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Molecular Ecology (2004) 13, 23332343

doi: 10.1111/j.1365-294X.2004.02227.x

Blackwell Publishing, Ltd.

Red to Mediterranean Sea bioinvasion: natural drift through the Suez Canal, or anthropogenic transport?

S I G A L S H E F E R , A V I G D O R A B E L S O N , O F E R M O K A D Y and E L I G E F F E N Institute for Nature Conservation Research, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

Abstract
The biota of the eastern basin of the Mediterranean Sea has experienced dramatic changes in the last decades, in part as a result of the massive invasion of Red Sea species. The mechanism generally hypothesized for the Red-to-Med invasion is that of natural dispersal through the Suez Canal. To date, however, this hypothesis has not been tested. This study examines the mode of invasion, using as a model the mussel Brachidontes pharaonis, an acclaimed Lessepsian migrant that thrives along the eastern Mediterranean coast. Our findings reveal two distinct lineages of haplotypes, and five possible explanations are discussed for this observation. We show that the genetic exchange among the Mediterranean, Gulf of Suez and the northern Red Sea is sufficiently large to counteract the build up of sequential genetic structure. Nevertheless, these basins are rich in unique haplotypes of unknown origin. We propose that it is historic secondary contact, an ongoing anthropogenic transport or both processes, that participate in driving the population dynamics of B. pharaonis in the Mediterranean and northern Red Sea.
Keywords: anthropogenic transport, dispersal, Lessepsian migration, marine bioinvasion Received 5 January 2004; revision received 5 April 2004; accepted 5 April 2004

Introduction
The invasion of Red Sea species into the eastern basin of the Mediterranean through the Suez Canal is known as Lessepsian migration (Por 1978). Since its opening in 1869, more than 300 Red Sea species of algae, invertebrates and fish have dispersed through the canal, generating dramatic modifications in the local communities (Por 1978; Galil 2000a). Some of these species have been defined as pests, causing serious damage to fishery, tourism and power plant functioning (e.g. Rhopilema nomadica and Charybdis longicollis; Spanier & Galil 1991; Lotan et al. 1992). Lessepsian migration is an ongoing process (Zibrowius 1991; Galil 2000a) that has been a case study for marine bioinvasion research. Studies on Lessepsian migration do not agree as to the means of dispersal of the migrating biota. A stepwise advance has been suggested, assuming that the 168 km of the Suez Canal cannot be traversed in one attempt, even by excellent swimmers (Por 1978). Others have suggested a one jump scenario, accounting for Red Sea species that have not been recorded from the Suez
Correspondence: E. Geffen. Fax: 972-3-6407689; E-mail: geffene@post.tau.ac.il 2004 Blackwell Publishing Ltd

Canal itself (Safriel & Lipkin 1972; Agur & Safriel 1981). Up to the 1990s it was accepted that the main conveyance mechanism of Red Sea species into the Mediterranean was that of the natural dispersal of adults or larvae through the Suez Canal. This view, however, has changed somewhat recently, with the realization that the growth in volume of traffic passing through the canal also increases the probability for introduction of nonindigenous species (NIS) in ballast water or as fouling organisms on ship hulls (Zibrowius 1991; Galil 2000a). Por (1978) hypothesized that Lessepsian migrants most probably constitute those species that have a continuous distribution from the Gulf of Suez to the Mediterranean. However, the presence of a species in the Red Sea, the Suez Canal and along the eastern Mediterranean coast does not necessarily imply a sequential path of migration. We would expect natural northbound dispersal to result in populations comprised of haplotypes from the Gulf of Suez and the Red Sea. Moreover, genetic distance should correlate with geographical distance, as predicted by the stepping stone model. However, where recent human-mediated introductions have occurred, haplotypes at any site may include those introduced from remote sources, hence reducing the correlation between genetic and geographical distance.

