showing European sampling sites (Identified in Table 1) 20 US: Florida Lake Alice, Gainesville 21 US: North Raleigh Carolina sampling consisted of thorough sweep-netting while the substrate and submerged vegetation were agitated through kicking as well as manual examination of not yield C. pseudogracilis, only sites which yielded submerged rocks, roots and wood. Populations of C. C. pseudogracilis have been included in the analysis. pseudogracilis were sampled from UK sites between North American sites were sampled for C. pseudo- October 2001 and March 2001 and from French and gracilis and sympatric amphipods during March 2004. Dutch sites during August 2003. In the UK, C. As the native range of C. pseudogracilis exceeds one pseudogracilis were collected from Middleton Park, million square kilometres (Fig. 1), it was not feasible Leeds. The Dutch sites included the site of the first to sample from all possible source populations within record of the C. pseudogracilis in continental Europe the range. Based on the finding of the microsporidian (Pinkster et al. 1980) in Boerakker at Matsloot (site 2) Fibrillanosema crangonycis in both the native and as well as two sites further south in de Eem at invasive range (Slothouber Galbreath et al. 2004), Eembrugge (site 3) and in de Aa at Rijsbergen (site 4). Louisiana was chosen as the primary sampling These sites were chosen as they were geographically location in the native range. Further sites were chosen and hydrologically distinct and C. pseudogracilis had based on published and unpublished records (data been collected from them during previous surveys (D. records housed at USDA/ARS, Center for Medical, Platvoet personal communication). In France, C. Agricultural and Veterinary Entomology, Gainesville, pseudogracilis had been collected as by-catch from Florida). Within Louisiana, sampling was focused the Loire (T. Rigaud personal communication), and so around Lake Charles and numerous potential sites preferred habitat types were sampled westwards from consistent with C. pseudogracilis preferred habitat Dijon along the Loire and its tributaries. As many type were sampled (N = 30). Unfortunately, in many (N = 15) of the potential sites that were sampled did of the sites from which C. pseudogracilis had been 123 196 J. G. M. Slothouber Galbreath et al. previously recorded there was no evidence of sources for parasites invading with C. pseudogracilis. C. pseudogracilis presence in the current study. In To test whether C. pseudogracilis have introduced Louisiana, only those sites which yielded C. pseudo- novel microsporidia to hosts in the invasive range, we gracilis have been included in the analysis. To collect looked for evidence of shared parasites between C. pseudogracilis from across its large geographic native C. pseudogracilis and sympatric European range, suitable locations in Wisconsin, Tennessee, and amphipods (data from Terry et al. 2004; Haine et al. Florida were also sampled. All amphipod species 2004). We also looked for evidence that C. pseudo- from these sites have been included in the analysis, gracilis had acquired new microsporidian parasites in including from sites which were not found to contain the invasive range by comparing parasite diversity in C. pseudogracilis. invasive C. pseudogracilis with that in sympatric Upon collection, amphipods were checked for European amphipods (data from Terry et al. 2004; visible signs of disease, anaesthetised using carbon- Haine et al. 2004). We tested for sex ratio distortion ated water (tap water, CO2), killed by decapitation by comparing the parasite prevalence in the gonads of and dissected for tissue specific screening. To prevent males and females under the null hypothesis that environmental contamination of the samples, the gut frequency of parasites should be the same in both was removed prior to further dissection. To screen for sexes (Terry et al. 2004). horizontally and vertically transmitted microsporidia, DNA was extracted for PCR of microsporidian tissue samples were taken from muscle (for horizon- small subunit ribosomal DNA, host COI mitochon- tally transmitted microsporidia) and gonads (for drial DNA (Terry et al. 2004), and host small subunit vertically transmitted microsporidia) and these were ribosomal DNA (Englisch et al. 2003) as previously fixed separately in 95% ethanol and stored at -20°C. described (Terry et al. 2004). PCR was undertaken In the case of gravid females, the eggs were also fixed using methods previously described in Terry et al. separately in 95% ethanol and stored at -20°C. (2004) using a suite of host and parasite specific We used morphological characteristics and molec- primers (Table 2). PCR products were cleaned using ular sequence data (host COI mitochondrial DNA and the WizardÒ SV Gel and PCR Clean-Up System small subunit ribosomal DNA) to identify the species (Promega, Southampton, UK) and sequenced by of amphipods collected and evaluate genetic diversity the Natural History Museum, London. All of the in the native and invasive range. We compared host and microsporidian sequence representatives molecular sequence data in native and invasive were deposited in GENBANK, accession numbers populations to identify the source population for the AJ966698-717, AJ968893-918, and AJ966718-726 invasion and to determine whether the invasive (Supplementary Tables 1, 2). For C. pseudogracilis population had experienced a change in post-invasion COI mtDNA and SSR rDNA, percentage sequence genetic diversity. similarity was calculated using the identity matrix We assessed the diversity and prevalence of function in BioEdit (Table 3; Hall 2001). microsporidian parasites in adult amphipods by The host sequences were rooted against distantly PCR and molecular sequence data (microsporidian related decapod malacostracan Crustacea. Microspo- small subunit ribosomal DNA) and determined the ridian sequences were obtained from at least one transmission route of the parasites by tissue specific individual from each of the native populations and screening (Terry et al. 2004). While horizontally two to four infected individuals from each invasive transmitted parasites may be found in any tissue, the population. Microsporidian sequences were rooted distribution of vertically transmitted microsporidia is against four species of Zygomycete Fungi. The generally limited to gonadal tissue (Terry et al. 2004). FASTA program (European Bioinformatics Institute, To test for enemy release, we compared the diversity Cambridge) was used to perform a homology search and prevalence of vertically and horizontally trans- before sequences were aligned by eye using BioEdit mitted microsporidia in the native and invasive (Hall 2001); analyses included only those regions that populations of C. pseudogracilis. In addition to could be unambiguously aligned. Regions of inclusion screening C. pseudogracilis, we also screened all generally began and ended on single conserved bases. sympatric and congeneric amphipods collected within To reduce branch lengths, sites with single autapo- the native range, as these also present potential morphic insertions were excluded. MODELTEST3.06 123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 197

