Peter Rehm

Description of a new subspecies Diastylis enigmatica rossensis (Crustacea: Peracarida: Cumacea) from the Ross Sea, Antarctica

Keywords: Antarctica, Crustacea, Cumacea, Diastylidae, Ross Sea, Victoria Land

Diastylis enigmatica rossensis n. ssp. is described and illustrated on the basis of new specimens from the Antarctic shelf in the Ross Sea. The material was collected oV the Victoria Land coast during the 19th Antarctic expedition of the Italian research vessel Italica in 2004. Diastylis enigmatica Ledoyer, 1993 was Wrst obtained during the EPOS 3 campaign at Halley Bay in the Weddell Sea. A redescription based on further material from the Weddell Sea was published by Petrescu and Wittmann (Zool Med Leiden 77:557-630, 2003), which shows several diVerences to the newly collected material from the Ross Sea. The most obvious diVerences from Diastylis enigmatica rossensis n. ssp. to Diastylis enigmatica enigmatica Ledoyer, 2003 are the absence of four spines on the merus of the second pereiopod, a lower number of setae on the uropodal endopod, a gap between spines on the peduncle article 1 of antenna 1, and a higher number of articles in both Xagella of antenna 1.

Introduction

Diastylis enigmatica [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF] was Wrst described from material collected during the EPOS 3 campaign [START_REF] Arntz | The expedition ANTARKTIS VII/4 (EPOS leg3) and VII/5 of RV "Polarstern" in 1989[END_REF]) at Halley Bay in the Weddell Sea. The original description is based on a juvenile male with incomplete uropods, which was found between 270 and 280 m water depth. In 1989/1990, Wve further specimens were found at a depth between 240 and 260 m during the Expedition Antarktis-VIII/5 with RV Polarstern. Based on this material [START_REF] Petrescu | Elements for a revision and notes on bionomy of the Cumacea (Crustacea: Peracarida) of the Weddell Sea (Antarctica). Material collected by the expedition ANTARK-TIS-VIII/5 of R.V. "Polarstern" 1998/90[END_REF] redescribed a subadult female of Diastylis enigmatica. Additional specimens from the Antarctic shelf in the Ross Sea (216-366 m) were obtained oV the Victoria Land coast during the 19th Antarctic expedition of the Italian research vessel Italica in 2004 [START_REF] Rehm | Composition and distribution of the peracarid crustacean fauna along a latitudinal transect oV Victoria Land (Ross Sea, Antarctica) with special emphasis on the Cumacea[END_REF]. A Wrst examination of the material from the Italica cruise already showed slight diVerences in some of the cumacean species to the original descriptions. With the present study the description of a marsupial female of the new subspecies Diastylis enigmatica rossensis and a detailed analysis of sexual and developmental diVerences in the subspecies of Diastylis enigmatica is provided.

Materials and methods

Material for this study was collected during the 19th Antarctic expedition of RV "Italica" to the Ross Sea [START_REF] Rehm | Distribution and composition of macrozoobenthic communities along a Victoria-Land Transect (Ross Sea, Antarctica)[END_REF]. In total 173 specimens were sampled at four stations and examined with a Leica MZ12 5 and an Olympus SZX12 dissecting microscope with camera (Olympus Colour View I). Dissected appendages were mounted on slides in glycerine and studied with a Zeiss AxioSkop 1 with attached camera (Olympus DP70). Drawings were created from digital photographs using a digital drawing tablet (Wacom Intous3 9 £ 12) as described by [START_REF] Co | Digital inking": How to make perfect line drawings on computers[END_REF][START_REF] Co | Substituting time-consuming pencil drawings in arthropod taxonomy using stacks of digital photographs[END_REF]. Material has been deposited in the collection of the Zoological Museum Hamburg (ZMH).

Communicated by P. Funch. P. Rehm (&) Animal Physiology, Universität Hamburg, Biozentrum Grindel, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany e-mail: peter.rehm@awi.de 123

The material was collected with a Rauschert dredge [START_REF] Rehm | Distribution and composition of macrozoobenthic communities along a Victoria-Land Transect (Ross Sea, Antarctica)[END_REF]) along the Victoria Land coast at four stations ranging from 216 to 366 m. For detailed data on the sampling stations and on species diversity and distribution found at these stations refer to [START_REF] Rehm | Composition and distribution of the peracarid crustacean fauna along a latitudinal transect oV Victoria Land (Ross Sea, Antarctica) with special emphasis on the Cumacea[END_REF].

Body length is measured from the tip of the pseudorostrum to the tip of the telson. Length of articles are measured according to [START_REF] Mühlenhardt-Siegel | New species of the family Nannastacidae (Crustacea: Peracarida: Cumacea) from the Angola Basin, south-eastern Atlantic. Deep-Sea Expedition DIVA-1. Addendum[END_REF] and given as relative length of peduncle (RLP) articles 1-3 of antenna 1 compared to total peduncle length. The ratio basis to rest (B/R) is given for maxillipeds and pereiopods, which is the proportion of the basis to the combined length from ischium to dactylus, not including terminal setae. RLA refers to the relative length of each article from ischium to dactylus, excluding terminal setae.

The following types of setae were distinguished: simple setae are slender and completely lack outgrowths on the setal shaft; pappose setae have irregularly distributed setules along the shaft; plumose setae have many setules arranged in two strict opposite rows, giving the setae a feather-like appearance; serrate setae have one or two rows of denticles in their distal half; setulate-serrate setae have long setules on proximal half and one or two rows of denticles on distal half; cuspidate setae are short setae that are wide at the base; broom setae are tiny and have a pedestal and extremely thin setules distally; and annulate setae are simple and have a distinct internal spiral structure in their distal half.

Results

Order: Cumacea Kröyer, 1846 Family: Diastylidea Bate, 1856 Genus: Diastylis Say, 1818

Diastylis enigmatica rossensis n. ssp. (Figs. 1,2,3,4,5) Material: was deposited in the Zoolical Museum Hamburg.

Holotype: incubating female (ZMH 42030; partially dissected); station R2 74°49.0ЈS/164°18.1ЈE, Wne sand, 364 m, 21 February 2004.

Paratypes: subadult females (ZMH 41273), both specimen were dissected; same station data as holotype. Two juvenile females, a juvenile male, and a subadult male (ZMH 41274), station R3 74°49.3ЈS/164°11.5ЈE, rocky sand substratum with mud and pebbles, 330 m, 20 February 2004. Juvenile male (ZMH 41275), station SMN 74°43.2ЈS/164°13.1ЈE, 366 m, sand with gravel and stones, 20 February 2004.

Description

Adult female. Body length 8.9 mm.

Carapace (Fig. 1). 1.6 times longer than high, with four denticulate transverse ridges; some very small simple setae, slightly more concentrated on frontal lobe and on anterior and ventral margin; minor denticulate ridges, with slightly varying structure on both sides of carapace. Eyelobe with two spines; six spines on frontal lobe as described in [START_REF] Petrescu | Elements for a revision and notes on bionomy of the Cumacea (Crustacea: Peracarida) of the Weddell Sea (Antarctica). Material collected by the expedition ANTARK-TIS-VIII/5 of R.V. "Polarstern" 1998/90[END_REF], further dorsal spines of varying size along three-fourths of carapace length. Pseudorostrum moderately produced about one-sixth of total carapace length (including pseudorostrum). Antennal notch shallow. Lateral medial margin serrated. Integument of segments with honeycomb-like structure; margins with Wne serrations. Sternites of last three thoracic segments with median spine decreasing in length. Last pleon segment ventrally with two rows of Wve denticles forming a 'v' opening to the posterior end. Carapace and free thorax segments approximately 1.2 times longer than abdomen.