2334 S . S H E F E R E T A L . The Indo-Pacific mytilid Brachidontes pharaonis (Fischer 1870; = Brachidontes variabilis Kraus 1848) offers an excellent model for the study of Lessepsian invasion. This species has a wide distribution, spanning a variety of tidal zone habitats over the Indo-Pacific, Red Sea and the Gulf of Suez. B. pharaonis successfully invaded the Mediterranean at the beginning of the 20th century (Por 1978). Over the last 30 years this mussel has become dominantly established in the rocky habitats of the Israeli Mediterranean coast, displacing the indigenous mytilid Mytilaster minimus (Felsenburg & Safriel 1974; Rilov et al. 2001). B. pharaonis displays several features that are typical of invading species, such as high fecundity and tolerance to variable environmental conditions, including high levels of pollution (Morton 1988; Shefer 2003). Safriel & Ritte (1986) and Lavee & Ritte (1994) studied the genetic variability of B. pharaonis using allozymes. Their sampling design comprised nine locations: three along the eastern Mediterranean coast, two in the Gulf of Suez and four in the Gulf of Aqaba (northern Red Sea). Populations were highly variable (number of loci = 17, mean number of alleles per locus was 5.41 and 5.47 in the Mediterranean and Red Sea, respectively), and most of the Red Sea haplotypes were also detected in the Mediterranean (8% unique alleles in the Mediterranean). In this study, we used mitochondrial DNA (mtDNA) data to explore genetic diversity and structure, and attempted to resolve the debate over the arrival modes of B. pharaonis into the eastern basin of the Mediterranean Sea.

Materials and methods Sample collection


Brachidontes pharaonis mussels were collected between August 1998 and May 2000 from four populations along the Mediterranean coasts of Israel [Achziv (Zv; 3303 N, 3506 E), Shemen (Sh; 3249 N, 3500 E), Tel Shiqmona (Ts; 3249 N, 3450 E) and Ashqelon (As; 3139 N, 3432 E)], eight populations along the Red Sea coasts of Egypt [Timsah Lake (Tl; 3033 N, 3218 E), Suez (Sz; 2958 N, 3233 E), Ras-Ruahmi (Rr; 2842 N, 3252 E), Guna (Gu; 2737 N, 3331 E), Port Safaga (Sf; 2643 N, 3356 E), Mangrove Bay (Mg; 2550 N, 3426 E), 60 km south of AlQuseir (60Q; 2531 N, 3439 E) and 90 km south of AlQuseir (90Q; 2516 N, 3453 E)] and two populations from Eritrea [Dahlak Archipelago (Da; 1520 N, 4014 E) and Irafala (Ir; 1507 N, 3921 E)] (Fig. 1). An additional population was sampled from Sri Lanka (Sr; 0656 N, 7950 E). Our sampling spanned 180 km along the eastern Mediterranean shores, and 2000 km of Red Sea coast. Tissue samples, taken from the adductor muscle, were preserved in DMSONaCl solution (Seutin et al. 1991). In total, 258 individuals were analysed with a sample size of 10 29 individuals per site.
Fig. 1 Map of the sampling sites. The eastern Mediterranean was sampled at sites 14 (Zv, Sh, Ts, As, respectively), the Gulf of Suez at sites 58 (Tl, Sz, Rr, Gu, respectively), and the northern Red Sea at sites 912 (Sf, Mg, 60Q, 90Q, respectively). The sampling sites in the southern Red Sea (Da and Ir) and Sri Lanka (Sr) are not shown. For site abbreviations see Materials and methods.

Molecular genetic analysis


Total genomic DNA was obtained by proteinase K extraction. A region of the mtDNA, comprising a fragment of the cytochrome oxidase subunit I (COI), was amplified by polymerase chain reaction (PCR) using the primer set of Folmer et al. (1994). Amplification was performed using 2.5 mm MgCl2, and consisted of initial denaturing at 94 C for 2 min and 40 cycles of 30 s at 94 C, 1 min at 45 C and 1 min at 72 C, followed by a final elongation at 72 C for 10 min. Fragments of COI were screened for individual haplotypic variation by examining single-strand conformation polymorphism (SSCP; Grompe 1993). SSCP distinguishes between equal-length DNA fragments differing in sequence based on their molecular conformation. Under the conditions used in SSCP, including low temperature
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 23332343

