Recent invasions of alien macroinvertebrates and loss of native species in the upper Rhine River , Germany

In the summers of 2003-2005 we sampled the macroinvertebrate community of the upper Rhine River at 28 locations between kilometer 351.9 and 399.5 with a dredge and a hydraulic grab from a ship, or by hand sampling. Additionally, 16 samples were obtained from the cooling water intake screens of a thermal power plant in 2003. A total of 133 species and higher taxa were identified from approximately 140,000 organisms. A total of 33 neozoa (alien) species were identified, including the first record of Chelicorophium robustum in the Rhine River. Neozoa contributed approximately 74% to the total number of organisms collected from the ship-based samples and 85% to those from the power plant cooling water. Almost 64% of all individuals from the cooling water intake belonged to the Ponto-Caspian amphipod Dikerogammarus villosus. Similarly, only 13 taxa, four native and nine alien species, numerically dominated in the ship-based samples, representing 95% and 98% of all organisms in 2003 and 2004, respectively. The nine alien species contributed 74.7% and 72.1%, respectively, to the number of organisms. Only six species occurred abundantly in the main channel of the Rhine River, five of which were neozoa: Jaera istri, Dikerogammarus villosus, Dreissena polymorpha, Chelicorophium curvispinum, and Hypania invalida. The six abundant species colonized nearshore substrates, whereas only few species were retrieved at low densities from the bottom gravel of the central shipping lane. Species diversity was generally higher and relative abundance of neozoa was lower in backwater areas than in the main river channel. Some pronounced changes in the abundance of three numerically dominant species, including the neozoa D. polymorpha and C. curvispinum, occurred between 2003 and 2004, which likely correlated with differences in water levels and temperatures. Overall, our results demonstrate that the macroinvertebrate community of the upper Rhine River has been severely altered by the invasion of several highly successful alien species and the disappearance or population decline of native species, and that these processes are still ongoing. Changes in species composition and relative numerical abundance indicate both a displacement of native species by invasive species, and a relative rapid succession in the numerical dominance of “old“ neozoa and “new“ neozoa.


Introduction
Macroinvertebrate species richness of the Rhine River has been substantially reduced during the last century, mainly because of anthropogenic impacts, such as declining water quality and alterations to river morphometry and hydrology (Kinzelbach 1982(Kinzelbach , 1983)).By the early 1970s, less than one fourth of the more than 160 species identified prior to 1920 could still be found in the river (Titizer et al. 1991).The water quality of the Rhine River has substantially improved over the last 30 years, and while macroinvertebrate species richness has largely recovered to former numbers, community composition has been substantially altered primarily by invasive species (IKSR 2002).With the opening of the Main-Danube canal in 1992, one further route for the exchange of biota between the Rhine River and other biogeographical regions was established.
Within a year, the first macroinvertebrate species of Ponto-Caspian origin were documented near the mouth of the Main River tributary (IKSR 2002).The aim of this study was to document the current species composition and abundance of macroinvertebrates over a 48 km section of the Upper Rhine River, including the mouths of some tributaries and backwaters.Particular focus was given to a complete inventory of neozoa and the dominace relationship between native and invasive species.