123

Antenna 1 (RLP 54/23/23) (Fig. 2e). First article of peduncle with three spines on distal margin, one slightly separated from others, with one pappose seta and one broom seta distally, many hair-like setae, row of hair-like setae along little more than distal third of article turning back along integumental fold and ending with a simple seta; second article with few hair-like setae, proximal many hair-like setae, Wve simple setae and one broom seta distally; third article with a simple and three broom setae distally; main Xagellum of six articles, article 4 with one simple seta, article 5 two simple setae and two aesthetascs distally, article 6 with three simple setae and a broom seta; accessory Xagellum with four articles, article 1 with one seta (was broken in the specimen), third article with one broom seta, fourth article with a broom seta and three simple setae.

Antenna 2 (Fig. 2f). Four articles, Wrst article with a pappose seta; second article with two pappose setae; fourth article bearing terminal a pappose seta.

Mandibles (Fig. 2b,c). Typical of genus; left mandible lacinia mobilis and 12 or 13 (one or two setae broken oV) setae of various shapes (simple and serrate); right mandible with 13 setae of various shape (serrate and bifurcate), most distal seta trifurcate.

Maxilla 1 (Fig. 2a). Lateral endite having 14 cuspidate simple or serrate setae, single subdistal seta on lateral mar- Maxilla 2 (Fig. 2d). A row of plumose setae parallel to medial edge of protopod slightly turning outward subdistally, medial edge with some hair-like setae and a serrate seta in distal third, medial distal edge with a setulate-serrate seta, distally various types and shapes of simple and serrate setae and a pappose seta at lateral edge; lateral lobe of endite with three medial serrate setae and three lateral simple setae distally; medial lobe with three medial serrate setae and one lateral simple seta distally, four simple setae distally on medial margin.

Maxilliped 1 (B/R 0.6; RLA -/27/32/30/11) (Fig. 3a). Basis with many hair-like setae (not drawn in the Wgure), one pappose seta at distal margin, projection of the basis with four pappose setae and four setulate-serrate setae at medial margin, two retinacula present, ischium absent; merus with three simple setae along distal half of lateral margin and 1 minute seta on a protuberance on lateral distal margin; carpus, medial margin with simple setae and a dense group of simple, trifurcate-pappose, and pappose setae close to medial and distal margin, medial margin with a lamelliform structure, three simple setae at lateral margin and one large pappose seta at lateral distal edge; propodus, simple and pappose setae mainly close to medial margin, three pappose setae and two serrate setae distally; dactylus with two terminal cuspidate and one serrated setae.

Maxilliped 2 (B/R 1.0; RLA 9/24/34/23/10) (Fig. 3c). Basis, eight simple and two pappose setae along distal lateral edge, hair-like setae at distal third of lateral margin and along three quarters of medial margin, four pappose setae distally and one simple seta subdistally; ischium, medial margin with hair-like setae; merus, lateral edge of distal margin with one pappose seta; carpus, along medial margin eleven setae (pappose and setulate-serrate), two pappose setae close to lateral distal edge; propodus having one large pappose seta proximally, eight setulate-serrate setae in distal half of medial margin, distal margin with three pappose setae; dactylus, terminal and subterminal four simple setae and one cuspidate seta.

Maxilliped 3 (B/R 1.4; RLA 14/12/24/28/22) (Fig. 3b). Basis curved, lateral margin with hair-like setation, proximal third without setation, along medial margin 20 pappose setae, medial distal edge with one pappose seta, distal lateral edge with one pappose seta and six large pappose setae, at distal third close to lateral margin 2 minute spines and a scale-like structure, one strong spine at medial distal edge (four spines in a row along medial margin in premature females); ischium, one pappose seta subdistally at medial margin, lateral margin bearing a process subdistally; merus, two pappose setae at medial margin, one large plumose setae at lateral margin, spine close to medial margin; carpus, three pappose setae, one medially, one subdistally, and one at lateral distal edge, one bifurcated seta at medial distal edge; propodus, four pappose setae at lateral margin, one pappose seta at medial distal edge; dactylus, six simple setae subterminally and terminally.

Pereiopod 1 (B/R 0.5; RLA 7/10/22/32/31) (Fig. 4a). Basis, hair-like setae along margins, four small simple setae at distal margin, a row of 16 pappose setae along lateral margin starting subdistally the position of the six proximal most setae shifting to midline of article, 45 pappose setae along medial and distal margin, small and minute simple setae along both rows of pappose setae, a row of four spines along midline, a second row of small spines in the middle of medial margin (some less developed, scale-like), a spine between seventh and eighth pappose seta of lateral margin (spine was not observed in premature females), a group of three spines at medial distal edge, separated from a larger spine in the middle of distal margin; ischium, few minute simple setae; merus, few minute simple setae, two small simple setae and two small pappose setae distally; carpus, six small simple setae along the article, three small simple setae at distal margin; propodus, 12 small simple setae along article, subdistally four simple setae; dactylus, 12 simple setae of varying size along the article, terminal and subterminal seven simple setae; exopod fully developed.

Pereiopod 2 (B/R 0.7; RLA 4/18/48/10/19) (Fig. 4b). Basis, few minute setae, 25 pappose setae along medial margin, small simple setae along medial margin, Wve pappose setae at distal fourth of lateral margin; one small simple setae on an elevation close to medial distal margin, one cuspidate simple seta with a tapered and curved tip (drawn with dotted lines, as it was broken oV, but was observed in subadult specimens of both sexes) at distal end of elevation, a second cuspidate seta of that morphology at medial distal margin (see enlargement), close to lateral distal margin one broom seta; ischium, small without setation, spine on medial margin; merus, some small and minute simple setae, lateral margin with one simple and one pappose seta, close to medial margin two pappose seta, distal edge with three pappose seta; carpus, some simple seta of varying size along article, distally Wve simple setae of varying size; propodus, medial margin distally with one simple seta and one small simple seta, one broken seta subdistally; dactylus, four seta along article (broken), distally nine simple setae of varying size.

Pereiopod 3 (B/R 1.0; RLA 15/36/21/12/15) (Fig. 5a). Basis, 11 pappose setae along medial margin, close to lateral margin in distal half three simple setae and one broken seta, integument of distal and proximal margin with honeycomb-like structure (not drawn, compare Fig. 5a); ischium, Wve small simple setae close to medial margin, three large annulated setae at medial margin, integument with honeycomb-like structure (partially drawn only; Fig. 5a); merus, four simple setae and two annulated setae at medial margin, one simple seta at lateral margin, four small simple setae; carpus; one simple seta at medial margin, two simple setae at lateral margin, lateral distal margin with four long and strong annulated setae; propodus, lateral distal margin with one strong annulated seta; dactylus, one simple seta and two cuspidate terminal setae; exopod, poorly developed with three articles, second articulation with one simple setae, third articulation terminating with two simple setae.

Pereiopod 4 (B/R?; RLA 14/39/17/14/16) (Fig. 5c). Basis (not completely preserved), few minute simple setae, a row of seven pappose setae increasing in size to distal lateral margin, two simple seta and one broom seta at medial margin, one broom and one pappose seta at distal margin, distal margin with honeycomb-like structure (compare Fig. 5a); ischium, four simple setae, integument with a honeycomb-like structure (compare Fig. 5a); merus, six simple setae on lateral distal margin, one simple seta on medial margin, few minute setae; carpus, three simple setae at distal half of lateral margin, two annulated setae at proximal half of lateral margin, six annulated setae along medial and distal margin, propodus, one strong annulated seta at medial margin and broom seta distally; dactylus, one simple seta, terminally one simple seta and one cuspidate seta; exopod, poorly developed with three articulations, second articulation with one simple seta, third articulation terminating with three simple setae.

