The clones are coming – strong increase in Marmorkrebs [ Procambarus fallax ( Hagen , 1870 ) f . virginalis ] records from Europe

We describe a new occurrence of parthenogenetic Marmorkrebs in southwestern Germany and give a synopsis of recent records of this species in Europe. Including the most recent records, 15 Marmorkrebs records are currently known, most of which are from Germany. At least six records represent established populations, which is an alarming increase beyond the one Marmorkrebs population known prior to 2010. Most established populations occur in lentic habitats near conurbations, typically in highly frequented secondary habitats, such as gravel pit lakes. In three instances, Marmorkrebs migrated over land, demonstrating their potential for active spread, and two invasive populations endanger indigenous crayfish populations. Most Marmorkrebs populations are large and are most likely several years old, suggesting a considerable lag between introduction and detection. Marmorkrebs populations in Europe are most likely the result of deliberate releases from aquaria, although secondary introductions may have occurred in one instance. Because Marmorkrebs are still widespread in the European pet trade, which most likely generates substantial propagule pressure, it is likely that the number of established populations will further increase over time. To mitigate the risk of further harmful crayfish releases, we suggest the prohibition of trading live high-risk crayfish species, including Marmorkrebs.


Introduction
The Marmorkrebs is an enigmatic crayfish species of North American origin.It was first discovered in the German pet trade in the mid 1990s, when aquarium enthusiasts reported an all-female crayfish species that reproduces without males (Lukhaup 2001).Due to their characteristic and conspicuous color pattern, these crayfish quickly became known as `Marmorkrebs´ (German, which translates into English as `marbled crayfish´).Scientists have puzzled for almost a decade about their phylogenetic position and status.Using genetic and morphological comparisons, Martin et al. (2010a) recently showed that Marmorkrebs are the parthenogenetic form of Procambarus fallax (Hagen, 1870) and proposed the tentative scientific name Procambarus fallax f. virginalis.The Marmorkrebs is unique because it is the only known decapod crustacean that obligatory reproduces by apomictic parthenogenesis: only females exist, which lay unfertilized eggs that develop into genetically identical offspring (Scholtz et al. 2003;Martin et al. 2007;Vogt et al. 2008).No males have been found in the laboratory or in introduced, wild populations (Seitz et al. 2005;Jones et al. 2009;Janský and Mutkovič 2010).
Marmorkrebs had circulated in the European pet trade for several years before the first freeliving individuals were captured in Europe and Madagascar in 2003 (Soes and van Eekelen 2006;Jones et al. 2009).The major pathway for Marmorkrebs introduction is the deliberate release of aquarium specimens (Soes and van Eekelen 2006;Souty-Grosset et al. 2006;Chucholl 2011).Their appealing coloration, undemanding nature and exceptional mode of reproduction make Marmorkrebs attractive to aquarium hobbyists.However, parthenogenesis permits a high reproductive potential, and Marmorkrebs can overpopulate an aquarium quickly.Aquarium hobbyists will likely offload their excess stock to either other aquarium hobbyists or natural habitats (Souty-Grosset et al. 2006;Chucholl 2011).Because Marmorkrebs reproduce by parthenogenesis, the risk that released Marmorkrebs may seed viable populations in the wild is considerably greater than that for sexually reproducing crayfish species -a single Marmorkrebs is sufficient to create a new population.
Marmorkrebs quickly became established in Madagascar and are considered to be `perfect invaders´ there (Jones et al. 2009).On the contrary, in Europe, almost all published Marmorkrebs records between 2004 and 2009 are accounts of single individuals, suggesting that Marmorkrebs might fail to establish selfsustaining populations in temperate zones (Marten et al. 2004;Souty-Grosset et al. 2006;Nonnis Marzano et al. 2009;Martin et al. 2010b).In 2010, the situation dramatically changed when the first informal evidence for an established Marmorkrebs population in Germany was published by newspapers (Privenau 2010): the local media repeatedly reported on Marmorkrebs emerging from a small pond in a village near Halle (Saxony-Anhalt; Wendt 2011).Shortly afterward, a research paper provided evidence that Marmorkrebs had established another stable, reproducing population in a small lake near the city of Freiburg (Baden-Württemberg; Chucholl and Pfeiffer 2010).Since then, numerous additional Marmorkrebs records from Europe were reported, including more reproducing populations.
The purpose of the present article is twofold: (1) to describe a new occurrence of Marmorkrebs in southwestern Germany that was discovered in 2011, and (2) to give a synopsis of the recent records of this species in Europe, including newly discovered populations in other parts of Germany and Slovakia.We then use the available information to discuss the introduction history, population trend, and invasiveness of Marmorkrebs in Europe.Because Marmorkrebs are primarily introduced from aquaria (Souty- Grosset et al. 2006;Chucholl 2011), we focus on the risks associated with aquarium introductions, a newly emerging introduction pathway of nonindigenous crayfish species (NICS) that may add yet another chapter to the notorious history of NICS introductions in Europe (cf.Holdich et al. 2009;Chucholl 2010).Our findings may therefore help with invasive species risk assessment and the prioritization of introduction pathway management.