Table 2 Oligonucleotides used for PCR and sequencing of host SSU rDNA (primer pair ‘A’), COI mtDNA (primer pair ‘B’), and microsporidian SSU rDNA (primer pairs ‘C’ and ‘D’) 12 3 45 6 7

A 52.5 1.5 F Small subunitF CCT A(CT)C TGG TTG ATC CTG CCA GT Englisch et al. (2003) A 52.5 1.5 R 1000r GAA CTA GGG CG(G/T) TAT CTG ATC G Englisch et al. (2003) B 40 2.0 F LCO1490 GGT CAA CAA CTC ATA AAG ATA TTG G Folmer et al. (1994) B 40 2.0 R HCO2198 TAA ACT TCA GGG TGA CCA AAA AAT CA Folmer et al. (1994) C 60 1.5 F V1f CAC CAG GTT TCT GCC TGA C Weiss et al. (1994) C 60 1.5 R 1492r GGT TAC CTT GTT ACG ACT T Weiss and Vossbrinck (1998) C 60 1.5 F 18f CAC CAG GTT GAT TCT GCC Weiss and Vossbrinck (1998) C 60 1.5 R 530SSUr CCG CGG CTG CTG GCA C Baker et al. (1994) C 60 1.5 R 981r TGG TAA GCT GTC CCG CGT TGA GTC Unpublished C 60 1.5 R 964r CGC GTT GAG TCA AAT TAA GCC GCA CA Terry et al. (2003) D 50 1.5 F 530SSUf GTG CCA GCA GCC GCG G Vossbrinck et al. (1993) D 50 1.5 R 580LSUr GGT CCG TGT TTC AAG ACG G Vossbrinck et al. (1993) D 50 1.5 F Hg4f GCG GCT TAA TTT GAC TCA A Gatehouse and Malone (1998) D 50 1.5 R Hg4r TCT CCT TGG TCC GTG TTT CAA Gatehouse and Malone (1998) 1 Primer pair: primers with the same letter were used in combination 2 Annealing temperature used: °C