Pereiopod 5 (B/R 0.5; RLA 16/31/26/12/15) (Fig. 5d). Basis, few minute setae; lateral and medial margin with one pappose seta each, two simple setae at medial distal margin, lateral distal edge and distal margin with one broom seta, honeycomb-like structure at distal and proximal margin (compare Fig. 5a); ischium, medial distal margin with one small simple and one simple seta, integument with honeycomb-like structure (compare Fig. 5a); merus, distally one simple seta at lateral margin, Wve simple setae in distal half of medial margin; carpus, a row of four annulated setae close to lateral distal margin, three of which are strong, two simple setae, three simple setae at medial distal margin; propodus, medial distal margin with one broom seta, lateral distal margin with one strong annulated seta; dactylus, with one simple seta and terminally two simple setae, one of which is strong; exopod, absent.

Uropod (Fig. 5b). All cuspidate setae with a setule; peduncle, three times longer than endopod, medial margin of peduncle with 22 short cuspidate setae, one small simple seta at distal margin, endopod triarticulate, medial margin of Wrst article with three short cuspidate setae, medial margin of second article with four short cuspidate setae, lateral margin of second article with two broom setae and one simple seta distally, medial margin of third article with four short cuspidate setae, lateral margin of third article with one broom seta, terminal with one small simple seta and one strong seta with a terminal Wlament; exopod, 1.2 times longer than endopod, lateral margin with two small simple setae and 2 minute simple setae, subterminal one small simple seta and three terminal simple setae.

Telson. 0.6 times length of peduncle, some small and minute simple setae on broader proximal part of telson, eight short cuspidate setae with Wlament on both sides of distal part, enlarging to distal end, two distal cuspidate setae.

Subadult males. Second antenna incompletely developed and lacking articulation and setae, reaching posteriorly to abdomen. Median row of spines of pereiopod 1 comprising about 15 spines; medial margin with six spines more developed than in female. Basis of maxilliped three with Wve spines along medial distal margin, instead of four as in the adult female. Exopods developing on maxilliped 3 and on pereiopods 1-4. Articles 2 of the uropodal endopod bearing four cuspidate setae with Wlament, instead of three as in the adult female. Pleopod buds present on abdominal segments 1 and 2, with 3 or 4 minute setae distally.

Juvenile males. The exopod of the third pereiopod is only biarticulate, whereas it is triarticulate in juvenile females.

Discussion

Subspecies of Diastylis enigmatica

In all specimens of the present study the ratio of telson length versus uropodal peduncle was 0.6, as described by [START_REF] Petrescu | Elements for a revision and notes on bionomy of the Cumacea (Crustacea: Peracarida) of the Weddell Sea (Antarctica). Material collected by the expedition ANTARK-TIS-VIII/5 of R.V. "Polarstern" 1998/90[END_REF]. In the description of [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF] the exopod of the third pereiopod has two segments, whereas in Petrescu and Wittmann the exopod is three-segmented. During the present study it was possible to explain this variation by sexual dimorphism, as juvenile males were collected, which have two-segmented exopods, whereas the exopods of juvenile females are three-segmented. From the number of segments and the large size of the exopod, the specimen of Ledoyer must have been a juvenile male. In Petrescu and Wittmann the length of the postanal part of the telson from Ledoyer's description is mentioned as being smaller than the preanal part. In the original drawing, both parts seem to be equal in length (in the text Ledoyer described the telson as being even three times longer than the preanal part). The ratio of postanal to preanal part of telson (Table 1) varied from 1.2 to 1.9 in the present study. The ratio in the specimens from the Weddell Sea material seems to be lower than in the same developmental stages from the Ross Sea. However, the ratios from the Weddell Sea are not precise enough to use this character to diVerentiate both subspecies.

Presently, two subspecies of Diastylis enigmatica are distinguished according to diVerences found from the description of [START_REF] Petrescu | Elements for a revision and notes on bionomy of the Cumacea (Crustacea: Peracarida) of the Weddell Sea (Antarctica). Material collected by the expedition ANTARK-TIS-VIII/5 of R.V. "Polarstern" 1998/90[END_REF] to the material of the Ross Sea (Table 1). Due to obscure or contradicting morphological characters of the Wrst description of Diastylis enigmatica [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF] it is impossible to assign it to one of the subspecies. This problem can be addressed in the future, when further material from the type locality (Weddell Sea), including adult males and females, is available. Nevertheless, the geographical distribution supports the assumption that both descriptions from the Weddell Sea refer to the same subspecies Diastylis enigmatica enigmatica [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF]. The second subspecies, Diastylis enigmatica rossensis n. ssp., was found only in the Ross Sea.

One of the most important diVerences between the two subspecies is the presence of four spines on the merus of pereiopod 2 in D. enigmatica enigmatica, whereas no spine was found in all developmental stages of both sexes from D. enigmatica rossensis (Table 1). The number of cuspidate setae on Wrst and second articles of the uropodal endopod of the subadult female of D. enigmatica enigmatica is Wve and four, respectively. The number of setae in D. enigmatica rossensis is three for both articles in subadult and adult females. The subadult male has tree and four setae which are still less than in D. enigmatica enigmatica. In contrast to D. enigmatica enigmatica, there is a gap between the spines of the peduncle article 1 of the Wrst antenna in D. enigmatica rossensis. Additionally, the number of articles of the Wrst antennas Xagella of specimens from the Weddell Sea is less than in most even less developed stages of specimens from the Ross Sea (compare Table 1). Further 123 characteristics which also support splitting D. enigmatica into two subspecies are diVerences in the ratios of "uropodal peduncle to last pleon segment" and "distal to proximal part of pereiopod 1". The subadult female of D. enigmatica enigmatica has Wve spines on the basis of maxilliped 3; only four spines were recorded in adult and subadult females of D. enigmatica rossensis. As these spines tend to break oV, this characteristic has to be treated carefully, even though in some specimens it seemed that no spine has broken oV.

Cryptic speciation and the possible eVects on the status of the subspecies of Diastylis enigmatica In the early stage of Antarctic exploration many benthic invertebrates were described to be widespread distributed on the continental shelf surrounding Antarctica. Thus, circumantarctic distribution was assumed for many invertebrate taxa [START_REF] Hedgpeth | Marine biogeography of the Antarctic regions[END_REF][START_REF] White | Marine benthos[END_REF][START_REF] Clarke | The origin of the Southern Ocean marine fauna. In: Crame JA (ed) Origins and evolution of the Antarctic Biota[END_REF][START_REF] Arntz | Antarctic zoobenthos[END_REF][START_REF] Dayton | Polar marine communities[END_REF]. During the last decade, however, cryptic species of several benthic invertebrate taxa in the Antarctic were discovered [START_REF] Bernardi | Molecular evidence for cryptic species among the Antarctic Wsh Trematomus bernachii and Trematomus hansoni[END_REF][START_REF] Allcock | Restricted gene Xow and evolutionary divergence between geographically separated populations of the Antarctic octopus Pareledone turqueti[END_REF][START_REF] Raupach | Distinguishing cryptic species in Antarctic Asellota (Crustacea: Isopoda) -a preliminary study of mitochondrial DNA in Acanthaspidia drygalskii[END_REF][START_REF] Linse | Is the Scotia Sea a centre of Antarctic marine diversiWcation? Some evidence of cryptic speciation in the circum-Antarctic bivalve Lissarca notorcadensis (Arcoidea: Philobryidae)[END_REF][START_REF] Wilson | Multiple lineages and absence of panmixia in the "circumpolar" crinoid Promachocrinus kerguelensis from the Atlantic sector of Antarctica[END_REF]). These Wndings point towards a higher species richness on the Antarctic shelf than formerly believed.

