The East Asian shore crab Hemigrapsus sanguineus ( Brachyura : Varunidae ) in the Mediterranean Sea : an independent human-mediated introduction *

During the past twelve years, two East Asian crab species of the genus Hemigrapsus (family Varunidae, see Martin and Davis, 2001; Schubart et al., 2002) have invaded almost simultaneously the temperate coasts of the eastern (H. penicillatus) and western (H. sanguineus) North Atlantic (Williams and McDermott, 1990; Noël et al., 1997). Previously, this genus was unknown in the Atlantic Ocean. Within a few years, both species were found to reproduce in their new environments (McDermott, SCI. MAR., 67 (2): 195-200 SCIENTIA MARINA 2003


INTRODUCTION
During the past twelve years, two East Asian crab species of the genus Hemigrapsus (family Varunidae, see Martin and Davis, 2001;Schubart et al., 2002) have invaded almost simultaneously the temperate coasts of the eastern (H.penicillatus) and western (H.sanguineus) North Atlantic (Williams and McDermott, 1990;Noël et al., 1997).Previously, this genus was unknown in the Atlantic Ocean.Within a few years, both species were found to reproduce in their new environments (McDermott,SCI. MAR., SCIENTIA MARINA

2003
The East Asian shore crab Hemigrapsus sanguineus (Brachyura: Varunidae) in the Mediterranean Sea: an independent human-mediated introduction* extended their distributional ranges and are now successfully established over several hundred kilometres of the respective coastlines (McDermott, 1999;Nijland and Beekman, 2000).
In their native East Asia, H. penicillatus and H. sanguineus are known to thrive in quite different ecological niches.The first species is more common along protected shores with low wave action and soft sediments (e.g.lagoons and estuaries), while H. sanguineus is typical on more exposed coasts with rocks and boulders (Fukui, 1988;Lohrer et al., 2000).The initial distribution of H. penicillatus and H. sanguineus in the Atlantic brought up the question whether the restriction of H. penicillatus to European waters and of H. sanguineus to American ones could be a consequence of possible differences in ecological suitability of North American versus European habitats.However, the recent findings of H. sanguineus along the European Atlantic coasts of France (Le Havre) and the Netherlands (Oosterschelde) with accounts of breeding animals in Le Havre (Breton et al., 2002) suggests that the second East Asian species of Hemigrapsus will also establish successfully along the French Atlantic and that both species of Hemigrapsus can potentially coexist in Europe.Continuing observations on the establishment and subsequent distribution of H. sanguineus in European waters, as well as on the number of independent introductions, are of crucial importance for understanding and predicting ecological scenarios that will determine successful invasion of alien species in the marine environment.

MATERIAL AND METHODS
The shallow subtidal and intertidal crustacean fauna of Uvala Slanik was studied along the rocky shore of the Ladin Gaj Campground in August 2001.U. Slanik is a small and shallow bay surrounded by flat rock terraces.It is located south of the small village of Lovrec ´ica (13°32'26"E 45°22'54"N), on the northwestern coast of Istra, between Umag and Novigrad in Croatia.
The collected crustaceans were killed on ice (to prevent loss of appendages) or transported alive to the University of Regensburg (Germany) for colour photography previous to preservation in 95% ethanol.Samples of the collected crab species were archived at the Forschungsinstitut und Museum Senckenberg, in Frankfurt a. M. (Germany).
Genomic DNA of Hemigrapsus sanguineus was isolated from the muscle tissue of one walking leg.Selective amplification of a fragment from the mitochondrial large subunit rRNA gene (16S mtDNA) was carried out by polymerase-chain-reaction (PCR) with the primer combination 16L2 (5'-TGC-CTGTTTATCAAAAACAT -3') and 1472 (5'-AGATAGAAACCAACCTGG -3') (see Schubart et al., 2000: Table 3).PCR products were purified and sequenced in an ABI Prism 310 Genetic Analyzer®.Sequences were aligned manually and compared with the multisequence editing program ESEE (Cabot and Beckenbach, 1989).
In addition to the specimen from Uvala Slanik, DNA of two other individuals of H. sanguineus from geographically distant populations was examined, one from Sippewisett (Massachusetts, USA) collected by Megan Tyrell in 1997 and deposited at the University of Louisiana at Lafayette (ULLZ 3793), and one from Taipei county (Taiwan) collected by Hung-Chang Liu on 7 June 1995 and deposited at the Senckenberg Museum, Frankfurt a. M. (SMF 27592).In addition, a third sequence corresponding to H. sanguineus from Japan (Kitaura et al., 2002) was obtained from Genbank under the accession number AB058630.New DNA-sequences were submitted to the European Molecular Biology Laboratory (EMBL) genomic library (AJ493053-AJ493054).The genetic comparisons were carried out to confirm identification of the specimen from Istra as well as to establish the intraspecific variation of this gene.Genetic variation at the mtDNA level can be useful for future population genetic studies to determine possible founder populations.