R E D T O M E D I T E R R A N E A N S E A B I O I N V A S I O N 2335 (15 C), separated strands remain folded in specific conformations dictated by their sequence, and migrate differentially in PAGE. Consequently, SSCP permits the screening of a large number of equal-size DNA fragments and differentiating between them without the need to sequence them all. PCR products were diluted and electrophoresed using the CleanGel DNA Analysis Kit (Pharmacia Biotec), according to the manufacturers instructions. Following silver staining, strands were scored by relative mobility. One representative of each SSCP pattern (= haplotype) was sequenced using an automated DNA sequencer (ABI 377). Pairwise sequence divergence between haplotypes was computed using the TamuraNei model of sequence substitution (Tamura & Nei 1993). The distance matrix was used to construct a spatial representation using multidimensional scaling (MDS). We selected this additional approach because it delineates the relative position of haplotypes in a two-dimensional space, without going through a tree algorithm (Borg 1981). The MDS approach was used to verify the major divisions of haplotypes in the study area. Newly established populations are expected to show low genetic variability and a state of expansion. Gene diversity (H; Nei 1987) and nucleotide diversity (; Nei 1987) were used as estimates for genetic variability within populations. To test whether the populations in the Mediterranean are less variable than populations in southern locations, we compared these indices between basins using one-way analysis of variance (anova). We tested whether populations are different in their haplotypic composition using a contingency table (Raymond & Rousset 1995). This is an exact test that compares the observed frequency table of basins haplotypes with other permutations of the table using a Markov chain process. This test is implemented in arlequin 2.0 (Schneider et al. 2000). To test for selective neutrality we used Tajimas D statistic (Tajima 1989). This test is based on the infinite-site model without recombination, making it suitable for mtDNA sequences. The method is based on the correlation between number of segregating sites and the average number of pairwise nucleotide differences. The significance of the D statistic is evaluated by generating random samples under the hypothesis of selective neutrality using a coalescent simulation algorithm (Tajima 1989). The shape of the distribution of the observed number of differences between pairs of haplotypes is a signature of the expansion process. Such a distribution is called mismatch distribution, and it is multimodal in samples drawn from a population at equilibrium. In a recently expanded population, this distribution is unimodal. We used the approach of Schneider & Excoffier (1999), in which a sudden expansion model is used for generating a large number of random samples. The sum of square deviations (SSD) between the observed and the simulated mismatch distributions are the test statistics. A significant result implies that the population is at equilibrium, whereas nonsignificance indicates an expanding population. The calculations were performed using arlequin 2.0 (Schneider et al. 2000). To evaluate the association between populations, based on genetic distance, we constructed a minimum spanning network (MSN). The shortest tree that connects between a set of populations is called a minimum spanning tree (MST; Kruskal 1956). Often, there is more than one tree that meets this criterion. A minimum spanning network, embedding all possible MSTs, is a more realistic model than a tree for connectivity between populations. A matrix of pairwise

Data analysis
We divided the sampling area into four distinct geographical basins: Mediterranean (populations of Zv, Sh, Ts and As; Fig. 1), Gulf of Suez (Tl, Su, Rr and Gu), northern Red Sea (Sf, Mg, 60Q and 90Q) and southern Red Sea (Ir and Da). Sri Lanka (Sr) was a distant, presumably disconnected, population that we used for comparison. Sequences were aligned using the program clustal x (Version 1.81, Thompson et al. 1997), adjusted by eye, and trimmed to a 594 bp data matrix. A rarefaction analysis was conducted to determine the expected total number of haplotypes. The average ( SD) expected number of haplotypes for a given sample size was calculated using 100 resampling iterations. The number of expected haplotypes (Nh) as a function of individuals sampled (Ni) is evaluated by fitting a nonlinear curve using the equation Nh = Ni/( + Ni), where is the total number of expected haplotypes and is the slope (Roy et al. 1994). Mytilus species are known to have relatively common mtDNA paternal inheritance in contrast to most other organisms (double uniparental inheritance; Zouros et al. 1992, 1994). To rule out the possibility that phylogenetic clades in B. pharaonis are attributed to female and male mtDNA lineages, we sequenced mtDNA COI fragments of 17 males and 18 females. Gonadal tissue was used as a DNA source, in order to ensure that we obtain the two mtDNA variants (Zouros et al. 1992). A maximum-likelihood analysis was used to test whether haplotypes cluster according to sex. Using blast, we screened COI sequences available in the databases for the most related species to B. pharaonis. Geukensia demissa and Septifer virgatus were found to be the closest mytilids, and were added to our analysis as a reference. A maximum-likelihood tree of all haplotypes was constructed using the quartet puzzling algorithm (Strimmer & von Haeseler 1996) in paup 4.0 (Swofford 2000), with the HKY85 sequence substitution model (Hasegawa et al. 1985). The probability for each branch to equal zero length was calculated for the generated tree using dnaml (phylip; Felsenstein 1993). The phylogenetic tree reconstruction approach was used to test for the presence of cryptic sibling species.
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 23332343