3 MgCl2:mM 4 Direction 5 Primer name 6 Sequence (50?30) was used to predict the best substitution model for model; 5,000,000 generations) and microsporidian maximum likelihood analysis (Posada and Crandall 16S rDNA (GTR?G substitution model; 3,500,000 1998). Maximum likelihood analyses were conducted and 3,000,000 generations), and nodal support was using PAUP*4.0b10 (Swofford 2002). Maximum assessed by posterior probabilities estimated from the likelihood trees were estimated based on the best final 80% (4,000; 4,000; 2,800; 2,400) sampled trees. substitution model predicted by ModelTest for host 18S SSU rDNA (Fig. 3; equal frequency Tamura–Nei model including estimates of among-site rate heter- Results ogeneity; TrNef?G), host COI mtDNA (Fig. 4; transversion model including estimates of invariant Host diversity sites and among-site rate heterogeneity; TVM?I?G), and microsporidian 16S SSU rDNA (Fig. 5, Tamura– Samples from the native range in North America Nei model including estimates of among-site rate included amphipod Crustacea putatively identified as heterogeneity; TrN?G) with nodal support estimated C. pseudogracilis, C. floridanus as well as sympatric by bootstrap analysis (distance analysis using the amphipods from the genera Synurella and Hyalella. maximum likelihood substitution model and base C. pseudogracilis and C. floridanus are morphologi- frequencies). Bayesian analysis was conducted four cally similar and so identification was confirmed using times (independent runs started from different, ran- molecular sequence data. Analysis of the 18S domly chosen trees) for each set of phylogenetic data SSUrDNA sequence data (Fig. 3) supported the mor- using MrBayes (Huelsenbeck and Ronquist 2001). phologically based taxonomic division of C. pseudo- The Bayesian analysis was inferred for host 18S SSU gracilis and C. floridanus (Zhang and Holsinger 2003). rDNA (GTR?I?G substitution model; 5,000,000 These two species were segregated geographically generations), host COI mtDNA (GTR?G substitution with C. pseudogracilis found primarily in the lower 123 198 J. G. M. Slothouber Galbreath et al.

Table 3 Similarity of Crangonyx sp. sequences amplified from European and North American populations in relation to the reference 18S SSUrDNA (1,149 bp) and COI mtDNA (626 bp) sequences from Crangonyx pseudogracilis isolated from site 1 Site Location 18S SSU rDNA n COI mtDNA n (%) similarity (%) similarity to C. pseudogracilis to C. pseudogracilis

1 United Kingdom C. pseudogracilis 100 4 100 3 2 Holland C. pseudogracilis 100 1 100 6 3 Holland C. pseudogracilis 100 1 100 1 4 Holland C. pseudogracilis 100 3 5 France C. pseudogracilis 100 3 6 France C. pseudogracilis 100 1 100 3 7 France C. pseudogracilis 100 2 100 3 8 France C. pseudogracilis 100 2 100 4 9 Louisiana C. pseudogracilis 100 1 10 Louisiana C. pseudogracilis 99.9–100 3 94.2–98.8 6 11 Louisiana C. pseudogracilis 100 1 82.1 1 14 Tennessee C. nr pseudogracilis 99.3 1 83.2–83.3 4 17 Florida C. floridanus 98.9 3 18 Florida C. floridanus 98.9 2 19 Florida C. floridanus 98.9 2 77.6–81.3 4 SSU sequence data were not generated for individuals from sites 4 to 5 as the, generally more variable, COI mtDNA sequence data was identical to sequence from individuals in sites 1–3 to 6–9