The genetic variability of the mitochondrial 16S ribosomal RNA gene gave Wrst evidence for cryptic speciation in the Antarctic isopods Glyptonotus antarcticus Eights, (1852) and Ceratoserolis trilobitoides, Eights, (1833) [START_REF] Held | Molecular evidence for cryptic speciation within the widespread Antarctic crustacean Ceratoserolis trilobitoides (Crustacea, Isopoda)[END_REF]Wägele 2005, Held 2003). Ceratoserolis trilobitoides was regarded as a single species with high morphological plasticity [START_REF] Wägele | Polymorphism and distribution of Ceratoserolis trilobitoides (Eights, 1833) (Crustacea, Isopoda) in the Weddell Sea and synonymy with C. cornuta (Studer, 1879)[END_REF]), but genetic and morphological data support a scenario of cryptic speciation with more than one species [START_REF] Held | Cryptic speciation in the giant Antarctic isopod Glyptonotus antarcticus (Isopoda: Valvifera: Chaetiliidae)[END_REF]. Also, the study of the Ross Sea cumacean fauna revealed minor morphological diVerences in some of the cumacean species to the original descriptions [START_REF] Rehm | Composition and distribution of the peracarid crustacean fauna along a latitudinal transect oV Victoria Land (Ross Sea, Antarctica) with special emphasis on the Cumacea[END_REF]. For the species Diastylis enigmatica these diVerences are interpreted as diVerences between subspecies, but future genetic analysis might show that genetic diversity is higher than would be expected to be intraspeciWc. First results from the examination of 16S rDNA sequences of the Antarctic cumacean species Leucon antarcticus [START_REF] Zimmer | Neue Cumaceen aus den Familien Diastylidae und Leuconidae von der Deutschen und Schwedischen Südpolar-Expedition[END_REF] have shown that populations from the Ross Sea and the Weddell Sea have been genetically separated for an extended period of time [START_REF] Rehm | Cumacea (Crustacea; Peracarida) of the Antarctic shelf -diversity, biogeography, and phylogeny[END_REF]. This is a Wrst indication for cryptic speciation in cumacean species based on molecular methods.

All Antarctic benthic invertebrate species which turned out to consist of a complex of cryptic species have a limited dispersal potential in common. In contrast, decapods with a broadcasting reproduction mode through planktotrophic larvae show a circumantarctic distribution [START_REF] Rehm | Cumacea (Crustacea; Peracarida) of the Antarctic shelf -diversity, biogeography, and phylogeny[END_REF]. Peracarid crustaceans display brood protection and release their young as mancae, which results in a reduced dispersal potential and might lead to reduced gene Xow. As isopods Table 1 DiVerences observed in Diastylis enigmatica between the Weddell Sea and the Ross Sea and between diVerent developmental stages A1 antenna 1, AF accessory Xagellum, MF main Xagellum, Mp maxilliped, dent denticulate, juv juvenile, P pereiopod, Ped peduncle, R ratio, sa subadult, UP uropodal peduncle; Data from the Ross Sea obtained from one specimen ($, sa #, juv $ d ), three specimens (sa $), or four specimens (juv $ c , juv #). Italic characters serve to diVerentiate the two subspecies of D. enigmatica a [START_REF] Petrescu | Elements for a revision and notes on bionomy of the Cumacea (Crustacea: Peracarida) of the Weddell Sea (Antarctica). Material collected by the expedition ANTARK-TIS-VIII/5 of R.V. "Polarstern" 1998/90[END_REF] b [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF] c Older stage than in [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF] d Same stage as in [START_REF] Ledoyer | Cumacea (Crustacea) de la campagne EPOS 3du R.V. Polarstern en mer de Weddell, Antarctique[END_REF] e The drawing of the original description diVers to the text, in which the postanal part is three times longer than the preanal part f The maximum number observed is given, but may be less as the spines seem to be broken oV or not developed (numbers may vary in the same specimen)

Fig. 1

 1 Fig. 1 Diastylis enigmatica rossensis n. ssp. adult female; (scale 1 mm)

Fig. 2

 2 Fig. 2 Diastylis enigmatica rossensis n. ssp. adult female; a maxilla 1, b left mandible, c right mandible, d maxilla 2, e antenna 1, f antenna 2 (scales 0.2 mm); 1 = a; 2 = d; 3 = e, f; 4 = b, c

Fig. 3

 3 Fig. 3 Diastylis enigmatica rossensis n. ssp. adult female; a maxilliped 1, b maxilliped 3, c maxilliped 2 (scales 0.5 mm; 1 = a, c; 2 = b)

Fig. 4

 4 Fig. 4 Diastylis enigmatica rossensis n. ssp. adult female; a pereiopod 1, b pereiopod 2 (scale 0.5 mm)

Fig. 5

 5 Fig. 5 Diastylis enigmatica rossensis n. ssp. adult female; a pereiopod 3, b uropod, c pereiopod 4, d pereiopod 5 (scale 0.5 mm; 1 = a, c, d; 2 = b)

Acknowledgments I am very grateful to Maya Robert for the translation of the original description of Diastylis enigmatica from French to English, to Ute Mühlenhardt-Siegel for her advice during the description, to Anna Kop and Sally Hall for their revision of the English, and to Sven Thatje and Christoph Held for reviewing the manuscript. Special thanks are due to Daniel Roccatagliata for his critical annotations and for discussing taxonomic problems, which arose during preparation of the manuscript.


Mads S Thomsen
email: m.thomsen@ecu.edu.au

Thomas Wernberg

Brian R Silliman

Alf B Josefson

H.-D M S Franke

Thomsen

Broad-scale patterns of abundance of non-indigenous soft-bottom invertebrates in Denmark

Keywords: North Sea, Baltic Sea, Non-indigenous marine invertebrates, Large-scale distribution patterns

Quantifying the broad-scale distribution and abundance of non-indigenous species (NIS) is necessary to provide accurate estimations on impacts of invasions, to prioritize research, and to guide national management. Sediment grab-sampling is a standardized method for monitoring marine benthos. In Denmark, »45,000 grab-samples were collected from 1970 to 2005. Using these samples, we compared densities of NIS and native species among 27 broad spatio-temporal groupings. Eight known NIS and one 'cryptogenic species' (the polychaete Neanthes succinea) were found in the samples. Most were present in low abundance, but the bivalve Mya arenaria, likely introduced by the vikings from North America, was particularly abundant. M. arenaria was found in ca. 20% of all samples and was among the 10 most common species in all of Denmark. M. arenaria's high abundance, high Wltration capacity and importance in food-web interactions, suggest that this species has dramatically impacted shallow coastal ecosystems in Denmark. The polychaete Marenzelleria viridis, the gastropod Potamopyrgus antipodarum and N. succinea were also widespread and abundant, and they too are likely to have had broad-scale impacts. In conclusion, 28% of grabsamples collected in Denmark over 35 years were aVected by some degree of NIS or cryptogenic species, suggesting that centuries of human-mediated transfer of organisms has had a profound impact on the ecology of soft-bottom systems in Denmark.