2336 S . S H E F E R E T A L . DA distances (see below) between populations was used to construct a MSN of populations using the program minspnet (Excoffier 1993). Isolation-by-distance was evaluated by plotting pairwise DA values (Nei & Li 1979) against geographical distance. DA is calculated between each two populations as: DA = Pxy (Px + Py ) 2 2.0 (Schneider et al. 2000). Mantels test (Mantel 1967) was used to compute the correlation between matrices of geographical distance and DA using the software permute! (Version 3.4a9; Casgrain 2002). We calculated the percent number of unique haplotypes, and relative frequency of unique haplotypes among individuals within each basin. A haplotype was considered unique if its distribution was limited to one basin.

where Pxy is the average number of pairwise differences between populations, and Px and Py are the average number of pairwise differences within population x and y, respectively (Nei & Li 1979). Geographical distance was calculated as the shoreline distance between sites. The calculations of the DA distance matrices were done using arlequin

Results
A total of 93 haplotypes were identified among 258 Brachidontes pharaonis individuals analysed from 15 populations. The rarefaction analysis outcome fits the curve Nh = 134.6Ni/(147.5 + Ni), where Nh is the number of expected
Fig. 2 A maximum likelihood tree for the 93 COI mtDNA haplotypes (transition/ transversion ratio = 2.66; ln Likelihood = 3756.0). An asterisk indicates branch length significantly larger than zero (P < 0.05). Symbols: star, eastern Mediterranean; circle, Gulf of Suez; square, northern Red Sea; triangle, southern Red Sea. Gdem = Geukensia demissa and Svir = Septifer virgatus. Lineages 1 and 2 are indicated on the right.

2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 23332343

R E D T O M E D I T E R R A N E A N S E A B I O I N V A S I O N 2337 All B. pharaonis haplotypes formed a monophyletic group in the maximum-likelihood analysis (Fig. 2). Both Geukensia demissa and Septifer virgatus were classified as separate units. Both MDS and maximum-likelihood analysis categorized haplotypes into three defined clusters (Fig. 2). The composition of haplotypes in each of the MDS clusters (Stress factor = 0.0129 and r 2 = 0.9997) was identical to the corresponding lineage observed in the haplotype maximum-likelihood tree (Fig. 2). Haplotypes that occur in the Mediterranean, Gulf of Suez and northern Red Sea were found in both lineages 1 and 2, whereas those from the southern Red Sea were associated only with lineage 1. Haplotypes from Sri Lanka were monophyletic (lineage 3). The ratio between the number of haplotype of lineage 2 and lineage 1 was similar for the three northern basins (0.58, 0.50 and 0.50 for the Mediterranean, Gulf of Suez and the northern Red Sea, respectively; Fig. 2). Haplotypes did not cluster by sex (Fig. 3). The 35 individuals that we sexed and sequenced were categorized on the maximum-likelihood tree into two lineages, corresponding to lineage 1 and 2 in Fig. 2, each containing males and females (64 and 31% females, respectively; Fig. 3). Gene diversity and nucleotide diversity were 0.948 0.012 and 0.040 0.020 in the Mediterranean, 0.968 0.011 and 0.026 0.013 in the Gulf of Suez, 0.948 0.010 and 0.034 0.017 in the northern Red Sea, and 0.869 0.021 and 0.006 0.003 in the southern Red Sea, respectively. Gene diversity was similar among basins (F3,10 = 2.07, P = 0.17). However, nucleotide diversity estimates were significantly different among basins (F3,10 = 5.75, P = 0.02). Student NewmanKeuls post-hoc test showed that the southern Red Sea population had significantly lower diversity than the other three basins (P < 0.04 for all cases). No significant difference in nucleotide diversity was detected among the Mediterranean, Gulf of Suez and northern Red Sea. To test for the effects of selection and population expansion, we conducted two neutrality tests. Tajimas D statistic did not significantly differ from the expected in a random sample, under the hypothesis of selective neutrality and population equilibrium, in all basins (Mediterranean: D = 0.51, P = 0.74; Gulf of Suez: D = 0.72, P = 0.23; northern Red Sea: D = 1.20, P = 0.91; southern Red Sea: D = 1.05, P = 0.87). The mismatch distribution fitted the sudden expansion model in the Mediterranean (SSD = 0.022; P = 0.38), Gulf of Suez (SSD = 0.012; P = 0.54) and southern Red Sea (SSD = 0.005; P = 0.85). Only the northern Red Sea had a signature of a population at equilibrium, significantly different from the sudden expansion model pattern (SSD = 0.031; P = 0.03). Basins were significantly different from each other in haplotype composition (exact test for contingency table; P < 0.001 in all pairwise comparisons). Percentage of unique haplotypes among individuals was high in the Mediterranean (79.5%; n = 78), Gulf of Suez (70.2%; n = 57)