Mississippi region and C. floridanus confined to the C. pseudogracilis (Figs. 4, 6; Table 3). Sequence South Atlantic Gulf region. Analysis of C. pseudo- similarity in both SSU rDNA (100% similarity) and gracilis 18S SSUrDNA sequence data revealed genetic COI mtDNA (98.8% similarity) demonstrate that variation between sites in the native range (Figs. 3, 6). Lake Charles, a large seaport in Louisiana, USA, was Further phylogenetic analysis with the more variable the likely source of invasive European populations marker COI supported the pattern of genetic variation (Table 3). detected within and between populations of C. pseudo- gracilis across the native range (Figs. 4, 6). Diversity of microsporidian parasites Of the potential invaders, only one species, C. pseudogracilis, appears to have invaded the sites in Phylogenetic analysis revealed eight novel microspo- North Europe. Field samples included C. pseudo- ridian sequences in North American amphipods gracilis and several amphipod species that are native (Figs. 5, 6). The microsporidia detected appeared to to North Europe. Although C. floridanus has previ- be host specific with one species (Dictyocoela sp. ously been reported as invasive in California and HYAL) in Hyalella sp., one (Microsporidium sp. SYN) Japan (Zhang and Holsinger 2003; Kanada et al. in Synurella sp. and five in Crangonyx sp. (Table 4). Of 2007), we found no evidence of invasions into the five parasites in Crangonyx, four (Microsporidium Europe by amphipods sympatric with C. pseudograc- spp. CRANA, CRANB, CRANFA, CRANFB) were ilis (C. floridanus, Hyalella sp., Synurella sp.) in the detected in multiple host tissues suggesting a mixed native range. In addition, sequence analysis revealed vertical/horizontal transmission strategy while the fifth a reduction in post-invasion genetic diversity for parasite, Microsporidium sp. CRANPA, was found invasive C. pseudogracilis and suggested a single primarily in gonadal tissue suggesting dominant ver- area of origin, Lake Charles, Louisiana (Fig. 3). In tical transmission. There was no evidence for host sex contrast to the diversity in native populations, there ratio distortion by these microsporidia (Table 4). was no genetic variation in the 26 individuals Although the vertically transmitted parasite Fibrillan- sequenced from the 8 invasive populations of osema crangonycis has previously been reported at low 123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 199

Fig. 3 Amphipod host 18S SSU rDNA sequence phylogeny. Single best tree (-lnL = 2237.4216) from maximum likelihood analysis assuming the TRNef?G model of evolution. Topology of the majority consensus tree derived from Bayesian analysis (GTR?I?G) was identical. The values at the nodes represent: the percent bootstrap derived from 1,000 iterations (distance analysis using the maximum likelihood substitution model and base frequencies)/the Bayesian posterior probabilities

prevalence (\10%) from the ovaries of C. pseudo- range, four microsporidia were detected of which three gracilis in Louisiana populations (Slothouber appear to be derived from the native range, with two of Galbreath et al. 2004), it was not detected in North these detected in the source population. The micro- American populations in the current survey. Although sporidian parasite assemblage was dominated by the not the main aim of the study, it should be noted that no vertically transmitted F. crangonycis. This parasite macroparasites were recorded from the body cavities was found at all sites sampled across Europe at during dissection for tissue specific screening of prevalences ranging from 36.7 to 100% (60.5%, of microsporidia. individuals infected overall, N = 291) and caused host There is no evidence that C. pseudogracilis in the sex ratio distortion at multiple sites (Table 4). A invasive range had undergone enemy release; there second microsporidium which appears to use vertical was no difference in the average number of microspo- transmission, identified from a single individual in a ridian parasite species in invasive sites versus the French population, was identical to Microsporidium probable source population (Mann Whitney U = 6.5, sp. CRANPA found in the source locale of P = 0.44). In C. pseudogracilis from the invasive C. pseudogracilis in Louisiana. The third parasite 123 200 J. G. M. Slothouber Galbreath et al.