Material and Methods
During April, July, and November of 2003 and April, August, and November of 2004 we took 43 and 55, respectively, macroinvertebrate samples from aboard the research vessel "Max Honsell" (LfU Baden-Württemberg) from 29 sites between Rhine-kilometer 351.9 and 399.5 (Figure 1).Ship samples were taken using three different methods: with a dredge, a grab, and by hand sampling.Invertebrate samples were obtained using one, two, or all three methods depending on the available substrates at a sampling site (Annex 1).The dredge consisted of a 100 cm long, 60 cm wide, and 25 cm high steel frame with a removable inside nylon mesh (1000 μm) bag.It was pulled behind the ship in areas of >1.6 m water depth over the bottom substrate for approximately 100 m at a speed of 3-5 km•h -1 .The drege content was sorted on deck through a set of 10, 5, and 1 mm sieves and washed.
The grab was mounted on a hydraulically operated arm with a 7 m reach from the ship deck and sampled an area of approximately 0.8 m 2 .
Larger pieces of wood, stones and boulders were separated from fines, sand, and gravel on deck and were hand-brushed inside of large water filled tubs.The water and a 20-30 L subsample of the smaller substrate fractions were treated similarly to the dredge material.Because of the fast current speeds (1.5-2 m•s -1 ) of the main river channel, grab samples could only be taken in sheltered areas or when the ship was fastened on shore.Hand sampling was performed in near-shore areas inaccessible to the ship or on substrates unsuitable to sampling by the other two methods.Areas of 0.5-1.0m 2 were sampled by kick-netting (0.3 x 0.3 m net opening, 200 μm mesh) and/or by picking organisms from large pieces of substrate with forecepts.
In addition, 16 macroinvertebrate samples were taken from the water intake screens of the thermal power plant in Karlsruhe (RDK) at Rhine-kilometer 359.3 in May 2003.Invertebrates and debris washed from stainless steel rotating screens with 1 mm 2 openings were sampled with small dip nets (200 μm mesh) for 2-9 minutes, depending on the total amount of debris collected on the screens.The screens filter 17 m 3 •s -1 of cooling water under normal operating conditions of the power plant.
On 21 July, 2005, the site at Rhine-kilometer 375.4 was re-sampled as part of a university field trip.This site is located within a section of old river bed and has been used for gravel extraction in the 1960s and 1970s.Five samples of invertebrates were taken at this site with dip-nets and by hand sampling.
All samples were preserved in 75% ethanol and identified and enumerated in the laboratory using dissecting and compound microscopes.Organisms were identified to species or (in a few instances) to genus, except for most Dipera and Tubificidae, which were identified to family.Numbers of juveniles that could not be identified to species (e.g., Gammaridae) were estimated according to the relative abundance of adults in the same sample.In this study, we synonymously refer to those species that historically have not been documented from the Rhine River basin as neozoa (Kinzelbach 1972, Arbeitsgruppe Neozoa 1996), or alien species.

Results and Discussion
We collected approximately 4,800 individuals (Table 2), belonging to 68 species or higher taxa from the cooling water intakes of the thermal power plant at Karlsruhe.Neozoa were represented with 17 species and comprised approximately 85% of all organisms identified (Table 1).The three most abundant species in the power plant samples were also highly abundant in the ship-based samples (see below).2).Conversely, five neozoa (Pectinatella magnifica, Caspiobdella fadejewi, Chelicorophium robustum, Gammarus tigrinus and Hemimysis anomala) were found with low abundance only in the 2004 samples.Thirteen invasive species were identified from the single shore-based hand sample taken in 2005, including one species (Orconectes immunis) that had not been captured in the previous two years (Table 2).Thus, a total of 33 alien macroinvertebrate species were identified in this study (Annex 2).This includes the first record of C. robustum from the Rhine River.This species originates from the Ponto-Caspian region.Similar to several other crustacea (Bij de Vaate et al. 2002), C. robustum likely entered the Rhine River drainage basin facilitated by ship traffic through the recently opened Main-Danube canal and was first reported in 2002 from the Main River at Frankfurt harbor (Bernerth and Stein 2003).The 52 specimen of C. robustum that we identified in November of 2004 all came from the harbor at Karlsruhe.Two months later, C. robustum was collected approximately 55 km downstream at Mannheim harbor (Peter Roos, Büro für Gewässerökologie, Karlsruhe, pers.commun.).These capture locations further suggest ship transport as a major mode of dispersal for C. robustum.
The relative abundance (approximately 74%)  2003 and 2004.For the proportion of species, this calculation assumes that the number of (unidentified) species of Tubificidae and Chironomidae did not change between the two years.
The macroinvertebrate community of the Rhine River was numerically dominated by nine alien species and four native taxa (Figure 2).These thirteen abundant species/taxa (found with >500 individuals in one of the two sampling years) contributed 94.5% and 98.4% to the total number of invertebrates collected in 2003 and 2004, respectively.The nine alien species contributed 74.7% and 72.1%, respectively.
Three of the native taxa, Chironomidae, Tubificidae, and the heteropteran Micronecta sp. were captured almost exclusively in (slow flowing) backwater areas, where they sometimes reached high densities.These results are in line with findings that the relative abundance of Chironomidae and Oligochaeta in the main (shippable) channel of the upper Rhine River has substantially decreased between 1990 and 2000, and that these two taxa (for which no species identification is done during routine monitoring) no longer numerically dominate the invertebrate community (IKSR 2002).In the backwater areas, species diversity was generally higher and relative abundance of neozoa was lower compared to the main channel.Particularly in the most downstream sections of tributaries, native mayfly species typical of large rivers, such Ephemera vulgata and Ephemera glaucops were still found regularly and often in association with the neozoa Limnomysis benedeni and Echinogammarus ischnus.
Ancylus fluviatilis was the only native species that occurred abundantly in the main channel of the Rhine River.This gastropod colonized mainly shoreline rip-rap up to the maximum extent of wave action from ship traffic.The other five species that were consistently found in the main channel at very high abundances were all neozoa: Jaera istri, Dikerogammarus villosus, Dreissena polymorpha, Chelicorophium curvispinum, and Hypania invalida.The large increase in the relative abundance of J. istri between 2003 and 2004 was due mainly to the fact that this species occurred at high densities (up to 17,000 individuals•m -2 ) at most sampling sites in 2004, whereas in 2003 its abundance was (still) relatively low at many locations.Similar changes in community composition, particularly the increase in density and relative abundance of D. villosus and Jaera sp. and the concomitant decreases of C. curvispinum and G. tigrinus in the upper Rhine River since the early 1990s have been observed in other studies (IKSR 2002, Haas et al. 2002).The reasons for the sometimes dramatic changes in community composition are not well understood.In addition to competition and predation effects (IKSR 2002, Haas et al. 2002), these changes may be due to between-year climatic variability, particularly in water temperature/levels.This notion is supported by the strong increase in the abundance of A. fluviatilis, D. polymorpha, and C. curvispinum between 2003 and 2004, which, at least for the first two species, was largely due to juvenile individuals.These three species were apparently negatively affected by the low water levels and high temperatures in 2003, when large numbers of dead C. curvispinum were often seen, and apparently recovered rapidly under the more favorable weather conditions of 2004.
Overall, our results demonstrate, that, likely mediated by changes in the physical and chemical conditions of the river, the macroinvertebrate community of the upper Rhine River has been severely altered by the invasion of several highly successful neozoa and the disappearance or population decline of native species.Although data were mainly collected during only two years, they further show that these changes are still ongoing.The documented changes in species composition and relative numerical abundance indicate both a displacement of native species by alien species, and also a rapid succession in the numerical dominance of "old" neozoa (mainly from North America, e.g.G. tigrinus) and "new" neozoa (mainly from the Ponto-Caspian area via the Main-Danube canal, e.g.J. istri).

