Genetic and Morphological Differentiation of Dikerogammarus Invaders and Their Invasion History in Central Europe

Freshwater Biology (2002) 47, 2039–2048

Genetic and morphological differentiation of Dikerogammarus invaders and their invasion history in Central Europe

JAKOB C. MU¨ LLER, STEPHANIE SCHRAMM and ALFRED SEITZ Institute of Zoology, University of Mainz, Mainz, Germany

SUMMARY 1. Biological invasions often involve close taxonomic relatives either as native ⁄ invader pairs or as invader ⁄ invader pairs. Precise identification and differentiation of species is therefore of paramount importance to reconstruct the invasion history. Genetic studies are indispensable in the case of morphologically conservative taxonomic groups. 2. We analysed the Pontocaspian freshwater amphipods Dikerogammarus that have successfully invaded the benthos of large Central European rivers. Taxonomic uncertainties were clarified by phylogenetic analyses of mitochondrial 16S and COI genes. The three-way partitioning of allozyme genotypes in a syntopic population further corroborated the taxonomic status of the three species Dikerogammarus haemobaphes, D. villosus and D. bispinosus. Dikerogammarus bispinosus had been prior misidentified as a subspecies of D. villosus. The conspicuous colour types of D. villosus, however, appeared to be conspecific. 3. The genetic identification of the previously more abundant D. haemobaphes individuals in old samples supported the ‘successive invasion wave’ hypothesis with D. haemobaphes as the first invader displaced by the second invader D. villosus. Dikerogammarus bispinosus could be a potential future invader. 4. Haplotype differentiation was apparent between two invasion lines of D. haemobaphes, but the occurrence of a single widespread haplotype indicates genetic impoverishment during rapid colonisation.

Keywords: biological invasions, colour types, Crustacea, freshwater amphipods, mtDNA

are in doubt (Marsden, Spidle & May, 1996). Only Introduction genetic studies could clarify the taxonomic problems Taxonomic misdetermination may obscure the true and stimulate further investigation to discover diag- history of invasion patterns when close relatives are nostic morphological traits. involved. The Dreissena invasion of North America is Concurrent immigration of closely related taxa is a good example. Between the first appearance of common in European waters (Kinzelbach, 1995). dreissenid mussels in 1988 and the genetic identifica- Examples are the species groups of Corbicula, Viviparus, tion of a second species Dreissena bugensis in 1992 Jaera and Dikerogammarus in the highly invaded Central (May & Marsden, 1992) all individuals were assumed European large rivers (Tittizer, 1996). Taxonomic to be Dreissena polymorpha. For this period, life history uncertainties are particularly conspicuous among taxo- experiments, specific invasion protocols and even nomic groups originating from remote areas when genetic inferences about founder and heterosis effects taxonomic literature is not readily available. In Central Europe, most of the riverine invaders come from the Correspondence: Jakob C. Mu¨ ller, Institute of Human Genetics, species-rich Pontocaspian region via either the Dnjepr GSF-National Research Center for Environment and Health, or Danube rivers. In particular, the systematics of 85764 Neuherberg, Germany. E-mail: [email protected] freshwater amphipods is difficult (Barnard & Barnard,

Ó 2002 Blackwell Science Ltd 2039 2040 J.C. Mu¨ller et al. 1983), because of their morphological conservatism The present study aims to clarify the diversity of and presumably high speciation rate (Mu¨ ller, 2000). Central European Dikerogammarus invaders by analy- Imprecise taxonomy makes inferences about the ses of genetic markers. In view of the uncertain original distributions of described taxa also doubtful taxonomic status of subspecies and colour morphs, we (Mordukhai-Boltovskoi, 1964; Jazdzewski, 1980). test whether those involved in the Central European Two species of the amphipod genus Dikerogamma- invasion merit species status. Dikerogammarus villosus rus are extraordinarily successful invaders in Europe. bispinosus, which was also recorded from the lower Dikerogammarus haemobaphes (Eichwald, 1841) invaded Danube (Ponyi, 1958) is suspected to be a potential the upper Danube around the middle of the 20th candidate. Because of the short documentation period century (Nesemann, Po¨ckl & Wittmann, 1995) and of D. haemobaphes in the Rhine area, a misdetermin- was the ﬁrst discovered in the Rhine drainage ation could not be excluded. The present genetic immediately following the 1992 opening of the analysis of historical samples may therefore corrobor- Main-Danube-canal (Schleuter et al., 1994; Tittizer, ate the hypothesis of successive invasion waves of the 1996). Dikerogammarus haemobaphes also immigrated two Dikerogammarus species. In addition, the geo- to Central Europe from the Dnjepr river (Jazdzewski graphical distribution of genotypes will allow some & Konopacka, 2000). Dikerogammarus villosus (Sowinsky, inferences to be made about the invasion process. 1894) was recorded in the upper Danube in the late 20th century and was found in the Rhine system about 1 year after D. haemobaphes (Nesemann et al., 1995; Bij Methods de Vaate & Klink, 1995; Scho¨ll, Becker & Tittizer, Sampling and morphological typing 1995). The larger D. villosus has now displaced D. haemobaphes at most locations in the upper Danube Dikerogammarus specimens were collected widely in and Rhine system and has successfully colonised the Central Europe, including the lower Danube (Fig. 1). large rivers in northern Germany and France (perso- The southern invasion route (Danube) was intensively nal observations; Grabow, Eggers & Martens, 1998; sampled around the Main-Danube-canal, and the nor- Devin et al., 2001). thern invasion route was considered by sampling a