Introduction

The introduction and invasion of non-indigenous species (NIS) is a serious threat to biodiversity conservation (Sim-berloV 2005). Well known examples of high-proWle invaders include rabbits in Australia and Dutch elm disease and water hyacinth in Europe and North America [START_REF] Lowe | 100 of the world's worst invasive alien species[END_REF]. Marine invasions are less well documented but may match terrestrial invasions in their extent and impacts. In marine systems, NIS are constantly moved around in ballast water, on ship hulls, and by aquaculture [START_REF] Verling | Supply-side invasion ecology: characterizing propagule pressure in coastal ecosystems[END_REF], and these vectors have relocated NIS to numerous new regions, potentially with large impacts on local ecosystems (e.g. the seastar Asterias amurensis Lutken 1871, or the oyster Crassostrea gigas Thunberg 1793, [START_REF] Ruiz | Global invasions of marine and estuarine habitats by non-indigenous species: mechanisms, extent, and consequences[END_REF][START_REF] Lowe | 100 of the world's worst invasive alien species[END_REF]. Ecological impacts of invasions are often inferred by assuming that the more abundant and widespread a NIS is, the more severe is its impact (Parker 123 et al. 1999). However, studies where distribution and abundance of NIS can be evaluated relative to native species, are typically only done at restricted spatio-temporal scales in areas known to be infested (e.g. [START_REF] Staehr | Invasion of Sargassum muticum in Limfjorden (Denmark) and its possible impact on the indigenous macroalgal community[END_REF]Thomsen et al. 2006a, b;Thomsen et al. 2007b), potentially over-estimating the importance of the NIS over broader scales. Here, we argue that large-scale quantitative assemblage studies may be particularly useful to (a) scale-up experimental results from speciWc locations to larger regions, (b) evaluate and compare region-to-national level importance of diVerent NIS, (c) identify general regions and habitats most threatened by NIS, (d) prioritize national management of widespread and abundant NIS, and (e) guide future research eVorts by identifying which NIS should be studied and where.

Explicitly targeting multiple NIS in broad-scale Weld surveys is expensive. Alternatively, NIS can be quantiWed from existing sampling programs where well-deWned methodological practices are in place such that sampling is relatively standardized. 'Grab-sampling', where a device is lowered from a boat to collect a sample of a known area from the seaXoor, is a prime example of such a sampling tool and grab-samples have been collected worldwide to quantify and compare assemblage structures of soft-bottom systems [START_REF] Petersen | A brief survey of the animal communities in Danish waters, based upon quantitative samples taken with the bottom sampler[END_REF][START_REF] Nybakken | Marine biology-an ecological approach[END_REF]. In Denmark, as part of a national monitoring program, soft-bottom invertebrate assemblages have been sampled regularly with grabs since 1970 [START_REF] Josefson | Species richness of benthic macrofauna in Danish estuaries and coastal areas[END_REF]. Large eVort has gone into identifying species and analyzing species lists with respect to anthropogenic pressures (eutrophication in particular, [START_REF] Conley | Characteristics of Danish estuaries[END_REF][START_REF] Josefson | Nutrient retention by benthic macrofaunal biomass of Danish estuaries: importance of nutrient load and residence time[END_REF][START_REF] Josefson | Species richness of benthic macrofauna in Danish estuaries and coastal areas[END_REF]. Marine invasions have however only recently caught the attention of managers and researchers in Denmark (Thomsen et al. 2007a), and these grab-samples provides a unique opportunity to assess the abundance of soft-bottom invertebrate NIS across broad temporal and spatial scales.

Management objectives in many marine systems aim at achieving near-pristine conditions. It is therefore important to document the extent to which marine systems are aVected by NIS. A broad-scale perspective is critical because the ultimate management units are at the national and international levels. Our objective was to quantify the abundance of soft-bottom macro-benthic NIS in Denmark over long time scales (decades) and across broad geographic regions. Our expectation was that, like other temperate marine systems with a long human history [START_REF] Lotze | Depletion, degradation, and recovery potential of estuaries and coastal seas[END_REF], Danish soft-bottom systems would be markedly aVected even decades ago, but also that the steadily increasing NIS pressure would be visible in the record. The intention was also for this study to set the stage for similar analyses of grab-samples collected throughout the world for large-scale comparisons between and across biogeographical regions, seas and oceans.

Materials and methods

Study area and soft-bottom taxa

Denmark is located between the North and Baltic Seas (Fig. 1) where salinity decreases from 34 psu in the North Sea to ca. 8 psu around Bornholm. Secondary salinity gradients are also seen within most Danish estuaries. Only the Wadden Sea has any signiWcant tidal inXuence (1-2 m). The taxonomic status of benthic invertebrate NIS in Danish waters was recently reviewed by [START_REF] Jensen | A summary of alien marine benthic invertebrates in Danish waters[END_REF] and we use their lists as a template for what species to include or exclude as NIS. However, it should be noted that the polychaete Neanthes succinea Frey and Leuckart 1847 is considered a native species in Europe [START_REF] Reise | Introduced marine species of the North Sea coast[END_REF]) but this species was observed for the Wrst time in Danish waters only in the 1940s and has therefore been suggested to be introduced to Denmark from nearby European regions [START_REF] Jensen | A summary of alien marine benthic invertebrates in Danish waters[END_REF]. Because its status as a NIS is uncertain, we included N. succinea as a 'cryptogenic' species with its own level of 'origin' (i.e. it was not included in pooled NIS abundance estimates). Clearly, this species needs biogeographical and molecular analysis before its origin can be concluded upon (cf. [START_REF] Reise | Introduced marine species of the North Sea coast[END_REF] for similar discussion on the polychaete Nereis virens Sars 1835). Also, the taxonomy of Marenzelleria species (also polychaetes) is complex, and it is likely that multiple Inner Baltic all the way to Bornholm (Bh). West is characterized by an 'oceanic' inXuence of high salinity (western fjords and sounds can have brackish conditions), Northeast by frequent Xuctuations in salinity, from fully marine to brackish conditions (depending on wind direction, currents, precipitation patterns and runoV), and Southeast by relatively constant and low salinities 123 sibling species have invaded the North and Baltic Seas in several independent events [START_REF] Blank | EVect of salinity on spatial distribution and cell volume regulation in two sibling species of Marenzelleria (Polychaeta : Spionidae)[END_REF]). In the present paper we refer the Danish records to M. viridis Verrill 1973, but note that this may represent a conglomerate of several morphologically and taxonomically very similar species.

Data collection

Grab-samples of sediments were collected by the Danish counties and The National Environmental Research Institute. The total data-set included ca. 45,000 assemblagelevel samples distributed around the Danish coastline (including samples collected in Northwestern Sweden) to a depth of 300 m and covering all major open and estuarine waters. The number of stations and samples varied from year to year (Table 1). Most samples were collected in summer months and all samples were standardized to unit area before data analysis (grab-sizes varied typically between 0.0085, 0.0123, 0.0143 or 0.1 m 2 , but not in any confounding way across years, regions or depth). Grab-samples normally sampled the sediment to depth of ca. 20 cm. Sediments were sieved through a 1 mm mesh, and the residual preserved in 80% ethanol. Species were identiWed and counted in the laboratory to provide estimates of grab-densities (converted to individuals per m 2 ). Densities were reported as both 'absolute' and 'relative' abundances, which focus on the individual NIS and on NIS in relation to the entire grab-assemblage across all samples (rankordered, with the most abundant species = number 1), respectively. In addition to calculating national averages, we also calculated abundances for 27 broad combinations of temporal (1970-1984, 1985-1994, 1995-2004), spatial (West = North Sea + Skagerrak + western fjords including Limfjorden, Northeast = Northeastern Kattegat + Swedish waters near Kullen and Goteborg, Southeast = Southeast of Aarhus + the Belts + Inner Baltic, see Fig. 1) and bathymetric (•5, 5-20, >20 m)