Fig. 3 A maximum likelihood tree for COI mtDNA of 35 sexed individuals collected in the Mediterranean. F, female; M, male. Ten haplotypes from Zv site were added for reference. The haplotype Sr1 from Sri Lanka was used as an outgroup. Transitions/transversions ratio = 2.23, and ln Likelihood = 2213.8. An asterisk indicates branch length significantly larger than zero (P < 0.05).

haplotypes and Ni is the number of individuals sampled (r2 = 0.999). This curve asymptotes at ~135, which is an estimate for the actual number of haplotypes. The 85 haplotypes we sampled (excluding Sri Lanka) represent 63% of the maximal haplotypic variation. An assorted sample of ~2800 individuals is required in order to sample 95% of haplotypes expected (128) in the Red Sea east Mediterranean area.
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 23332343

2338 S . S H E F E R E T A L .

Fig. 4 The proportion of individuals belonging to lineage 2 in different populations as a function of distance from the most northern collection site (Zv; r = 0.723, P = 0.003).

and northern Red Sea (57.7%; n = 78), and reached 100% (n = 32) in the southern Red Sea. A higher percentage of individuals with unique haplotypes was observed in lineage 2 in the Mediterranean (lineage 1: 76.6%, n = 47; lineage 2: 83.9%, n = 31), Gulf of Suez (lineage 1: 62.5%, n = 40; lineage 2: 88.2%, n = 17) and northern Red Sea (lineage 1: 50.0%, n = 60; lineage 2: 83.3%, n = 18). The proportion of individuals belonging to lineage 2 decreased in populations from north to south (r = 0.723, P = 0.003; Fig. 4). The MSN of populations showed no clustering of populations by basins, except for the two populations of the southern Red Sea (Fig. 5). Suez, the largest port in the Gulf of Suez basin, was most closely related to two localities in the eastern Mediterranean where ports are located nearby (Shemen and Ashqelon). Isolation-by-distance was detected by the significant correlation between population pairwise genetic distance ( DA) and geographical distance (r = 0.951, P < 0.001). Because Sri Lanka and the southern Red Sea populations were located at a great distance from the other localities we were concerned that the high correlation might be an outcome of data clumping at the two tails of the distance distribution. To eliminate this possibility we excluded the populations of Sri Lanka and the southern Red Sea from the analysis, and obtained a low correlation that explains only 6.1% of the variance observed (r = 0.246, P = 0.04).

Fig. 5 A minimum spanning network (MSN) for the 14 populations sampled (Sri Lanka not included), constructed from a matrix of population pairwise DA distances. Dotted circles indicate populations from the Mediterranean basin, hatched for the Gulf of Suez, empty circles for the northern Red Sea, and filled for the southern Red Sea populations. Circle diameters are proportional to sample size.

Discussion
Extended capes, peninsulas and straits may act as barriers to dispersal for marine organisms, and long and shallow canals even more so (Palumbi 1994). One key question is that of whether the Suez Canal is an effective barrier to gene flow? The flow along the coast of the Gulf of Suez follows a negative estuarine circulation, with surface currents (up to 30 m depth) flowing northwards, against north to north-

easterly prevailing winds (Morcos 1970; Rady et al. 1998). The currents are also mostly northbound in the Suez Canal (Morcos 1970; Agur & Safriel 1981). The eastern Mediterranean coast is characterized by two major flow patterns: a dominant thermo-haline northbound flow, and north south alternating longshore currents (Rozentraub 1999). These flow regimes suggest that larvae drift northward along the canal and into the eastern Mediterranean. Dispersal through the canal is almost unidirectional; more than 300 Red Sea species of algae, invertebrates and fish have dispersed north into the Mediterranean Sea, whereas only a few have dispersed in the opposite direction (Por 1978; Galil 2000a). Species that are capable of efficient swimming (e.g. fish) may disperse against currents and over other obstacles, unlike those that merely drift (e.g. algae and plankton). Between the northern Red Sea and the Mediterranean we detected weak isolation-by-distance and
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 23332343