RESULTS
The following species of brachyuran crabs were commonly encountered in shallow subtidal and intertidal habitats of Uvala Slanik (Istra, Croatia): Pachygrapsus marmoratus (Fabricius, 1787), Eriphia verrucosa (Forskål, 1775), Carcinus aestuarii Nardo, 1847, Maja crispata Risso, 1827, and Xantho poressa (Olivi, 1792).On 27 August 2001, one male specimen of Hemigrapsus sanguineus (de Haan, 1835) was collected from under a large boulder on a rocky platform at about sea level in the inner part of the bay.The crab was identified based on carapace and chelar morphology as well as on the characteristic punctuation pattern of the chelae (Fig. 1, colour picture in Galil et al., 2002).This speci-men is the first record of this species and genus from the Adriatic Sea as well as from the entire Mediterranean Sea.The specimen is a small but adult male with a maximum carapace width (cw) of 17.2 mm, a carapace length (cl) of 15.4 mm and a chelar propodus height (prh) of 5.9 mm.It is deposited at the Senckenberg Museum, Frankfurt a. M. (SMF 27591).For comparison, the Taiwanese male crab (SMF 27592) measured 27.0 mm cw, 23.78 mm cl, 10.6 mm prh and the crabs reported from Le Havre by Breton et al. (2002) between 19 mm (ovigerous female) and 31.5 mm cw.According to Fukui (1988), this species can reach ~40 mm cw.Subsequent efforts to find additional specimens of Hemigrapsus sanguineus at the same locality in Istra were unsuccessful.
Comparison of DNA-sequences corresponding to the 16S mtDNA revealed that among the studied 525 DNA-nucleotides, the Adriatic representative of H. sanguineus was genetically identical to the specimen from Massachusetts and to 474 nucletotides from a specimen of Japan.In contrast, the individual from Taiwan differed in two positions (two transitions) from the other three specimens (Fig. 2).

DISCUSSION
The present record of Hemigrapsus sanguineus from the Adriatic Sea together with the newly documented presence of a breeding population in Le Havre (Breton et al., 2002) gives evidence for a current invasion of European waters by this originally East Asian species.D'Udekem d'Acoz (1999) suggested that the presence of H. sanguineus in the northwestern Atlantic might facilitate its introduction into European waters via ballast waters from ships crossing the Atlantic Ocean.This, however, should not be regarded as the only possible way of introduction of H. sanguineus into the eastern Atlantic.The first European record of this species is from Le Havre (Breton et al., 2002), where several other introductions were reported, many of them originating from the western Pacific (Breton and Vincent, 1999;Breton et al., 2002).Ballast water is only one of many means of potential introduction of alien crabs (Carlton, 1985).Gollasch (1999) found H. penicillatus among fouling organisms of a ship hull in the harbour of Bremerhaven.
The larval development of Hemigrapsus sanguineus has been described by Hwang et al. (1993).It lasts a minimum of 16 days to the megalopa stage and 25 days to the first crab stage.Epifanio et al. (1998) report that the planktonic larval phase will last for about one month under optimal conditions.Thus, as in all other Atlantic coastal crab species, larval development in the plankton is not long enough to allow natural dispersal across the North Atlantic (Scheltema, 1986).Human-mediated transport therefore appears to be responsible for the intro- ish and Portuguese Atlantic coast as well as in the western Mediterranean strongly suggests that the single record from Istra is based on an independent introduction.If colonisation were carried out by a larval stage, our individual would have settled in Uvala Slanik approximately in autumn of 1999, based on Fukui's (1988) observations that H. sanguineus grows to 20 mm cw in about two years.
Although DNA sequences from American and European populations of Hemigrapsus sanguineus are identical to a Japanese sequence and two nucleotides differ from a Taiwanese sequence, it would be premature to interpret the molecular results obtained in this study with respect to the possible origin of the introduced populations.For this purpose, populations from the entire range of this species in East Asia should be sampled and more individuals should be studied to establish intra-and interpopulation genetic variation (see for example Fratini and Vannini, 2002).Only if the analysed molecular marker shows a complete lineage sorting among East Asian populations, it can be used to determine, or at least exclude, possible founder populations from the entire range of the invasive species.Geller et al. (1997) have shown that in some cases marine invasions may originate from more than one source population.
There have been several ecological studies monitoring the range extension and biotic interactions of the two species of Hemigrapsus in their new environments (Noël et al., 1997;Lohrer and Whitlatch, 1997;McDermott, 1998bMcDermott, , 1999;;Gerard et al., 1999;Tyrrell and Harris, 2000).In western Europe as well as in the USA, the most important potential competitor is believed to be the green crab Carcinus maenas (Linnaeus, 1758) (introduced in the USA) and the most important prey, despite a predominantly algal diet, molluscs of the genera Mytilus and Littorina (see Gerard et al., 1999;Tyrrell and Harris, 2000).To date, no measurable impact on the native (or previously introduced) fauna and flora has been reported (Breton et al., 2002).Noël et al. (1997) predicted that H. penicillatus may have a future range from Norway to North Africa, including the Mediterranean and Black Seas, based on its distribution across climatic regions in native East Asia.H. sanguineus has a similar native distribution and shows wide physiological tolerances (Watanabe, 1982;Gerard et al., 1999).In the more southern regions of the potential European range, the grapsid crab Pachygrapsus marmoratus and the varunid crab Brachynotus atlanticus Forest, 1957 may be postulated as the main competitors of the two species of Hemigrapsus (see also d'Udekem d 'Acoz, 1999).The first record of Hemigrapsus sanguineus from the Mediterranean Sea supports the assumption of potential competition with Pachygrapsus marmoratus, as the single specimen was collected under rocks, in a habitat shared with P. marmoratus.The two species did differ in their escape response, which was more sluggish in Hemigrapsus sanguineus.Feeding competition experiments as carried out between H. sanguineus and Carcinus maenas in North America might help to predict the outcome of a potential competitive exclusion.The dominant role of P. marmoratus as a herbivore and scavenger of the rocky intertidal in southern European waters casts doubt on the hypothesis that this species could be endangered by the invasive H. sanguineus.It is even possible that P. marmoratus will prevent the establishment of Hemigrapsus in southern Europe.Additional records of H. sanguineus from the Mediterranean Sea, especially of ovigerous females, will be needed to determine whether this species will become established and can be considered a new member of the Mediterranean fauna.