Results

Of the 1,121 species recorded eight were NIS and one cryptogenic (N. succenea), corresponding to <1% of the full species list. NIS and cryptogenic species accounted for 3.9% of all individuals, and at least one NIS or cryptogenic species was found in 28% of the samples. 1970-1984 in West (5-20 m) in West (5-20 m) where it was ranked 66. P. antipodarum was much more common with an overall mean density of >40 ind. m ¡2 and was ranked 24 overall. In 1970-1984 it was registered in Southeast (•20 m), in 1985-1994 in West (•5 m) and Northeast (•20 m), and in 1995-2004 in both West (•5 m), Northeast (at all depths) and Southeast (•20 m). This species was particularly common in 1970-1984 and 1985-1994 in Southeast (•5 m) where it was found with very high densities and was ranked respectively number 4 and 6, i.e. was amongst the 10 most abundant soft-bottom species. The third old NIS, the barnacle Balanus improvisus Darwin 1854, had a mean density of 2.3 ind. m ¡2 with rank 152, but was only found in the last two time periods and with increasing abundances (Figs. 2,3). In 1985-1994 it was found in West and Southeast (•20 m) and in 1995-2004 in the same two regions (but only at 5-20 m in Southeast). This NIS was most common in 1985-1994 in West (•5 m), where it was ranked 45. The last of the old NIS, the bivalve Mya arenaria Linnaeus 1758, was by far the most common of any NIS. M. arenaria was observed in all 26 spatio-temporal groupings, was found in 21% of all samples, with an overall mean density of 131.5 ind. m ¡2 . Overall, M. arenaria was the 9th most abundant soft-bottom invertebrates based on all 45,000 samples. M. arenaria was particularly abundant in shallower waters, and was the second most abundant species in Denmark in Southeast (•5 m). The combined abundances of all old NIS showed variable temporal patterns; some depth strata and regions were increasingly impacted by NIS (e.g. Northeast >20 m), some groupings had highest impact in the mid-time interval (e.g. West >20 m), and a single grouping had decreasing impacts over time (West •20 m). These patterns were primarily driven by Xuctuations in two highly abundant NIS, M. arenaria and P. antipodarum.

Of the new NIS the gastropod Crepidula fornicata Linnaeus 1758 was found in all three time periods, but without any clear temporal trends (Figs. 2,3). C. fornicata had a mean density of 0.9 ind. m ¡2 and was ranked 234. In 1970In -1984 Finally, the cryptogenic N. succinea, was found in all three time periods with increasing abundances and in increasing number of regions and depth intervals (Figs. 2,3). N. succinea had a density of 9.7 ind. m ¡2 and was ranked 61. In 1970-1984 N. succinea was registered in West andSoutheast (•20 m), in 1985-1994 in West (•20 m), Northeast (•5 m) and Southeast (all depth) and in 1995-2004 in almost all regions and depths (except >20 m in West). N. succinea was most common in 1995-2004, particularly in Northeast •5 m where it was the 14th most common soft-bottom invertebrate (Fig. 3).

Summing old and new NIS demonstrated that the spatiotemporal groupings most impacted by NIS, with total NIS densities of >300 ind. m ¡2 , were shallow water regions 123 from the West and Southeast. This pattern was primarily driven by high abundances of the two old and very abundant invaders, M. arenaria and P. antipodarum (Fig. 4).

Discussion

We used a large dataset, collected using a relatively standardized sampling methodology, to quantify the abundance of marine benthic NIS in Danish soft-bottom systems. NIS were found in 28% of all samples, documenting the ubiquity of invader impacts in Danish softbottom systems. Shallow waters are particularly impacted with total NIS densities routinely reaching >300 ind. m ¡2 , and one NIS, M. arenaria, was found in high abundance at all time intervals, depth, and geographical regions.

General spatio-temporal patterns

The distribution of benthos in Danish coastal waters varied between years, regions and depth intervals. Many key drivers could contribute to these patterns of distribution, including hydrodynamic conditions, nutrient input, water temperature, solar irradiance, salinity, sedimentation, sediment characteristics, hypoxia events and biotic interactions such as competition and predation [START_REF] Conley | Characteristics of Danish estuaries[END_REF][START_REF] Josefson | Species richness of benthic macrofauna in Danish estuaries and coastal areas[END_REF]). Superimposed on this environmental variability are patterns relating to time of NIS introduction, and dispersal and colonization capability. Here, we did not attempt to relate temporal variation in abundances to environmental causes (work in progress), but taking the invasion history into account; our results suggest an increasing abundance, particular of the new NIS. Thus, the three new NIS and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Southeast <5m

SouthEast >20m

Southeast 5-20m ND also the cryptogenic N. succinea appear to be spreading. This contrasts the old NIS which has had a long opportunity for secondary dispersal into suitable habitats throughout Denmark. Most of the new NIS are eurohaline and tolerant to sedimentation and anoxia (see later), and they will most likely continue their spread into other Danish estuaries and become even more abundant. Most NIS were found at shallow depths in West, suggesting that (a) this grouping is exposed to high propagule pressure (e.g. secondary introductions via the English Channel and the North Sea) [START_REF] Lockwood | The role of propagule pressure in explaining species invasions[END_REF], (b) that the declining salinity through the Northeast is a barrier to stenohaline marine organisms (e.g. Crepdula fornicata) [START_REF] Leppakoski | The Baltic-a sea of invaders[END_REF], and (c) that elevated wave disturbance and primary production in West create space and food for invaders [START_REF] Mack | Impacts of biological invasions on disturbance regimes[END_REF].