Fig. 4 Amphipod host 18S COI mtDNA sequence phylogeny. Single best tree (-lnL = 5833.9673) from maximum likelihood analysis assuming the TVM?I?G model of evolution. Topology of the majority consensus tree derived from Bayesian analysis (GTR?G) was identical. The values at the nodes represent: the percent bootstrap derived from 1,000 iterations (distance analysis using the maximum likelihood substitution model and base frequencies)/the Bayesian posterior probabilities

(Microsporidium sp. CRANPC) was detected from a evidence to support the hypothesis that the invading single UK site. This sequence had no exact match to C. pseudogracilis has acquired microsporidian para- parasites from the native range but was most closely sites from European amphipods. Furthermore, there is related to a sequence (Microsporidium sp. CRANFA; no evidence that parasites described here from 7% divergence) retrieved from C. floridanus in C. pseudogracilis have been detected in European Florida. The final parasite (Microsporidium sp. native amphipods (Terry et al. 2004), suggesting that CRANPB), found in the same UK site, was most the threat of emerging disease is currently low. similar to sequences from microsporidia from UK populations of the winter moth Operophtera brumata (Orthosomella operoptherae 2% divergence) and the Discussion freshwater snail Planorbis vortex (Microsporidium sp. PLA, 4% divergence) raising the possibility that these The invasive Crangonyx pseudogracilis has experi- are generalist microsporidia. Comparison with micro- enced a post-invasion genetic bottleneck and appears sporidia described in Terry et al. (2004) revealed no to have been introduced from a population in the 123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 201

Fig. 5 Microsporidian parasite 16S COI mtDNA sequence phylogeny. Majority consensus tree derived from Bayesian analysis (GTR?G). The topology of the maximum likelihood analysis assuming the TrN?G model of evolution (single best tree - lnL = 7348.2025) was highly similar within well supported clades but different at the higher level, poorly supported nodes. The values at the nodes represent: the percent bootstrap derived from 1,000 iterations (distance analysis using the maximum likelihood substitution model and base frequencies)/the Bayesian posterior probabilities. Parasite sequences from Crangonyx pseudogracilis, C. floridanus, Hyalella sp., Synurella sp. and European amphipod spp. are highlighted

Lake Charles, LA area (Table 3; Fig. 6). Whilst both with both of these markers in relatively small sample host and parasite molecular evidence support the sizes in the native range (Table 3; Figs. 3, 4). The conclusion that Lake Charles was the source popu- success of a single haplotype in the invaded range is lation, the distribution and size of C. pseudogracilis not unusual as many other successful invasive species populations in the native range has decreased since have experienced severe post-invasion genetic bot- the introduction of this species to Europe, therefore tlenecks, including the freshwater amphipods not all potential source populations still exist. Hence Echinogammarus ischnus (Cristescu et al. 2004) we cannot rule out other possible sources of invasion. and Dikerogammarus ischnus (Muller et al. 2002). Although molecular sequence variability for the COI Conversely, invasive populations of Dikerogamma- mtDNA and SSU rDNA markers was low in the rus villosus show no evidence for genetic bottlenecks, invaded range of C. pseudogracilis, we were able to possibly reflecting recurrent invasions via canals detect both within and between population variation (Wattier et al. 2007); whilst invasive populations of 123 202 J. G. M. Slothouber Galbreath et al.

Fig. 6 Distribution of host genotypes and parasites across designations are identified in Table 4. A* indicates that F. North America and Europe. Boxes show sample sites (1–20); crangonycis was only identified in this population from host genotype according to SSU rDNA sequence (left box) and archived material and that it was not found in the current COI mtDNA sequence (right box). Parasite species survey