New occurrence of established Marmorkrebs in southwestern Germany
On September 14, 2011, one of the authors (KM) discovered a live Marmorkrebs at a pedestrian underpass, 130 m from a gravel pit lake (Lake Epplesee; Figure 1A, B; Table 1, Nr. 13).Lake Epplesee is a shallow, eutrophic lake with a surface area of 26.6 ha and a maximum depth of 5 m.It lies 302 m above sea level, in close proximity to the River Neckar.The lake is popular for several recreational activities, including swimming, sunbathing, and angling, and is highly frequented because of its easy accessibility from a nearby highway.Lake Epplesee is situated in the vicinity of Tübingen but also attracts people from Reutlingen and Stuttgart.
To verify the sighting, the northern lake margin was sampled using a hand-held net on September 30, 2011.Within five minutes, two Marmorkrebs were captured within an area of approximately 2 m 2 , and a third individual was spotted, but escaped.One of the captured females carried hatchlings, while the other female exhibited active glair glands on the ventral side of the pleon, indicating breeding condition.In addition, two dead individuals were found at the pedestrian underpass, which is separated from the northern lake margin by a 100 m stretch of sunbathing area (Figure 1C, D).The Marmorkrebs captured from the lake measured 29.6 and 50.2 mm in carapace length (CL: measured from the tip of the rostrum to the dorsal posterior margin of the cephalothorax with a digital slide caliper), and the two dead individuals measured 47.1 and 44.5 mm CL.
The occurrence of a `new crayfish´ in Lake Epplesee has been known to recreational fishermen since at least 2010, when the species was reportedly already very abundant.Close to Lake Epplesee is the brook Schlierbach (cf. Figure 1C), which is inhabited by indigenous stone crayfish [Austropotamobius torrentium (Schrank, 1803)], an endangered species in the federal state of Baden-Württemberg (Chucholl and Dehus 2011).The environmental and fisheries agencies currently attempt to control and to contain the Marmorkrebs population, e.g., through stocking and managing of predatory fish and discouraging the public from transplanting and fostering Marmorkrebs (M.Konrad, pers. comm. 2011).

Synopsis of Marmorkrebs records from Europe
The year of the initial recording, country, location, habitat, population status, collection method, and additional comments are summarized for each of the known Marmorkrebs records from Europe in Table 1, and the present European distribution of Marmorkrebs is shown in Figure 2.
In total, 15 Marmorkrebs records are currently known in Europe, excluding putative records that could not be verified by voucher specimens or photos.Most of the records were initially discovered after 2005, and 13 of the 15 records were only discovered within the past four years (Figure 3).The vast majority of the records were reported in Germany (12), while one record each was reported from the Netherlands, Italy and  1.
Slovakia (Figure 2).Marmorkrebs were found in both lotic and lentic freshwater habitats, including brooks, rivers, canals, natural and artificial lakes, and ponds; however, established populations have only been found in lentic habitats (cf.Apart from the records summarized above, at least four additional putative records of Marmorkrebs in Germany have been reported and cover three federal states; however, these records have not been verified yet (Chucholl and Dehus 2011;H. Groß, unpublished data).In Slovakia, Marmorkrebs apparently also formed breeding populations in garden ponds (Stloukal 2009).Dead Marmorkrebs found on the land were repeatedly reported from North Rhine-Westphalia (Germany; H. Groß unpublished data), suggesting that the estimated number of unreported Marmorkrebs occurrences is still high (cf.Chucholl and Pfeiffer 2010).