Fig. 1 Sampling locations mapped on the Central European waterways.

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 Differentiation of Dikerogammarus invaders 2041 population in the river Vistula. Most sampling was gene we used the primers COIa-H (Simon, Franke & performed in May–August 1997 (RHI, MA1-MA8, Martin, 1991) and COI-Gf (Meyran, Monnerot & KA1-KA2, DA1-DA4, DUN and BUN) and in April– Taberlet, 1997). The PCR products were purified October 1998 (VIS, DEV, NU¨ R, LIN). Additional sam- using a microspin method and sequenced using the ples were collected in March ⁄ April ⁄ August 2000 (SAO, dye terminator cycle method (ABI PRISM BigDye kits) BRA, BSC) and spring 1996 (M96 near location MA2). with following gel electrophoresis on an automated Adult individuals were randomly collected from all sequencer (ABI PRISM 377). The haplotype sequences potential microhabitats near the riverbanks of each were submitted to the EMBL ⁄ GenBank ⁄ DDBJ data- site. Individuals from RHI, MA1-MA8, KA1-KA2, base (Accession Nos. AJ440887–AJ440921). DA1-DA4 and DUN were frozen in liquid nitrogen to be used for both allozyme and mtDNA analysis. The Statistics other specimens were stored in pure ethanol and were typed for mtDNA only. Specimens were identified to Genetic differentiation between the D. villosus colour species using the diagnostic morphological features types was tested with an exact test using GENEPOP according to Carausu, Dobreanu & Manolache (1955) (Raymond & Rousset, 1995) after all populations in and Ponyi (1956) prior to genetic analyses. Frozen the allozyme analysis were pooled and then sorted by D. villosus were classified according to their colour colour types. types (Nesemann et al., 1995). Based on the ideas of Buth (1984), we performed a parsimony cluster analysis on the syntopic population DUN to test the genetic three-way partitioning. Allozyme and mtDNA procedures Different multilocus-genotypes were listed and sin- Homogenates of half individuals were used for allo- gle-locus-genotypes were coded to allow one consis- zyme electrophoresis according to the methods of tent symmetric stepmatrix. The number of steps Hebert & Beaton (1989) and Mu¨ ller (1998). After between character states were given values between screening 30 enzyme systems, the following 9, well- 0 and 2: 0 means no change of single-locus-genotype; interpretable, enzyme loci were selected for the 1 means only one allele is different (e.g. between analysis: ACON (E.C. 4.2.1.3), APK (E.C. 2.7.3.3), genotypes aa and ab) and 2 means both alleles are GOT (E.C. 2.6.1.1) with the faster locus GOT-I and different (e.g. aa and bc). Most-parsimonious trees of the slower locus GOT-II, GPI (E.C. 5.3.1.9), G3PDH all multilocus-genotypes were generated from 500 (E.C. 1.2.1.12), MPI (5.3.1.8), PGM (E.C. 5.4.2.2) with replicates of random addition sequences for heuristic the faster PGM-f and the slower PGM-s. About 50 searches using PAUP (Swofford, 1993). Bootstrap individuals per population were typed for these frequencies were obtained by 1000 replicates of allozymes. heuristic searches with simple addition sequences. Two to 11 individuals of each of the 16 populations Mitochondrial DNA sequences were aligned by the representing the whole sample area were sequenced Clustal W program according to the algorithm des- at two mitochondrial genes. Animals were homo- cribed in Thompson, Higgins & Gibson (1994). The genised with a microspatula and total DNA was most-parsimonious trees were found by exhaustive extracted by a silica gel-based spin column procedure. searches using PAUP. Gaps were treated as fifth base The conditions of the subsequent polymerase chain and all mutations were weighted equally. Bootstrap reaction (PCR) are outlined in Mu¨ ller (2000) although values were revealed by 1000 replicates of branch- the annealing temperature was 50 °C instead of 45 °C. and-bound searches. We used a common type B We designed new conserved primers for the 16S sequence of Gammarus fossarum as the outgroup (B1 in rRNA gene, which performed equally well in the Mu¨ ller, 2000). genus Gammarus and Dikerogammarus: LR-J-Gam (5¢- ATTTTAATTCAACATCGAGGTTGC-3¢) and LR-N-Gam Results (5¢-TTTAACGGCTGCGGTATTTTGAC-3¢). These primers match conserved regions within sequences produced The two mitochondrial fragments (16S and COI) were by the universal primers LR-J-12887 and LR-N-13398 analysed separately, but revealed similar results (Simon et al., 1994). For the amplification of the COI (Fig. 2). The 322 bp (after alignment) of the 16S gene