G. tigrinus show higher genetic variation and accel- 3 sites) was much lower compared to the samples in erated range expansion in those populations which the invasive range (mean = 34.4 ± 5.8 SE, 8 sites). are derived from multiple sources (Kelly et al. 2006). Without further study of the system in this area, it is We also found no evidence for cryptic introductions difficult to determine why there were so few (Muller et al. 2002; Miura 2007) of other sympatric C. pseudogracilis. While sampling, it was noted that North American amphipods in Europe. C. pseudogracilis was only present in those sites Eight novel species of microsporidia were detected which had relatively few crayfish, particularly juve- in amphipods from the native range of C. pseudo- nile crayfish, which have been shown to have a gracilis. This diversity is of similar magnitude to the negative impact on macroinvertebrate diversity (Cor- diversity of microsporidia isolated from European reia and Anastacio 2008). Further causes of decline amphipods (Terry et al. 2004). Unlike the 12 may include general changes in the habitat over time, microsporidia described from native European am- recent environmental disturbances, changes in pest phipods, which often infect multiple host species (in particular mosquito) management, or even the (Haine et al. 2004; Terry et al. 2004), the microspor- result of a feminising microparasite driving local idia isolated from North American amphipods appear populations to extinction (Hatcher et al. 1999). to be host specific. Each of the microsporidia detected Predicting parasite diversity and prevalence at low in the native and invasive ranges appears to be sample sizes can be difficult as uncertainty about restricted to a single host species, even when different prevalence is greatest when sample sizes are low or infected amphipod species shared the same sampling when actual prevalence is around 50% (Jovani and sites. Tella 2006). However, it would be inappropriate to Although we revisited all sites in the Lake Charles leave out samples with very low or very high area from which C. pseudogracilis had been previ- prevalence, regardless of sample size. Indeed, low ously reported and sampled many sites with suitable sample numbers in the native range did not preclude habitats, the actual number of C. pseudogracilis Stahluhut et al. (2006) from demonstrating that collected in the native range (mean = 12.0 ± 9.0SE, Wolbachia infection prevalence in invasive and 123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 203

Table 4 Parasite diversity and prevalence and sex ratio distortion in invasive (1–8, European) and native (9–21, North American) amphipod populations Site Native/ Amphipod Microsporidian Prevalence Female Male Population Broods P invasive Name Code

1I C. pseudogracilis Fibrillanosema crangonycis A Gonad 27/29 4/23 Somatic 21/28 3/26 Total 28/29 6/28 34/57 6/7 ** 1I C. pseudogracilis Microsporidium sp. CRANPB C Gonad 0/29 0/25 Microsporidium sp. CRANPC D Somatic 7/28 2/25 Total 7/29 2/25 9/54 0/7 NS 2I C. pseudogracilis Fibrillanosema crangonycis A Gonad 28/37 5/14 Somatic 11/35 0/14 Total 28/37 5/14 33/51 8/10 * 3I C. pseudogracilis Fibrillanosema crangonycis A Gonad 7/7 – Somatic 0/7 – Total 7/7 – 7/7 1/2 – 4I C. pseudogracilis Fibrillanosema crangonycis A Gonad 16/22 2/16 Somatic 13/22 1/9 Total 19/23 3/16 22/39 3/4 ** 5I C. pseudogracilis Fibrillanosema crangonycis A Gonad 12/14 3/15 Somatic 1/14 0/15 Total 12/14 3/15 15/29 1/3 ** 6I C. pseudogracilis Fibrillanosema crangonycis A Gonad 14/18 4/8 Somatic 1/18 0/8 Total 14/18 4/8 18/26 2/3 NS 7I C. pseudogracilis Fibrillanosema crangonycis A Gonad 9/12 6/10 Somatic 5/12 2/10 Total 9/12 7/10 16/22 2/2 NS Microsporidium sp. CRANPA B Total 1a 1a –/– 8I C. pseudogracilis Fibrillanosema crangonycis A Gonad 29/31 5/13 –/– Somatic 15/30 0/13 –/– Total 29/31 5/13 34/44 6/9 ** 9N C. pseudogracilis Microsporidium sp. CRANPA B Gonad 1/2 0/1 –/– Somatic 1/2 0/1 –/– Total 1/2 0/1 1/3 –/– NS F. crangonycis Ab 10 N C. pseudogracilis – Total 0/30 –/– Synurella sp. Microsporidium sp. SYN I Total 0/1 1/1 1/2 –/– Hyalella sp. Dictyocoela sp. Hyal J Total 1/1 0/4 1/5 –/– NS 11 N C. pseudogracilis – Total 0/3 12 N Synurella sp. – Total 0/4 –/– 13 N Synurella sp. – Total 0/2 –/– 14 N C. near pseudogracilis Microsporidum CRANB F Total 21/21 3/4 24/25 7/16 NS 15 N Hyalella sp. Dictyocoela sp. Hyal J Total 1/2 –/– 16 N Hyalella sp. – 0/1 –/– 17 N C. floridanus Microsporidium sp. CRANFA G Total 5/9 1/2 6/11 –/– NS

123 204 J. G. M. Slothouber Galbreath et al.