Discussion
The Marmorkrebs is an extraordinarily successful crayfish species.It was first discovered in the German pet trade in the mid-1990s (cf.Werner 1998; Figure 3) and was first mentioned in a scientific publication in 2002 (Scholtz et al. 2002).By this time, it was already very popular as an aquarium pet (Lukhaup 2001; Figure 3).Only 10 years later, it has become a highly invasive species in Madagascar (Jones et al. 2009), was introduced into freshwater habitats in Japan (Kawai and Takahata 2010), and is well established in various parts of Europe (summarized in Table 1; Figures 2 and 3).Including the new occurrence described here, at least six established Marmorkrebs populations are currently known in Europe, representing an alarming increase beyond the one population known prior to 2010 (cf. Figure 3).Most populations are large and are most likely several years old, suggesting a considerable lag between introduction and detection.Given this probable lag between introduction and detection, it is likely that the currently observed trend of increasing population numbers is a result of introductions that occurred several years ago, presumably around the time that Marmorkrebs became popular in home aquaria (cf. Figure 3).This interpretation is in accordance with the propagule pressure hypothesis, which states that a major determinant of the invasion success of a non-indigenous species is the number of independent release events (propagule number) plus the number of individuals released in any one event (propagule size), that is, introduction effort or propagule pressure (Lockwood et al. 2005;Lockwood et al. 2007).
The proliferation of Marmorkrebs as aquarium pets likely increased propagule pressure, and thereby the likelihood of establishment in nature (Lockwood et al. 2005;Duggan et al. 2006;Lockwood et al. 2007).Specifically, the great popularity of Marmorkrebs as aquarium pets probably results in many independent release events, i.e., a high propagule number, which serves to overcome negative forces that are spatially structured, such as an unsuitable habitat at the site of introduction (Lockwood et al. 2005;Duggan et al. 2006).For instance, Marmorkrebs apparently failed to establish in cool, rapidflowing brooks but eventually invaded lentic habitats.Marmorkrebs propagule pressure and, thus, the likelihood of establishment in nature, is probably highest in countries where (1) Marmorkrebs are popular aquarium pets and (2) human population density is high (cf.Lockwood et al. 2005;Perdikaris et al. 2012).Both of the criteria apply to Germany (Lukhaup 2001;Perdikaris et al. 2012), where five out of the six currently known Marmorkrebs populations in Europe are located (Figure 2).Because Marmorkrebs are still abundant in the European pet trade (Stloukal 2009; Chucholl 2010; Soes and Koese 2010) and there is potentially a lag between introduction and detection, it is likely that the number of records of established populations will further increase with time.Marmorkrebs are acquired through retail pet shops, various online sources, and personal contacts between aquarium hobbyists (Stloukal 2009;Faulkes 2010;Soes and Koese 2010).Retail stores offer Marmorkrebs at approximately €5 per animal, whereas aquarium hobbyists often give their excess stock away for free (cf.Chucholl 2011).For instance, a search for `Marmorkrebs´ at a German online marketplace (http://www.kleinanzeigen.ebay.de) on January 12, 2012 resulted in 36 hits, and most of these vendors offered the animals for free or at prices below €5 per crayfish.In recent years, Marmorkrebs have also become popular as live food for predatory aquarium fish and ornamental turtles owing to their undemanding nature and high reproductive potential.Because ornamental turtles are frequently kept in outdoor ponds, using Marmorkrebs as a food source may facilitate accidental introductions.It is an even greater concern that Marmorkrebs are deliberately released into garden ponds (Stloukal 2009), from which they can easily escape to nearby freshwater habitats.
Introduced Marmorkrebs have been found in both lentic and lotic freshwater habitats (e.g., Marten et al. 2004;Martin et al. 2010b).However, populations in Europe have been found in lentic habitats only (cf.Table 1), which agrees with Chucholl's and Pfeiffer's (2010) suggestion that Marmorkrebs are most likely able to colonize summer-warm, lentic habitats in most parts of Europe.In Madagascar, Marmorkrebs were reported from a great variety of habitats, including rice paddies, rivers, lakes and swamps (Heimer 2010), as well as brick pits, drainage ditches and fish ponds (Jones et al. 2009).Most established populations in Europe occur in habitats near conurbations, typically in highly frequented secondary habitats, such as gravel pit lakes.Introductions from aquaria are more likely to occur in highly frequented habitats than in remote habitats because the latter are usually more easily accessible and may already be known to the potential pet releaser.However, the probability of detection is presumably also higher in highly frequented habitats, which may confound this effect.
In at least three instances, Marmorkrebs were observed to migrate over land (Table 1,Nr. 2,11,and 13; Figure 1), and single dead Marmorkrebs found on the land were repeatedly reported from North Rhine-Westphalia (H.Groß, unpublished data).The frequent observation of Marmorkrebs migrating over land suggests that this behavior is most likely an inherent dispersal mechanism for this species, rather than an escape mechanism in response to adverse environmental conditions.Established Marmorkrebs populations may therefore act as latent `bridgeheads´ for a further active range expansion both via waterways and land.Any attempts to contain local populations should take both pathways into account.A further potential pathway for spreading is translocation by humans, i.e., secondary introductions.This mechanism of spreading most likely occurred in Saxony-Anhalt (Table 1, Nr. 11), where people collected Marmorkrebs migrating over land and most likely transplanted them to garden ponds (Wendt 2011).
During the last decade, Marmorkrebs were primarily known as aquarium pets.Most research on these organisms was carried out in the laboratory and was concerned with their exceptional mode of reproduction or use as a model organism for development, epigenetics and toxicology (e.g., Seitz et al. 2005;Martin et al. 2007;Vogt 2008).It is only more recently that Marmorkrebs have been considered as an invasive species, and in fact their life history and trophic ecology in nature are almost unknown.For instance, nothing is known about the trophic position and ecological impact of Marmorkrebs.Given the major impacts of the related species P. clarkii on recipient ecosystems (Souty-Grosset et al. 2006, and citations therein), Marmorkrebs may have a profound impact on the species richness, functioning and integrity of ecosystems.
Marmorkrebs most likely pose a serious threat to the indigenous European crayfish species because they may compete with other species for food and space, and they may transmit crayfish plague (Jones et al. 2009;Chucholl and Pfeiffer 2010).Jimenez and Faulkes (2011) studied direct aggressive interactions between Marmorkrebs and P. clarkii and concluded that Marmorkrebs have the potential to compete with other crayfish species.Furthermore, Marmorkrebs differ ecologically from the more K-selected indigenous European crayfish because Marmorkrebs have a fast growth rate, very high fecundity and an extended breeding period (Seitz et al. 2005;Jones et al. 2009;Chucholl and Pfeiffer 2010), all of which might give an additional competitive advantage to Marmorkrebs.The risk of devastating consequences for indigenous crayfish would dramatically increase if Marmorkrebs were infected with the causative agent of crayfish plague, Aphanomyces astaci Schikora, 1903: any contact between Marmorkrebs and the susceptible European crayfish would almost certainly result in mass mortalities among the susceptible species.This potential threat to indigenous crayfish is alarming, especially because at least two of the six established Marmorkrebs populations already endanger indigenous crayfish populations (Table 1, Nr. 6 and 13).