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 2042 J.C. Mu¨ller et al. compared among the 89 typed Dikerogammarus indi- position is not clearly resolved: it clusters with viduals yielded nine unique haplotypes. Fig. 2a shows D. haemobaphes in 57% of the bootstrap trees (Fig. 2a), the strict consensus tree of the five equally most- but in 39% of trees D. haemobaphes and D. villosus parsimonious trees found. The high bootstrap percent- without D. villosus bispinosus cluster together. The ages and high numbers of mutational steps indicate main clusters are dominated by widespread haplo- three major groups of Dikerogammarus. The uppermost types (e.g. the villosus haplotype occuring from SAO to group is identified morphologically as D. haemobaphes BRA), although some local variation appears (LIN and and the lowermost lineage as D. villosus. One haplo- NU¨ R). type bears long and dense setae on the peduncle Amplified COI gene revealed 383 bp in the 23 typed segment and the flagellum of the second antenna and individuals and yielded six COI haplotypes (Fig. 2b). cylindrical, pointed protuberances with its two main The strong differentiation and bootstrap support spines at the back of the first and second urosome. This again suggest three major lineages, which can be haplotype is refered to as D. villosus bispinosus by named based on morphology. There is increased Carausu et al. (1955) and Ponyi (1956, 1958). It is resolution about the branching position of D. villosus strongly differentiated at 16S, but its phylogenetic bispinosus (73% bootstrap value). The support for the

(a)

(b)

Fig. 2 (a) Strict consensus tree of the ﬁve most parsimonious trees based on 16S haplotypes; (b) single most parsimonious tree based on COI haplotypes; EMBL ⁄ GenBank ⁄ DDBJ identiﬁcation labels for haplotypes are given before the population names; in parentheses are numbers of typed individuals; number of mutational steps >3 are given below the branches and bootstrap percentages >50 are given above the branches in boldface.

