The Baltic prawn Palaemon adspersus Rathke , 1837 ( Decapoda , Caridea , Palaemonidae ) : first record , possible establishment , and illustrated key of the subfamily Palaemoninae in northwest Atlantic waters

This study documents the introduction of the European Baltic prawn, Palaemon adspersus Rathke, 1837 to the coastal waters of northeastern North America, specifically the west coast of Newfoundland and the Magdalen Islands in the Gulf of St. Lawrence, Canada. Species identification was verified using morphological and genetic criteria. In September 2011, the first specimens of P. adspersus were collected in Gulf of St. Lawrence waters near Stephenville Crossing, Newfoundland, Canada. In 2012, additional P. adspersus specimens were collected in this area and at St. Andrew’s, located further south in western Newfoundland and in 2013 several egg-bearing females were collected further north in York Harbour. Accidental transport by ballast water of ships seems the likely vector for transport of Baltic prawn to the Gulf of St. Lawrence from Northern Europe or the Caspian Sea. It is possible that this shrimp has a wider presence in Atlantic Canadian waters but, due to its close resemblance to native shrimp species, it may have been previously misidentified, as occurred with specimens collected from the Magdalen Islands. We further expect that other species of the genus Palaemon, including P. elegans Rathke, 1837 from the Baltic Sea or northeastern United States, and P. macrodactylus Rathbun, 1902 from the northeastern United States, may invade the Gulf of St. Lawrence. We provide an illustrated key for the identification of these exotic Palaemon species and to differentiate them from native members of the subfamily Palaemoninae.


Introduction
The growing extent of biological invasions in coastal waters is widely recognised, with new introductions of exotic species resulting from increased human-mediated global dispersal and global warming (Cohen and Carlton 1998;Ruiz et al. 2000;Hiddink et al. 2012).Long distance dispersal of aquatic species facilitated by pathways between particular regions is defined as an invasion corridor (Ricciardi and Mclsaac 2000).Species movement through invasion corridors mainly occur via ballast water transport (Cohen and Carlton 1998;Cristescu et al. 2001).Ballast water management regulations have been considered ineffective, especially for macroinvertebrates (Ricciardi and Mclsaac 2000;Briski et al. 2012) and are currently under review in Canada (C. H. McKenzie, Dept. of Fisheries and Oceans, St. John's, NL, Canada, pers. com.).In particular, invasion corridors linking Eurasia with the North American Great Lakes have been reported to be delivering organisms at high rates (Ricciardi and Mclsaac 2000).These invasion rates are generally underestimated, as many species may remain undetected or misidentified for years, and therefore detailed histories of these invasions remain unclear.Therefore, early detection of non-indigenous species will enhance our understanding of their invasion histories.
Successful establishment of a non-indigenous species will be largely determined by its biological requirements and the availability of suitable habitat, particularly if they originate from similar latitude (Carlton 1985;Carlton and Cohen 2003;Niimi 2004).Salinity is an important limiting environmental factor on the distribution ranges of most aquatic species (Paavola et al. 2005).Species with high tolerance to salinity changes, including many crustaceans, are potentially better colonizers, and one of the most successful groups of historic invasions around the world (e.g.Ruiz et al. 2000).One such example is the European green crab, Carcinus maenas Linnaeus, 1758 (Carlton and Cohen 2003), which due to multiple introductions from different source populations and different genetic lineages, has recently expanded its distribution range northward in coastal waters of eastern Canada (Roman 2006).
The introduction of Palaemon adspersus Rathke, 1837 reported here represents the second European species of Palaemon detected in coastal North American waters.The first European species detected was the rockpool prawn, Palaemon elegans Rathke, 1837, reported from the northeast coast of the United States in 2010 (JT Carlton, Williams College, Mystic, CT, USA, pers.com.).In addition, the oriental shrimp, Palaemon macrodactylus Rathbun, 1902, a species originating from Southeast Asia (Newman 1963), that has spread globally (see González-Ortegón et al. 2007), has been observed in the northeastern United States since 2001 (Warkentine and Rachlin 2010).
In the present study, the possible establishment of the Baltic prawn in western Newfoundland is reported and assessed based on sample collections in this area (2009)(2010)(2011)(2012)(2013)(2014) and the potential vector for this new introduction is discussed.DNA analysis was used to verify the morphological identification of P. adspersus.In addition, an illustrated key is provided to facilitate the identification of palaemonid shrimps in northeastern North America that includes the three most recent introductions not included in previous keys (Holthuis 1952;Williams 1984;Squires 1990Squires , 1996)).