Annex 1
List of ship-based sampling sites with river kilometer (R-km), waterbody (Rhine River mainstem or backwaters, including the mouth of tributary streams and the old river channel), and location (left or right bank in downstream direction).The substrate(s) sampled and the method(s) of sampling are given

Figure 1 .
Figure 1.Map of Germany with river systems.The sampling area on the upper Rhine River is indicated by a rectangle

Figure 2 .
Figure 2. Relative numerical abundance of the 13 most abundant taxa collected from the ship-based samples in 2003 and 2004.The last nine species are all neozoa.For full species names see Table 2 Only a few species were collected between the wing dams or in the central shipping lane of the river where the bottom substrate consists mainly of relatively unstable gravel: the neozoa Dendrocoelum romanodanubiale, Potamopyrgus antipodarum, and Echinogammarus trichiatus, and the native trichoptera Hydropsyche bulgaromanorum and Psychomyia pusilla, which both occured at relatively low abundance.IKSR (2002) andHaas et al. (2002) reported that the relative abundance and density of P. pusilla in the upper Rhine has increased from1990-2000,  and 1993-2001, respectively.Overall, with a total of 21 species the macroinvertebrate diversity of the main channel (including shoreline areas) of the Rhine River between Speyer and Karlsruhe was very low.Compared to results from similar sampling in 1993(Bernauer 1995), the abundance of erbobdellid leeches and of the native mayfly Heptagenia sulphurea has noticably decreased, and the formerly abundant native (Aphelocheirus aestivalis, Hydropsyche contubernalis) and invasive (Gammarus tigrinus, Dugesia tigrina) species have largely disappeared.

Table 1 .
Number of individuals of the three most abundant species, of all neozoa, and of all taxa collected from the cooling water of the thermal power plant at Karlsruhe in May 2003

Table 2 .
(Dehus et al. 1999, Tittizer et al. 2000, Gleiter et al. 2002h year of first detection in the Rhine River(Dehus et al. 1999, Tittizer et al. 2000, Gleiter et al. 2002, IKSR 2002)and geographical area of origin.If the location was not the Rhine River, the name of the tributary stream is given in brackets.Two individuals of the species complex Polycelis nigra/tenuis that were found in 2003 are not listed as neozoa Full reference to the data:Bernauer D and Jansen W (2006)Recent invasions of alien macroinvertebrates and loss of native species in the upper Rhine River, Germany, Aquatic Invasions 1(2): 55-71 **For geographic coordinates of the sampling location see Annex 1 *