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 Differentiation of Dikerogammarus invaders 2043 grouping of D. haemobaphes with D. villosus is only MPI loci. Thus MPI discriminates among all three 22%. mtDNA lineages. Table 1 lists the mean Kimura two-parameter dis- All three colour types of D. villosus (unicoloured, tances, assuming equal evolutionary rates of variable spotted and longitudinal striped) described by Nese- sites and excluding gaps and missing values. Trans- mann et al. (1995), and one additional colour type ition to transversion ratios of the 16S fragment range (cross striped), occurred in populations between RHI from 2.01 ± 1.60 (SD) within taxonomic units (major and DUN. They appeared in equal proportions in lineages) of Dikerogammarus to 1.26 ± 0.18 among each population (homogeneity of colour type propor- Dikerogammarus taxa. Between Dikerogammarus and tions across sample sites was tested by pairwise R · C Gammarus, transition ⁄ transversion ratios average tests of independence). Forty per cent unicoloured, 0.68 ± 0.06 and are thus close to saturation of 0.5. 29% spotted, 16% longitudinal striped and 15% cross The nucleotide substitutions of the COI fragment striped individuals were recorded on average. Table 2 resulted in only two amino acid substitutions of the shows the results of the tests for genetic homogeneity 127 coded amino acids within D. haemobaphes (resp. 0 among pooled colour types of D. villosus. According to in D. villosus) and 5–9 amino acid changes among the sequential Bonferroni method (Rice, 1989), no Dikerogammarus taxa (Table 1). Here, we used the significant table-wide differentiation was found, sug- genetic code of insect mitochondria (Lewin, 1997). gesting panmixia among these colour morphs. The DUN population contained all three major mtDNA lineages (see Fig. 2a). A parsimony analysis of the nuclear-coded allozyme genotypes was per- Discussion formed to test the current genetic isolation of these Taxonomic significance of the morphological variation lineages. We found three clusters consistent with the morphological and mtDNA typing (Fig. 3). The boot- The three distinct mitochondrial lineages of Central strap support for the bispinosus group is weak (45%) European Dikerogammarus invaders coincide with the indicating low allozyme differentiation from D. villo- morphological features described by Carausu et al. sus. However, at the MPI locus, D. villosus is fixed for (1955) and Ponyi (1956, 1958) as D. haemobaphes, the allele ‘100’, whereas D. bispinosus is fixed for the D. villosus and D. villosus bispinosus. All three taxa are allele ‘110’. No heterozygotes of these alleles occur in syntopic at DUN. It was therefore possible to test for the DUN population. Differentiation between hae- reproductive isolation according to the biological mobaphes and bispinosus is stronger and fixed at the species concept (Mayr, 1942). Mitochondrial genes GPI, G3PDH and MPI loci. The groups of haemobaphes are not qualified for the detection of hybrid genotypes and villosus are different at the GOT-I, G3PDH and as a criterion for reproductive isolation, because

Table 1 Mean (±SD) Kimura two-parameter distances of 16S and COI haplotypes D. haemobaphes D. villosus D. bispinosus within (diagonal) and among taxonomic D. haemobaphes units of Dikerogammarus spp. and G. fos- 16S 0.012 ± 0.009 sarum; number of resultant amino acid COI 0.006 replacements at COI are given below in 2 italics D. villosus 16S 0.109 ± 0.007 0.008 ± 0.005 COI 0.167 ± 0.007 0.004 ± 0.002 5–7 0 D. bispinosus 16S 0.161 ± 0.010 0.158 ± 0.013 – COI 0.204 ± 0.010 0.195 ± 0.005 – 5–7 9 G. fossarum B 16S 0.375 ± 0.004 0.322 ± 0.004 0.380 COI 0.328 ± 0.019 0.308 ± 0.003 0.351 25–27 22 24

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 2044 J.C. Mu¨ller et al.

Fig. 3 Strict consensus tree of 437 most parsimonious trees based on multilocus- genotypes of nine allozyme loci in the population DUN; number of mutational steps >3 are given below the branches and bootstrap percentages are given above the branches in boldface. recombination is suggested to be rare (Harrison, 1989; results. The COI analysis revealed D. haemobaphes and Ladoukakis & Zouros, 2001). The selected allozyme D. bispinosus to be closer relatives, whereas the loci, however, are coded by the recombining nuclear allozyme analysis put D. villosus and D. bispinosus genome. The lack of heterozygotes combining fixed together genetically. The genetic distances also sug- alleles indicates genetic and reproductive isolation gest that D. haemobaphes and D. villosus are the closest among all three taxa in the syntopic DUN sample. It is relatives (see Table 1). An explanation could be thus justified to regard D. haemobaphes, D. villosus and different random lineage sorting or different selective D. bispinosus as true species. This concept of discrimi- regimes in different genes (Avise, 2000). nation was proposed by Barnard & Barnard (1983) and The mean genetic distances of the 16S gene among Jazdzewski & Konopacka (1988), but is usually not the Dikerogammarus taxa (14%) fall within the range of adopted. Dikerogammarus bispinosus was first described other congeneric amphipod species (e.g. 4–18% as a subspecies by Martynov (1925) from the lower among postulated sibling species of the marine Dnjepr. Table 3 lists the prominent morphological amphipod Eurythenes gryllus; France & Kocher, features of the three invading Dikerogammarus species. 1996). This also holds true for the COI gene (19%), In contrast to the specific status, phylogenetic in which distance values among Gammarus species are relationships could not be clearly resolved with our between 9 and 35% (Meyran et al., 1997) and between

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 Differentiation of Dikerogammarus invaders 2045

Table 2 P-values of exact tests for genetic homogeneity among colour types of D. villosus; based on allele distributions of polymorphic allozyme loci; empty ﬁelds signify that no test was possible because of monomorphy