Study area and identification of species
As part of an ongoing project investigating the biodiversity and distribution ranges of several marine fishes along the west coast of insular Newfoundland, pole seining has been conducted annually since 2009 (Table S1).A pole seine (1.4 m H × 10.0 m L, 7 mm stretched mesh) was pulled 60 -100 m parallel to the shoreline at 0.3 -1.4 m depths at nine sampling stations (Figure 1).Catches were identified, counted, and measured before being returned alive to seine sites.In addition, shrimp specimens from an eel fisherman (D.Hynes) in St. Andrew's, caught by fyke net, (3 m bag, 20 m leader, 7 mm stretched mesh) were examined.
Specimens not readily identified to species on site were retained and preserved in 5% buffered formalin for later detailed examination and identification.In 2011, 5 specimens of an unidentified shrimp species were retained from Site S2 (Figure 1) near the community of Stephenville Crossing.Additional shrimp specimens from this area were collected for DNA analysis in 2012.Morphological features of live specimens were first examined and photographed before being preserved in anhydrous ethanol.Environmental parameters, including salinity, water temperature, substrate types and associated vegetation were recorded annually at each site, when possible.
Pole seining was not conducted by the authors in 2013.However, preserved shrimp samples collected by beach seine from Stephenville Crossing Site S2 of that year were provided by C. McKenzie (unpubl.data) from aquatic invasive species surveys.A 25 m long beach seine (19 mm stretched mesh in the wings and belly, and 13 mm in the codend) was deployed by boat 55 m from shore and pulled perpendicular to the shore.
Additional shrimp samples from the southern portion of the Magdalen Islands were collected by N. Simard (Dept. of Fisheries and Oceans, Mont-Joli, QC, Canada) with a pole seine in 2011 (Figure 1, Table S1).
Using morphological and molecular evidence De Grave and Ashelby (2013) have recently indicated that the genus Palaemonetes is a junior synonym of Palaemon.As such, they suggested the transfer of all Palaemonetes species to the genus Palaemon.Thus, Palaemonetes intermedius, P. pugio, and P. vulgaris should be within the genus Palaemon.Here we have adopted this change using Palaemon (= Palaemonetes) for these three species.Also, in keeping with De Grave and Ashelby (2013), we use Palaemon mundusnovus as a replacement name for the North American Palaemonetes intermedius that is a homonymy with an earlier-named Australian species of the same name.
Key morphological and meristic characters are presented to differentiate the exotic species  S1 for further details about the sampling sites in the study area.* European locations from which P. adspersus 16S DNA sequences were compared with Stephenville Crossing (Canada) sequences.
To verify the morphological identification of P. adspersus, 16S rDNA sequences were obtained from GenBank (Table S2); one from San Pedro River, Spain (accession number JN674330) and one from Algarve, Portugal (accession number JQ042293).We used the 16S rDNA based method to compare sequences between the nonnative Stephenville Crossing population and the European population published in GenBank.