Species-speciWc patterns

Mya arenaria has a remarkable invasion history. The species is of PaciWc origin, but dispersed to Atlantic North America and Europe in the Miocene before becoming extinct in Europe, probably due to climatic changes [START_REF] Strasser | Mya arenaria-an ancient invader of the North Sea coast[END_REF]. It was likely re-introduced from North America by the vikings ca. 1,000 years ago, potentially as a food source [START_REF] Petersen | Clams before Columbus?[END_REF][START_REF] Strasser | Mya arenaria-an ancient invader of the North Sea coast[END_REF][START_REF] Behrends | Earliest Holocene occurrence of the soft-shell clam, Mya arenaria, in the Greifswalder Bodden, Southern Baltic[END_REF]. M. arenaria is characterized by a large size, relatively fast growth rate, high burial capacity aVecting sediment characteristics, high water Wltration rates aVecting water-column characteristics and by being an important food source for Wsh and birds [START_REF] Hansen | Impact of the soft shell clam Mya arenaria on sulfate reduction in an intertidal sediment[END_REF][START_REF] Riisgard | Filtration rates in the soft clam Mya arenaria: eVects of temperature and body size[END_REF]. The high abundances, wide distributions and 'ecosystem engineering' characteristics of M. arenaria [START_REF] Palacios | Fate and legacy of an invasion: extinct and extant populations of the soft-shell clam (Mya arenaria) in Grays harbor (Washington)[END_REF] suggest that this species has had a Fig. 3 Densities of 'new' NIS (<100 years in Danish waters, §SE) and cryptogenic species (N. succenea) for 26 broad spatio-temporal groupings (cf. Table 1). White bars correspond to 1970-1984 samples, grey bars to 1985-1994 samples and black bars to 1995-2004 samples. Note the diVerent scales on Y-axis and that no samples were collected from >20 m at West in 1970-1984 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 large impact on the present-day ecosystems not only in Denmark, but potentially throughout invaded locations in Europe. Another important NIS, M. viridis, is today, only 15 years after its Wrst recording, abundant in several shallow fjords, with >140 ind. m ¡2 at shallow depths in the West, a dramatic invasion as also observed in the Gulf of Riga [START_REF] Cederwall | Long-term changes in the soft-bottom macrofauna of the Gulf of Riga[END_REF]) and other locations in the southern Baltic Sea [START_REF] Bastrop | Multiple invasions-a polychaete genus enters the Baltic Sea[END_REF]. Impacts in the Baltic include competition with native infauna and increased benthic chlorophyll levels, probably via excretion of waste products that facilitate growth of benthic diatoms [START_REF] Kotta | Competition for food between the introduced polychaete Marenzelleria viridis (Verrill) and the native amphipod Monoporeia aYnis Lindstrom in the Baltic Sea[END_REF][START_REF] Neideman | Competition between the introduced polychaete Marenzelleria sp. and the native amphipod Monoporeia aYnis in Baltic soft bottoms[END_REF]. The success of Marenzelleria species has been attributed to a high tolerance to low salinity, sedimentation, anoxia and nutrient enrichment [START_REF] Blank | EVect of salinity on spatial distribution and cell volume regulation in two sibling species of Marenzelleria (Polychaeta : Spionidae)[END_REF]). We expect the invasion to continue, as the polychaetes are given the opportunity to disperse to estuaries throughout Denmark. A third successful species, N. succinea, is cryptogenic, and is generally not considered introduced in Europe [START_REF] Reise | Introduced marine species of the North Sea coast[END_REF]. N. succinea has increased dramatically in abundance and distribution since its Wrst observation in Denmark in the 1940s [START_REF] Jensen | A summary of alien marine benthic invertebrates in Danish waters[END_REF]. Still, historical, biogeographic and molecular analyses are needed to clarify why this conspicuous species was not observed in Denmark until the 1940s. If these analyses suggest a natural distribution in Denmark, alternative explanations are needed to explain its late Wrst description and dramatic population expansion. Thus, natural range expansion from nearby regions could have co-occurred with climatic changes and eutrophication and thereby both facilitated new arrivals and increased ecological performance. For example, N. succinea is an eurohaline predator that is known to be resistant to hypoxia [START_REF] Sagasti | Epifaunal communities thrive in an estuary with hypoxic episodes[END_REF], important traits in eutrophic brackish waters. We are not aware of any impact studies of N. succinea, but with an average density of >70 ind. m ¡2 in shallow water, this species is likely an important trophic link between small consumers and top-predators such as Wsh. P. antipodarum is a freshwater and estuarine species in its native New Zealand. It is tolerant to environmental stressors such as ammonia pollution and anoxia [START_REF] Alonso | Short-term toxicity of ammonia, nitrite, and nitrate to the aquatic snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)[END_REF], making it robust to environmental conditions in Danish coastal ecosystems. Large impacts have been documented in invaded freshwater systems in Europe and North America, including competition with native snails, altering biogeochemical cycles and increasing secondary production [START_REF] Hall | Exotic snails dominate nitrogen and carbon cycling in a highly productive stream[END_REF][START_REF] Hall | Extremely high secondary production of introduced snails in rivers[END_REF]. P. antipodarum is common at shallow depths in both the Southeast and West, where high abundances match the brackish conditions of the Southeast and estuaries and semi-enclosed lagoons in West. Given the high abundances at shallow depths and its known impacts on invaded freshwater systems, we expect this fast growing gastropod to have a major impact on secondary productivity and trophic transfers in Danish brackish waters.

Crepidula fornicata

Less common NIS and sampling bias

The data presented are not representative for all benthic NIS and have several types of potential bias. First, grabsamples can only be collected in soft-bottom systems, excluding boulder Welds, rocky coasts and seagrass beds. In addition, grab-samples are diYcult to collect in very shallow water (e.g. <1 m) and under stormy conditions. Grabsamples may also have diVerent collection eYciencies for diVerent sediment textures and in the presence of biogenic structures (e.g. reefs, large shells). Furthermore, fewer ) and therefore has had amble time to disperse between regions. However, P. pholadiformis exhibits high substratum selectivity, primarily burrowing into hard clays or limestone [START_REF] Alexander | InXuence of sediment grain-size on the burrowing of bivalves-correlation with distribution and stratigraphic persistence of selected neogene clams[END_REF]) and is therefore only found in low abundances in Denmark.

Perspectives

Most of the benthic soft-bottom NIS in Denmark are thought to be secondary introductions which have dispersed from founder populations in neighboring countries [START_REF] Jensen | A summary of alien marine benthic invertebrates in Danish waters[END_REF]. It is therefore unlikely that a national action program could have prevented these introductions, highlighting the need for international collaboration and regulation. The primary introductions for many of the founder populations in adjacent and nearby countries have been via oyster-transplantations or by fouling of ship hulls (Jensen and Knudsen 2005). Future introductions can to some extent be controlled, as the oyster-industry is increasingly controlled, new antifouling agents are developed and oceanic ballast water exchange become increasingly utilized.

In a recent companion paper we analyzed »10,000 samples for introduced macroalgae in Denmark (Thomsen et al. 2007a). We found that Danish macroalgal assemblages have been invaded by ca. 10 NIS, and that two species, Sargassum muticum (Yendo) Fensholt 1955 and Bonnemaisonia hamifera Hariot 1891, constituted a large part of the algal Xora. Collectively, these two large-scale distribution studies document a substantial inXuence of NIS throughout Danish benthic assemblages, although the inXuence of animal invaders on stone reefs remains unknown. Still, marine NIS must be recognized as a fundamental human-associated inXuence which, in concert with other and better described anthropogenic pressures, impact marine ecosystems in Denmark and elsewhere. Finally, it is important to emphasize that grab-samples have been collected throughout the world, bridging >50 years of monitoring programs and scientiWc investigations across institutions, nations and biogeographic regions. Today, retrospective analysis of such grab-samples presents a rare opportunity to investigate the temporal trajectory and spatial extent of marine invasions in soft-bottom systems throughout the world and at many levels of hierarchical scales. No similar globally available data-set and based on a single relatively standardized methodology, exist for any other marine group of organisms or habitat. Our study should therefore be straightforward to duplicate in other regions. We suggest to utilize already existing grab-based datasets for more detailed distributional studies, to compare broad-scale patterns between seas, oceans and biogeographic regions, and, in conjunction with demographic research and manipulative experiments, to test for ecological impacts and theories of invasions (e.g. 'propagule pressure', 'diversity-resistance' or 'invasional meltdown' theories, Sim-berloV and Von Holle 1999; [START_REF] Lockwood | The role of propagule pressure in explaining species invasions[END_REF].