R E D T O M E D I T E R R A N E A N S E A B I O I N V A S I O N 2339 populations clustered independent of basins. Our findings show that the northern Red Sea, Gulf of Suez and eastern Mediterranean are not isolated, but share a substantial proportion of haplotypes (20.5% of the haplotypes found in the Mediterranean are also present in the other two basins). However, populations in the southern Red Sea and Sri Lanka are isolated from the northern pool (Figs 2 and 5), and share no haplotypes. The most southern population in the northern Red Sea (90Q) is located 700 km south of the canal, but is genetically most similar to two northern sites in the eastern Mediterranean (Zv and Ts, Fig. 5). We can conclude that larvae of Brachidontes pharaonis from the northern Red Sea drift freely through the Suez Canal, whereas those from the more distant populations (i.e. southern Red Sea, 2000 km) do not. Studies of fish have reported no genetic differentiation between Mediterranean and Red Sea populations with 90% or more of haplotypes shared among basins (Bucciarelli et al. 2002; Hassan et al. 2003). By contrast, we detected significant differences among basins in haplotype composition and most haplotypes were not shared (up to 80%). The results of the above fish studies fit the expected model predicting a large number of migrants colonizing the Mediterranean. However, B. pharaonis shows substantially lower mixing and rate of exchange. Our study also revealed the presence of two major phylogenetic lineages of haplotypes of the Indo-Pacific mussel B. pharaonis in the Levant basin (Figs 2 and 3). Lineages were not associated with the sex of individuals or with geographical region (except in the case of haplotypes from the southern Red Sea). Below we examine five possible hypotheses for this division. The neutrality test using the mismatch distribution showed that the Mediterranean Sea and Gulf of Suez populations are in a state of expansion. Furthermore, we did not detect any significant negative values of Tajimas D or decrease in genetic variance, the signature for bottleneck, in any basin. Recent increase in population size and range do not present favourable conditions for the formation of phylogeographical breaks (Irwin 2002). In fact, B. pharaonis is an organism that does not display the typical characteristics conducive to phylogeographical breaks without a true barrier. This species has an Indo-Pacific distribution, spanning a wide variety of tidal zone habitats, indicating its tolerance and dispersal abilities. During the past 30 years this mussel has become dominantly established in the rocky habitats of the Israeli Mediterranean coast, and can be found in very high densities in the northern Red Sea, Gulf of Suez and eastern Mediterranean at almost every suitable location (Safriel, Felsenburg & Gilboa 1980; Rilov et al. 2001). Furthermore, mytilid larvae are planktotrophic, spending considerable time in the water column (at least 1020 days; Olson & Olson 1989; Widdows 1991). During the planktonic phase, larvae are borne away by sea currents and can settle at great distances from their place of origin. For example, an ocean current of only 0.5 km/h can carry a larva 150 500 km prior to settlement (Scheltema 1977). B. pharaonis is reproductive throughout most of the year, and larvae are released into the water even during mid-winter (Shefer 2003). Although we cannot exclude the possibility that the two lineages of haplotypes of B. pharaonis are the result of stochastic events, this explanation is unlikely in light of the expansion mode, enormous population size and large dispersal distance exhibited by this mussel.

Stochasticity
Irwin (2002) showed that deep phylogeographical breaks, which are usually assumed to be the result of long-term barriers to gene flow, can form within a continuously distributed species even when there are no barriers present. Species with short dispersal distance and small population size are more likely to show phylogeographical breaks without barriers to gene flow (Irwin 2002). The lack of recombination forces mtDNA haplotypes to evolve through bifurcating genealogies, making this type of genetic marker the most likely to show evidence of genuine barriers to gene flow but also the most likely to show false evidence for nonexisting barriers. Furthermore, simulations showed that neutral genealogies often produce two clades of haplotypes just by chance because the stochastic variance of the coalescence process in mtDNA is large (Hudson & Turelli 2003). Conversely, large dispersal distance and population size can rapidly counteract phylogeographical breaks, even those that were originally created by a true barrier that has since dissipated (Irwin 2002).
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 23332343

Cryptic sibling species


The appearance of two monophyletic clades within a species may indicate the existence of cryptic sibling species, i.e. species that are difficult to differentiate using traditional techniques (Knowlton 2000; Bucciarelli et al. 2002). Although many sibling species continue to be discovered, in sympatry they often have characteristic differences in ecology or life history, and can, in retrospect, be identified by subtle differences in morphology or colour pattern. No differences in morphology, microhabitat or reproductive cycle between the discussed lineages have been observed in B. pharaonis (Shefer 2003). Furthermore, we used homologous sequences of two mytilid species, currently recognized as the most closely related to B. pharaonis, as a reference (Fig. 2). Yet, all the haplotypes of B. pharaonis, even those sampled 7000 km away (i.e. Sri Lanka) were monophyletic. These findings thus provide no support for the hypothesis that the two lineages of haplotypes are in fact two separate species.