Conclusion
Without much doubt, all Marmorkrebs introductions trace back to this species' first occurrence in the German pet trade in the mid-1990s.It circulated in the European pet trade for several years before the first free-living individuals were captured in Germany and the Netherlands (cf.Table 1 and Figure 3).The currently observed and alarming increase in established populations is likely to continue over time, unless Marmorkrebs propagule pressure from the pet trade decreases.The introductions occurred despite public education efforts, explicitly advising to not release Marmorkrebs into nature (e.g., Lukhaup 2001;Lukhaup and Pekny 2005;Lukhaup and Pekny 2007;Edelkrebsprojekt NRW 2009).In fact, one of the first articles on Marmorkrebs in a popular German aquarium magazine (Lukhaup 2001) and the first scientific mention of Marmorkrebs, published in Nature (Scholtz et al. 2002), both implied warning that Marmorkrebs may become a pest if released from captivity.Unfortunately, there is no magic "silver bullet" to eradicate established NICS populations (Gherardi et al. 2011) and Marmorkrebs do not need to maintain a minimum viable population size.Their resilience against small population size renders eradication of established Marmorkrebs populations even more difficult or even impossible.The prevention of new introductions is therefore imperative.Given the apparent ineffectiveness of public education efforts to halt exotic crayfish releases, we suggest the prohibition of trading live high-risk crayfish species, including Marmorkrebs.

Figure 1 .
Figure 1.Lake Epplesee (A) and the pedestrian underpass (C), where Marmorkrebs were first found (B: live individual on September 14, and D: dead individual on September 30, 2011; red arrows mark the locations where the two dead Marmorkrebs were found).The pedestrian underpass is located 130 m northeast of Lake Epplesee, and it is separated from the lake by a 100 m stretch of sunbathing area (visible in the background of C).Behind the metal boarding on the left side of the walkway is the brook Schlierbach, which is inhabited by the endangered stone crayfish (Austropotamobius torrentium).Photographs A, C, and D by CC, photograph B courtesy of KM.

Figure 2 .
Figure 2. Current distribution of Marmorkrebs in Europe, shown as presence in CGRS grid squares.Numbers refer to the records summarized in Table1.

Table 1 )
. Out of the 15 records, six clearly represent established populations, and on the basis of the collection of several individuals, five additional records might also represent established populations.Single individuals that most likely failed to propagate, i.e., failed introductions, were reported in three cases, of which two involved captures from small brooks (

Table 1 .
Current records of Marmorkrebs in Europe.Only records verified by voucher specimens or photos are included.NA means not available.