Unicoloured– Unicoloured– Unicoloured– Spotted– Spotted– Long. striped– Locus spotted long. striped cross striped long. striped cross striped cross striped

ACON 0.163 0.796 0.281 0.467 0.033 0.302 GOT-II 0.511 >0.999 >0.999 GPI 0.280 0.398 >0.999 0.093 0.418 0.514 G3PDH 0.290 0.354 >0.999 PGM-s 0.186 0.295 >0.999 0.556 >0.999

Table 3 Prominent morphological features for the discrimination of invading Dikerogammarus species; these features apply mostly to adult males; in females and juveniles these traits are less diagnostic

Antenna 2 Urosome 1 and 2 Propodus (on each) Peduncle Flagellum of gnathopods

D. haemobaphes Shallow dorsal protuberance with Short setation Short setation Short setation mostly two apical main spines D. villosus Pointed dorsal protuberance with Short setation Dense and Long setation (conspeciﬁc colour types) 3–5 main spines long setation D. bispinosus Pointed dorsal protuberance with Dense and Dense and long Long and dense two main spines long setation setation setation

9 and 28% among cryptic Hyalella species (Witt & tion pressure by fish. The polymorphism should be Hebert, 2000). If we assume the 16S clock rate of based on only few genes, because colour groups are between 0.4 and 0.9% sequence divergence per mil- discrete and lack intermediates. Secondly, the colour lion years (Cunningham, Blackstone & Buss, 1992; types could simply represent different stages of the Sturmbauer, Levinton & Christy, 1996; Schubart, moulting or life cycle. We tend to prefer the second Diesel & Hedges, 1998), the estimated minimum time hypothesis, because the colour types are found in since common ancestry for the three Dikerogammarus equal proportions across a wide geographical range. It taxa is between 16 and 35 million years. The COI is unlikely that different drainage systems (Rhine and clock rate of 2.2–2.6% divergence per million years Danube) and the canal have similar proportions of (Knowlton et al., 1993) suggests that Dikerogammarus microhabitats or substrates. taxa last shared a common ancestor 7–9 million years ago. In view of this large discrepancy, we conclude that Genetic inferences about the invasion process the Dikerogammarus species are old and probably of Tertiary origin. The spatio-temporal distribution patterns of the In contrast to other Dikerogammarus species, D. villosus haplotypes allow some inferences to be made about exhibits a conspicuous colour variation (Nesemann the history and process of the Dikerogammarus invasion. et al., 1995). Our tests demonstrated that the different Dikerogammarus villosus, D. haemobaphes and probably colour types do not differentiate at the allozyme level. also D. bispinosus, originate from the lower reaches of This strongly indicates panmixia among these types rivers draining into the Black or Caspian Sea that had and thus conspecificity. Two possible explanations for been interconnected in geological times (Mordukhai- their origin are proposed. First, the colour variation Boltovskoi, 1964). Dikerogammarus villosus appears could reflect a balanced polymorphism that is main- now to be the most successful invader in Central tained by different adaptations to substrates in a Europe, at least along the southern invasion route (up diverse environment of sand, stones, macrophytes the Danube). It is the only species found between 1997 and roots. The colour matching might reduce preda- and 2000 in our samples from the upper Danube, the