2340 S . S H E F E R E T A L .

Natural secondary contact


The most recent glacial periods impacted upon the Red Sea biota by lowering the sea level and creating a barrier to dispersal in the straits of Bab el Mandeb (1242 N, 4310 E), which separate the Red Sea from the Gulf of Aden (Por 1978; Sirocko 2003). These shallow straits (137 m) have been land bridges at various times, offering several terrestrial African mammals a passageway to the southwestern corner of the Arabian Peninsula (Tchernov 1992). This barrier may have formed the two separately evolving lineages documented in our data. Under this scenario, migration would have partly erased the spatial differentiation after the reopening of the straits, but the signature of past allopatric differentiation may have remained in the gene genealogy. The prevailing flow in the Red Sea is mostly northwards (Sofianus et al. 2002). Therefore, the haplotypic frequency gradient with distance suggests that lineage 1 is the population from the Arabian Sea that infiltrated into the Red Sea as a result of the rise in sea level, while lineage 2 is the ancestral Red Sea population. Indeed, lineage 1 includes the unique haplotypes sampled in the Dahlak Archipelago, a shallow area (15 30 m) near the straits of Bab el Mandeb that may have remained above water during periods of peak glaciation.

Selection
Recent empirical and theoretical studies contest the neutrality-based predictions associated with mtDNA variation and divergence (Hudson & Turelli 2003). Although selection may have provided the drive for the split in haplotypes of B. pharaonis, in none of the basins was Tajimas D statistic significantly different from that expected under selective neutrality. We can thus conclude that selection is not the cause that has maintained the two haplotypic lineages in the Red Sea.

Human-mediated introduction (anthropogenic transport)


The introduction of haplotypes from a source other than the Red Sea could generate two clusters of haplotypes within a region. Human-introduced haplotypes may outcompete local haplotypes and increase their population size via selection. However, we did not detect any signature of selection. The alternative to selection is that the humanintroduced haplotypes are carried across into the study area in sufficient numbers to appear within our samples. The following data suggest how large mussel populations may have been established as a result of larvae transport in ballast water. A large number of cargo ships and oil tankers (~15 000 ships per year; Galil 2000b) pass through the shallow Suez Canal (20 m deep). About 5000 vessels reach the two east Mediterranean ports on the