Table 4 continued Site Native/ Amphipod Microsporidian Prevalence Female Male Population Broods P invasive Name Code

17 N C. floridanus Microsporidium sp. CRANFB H Total 18 N C. floridanus – 0/6 –/– 19 N C. floridanus Microsporidium sp. CRANFA G Total 32/40 7/9 39/49 11/11 NS 19 N C. floridanus Microsporidium sp. CRANFB H Total 20 N Hyalella sp. – 0/6 –/– 21 N Crangonyx sp. Microsporidium sp. CRANA E Total 2/2 –/– Novel parasite sequences have been assigned to the holding genus ‘‘Microsporidium’’. To facilitate cross referencing of tables and figures, the microsporidia detected have also been given codes A–J. Microsporidium CRANPB and CRANPC have been counted together as have Microsporidium CRANFA and CRANFB. Comparison of parasite prevalences in males and females (Fisher’s exact tests, 2-sided) provides evidence for sex ratio distortion by Fibrillanosema crangonycis * P \ 0.05; ** P \ 0.01 NS not significant a Population screen not conducted for this parasite b Although not recovered in the current survey, Fibrillanosema crangonycis has been reported from this site in the past (Slothouber Galbreath et al. 2004). In the majority of females with somatic infections (site 2: 8/11; site 4: 8/13; site 6: 1/1; site7: 5/5; site 8: 13/15), these were detected primarily when the gonadal infection was at a very advanced state (this information was not collected for site 1) native populations of the European paper wasp, were both detected in the invasive range. Similarly, Polistes dominulus, was similar between the two Colautti et al. (2005) found that, taking into account ranges. As we were able to detect microsporidian the source of invasion, there was no evidence for a infection in five populations where the number of reduction in helminth diversity in introduced popu- individuals screened was less than five, we feel lations of starlings in North America. We found no confident that we have not underestimated the evidence for the acquisition of microsporidia from diversity and prevalence of microsporidia in the European amphipods by C. pseudogracilis. However, invasive range. C. pseudogracilis appears to have acquired a gener- Our data demonstrates that, although a diverse alist microsporidium in the UK, in discordance with range of microsporidian parasites was present across predictions that invaders should be less likely to the geographic range of Crangonyx, the parasite acquire local parasites (Torchin et al. 2003). Finally, distribution was highly structured within the host there was no evidence that invasion by C. pseudo- distribution. Many previous studies of parasites in gracilis has lead to transmission of microsporidia to invasive species may have over-estimated enemy native European amphipods in the invaded habitat; release as they did not restrict comparison to the none of the microsporidia that were detected in North invasive population and the source populations from American C. pseudogracilis, C. floridanus, Hyalella which it was founded (Colautti et al. 2004). In the sp., or Synurella sp. have been found to infect native current study, to test for enemy release, whilst taking European amphipods, leading us to conclude that the into account the source of invasion and restriction in risk of emergent disease is currently low. host genetic diversity in the invasive versus native We were able to detect nuclear and mitochondrial range, the comparison of C. pseudogracilis micro- genetic diversity even in small, seemingly isolated sporidian parasite diversity was conducted only native populations of C. pseudogracils. Thus, while between invasive populations and native source there appears to have been recent fragmentation of populations matched by host molecular sequence C. pseudogracilis populations, genetic diversity in data. This comparison did not provide any evidence these populations has not been as severely reduced as for a reduction in prevalence or diversity of micro- in populations in the invaded range. Furthermore, sporidia; two microsporidia were isolated from historical data supports the introduction of C. pseudo- C. pseudogracilis in the source population and these gracilis from the native range before the recent