Field collections and morphology-based identification of P. adspersus
In September 2011, five specimens of an unidentified shrimp species were collected just west of Stephenville Crossing, Newfoundland, Canada (Site S2; Figure 1).Morphological characteristics of these specimens were inconsistent with those of native palaemonids in Atlantic Canada and consistent with those of the European species, Palaemon adspersus.Specimens exhibited red spots along the lower edge of the rostrum, yellow bands on the joints of the pereiopods (Figure 2A), and antennules with strong red pigmentation (Figure 2B).Further, total length of these five specimens ranged from 49.0 -53.8 mm, such that all but one specimen exceeded the approximate maximum total length previously recorded for native Palaemonidae species (Anderson 1985;Squires 1990).Substrate and environmental conditions at this collection site (Table S1) were also consistent with known habitat for P. adspersus (Berglund 1980;Cuesta et al. 2006;Janas et al. 2013).
The presence of P. adspersus was not detected in earlier surveys (2009 to 2011) at other sites along the west coast of Newfoundland, including a site just south of Stephenville Crossing (Site S1; Figure 1).At Site S1, the only crustacean species collected were mysids Mysis sp., sand shrimp, Crangon septemspinosa Say, 1818, and green crab, Carcinus maenas.
In September 2012, 9 and 64 specimens of P. adspersus, of which 30 were retained for DNA analysis, were collected at Sites S2 and S3, respectively, near Stephenville Crossing (Figure 1; Table S1).In addition, 107 sand shrimp were collected at Site S3.However, as in the previous years (2009 and 2010), the only crustacean species collected at Site S1 were mysids, sand shrimp, and green crab.
In August 2012, one additional P. adspersus specimen was collected in St. Andrew's by eel fisherman D. Hynes (Figure 1).Earlier observations by D. Hynes suggested this species had been present for several years in this area.Additional pole seining in September 2012 adjacent to this area (Table S1) did not result in the collection of any Baltic prawn but mysids and sand shrimp were collected.
In July 2013, four Baltic prawn specimens, along with sand shrimp and green crab, were retained from a beach seine hauled at Site S2 while sampling for aquatic invasive species (C.H. McKenzie et al., unpubl. data).
In August 2013, three berried female Baltic prawn specimens (Figure 2C) were collected in minnow traps while surveying for banded killifish Fundulus diaphanous Lesueur, 1817, in York Harbour, Newfoundland (Figure 1, Table S1).This observation was the first indication that reproduction of Baltic prawn in the Gulf of St. Lawrence could be occurring.
In August 2014, while pole seining, sand shrimp and green crab were collected at Site S2 but no Baltic prawns were detected.However, five Baltic prawn specimens were collected at St. George's, Newfoundland (Figure 1, Table S1), while sand shrimp and green crab were not collected.
A re-examination of a shrimp collection provided by N. Simard from the Magdalen Islands, Quebec, in 2011 (Figure 1, Table S1), showed two specimens originally identified as Palaemon (= Palaemonetes) pugio were actually P. adspersus, providing the first record of Baltic prawn in the southern Gulf of St. Lawrence.

16S ribosomal DNA-based identification of P. adspersus
The 16S rDNA region of eight specimens from Stephenville Crossing, four from Limfjorden (Denmark), one from Île de Ré (France) were sequenced and two GenBank sequences of P. adspersus, from Spain and Portugal, were used to confirm the morphological identification of P. adspersus.These 15 16S rDNA sequences were aligned with 66 16S DNA sequences of 22 species of the genus Palaemon and three other 16S DNA sequences from three species of the genus Macrobrachium available from GenBank (Table S2).The topology of the maximumlikelihood tree is consistent with a maximum parsimony tree reconstructed from the same data set.That is, the best selected model consistently clustered all sequenced samples of Palaemon specimens from Stephenville Crossing within the P. adspersus clade (Figure 3).This result strongly supports the conclusion that the specimens collected from Stephenville Crossing belong to P. adspersus.An illustrated key to identification of the Palaemoninae species in northwest Atlantic waters from New Jersey (US) to Canada (adapted from Holthuis 1952;Williams 1984;Squires 1990) Family Palaemonidae: Caridea having first 2 pairs of legs chelate, second pair usually larger than first, carpus of second pair not subdivided.Rostrum usually armed with non-movable teeth.Mandibles usually with an incisor process.Subfamily Palaemoninae: Upper antennular flagellum with both rami fused in basal part.Appendix masculina generally present on second pleopod of male, appendix interna on second pleopod of female.Pleurobranch present on third maxilliped segment.Posterior margin of telson with 2 pairs of spines and 1 or more pairs of setae (Holthuis 1952).The technical terms used in the key are illustrated in Figures 4A and 5. Morphological differences among these species are further summarized in Table S3.Rostrum with 5 to 7 dorsal teeth, usually 5 and 1 postorbital tooth; inferior half of rostrum with dark red spots (Figures 2A, 4D); body with a uniform yellowish grey colour and yellow bands at the joints of the pereiopods; mandible with palp (Figure 5A) ………………………………………………………………………….……………Palaemonadspersus ─ Rostrum with 7 to 10 dorsal teeth, usually 8 to 9 (including the postorbital tooth); rostrum without dark red spots on its lower half (Figure 4E-I); body generally almost colourless and translucent; mandible without palp (Figure 5B) ………5 5.

