Introduced alien ringed crayfish ( Orconectes neglectus neglectus [ Faxon , 1885 ] ) threaten imperiled coldwater crayfish ( Orconectes eupunctus Williams , 1952 ) in the Eleven Point River drainage , Missouri , USA

The Eleven Point River drainage in the Ozarks ecoregion of North America contains the largest known populations of and critical habitat for the globally imperiled coldwater crayfish (Orconectes eupunctus). We present here the discovery of an introduced population of alien ringed crayfish (Orconectes neglectus neglectus) in Jolliff Spring Branch in the upper reaches of the drainage. Sampling was conducted at eight sites throughout the tributary to determine the extent of the alien species’ range in the stream. Orconectes n. neglectus were found at four sites extending along 3.4 km of stream, with reproducing populations confirmed at two upstream sites, and few individuals found at two others. This invasion represents a threat to O. eupunctus, which occurs a few kilometers further downstream in the drainage and has been recorded from only the lower reaches of Barren Fork and throughout the mainstem Eleven Point River. Introduced O. neglectus were previously associated with localized extirpation of O. eupunctus in another drainage. Study results warrant monitoring of the invasion and possible reassessment of the conservation status for O. eupunctus.

Nearly half of the native crayfish fauna in the United States and Canada are considered imperiled or in need of conservation (Taylor et al. 2007;NatureServe 2010), and the introduction of invasive alien crayfish has been cited as the most significant threat to crayfish biodiversity in North America and worldwide (Lodge et al. 2000). Invasive crayfishes have displaced native crayfishes (Lodge et al. 1986;Light et al. 1995;Daniels 1998); adversely affected or displaced native amphibians (Gamradt and Kats 1996;Kats and Ferrer 2003), reptiles (Fernandez and Rosen 1996), and fishes degraded recreational and commercial fisheries (Hobbs et al. 1989;Guan and Wiles 1997;Dorn and Mittelbach 1999;Wilson et al. 2004;Peay et al. 2009); and altered the structure and function of stream, lake and marsh communities (Feminella and Resh 1989;Olsen et al. 1991;Charlebois and Lamberti 1996).
The ringed crayfish, Orconectes neglectus neglectus (Faxon, 1885;Figure 2), is native to streams in southwestern Missouri as well as portions of Arkansas, Colorado, Kansas, Nebraska, Oklahoma and Wyoming (Taylor et al. 2007). Alien populations of this species are established in New York (Taylor et al. 2007) and in the Rogue River of Oregon, where they have displaced the native Klamath signal crayfish, Pacifastacus leniusculus klamathensis (Stimpson, 1857) (Bouchard 1977). An introduced population of O. n. neglectus was also recently documented in Little Tebo Creek, a tributary to Harry S. Truman Reservoir (Osage River) in Missouri (Jones 2009). The subspecies, the gap ringed crayfish Orconectes neglectus chaenodactylus Williams, 1952, was introduced and appears to be displacing two native crayfishes including the "Vulnerable" (DiStefano et al. 2010) and "Threatened" (Taylor et al. 2007) coldwater crayfish, Orconectes eupunctus Williams, 1952 (Figure 3), in the Spring River of Missouri and Arkansas (Magoulick and DiStefano 2007 Jolliff Spring Branch averages approximately 3 m in width (1-8 m range) and the downstream reaches of Barren Fork average 6 m in width (3-9 m range); both streams are generally <1 m in depth. Land use surrounding the streams is generally livestock pasture with forested riparian corridors. Eight sampling sites were selected at regular intervals corresponding to stream access points, throughout the length of Jolliff Spring Branch and the reach of Barren Fork between its confluences with Jolliff Spring Branch and the Eleven Point River (Table 1; Figure 1). Additionally, we sampled three small ponds located immediately north of site 1 to determine if they might be possible sources of the invasion.