123 Reduction in post-invasion genetic diversity in Crangonyx pseudogracilis (Amphipoda: Crustacea) 205 alterations to habitat and climate in the probable haplotypes with vertically transmitted microsporidia source area. It is likely, therefore, that the introduc- may result in an increase of the frequency of the tion came from a population as diverse as that infected host genotype (Ironside et al. 2003). This currently sampled, although we cannot rule out the contrasts with horizontally transmitted parasites possibility of a source population with low genetic where selection may remove the susceptible host diversity. Therefore, it would appear that C. pseudo- genotypes from the invasive population. gracilis has been very successful in its invaded range, Even with a genetic bottleneck and potential in spite of experiencing a post-invasion a genetic associated reduction in its capacity for adaptive bottleneck. The lack of C. pseudogracilis genetic evolution, Crangonyx pseudogracilis has rapidly diversity together with the microsporidian parasite spread across Northern Europe. As there is substantial distribution in the invaded range is supportive of the within and between population diversity in the source historical data that suggests a ‘stepping stone’ pool despite the small sample size in the native range, invasion in which the non-indigenous UK population it is perhaps surprising that C. pseudogracilis, which served as a source for further introduction into The should have had ample opportunity for multiple Netherlands. C. pseudogracilis was first reported in introductions and diversification in its invasive hab- the UK in 1937 (Crawford 1937) and subsequently in itat, appears to be genetically homogenous across its The Netherlands in 1979 (Zhang and Holsinger 2003) invasive range. It is also of interest that we have not from where it has spread through Belgium and found evidence of invasion by other species from France, aided by the extensive canal systems that join the genus Crangonyx which are sympatric with the catchment areas of the different river systems of C. pseudogracilis in the native range, particularly this region. The greater diversity of microsporidia in given C. floridanus is a successful invader of Japan the UK as compared to The Netherlands and France and areas of the North American Pacific Seaboard is in keeping with this invasion pattern. (Kanada et al. 2007). Given the morphological The invasive microsporidian parasite assemblage similarity of these two species (Zhang and Holsinger is dominated by a single, vertically transmitted 2003), our study highlights the importance of com- species, Fibrillanosema crangonycis which has also bining morphological and molecular tools when been reported from the source locale (Slothouber investigating amphipod invasions. Galbreath et al. 2004). The high frequency of this The lack of genetic diversity in C. pseudogracilis parasite in all UK and continental European popula- in the invasive range raises the question as to whether tions suggests that it has been retained during there has been a single introduction event or whether successive invasion events. This is consistent with a higher population growth rate under sex ratio the proposal that vertically transmitted parasites will distortion has driven infected genotypes through the be less affected by the selective pressures acting on population or prevented subsequent invasions by horizontally transmitted parasites and will thus not be uninfected host genotypes. Indeed, Hurst and Jiggins lost during invasion. Furthermore, vertically trans- (2005) note that the use of mitochondrial DNA mitted parasites, including F. crangonycis, are markers in invasion studies may be problematic as generally limited to maternal (cytoplasmic) transmis- low mtDNA diversity may be due to a symbiont- sion and often manipulate host reproduction to driven selection sweep rather than a bottleneck or increase their own transmission success by mecha- founder event associated with the invasion process. nisms including feminisation (Bandi et al. 2001; Using only mtDNA markers, it is not possible to Dunn et al. 2001; Slothouber Galbreath et al. 2004; determine whether low host genetic diversity within Terry et al. 2004). Parasite-induced feminisation may and between invasive populations is due to invasion result an increase in host population growth rate processes such as bottlenecks, recent isolation of (Hatcher et al. 1999), hence invading hosts may populations, and stepping stone invasions or due to a benefit from infection by these parasites especially recent spread of a symbiont (Hurst and Jiggins 2005). during the establishment period of the invasion event. COI mtDNA and SSU rDNA sequence data have Furthermore, as these parasites are cytoplasmically been commonly used in studies of biological invasion inherited and spread through the population under sex of amphipods (Cristescu et al. 2004; Muller et al. ratio distortion, the association of host mitochondrial 2002). However, the tracking of host mtDNA 123 206 J. G. M. 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