Israeli coast annually (Galil 2000b; Israel Ports Authority Annual Reports for cargo vessel traffic between 1994 and 2003; http:/ /www.israports.org.il/cargo/Statistic.asp). Close to 20% of the total ballast water on board these vessels originates from the Indo-Pacific Ocean (Galil 2000b). This continuous flow of ballast water may result in the establishment of large populations of invading species, especially in the ports. Larvae produced at the ports may then drift with the currents and populate suitable sites along the coast. This scenario is not unrealistic because some of the most successful invading species, which now maintain huge populations, were initiated by the arrival of only a few individuals (Holland 2000). For example, the Eurasian mussels (Dreissena polymorpha and D. bugensis) are two species that were introduced via ballast water into the Great Lakes of North America. Within 10 20 years these species had rapidly spread and reached high population densities (Holland 2000). The other line of evidence for human-mediated introduction comes from the presence of unique haplotypes. Populations situated near the edge of distribution are commonly characterized by a lower population size, higher temporal variability in abundance, confinement to isolated patches of suitable habitat and a lower migration rate than populations at the core of distribution (Smith et al. 1997; Widmer & Lexer 2001). Population genetics theory, based on such biogeographical characteristics, predicts lower effective population size (Ne) and higher genetic drift at the edge populations than near the core of distribution (Wright 1931; but see Vucetich & Waite 2003). The frequency of unique haplotypes is expected to increase with proximity to edge populations, due to increased isolation and lower rate of migration from the core. According to the theory, therefore, the similarity in genetic diversity and the significant difference in haplotype composition among the Mediterranean, Gulf of Suez and northern Red Sea are inconsistent with the notion that the Mediterranean is a typical edge population. However, empirical studies have shown that edge populations may exhibit a continuum in the level of genetic diversity, from much lower to much higher than in the core (Schnabel & Hamrick 1990; Lesica & Allendorf 1995; Walter & Epperson 2001). Moreover, populations of B. pharaonis in the Mediterranean are large and continuous along the coast, and not restricted to isolated patches. Indeed, we observed a decline in proportion of unique haplotypes from the edge (Mediterranean) towards the core (northern Red Sea) in lineage 1, but the proportion of unique haplotypes in lineage 2 was similar across basins. Selection is an unlikely cause for these observations, because individuals of both lineages were always mixed on mussel beds at the collection sites (excluding the sites in Eritrea and Sri Lanka). Moreover, Tajimas D did not detect any deviation from selective neutrality. Demography is another unlikely cause for the observed pattern, because
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R E D T O M E D I T E R R A N E A N S E A B I O I N V A S I O N 2341 the ratio in number of haplotypes between lineages was similar in the Mediterranean, Gulf of Suez and northern Red Sea. We sampled a roughly equal number of individuals from the Mediterranean, Gulf of Suez and northern Red Sea, making it unlikely that the richness in unique haplotypes is rooted in a sampling bias. Finally and most importantly, the time frame for the Mediterranean invasion by B. pharaonis is < 150 years (Por 1978; Safriel, Felsenburg & Gilboa 1980). The average rate of its expansion eastwards from the canals Mediterranean end was ~10 km/year and it is only in the past 30 years that a dramatic increase in population size has been reported (Felsenburg & Safriel 1974; Safriel, Felsenburg & Gilboa 1980; Rilov et al. 2001). This time frame is undoubtedly not long enough for reproductive isolation and a high proportion of uniqueness (up to 80%) to evolve. Founders can arrive from several isolated sources, and can produce a genetically highly diverse collection of offspring (Holland 2000; Walter & Epperson 2001). We propose two sources of haplotypes in the Red Sea: lineage 1, which corresponds to the ancestral Red Sea population; and lineage 2, which represents haplotypes introduced by maritime transportation from remote sources. Lineage 2 shows an equally high frequency of unique haplotypes independent of edge or core, as if introduction has operated evenly along the route from the northern Red Sea to the eastern Mediterranean. Because cargo ships and tankers taxi between ports (Galil 2000b), exchanging ballast water at various stops, the Gulf of Suez and northern Red Sea could have been invaded from the same sources as those that invaded the eastern Mediterranean. Considering the northbound current flow, larvae from remote sources dumped from ships in the northern Red Sea and Gulf of Suez will tend to drift and accumulate along the eastern Mediterranean coast. Under these conditions, the pattern observed in Fig. 4 could provide further support for an association of lineage 2 with anthropogenic transport because it demonstrates an accumulation of lineage 2 haplotypes northwards on a gradient from the northern Red Sea to the eastern Mediterranean. At present, we are unable to single out one particular cause for the haplotype split in the Red Sea, Gulf of Suez and Mediterranean Sea. We have ruled out hypotheses based on selection and the existence of cryptic sibling species, and argued against stochasiticity on the basis that a stochastic split cannot be maintained considering the large population size and dispersal distances typical of B. pharaonis. The two most probable explanations are those of secondary contact and anthropogenic transport. However, natural secondary contact is in line with the southward decrease in the proportion of lineage 2 haplotypes, but not with the high number of unique haplotypes in the Mediterranean. The hypothesis based on human-mediated introduction has one major drawback. The source populations for the
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unique haplotypes are, as yet, unknown. It is possible that both secondary contact and anthropogenic transport act simultaneously, rather than a single process accounting for the observed pattern. Resolution of this issue may benefit from additional analyses of highly variable neutral markers (e.g. microsatellites), as well as sampling from the Red Sea coast of Sudan, the Arabian Sea (e.g. Oman) and the Persian Gulf.

Acknowledgements
We would like to thank Adel Ibrahim (Department of Biological and Geological Sciences, Suez Canal University, Ismailia, Egypt) for his hospitality and assistance with the collection of samples in Egypt. We also thank I. Brickner, I. Ayalon, J. Shoshani and R. Sponer for help with the collection of mussel samples. A. Ibrahim, U. Motro, M.S. Roy and three anonymous referees provided useful comments and N. Paz the final editorial touch. The KKL Interuniversity fund for Ecology, The Internal Tel.Aviv University Fund, The Reiger Foundation for Environmental Studies, and The Ford European Conservation Award supported this research.

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This study was part of the PhD research of Sigal Schefer, jointly supervised by Eli Geffen and Avigdor Abelson. Avigdor Abelson studies the ecology of the coastal environment, with emphasis on the processes of dispersal and recruitment of sessile invertebrates. Eli Geffen and Ofer Mokady use molecular markers to address ecological and evolutionary questions at levels varying from the individual to the species, in a number of model systems.

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