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 2046 J.C. Mu¨ller et al. Rhine drainage with the adjoining canals and the rivers (Straskraba, 1962; Jazdzewski & Konopacka, Rhone drainage. The only exception was at the 1988). harbour of NU¨ R, where two D. haemobaphes individuals were collected in 1998. From a faunistic survey in Acknowledgments 1996 (Tiefenthaler, personal communication) we could genetically identify D. haemobaphes from several loca- We are very grateful to the following people, who tions on the lower river Main (M96) indicating its supplied us with additional samples: Alexandre more widespread presence before 1997. Two succes- Bauer, Bourgogne, France; Angela Tiefenthaler, Elt- sive invasion waves of D. haemobaphes (first) and ville, Germany; Marc Reinhold, Berlin, Germany; D. villosus (second) are thus likely. Dikerogammarus Thomas Schmitt, Mainz, Germany; Ilona B. Musko, villosus probably displaced D. haemobaphes (Weinzierl, Tihany, Hungary; Gheorghe Ignat and Sergiu Cristo- Potel & Banning, 1996; Van der Velde et al., 2000; fer, Braila, Romania and Alicja Konopacka, Lodz, Haas, Brunke & Streit, 2002). Further studies about Poland. Simon J. Hadfield, two anonymous referees competitive properties under different environments and the editor made valuable comments on the are necessary to understand why these Dikerogam- manuscript. This work was supported by the Centre marus species can coexist at some localities (DUN), but for Environmental Studies, Mainz. displace each other at others (Dick & Platvoet, 2000). Populations of recently invading species are often References characterised by low genetic diversity, particularly of the mitochondrial genome (Davies, Villablanca & Avise J.C. (2000) Phylogeography. Harvard University Roderick, 1999; Mu¨ ller & Griebeler, 2002). The low Press, Cambridge, MA. number of haplotypes found for Dikerogammarus in Barnard J.L. & Barnard C.M. (1983) Freshwater Amphipoda comparison with the native Gammarus fossarum of the World. Hayfield Ass., Mt. Vernon, VA. Bij de Vaate A. & Klink A.G. (1995) Dikerogammarus (Mu¨ ller, 2000) confirms this. Additional habitat villosus Sowinsky (Crustacea: Gammaridae) a new isolation in G. fossarum may enhance these differ- immigrant in the Dutch part of the lower Rhine. ences. We found only one widespread 16S haplo- Lauterbornia, 20, 51–54. type in each Dikerogammarus species. For example, Buth D.G. (1984) The application of electrophoretic data the D. villosus populations of SAO and BRA showed in systematic studies. Annual Review in Ecology and the same haplotype. Additional rare haplotypes Systematics, 15, 501–522. occurred in the harbours of LIN and NU¨ R, indicat- Carausu S., Dobreanu E. & Manolache C. (1955) Amphi- ing that single long-distance colonisation events poda, forme salmastre si de apa dulce. Fauna Republicii occur, but are not effective to change the predomi- Populare Romine, Crustacea, Vol. IV (4), pp. 1–407 nant genetic structure. The northerly invasion route (Editura Academiei Republicii Populare Romine). of D. haemobaphes (VIS) is genetically differentiated Cunningham C.W., Blackstone N.W. & Buss L.W. (1992) from the southern populations. Similar genetic Evolution of king crabs from hermit crab ancestors. Nature 355 patterns occur in two Dreissena polymorpha invasion , , 539–542. Davies N., Villablanca F.X. & Roderick G.K. (1999) De- lines that are currently mixing at the Main-Danube- termining the source of individuals: multilocus geno- canal (Mu¨ ller et al., 2001; Mu¨ ller, Hidde & Seitz, typing in nonequilibrium population genetics. Trends 2002). in Ecology and Evolution, 14, 17–21. Our genetic analysis clearly identified a third Devin S., Beisel J.N. & Bachmann V. & Moreteau J.C. potential Dikerogammarus invader: D. bispinosus.It (2001) Dikerogammarus villosus (Amphipoda: Gammar- was found in large populations in lake Balaton and idae): another invasive species newly established in the the middle Danube (DUN, DEV). One individual Moselle river and French hydrosystems. Annales de found on a ship in LIN and a report from the German Limnologie, 37, 21–27. Danube (K. Rachl, personal communication) demon- Dick J.T.A. & Platvoet D. (2000) Invading predatory strates its dispersal abilities (Reinhold, 1999). Coloni- crustacean Dikerogammarus villosus eliminates both sation further upstream can be suspected, because it is native and exotic specie.s. Proceedings of the Royal Society, London, Series B 267 reported from upper reaches in other Pontocaspian , , 977–983.

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 Differentiation of Dikerogammarus invaders 2047