Taxonomic and ecological accounts of nonindigenous Palaemon species
Palaemon adspersus Rathke, 1837 Colour: Body with a uniform yellowish grey colour with yellow bands at the joints of the pereiopods.The lower half of the rostrum is covered with chromatophores in the form of red spots.The long flagella and the peduncle of the antennules bear strong red pigmentation.

Palaemon elegans Rathke, 1837
Colour: Cephalothorax and abdomen with a black striped pattern.The pereiopod joints are marked by yellow bands and the palms of the chela on the second pereiopods are blue.However, such colouration may be different in specimens living in turbid waters, in which the stripes may be almost completely faded away.
Habitat: Common in tidal rockpools, and in Zostera, Posidonia and Cymodocea seagrasses.Also, it can be found in slightly brackish water close to river mouths (Lagardère 1971).

Palaemon macrodactylus Rathbun, 1902
Colour: Translucent, with reddish spots covering the entire body surface, pereiopod joints are marked by yellow bands, the rostrum covered with chromatophores in the form of red blotches and a very distinctive dorsal colourless stripe running along its cephalothorax and abdomen.This colour pattern is sometimes stronger in females.
Habitat: Estuaries, protected harbours, bays, ponds, tidal creeks.This species tolerates a very broad range of ecological conditions (Newman 1963).