Fig. 1

 1 Fig. 1 Geographical regions. West = North Sea + Skagerrak + Wadden Sea (Ws) + western fjords (e.g. Ringkøbing Fjord = Rf) and Limfjorden (Lf, a sound connecting the western North Sea to eastern Kattegat). Northeast = Northeastern Kattegat + Swedish waters near Kullen and Goteborg. Southeast = Southeast of Aarhus + the Belts + Inner Baltic all the way to Bornholm (Bh). West is characterized by an 'oceanic' inXuence of high salinity (western fjords and sounds can have brackish conditions), Northeast by frequent Xuctuations in salinity, from fully marine to brackish conditions (depending on wind direction, currents, precipitation patterns and runoV), and Southeast by relatively constant and low salinities

  it was registered in West(•20 m), in 1985-1994 in West (•20 m) and in 1995-2004 in West (•20 m) and Northeast (5-20 m). C. fornicata was most common in1995 -2004 in West (5-20 m) in West (5-20 m) where it was ranked 41. The two other new NIS, the bivalve Ensis americanus Gould 1870 and M. viridis, were only found in the last two time periods, clearly with highest abundances in the last period (Figs.2, 3). E. americanus had a mean density of 0.4 ind. m ¡2 and was ranked 331. In 1985-1994 it was found in West (•20 m) and in 1995-2004 in both West and Southeast (all depth). E. americanus was most common in 1995-2004, in West (5-20 m) being ranked 75. M. viridis had a mean density of 25.4 ind. m ¡2 (rank 30). It was found in both time periods in both West (•5; •5 and >20 m) and Southeast (>20 m; •20 m). M. viridis was most common in 1995-2004, in West •5 m with densities of 152.8 ind. m ¡2 and was ranked 11. In contrast to the old NIS the new NIS, when added together, generally increased in abundances in most regions and depth intervals. This pattern was driven by all three invaders in West; C. fornicata at intermediate depth, E. americanus in intermediate and deep water, and M. viridis in shallow and deep water.

Fig. 2

 2 Fig.2Densities of 'old' NIS (>100 years in Danish waters, §SE), for 26 broad spatio-temporal groupings (cf. Table1). White bars correspond to 1970-1984 samples, grey bars to 1985-1994 samples and black bars to 1995-2004 samples. Note the diVerent scales on Y-axis and that no samples were collected from >20 m atWest in 1970West in -1984

Fig. 4

 4 Fig. 4 Summarized densities of NIS for 26 spatio-temporal groupings (cf.Table 1, §SE). White bars correspond to 1970-1984 samples, grey bars to 1985-1994 samples and black bars to 1995-2004 samples

  variability. No samples were collected from the deep West in 1970-1984 resulting in 26 Wnal groupings (i.e. only 26 groupings are shown on Wgures

	Table 1 Number of grab-sam-nance rankings (no. 1 = most ples and species-speciWc domi-	Period	Region	Depth	samples No. of	Bal	Cre	Ens Mar Mya Nea Pet	Pot
	abundant) of NIS compared to the entire assemblage within 26 spatio-temporal groupings (no	1970-1984 West 1970-1984 West	<5 m 5-20 m	411 1,341		118 99		8 12	16 35	35 66
	rankings could be provided for	1970-1984 Northeast <5	43				22
	groupings where the species was not recorded at all)	1970-1984 Northeast 5-20 m 1970-1984 Northeast >20 m	165 884				64 477
		1970-1984 Southeast <5	640				20	87	4
		1970-1984 Southeast 5-20 m	240				9	117	29
		1970-1984 Southeast >20 m	44				12
		1985-1994 West	<5	5,335	59	71	187	12	94	124	18
		1985-1994 West	5-20 m	4,734		116 147	20	59	137
		1985-1994 West	>20 m	130				6
		1985-1994 Northeast <5	156				17	38	10
		1985-1994 Northeast 5-20 m	1,441				56	0	293
		1985-1994 Northeast >20 m	1,459				261	0
		1985-1994 Southeast <5	3,048	45			2	39	134	6
		1985-1994 Southeast 5-20 m	6,391	303			13	41	273	54
		1985-1994 Southeast >20 m	1,079			301	21	250
		1995-2004 West	<5	5,249	54	114 122	6	47	93	25
		1995-2004 West	5-20 m	2,187	209	41	75	43	32	65
		1995-2004 West	>20 m	135			160	48
		1995-2004 Northeast <5	106				15	30	64
		1995-2004 Northeast 5-20 m	3,056		313		31	150	295
		1995-2004 Northeast >20 m	1,619				68	265
		1995-2004 Southeast <5	1,985			125	3	14	0.74	25
											134
		1995-2004 Southeast 5-20 m	5,628	186		241 344	16	40	169
		1995-2004 Southeast >20 m	1,845			244	31	78	15

Bal balanus improvisus, Cre crepidula fornicata, Ens ensis americanus, Mar marenzellaria viridis, Mya mya arenaria, Nea neanthes succinea, Pet petricola pholadiformis, Pot potamopyrgus antipodarum and tables). The database contained 1,543 taxa, including 422 taxa registered on taxonomic levels above the species level (e.g. Polychaete indet.).

  The tunicate Styela clava Herdman 1882 was only found in four samples (0.01 ind m ¡2 , rank 964), and due to its low abundance and lifestyle (sessile hard-substratum species) its distribution was not analyzed further. Half of the NIS have been known from Danish waters for more than a century, the other half are more recent introductions (one of them being S. clava). These two groups are here classiWed as 'old' and 'new' NIS and represent a (somewhat arbitrary) point in time to separate species that have had long vs. short time to disperse in Danish waters.Of the old NIS, the bivalve Petricola pholadiformis Lamarck 1818 and gastropod Potamopyrgus antipodarum Gray did not show any clear temporal trends(Figs. 2, 3). P. pholadiformis was found with an overall mean density of 0.7 ind. m ¡2 and was rank no. 262 of all Danish soft-bottom invertebrates on the entire data-set.

	In 1970-1984 it
	was found in West (•20 m), in 1985-1994 in West and
	Southeast (•20 m) and in 1995-2004 again in West
	(•20 m) and Southeast (•5 m). This NIS had highest abun-
	dances in

Table 1 ,

 1 §SE). collected from deep waters, the early sample period and the North West region. This implies that rare NIS are more likely to be missed from these groupings. Finally, and most importantly, highly mobile NIS (e.g. the decapod Eriocheir sinensis Milne-Edwards 1854) are often missed by the grab. For example, the abundance of E. americanus, introduced to the North Sea in the late 1970s[START_REF] Jensen | A summary of alien marine benthic invertebrates in Danish waters[END_REF], is probably substantially underestimated due to its capacity for fast and deep burial. It is relatively common in the West, and has recently been observed in the Southeast, indicating tolerance to low salinities and therefore a potential to inhabit most regions in Denmark. Its disjunct distribution (absent from the Northeast) may be attributed to a lack of dispersal rather than adverse environmental conditions. This, in turn, suggests that Southeast populations have bypassed the entrance to the Baltic Sea either via ballast water transport or via The Kiel Canal. E. americanus share several of the characteristics of M. arenaria; it can occur in high densities in the Wadden Sea, it is large and it has a high Wltration capacity[START_REF] Armonies | On the population development of the introduced razor clam Ensis americanus near the island of Sylt (North Sea)[END_REF]. It is therefore likely it will have considerable impacts on biogeochemical processes and biological interactions in the near-future. A relatively uncommon NIS is P. pholadiformis which was probably introduced with oysters >100 years ago[START_REF] Jensen | A summary of alien marine benthic invertebrates in Danish waters[END_REF]

			1000
		)	All NIS combined
	White bars correspond to 1970-1984 samples, grey bars to 1985-1994 samples and black bars to 1995-2004 samples	Densities (m -2	400 600 800
			200
			10
			1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 NIS abundance per abundance of all species
			8
		Percent	4 6
			2
			0		ND
			West	West	West	Northeast	Northeast	Northeast	Southeast	Southeast	SouthEast
			<5 m	5-20 m	>20 m	<5m	5-20m	>20m	<5m	5-20m	>20m

Acknowledgments Mads Thomsen was supported by the Danish Research Academy. We thank colleagues from the Danish counties and The National Environmental Research Institute, who collected this enormous dataset.