France S.C. & Kocher T.D. (1996) Geographic and Martynov A.W. (1925) Gammaridae des unteren Laufes bathymetric patterns of mitochondrial 16S rRNA des Dnjepr. Arbeiten der All-Ukarainischen Wissenschaft- sequence divergence among deep-sea amphipods, lich-Praktischen Staats-Station des Schwarzen und Asow Eurythenes gryllus. Marine Biology, 126, 633–643. Meeres, B1, 133–153. Grabow K., Eggers T.-O. & Martens A. (1998) Diker- May B. & Marsden J.E. (1992) Genetic identiﬁcation and ogammarus villosus Sovinsky (Crustacea: Amphipoda) implications of a second invasive species of dreissenid in navigable canals and rivers of northern Germany. mussel in the Great Lakes. Canadian Journal of Fisheries Lauterbornia, 33, 103–107. and Aquatic Sciences, 49, 1501–1506. Haas G., Brunke M. & Streit B. (2002) Fast turnover in Mayr E. (1942) Systematics and the Origin of Species.Columbia dominance of exotic species in the Rhine River deter- University Press, New York. mines biodiversity and ecosystem function: an affair Meyran J.-C., Monnerot M. & Taberlet P. (1997) Taxo- between amphipods and mussels. In: Invasive Aquatic nomic status and phylogenetic relationships of some Species of Europe (Eds E. Leppa¨koski, S. Olenin & species of the genus Gammarus (Crustacea, Amphipo- S. Gollasch), pp. 426–432. Kluwer Academic Publ., da) deduced from mitochondrial DNA sequences. Dordrecht, Netherlands. Molecular Phylogenetics and Evolution, 8, 1–10. Harrison R.G. (1989) Animal mitochondrial DNA as a Mordukhai-Boltovskoi P.D. (1964) Caspian fauna beyond genetic marker in population and evolutionary biol- the Caspian Sea. International Revue ges. Hydrobiology, ogy. TREE, 4, 6–11. 49, 139–176. Hebert P.D.N. & Beaton M.J. (1989) Methodologies for Mu¨ ller J. (1998) Genetic population structure of two Allozyme Analysis using Cellulose Acetate Electrophoresis: a cryptic Gammarus fossarum types across a contact zone. Practical Handbook. Helena Laboratories, Beaumont, TX. Journal of Evolutionary Biology, 11, 79–101. Jazdzewski K. (1980) Range extensions of some gammar- Mu¨ ller J. (2000) Mitochondrial DNA variation and the idean species in European inland waters caused by evolutionary history of cryptic Gammarus fossarum human activity. Crustaceana Supplement, 6, 86–107. types. Molecular Phylogenetics and Evolution, 15, 260–268. Jazdzewski K. & Konopacka A. (1988) Notes on the Mu¨ ller J. & Griebeler E. (2002) Genetics on invasive gammaridean amphipoda of the Dniester River Basin species. In: Invasive Aquatic Species of Europe (Eds and Eastern Carpathians. Crustaceana Supplement, 13, E. Leppa¨koski, S. Olenin & S. Gollasch), pp. 173–182. 72–89. Kluwer, Dordrecht, Netherlands. Jazdzewski K. & Konopacka A. (2000) Immigration Mu¨ ller J.C., Hidde D. & Seitz A. (2002) Canal construction history and present distribution of alien crustaceans destroys the barrier between major European invasion in Polish waters. In: The Biodiversity Crisis and lineages of the zebra mussel. Proceedings of the Royal Crustacea. Proceedings of the Fourth International Crusta- Society of London: Biological Sciences, 269, 1139–1142. cean Congress (Eds J.C. von Vaupel Klein & Mu¨ ller J., Wo¨ll S., Fuchs U. & Seitz A. (2001) Genetic F.R. Schram), Amsterdam, Netherlands, 20–24 July 1998, interchange of Dreissena polymorpha populations across Vol. 2, pp. 55–64. Balkema Publishers, Rotterdam, The a canal. Heredity, 86, 103–109. Netherlands. Nesemann H., Po¨ckl M. & Wittmann K.J. (1995) Kinzelbach R. (1995) Neozoans in European waters – Distribution of epigean Malacostraca in the middle exemplifying the worldwide process of invasion and and upper Danube (Hungary, Austria, Germany). species mixing. Experientia, 51, 526–538. Miscellanea Zoologica Hungarica, 10, 49–68. Knowlton N., Weigt L.A., Solorzano L.A., De Mills E.K. Ponyi E. (1956) O¨ kologische, erna¨hrungsbiologische und & Bermingham E. (1993) Divergence in proteins, systematische Untersuchungen an verschiedenen mitochondrial DNA, and reproductive compatibility Gammarus-Arten. Archiv fu¨r Hydrobiologie, 52, 367–387. across the Isthmus of Panama. Science, 260, 1629– Ponyi E. (1958) Neuere systematische Untersuchungen 1632. an den ungarischen Dikerogammarus-Arten. Archiv fu¨r Ladoukakis M. & Zouros E. (2001) Recombination in Hydrobiologie, 54, 488–496. animal mitochondrial DNA. Eighth Congress of the Raymond M. & Rousset F. (1995) GENEPOP (Version European Society for Evolutionary Biology, Arhus, 1.2): population genetics software for exact tests and Denmark, 20–26 August 2001. ecumenicism. Journal of Heredity, 86, 248–249. Lewin B. (1997) Genes VI. Oxford University Press, Reinhold M. (1999) Verschleppung Durch Binnenschiffe Als Oxford. Mo¨glichkeit Anthropochorer Ausbreitung Von Makroinver- Marsden J.E., Spidle A.P. & May B. (1996) Review of tebraten in Zusammenhang Mit Dem Faunenaustausch genetic studies of Dreissena spp. American Zoologist, 36, Rhein ⁄ Main ⁄ Main-Donau-Kanal ⁄ Donau. Cuvillier 259–270. Verlag, Go¨ttingen.