Discussion
The euryhaline species Palaemon adspersus Rathke 1837, native in European waters from the Baltic Sea to Mediterranean Sea (González-Ortegón and Cuesta 2006), was collected in eastern Canadian waters along ca.200 km of the western Newfoundland coast.DNA analysis verified the morphological identification of this non-native species and this introduction represents the first record of the Baltic prawn in North American waters.Previous records of this species outside Europe were from Eurasian waters: the Caspian Sea in the 1930s and 1980s and the Aral Sea in the 1950s (Zenkevich 1963;Holthuis 1980).
The detection of Baltic prawn along the west coast of Newfoundland for four consecutive years (2011 to 2014), a species with longevity estimated at < 3 years (Bilgin et al. 2009), along with berried females, and detection in the Magdalen Islands suggest this European species is established in Atlantic Canadian waters.However, given the extent of commercial shipping within the Laurentian Channel, the presence of this non-native species in Canadian waters from repeated ship ballast water discharges cannot be excluded.
Accidental transport in ballast water of ships from the Caspian Sea or northern European waters seems to be the most likely vector for this first documentation of P. adspersus in Canadian waters.A series of invasions have been reported in the North American Great Lakes by euryhaline species originating from the Caspian Sea via possible invasion corridors through the Baltic, North, or Mediterranean Seas (Ricciardi and Maclsaac 2000;Cristescu et al. 2001).Freighters from the Ponto-Caspian region bound for ports along the Great Lakes often take on ballast water near St. Petersburg in the Baltic Sea (Sprules et al. 1990).A clear example of this phenomenon is the cladoceran Cercopagis pengoi Ostroumov, 1891, which has extended its range over the past decade from the Ponto-Caspian basin into the Baltic Sea and the North American Great Lakes (Cristescu et al. 2001).Northern European waters have also been reported as the most likely source of a cryptic secondary introduction event of the European green crab, Carcinus maenas, to Canada (Roman 2006).This conclusion was based on frequent cargo vessel traffic from the North Sea to the Strait of Canso, one of the largest ice-free harbours in northeastern North America (Roman 2006).As well, considering that most successful introductions originate from regions with similar latitudes and climates (Carlton 1985), northern European populations of P. adspersus are a more likely candidate to establish in Canadian waters than southern European populations as the former should be more tolerant to low water temperatures of western Newfoundland.
Local vessel traffic from other Atlantic Canadian ports may be another means for Palaemon adspersus to have reached the west coast of Newfoundland, as was reported for European green crab, Carcinus maenas, first detected in Placentia Bay, southeast Newfoundland, in 2007 (Blakeslee et al. 2010).Placentia Bay is an area heavily used by ballast-carrying commercial vessels (Klassen and Locke 2007) and is where numerous nonnative species have been recently found (McKenzie et al. 2010;Matheson 2013;Sargent et al. 2013).On the west coast of Newfoundland, green crab was detected near Stephenville in 2008 (DFO 2011), but it is yet undetermined whether green crab was introduced there by means of local vessel traffic or by larval dispersal from a neighbouring location.For the Baltic prawn to have been transported from elsewhere in Atlantic Canada would require it to have been established somewhere else before 2011.While there is presently no evidence for this scenario, the possibility cannot be discounted.Failure to detect P. adspersus in other Atlantic Canadian regions could be the result of a combination of factors including: 1) general unawareness of this species, resulting in no one looking for it; 2) similar appearance to native Palaemoninae species, so that it may have been misidentified; 3) sampling methods may have been inappropriate to collect specimens; and 4) the preferred habitat of this species may not have been sampled.Misidentification of P. adspersus as Palaemon (= Palaemonetes) pugio in the Magdalen Islands and lack of sampling of Sites S2 and S3 before 2011 could support the hypothesis that this species was present elsewhere in Atlantic Canada prior to 2011.
The occurrence of P. adspersus in Newfoundland waters suggests the possibility of introductions of additional shrimp species to the Gulf of St. Lawrence.Recent introductions of Palaemon macrodactylus, and P. elegans in the northeastern United States (Warkentine and Rachlin 2010; J. Carlton, pers. com.) and warming water temperatures may facilitate the spread of these and other non-indigenous decapod crustaceans northward into Gulf of St. Lawrence waters (Jamieson 2000;Stachowicz et al. 2002).Other European palaemonid shrimp species recently found colonizing new regions, such as P. longirostris H. Milne Edwards, 1837 (Grabowski 2006;Sezgin et al. 2007), P. serratus (Pennant, 1777) (Gönlügür-Demirci 2006) and Palaemon (= Palaemonetes) varians (Leach, 1814) (Grabowski 2006), may follow the same invasion corridor as P. adspersus and establish populations in the Gulf of St. Lawrence.
The recent introduction of Baltic prawn reported here underlines the need for reconsideration of ballast water management practices as previously suggested by Miller et al. (2005) and Roman (2006).Currently, mid-ocean ballast water exchange regulations for transoceanic and coastal vessels arriving at ports on the Atlantic coast of Canada are ineffective for controlling introductions of macroinvertebrates (Briski et al. 2012).Existing Canadian regulations state that all vessels entering Canadian waters exchange or treat their ballast 200 nautical miles from shore and in at least 2000 m of water before discharging, except under exceptional circumstances (Transport Canada 2007).Due to safety considerations (e.g. during storms), however, vessels may exchange their ballast in alternative ballast water exchange zones, one of which is located within the Laurentian Channel in the Gulf of St. Lawrence (DFO 2009).At such times, vessels may release propagules of non-indigenous species directly into the Gulf of St. Lawrence; most of these vessels originate from the northeastern United States and Europe (Simard and Hardy 2004;McKenzie et al. 2010;Lo et al. 2012).In addition, vessels operating exclusively within waters of Canadian jurisdiction are exempt from ballast water exchange regulations (Canada Shipping Act 2006;Transport Canada 2007).Thus, such vessels travelling between Canadian ports may facilitate the secondary introduction of non-indigenous species.Interregional vessel transportation is a potentially important vector (Lavoie et al. 1999;Simkanin et al. 2009) of secondary spread and is a growing concern as a means for the spread of invasive species, especially to Newfoundland (Blakeslee et al. 2010;McKenzie et al. 2010).The recent detection of P. adspersus in the present study provides additional evidence of the ineffectiveness of Canada's current ballast water regulations (Smith et al. 2014).Consequently, the spread of non-indigenous species is likely to increase unless changes are made to current regulations regarding ballast water discharge and vessel movement between ports (Lambert and Lambert 1998).
Currently, the Baltic prawn appears merely as a non-indigenous species in Atlantic Canada and does not appear to be invasive.Further research on the introduction of P. adspersus in Atlantic Canada should include efforts to determine the initial area of introduction and current distribution Atlantic Canada, the European source population, and an assessment of its ecological interaction with native species.The determination of the source population location and the point of initial introduction of P. adspersus is of considerable importance, as this information may both aid in predicting future species invasions from the same region(s) and reveal the invasion corridor and transmission vector through which this non-indigenous species was transported.Finally, potential interactions between Baltic prawn and those congenerics native to Atlantic Canada (e. g.P. pugio and P. vulgaris) inhabiting similar habitats (Köhn and Gosselck 1989; this study), should be examined to ascertain whether P. adspersus will persist to become another problematic invasive species in this region, or will integrate within the ecosystem to enrich biodiversity.