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048 2048 J.C. Mu¨ller et al.

Rice W.R. (1989) Analysing tables of statistical tests. Swofford D.L. (1993) PAUP: Phylogenetic Analysis Using Evolution, 43, 223–225. Parsimony, Version 3.1.1. Computer program distributed Schleuter M., Schleuter A., Potel S. & Banning M. (1994) by the Illinois Natural History Survey, Champaign, IL. Dikerogammarus haemobaphes (Eichwald 1841) (Gam- Thompson J.D., Higgins D.G. & Gibson T.J. (1994) maridae) aus der Donau erreicht u¨ ber den Main- CLUSTAL W: improving the sensitivity of progressive Donau-Kanal den Main. Lauterbornia, 19, 155–159. multiple sequence alignment through sequence Scho¨ll F., Becker C. & Tittizer T. (1995) Das Makrozoo- weighting, position specific gap penalties and weight benthos des schiffbaren Rheins von Basel bis Emmer- matrix choice. Nucleic Acids Research, 22, 4673–4680. ich 1986–1995. Lauterbornia, 21, 115–137. Tittizer T. (1996) Vorkommen und Ausbreitung aquati- Schubart C.D., Diesel R. & Hedges S.B. (1998) Rapid scher Neozoen (Makrozoobenthos) in den Bundes- evolution to terrestrial life in Jamaican crabs. Nature, wasserstraßen. In: Gebietsfremde Tierarten (Eds 393, 363–365. H. Gebhardt, R. Kinzelbach & S. Schmidt-Fischer), pp. Simon C., Franke A. & Martin A. (1991) The polymerase 49–86. Ecomed, Landsberg, Bavaria. chain reaction: DNA extraction and amplification. In: Van der Velde G., Rajagopal S., Kelleher B., Musko I.B. & Molecular Techniques in Taxonomy (Eds G.M. Hewitt, bij de Vaate A. (2000) Ecological impact of crustacean A.W.B. Johnston & J.P.W. Young), Vol. H57, pp. 329– invaders: general considerations and examples from 355. Nato ASI Series H, vol. 57, Springer Verlag, the Rhine river. In: The Biodiversity Crisis and Crustacea Berlin. – Proceedings of the Fourth International Crustacean Simon C., Frati F., Beckenbach A., Crespi B., Liu H. & Congress (Eds J.C. Von Vaupel Klein & F.R. Schram), Flook P. (1994) Evolution, weighting, and phylogenetic Amsterdam, Netherlands, 20–24 July 1998, Vol. 2, pp. 3– utility of mitochondrial gene sequences and a compila- 33. Balkema Publishers, Rotterdam, The Netherlands. tion of conserved polymerase chain reaction primers. Weinzierl A., Potel S. & Banning M. (1996) Obesogam- Annals of the Entomological Society of America, 87, 651–701. marus obesus (Sars 1894) in der oberen Donau (Amphi- Straskraba M. (1962) Amphipoden der Tschechoslowakei poda, Gammaridae). Lauterbornia, 26, 87–88. nach den Sammlungen von Prof. Hrabe. Vestnik Witt J.D.S. & Hebert P.D.N. (2000) Cryptic species Ceskoslovenske Zoologicke Spolecnosti, 26, 117–145. diversity and evolution in the amphipod genus Sturmbauer Ch., Levinton J.S. & Christy J. (1996) Hyalella within central glaciated North America: a Molecular phylogeny analysis of fiddler crabs: test of molecular phylogenetic approach. Canadian Journal of the hypothesis of increasing behavioral complexity in Fisheries and Aquatic Sciences, 57, 687–698. evolution. Proceedings of the National Academy of Sciences, USA, 93, 10855–10857. (Manuscript accepted 15 April 2002)

Ó 2002 Blackwell Science Ltd, Freshwater Biology, 47, 2039–2048