Figure 1 .
Figure 1.Worldwide distribution of Palaemon adspersus Rathke, 1837, and a magnification of the study area in Newfoundland and Magdalen Island (inset); 1. Original distribution; 2. Caspian Sea; 3. Aral Sea; 4. Newfoundland and Magdalen Islands, Gulf of St. Lawrence (Canada): Sampling sites in the western Newfoundland (S1, S2, and S3 are Stephenville Crossing Site 1, Site 2, and Site 3, respectively).Black stars and dark grey points indicate sites where P. adspersus have been detected and not detected, respectively in this study.See TableS1for further details about the sampling sites in the study area.* European locations from which P. adspersus 16S DNA sequences were compared with Stephenville Crossing (Canada) sequences.

Figure 2 .
Figure 2. Photographs of Palaemon adspersus specimens collected in western Newfoundland, Canada.A -specimen showing red spots on rostrum and yellow bands at the joints of the pereiopods; B -specimen showing yellow bands along edges of abdomen segments, and red pigmentation on antennules; C -berried female from York Harbour.Photographs A and B by V. Ramírez-Luna and photograph C by P. Sargent.

Figure 3 .
Figure 3. Maximum likelihood phylogenetic relationships based on the 16S DNA sequences of Palaemon spp.and Macrobrachium spp., which were used as the outgroup.Nucleotide substitution model: TN93+G.All P. adspersus sequences cluster into a single clade with high support.The topology of the tree is consistent with a maximum parsimony tree reconstructed from the same data set (not shown).
identified a single record of Macrobrachium sp.just inside Atlantic Canadian waters in the Gulf of Maine.**recent evidence (De Grave and Ashelby 2013) indicates that the genus Palaemonetes is a junior synonym of Palaemon resulting in the transfer of all Palaemonetes species in the present study to Palaemon; also Palaemonetes intermedius has been renamed Palaemon mundusnovus as the North American name (Palaemonetes intermedius) fell to homonymy with an earliernamed Australian species of the same name.