Each study site was sampled once from June 24 through October 8, 2010, during low stream flows when our sampling method was most effective and consistent. Eight habitat sampling units (four riffles and four runs) were selected at each site, and upstream and downstream ends of the site were marked with a Global Positioning System unit. A random number table was used to select locations for three kick-seine subsamples within each habitat sampling unit (total n = 4 samples per habitat type per site). Crayfish species and densities were determined by disturbing the substrate inside a 1 m 2 pvc quadrat frame located directly upstream of a kick seine (1.5 m length  1.5 m height) with 3 mm delta mesh (Flinders and Magoulick 2005). We identified crayfish to species and returned them to the stream unharmed.
Three ponds adjacent to site 1 ( Figure 1) were sampled with wire mesh minnow traps baited with canned dog food. This method has been used previously to collect O. neglectus and typically outperforms kick seining methods in deeper lentic environments. Traps were set in the evening, evenly spaced along the shoreline about   2-3 m from shore, and harvested the following morning. Thirty traps were placed in the largest of the ponds (~ 1400 m 2 ), and ten and six traps were placed in two smaller ponds (< 1100 m 2 each). Orconectes n. neglectus populations were established in the upstream-most reaches of Jolliff Spring Branch, but had not yet occupied the entire stream. Orconectes n. neglectus were found at four sites spanning the upstream 3.4 km of the stream (Table 2; Figure 1). The species appeared to be well-established only at sites 1 and 2, where it constituted 45% and 63% of the crayfish community, respectively, and occurred at higher densities than the native Ozark crayfish (Orconectes ozarkae Williams, 1952) and spothanded crayfish (Orconectes punctimanus [Creaser, 1933]). Very few O. n. neglectus individuals were found (four and one) at sites 3 and 4, and none were found at sites 5 through 8. The source and timing of the invasion is unknown.
"Baitbucket introductions" by recreational fishers are generally considered the primary vector for alien crayfish introductions in North America (Lodge et al. 2000), but Orconectes n. neglectus is not on the state of Missouri's Approved Aquatic Species List (AASL; State of Missouri 2011) for commercial trade, and thus not legally sold in fishing bait shops. However, 62% of Missouri's known crayfish invasions involve species not on the AASL (R.J. DiStefano, unpublished data). Missouri fishing license holders are permitted to catch wild crayfish for several uses (bait, human consumption, pets, etc.; State of Missouri 2011), and we suspect that wild-caught crayfish are being transported across drainage basin boundaries and released to the wild. Our data suggest that O. n. neglectus were introduced in the vicinity of sites 1 and 2, possibly directly to the stream at a lone public road crossing immediately upstream of site 1 or into recreational fishing ponds at site 1, or into additional ponds (which we were unable to sample due to lack of landowner permission) at site 2.
The imperiled O. eupunctus is one of the world's most geographically limited crayfishes, endemic to only portions of the Spring, Strawberry, and Eleven Point rivers of southern Missouri and northern Arkansas (Pfleiger 1996). Population declines and localized extirpations of O. eupunctus and native Hubbs' crayfish, Cambarus hubbsi (Creaser, 1931) in many kilometers of the nearby Spring River drainage have followed a previous invasion by the O. n. chaenodactylus subspecies (Magoulick and DiStefano 2007). Magoulick and DiStefano (2007)  DiStefano, personal observation). These agencies are also concerned about the potential threat of this invasion to another "critically imperiled" (Missouri Natural Heritage Program 2011) native crayfish, the Mammoth Spring crayfish (Orconectes marchandi Hobbs, 1948) that occurs in the Spring River drainage, close to the Eleven Point River. Therefore, the discovery of the O. n. neglectus invasion in Jolliff Spring Branch, in the Eleven Point River drainage, is cause for concern.
Orconectes n. neglectus dominated the crayfish community at sites 1 and 2. Densities of the native O. ozarkae at these two sites were significantly lower than at several downstream sites, but we have no direct evidence that the invader has negatively affected this native species or the native O. punctimanus. Our study establishes a baseline for potential comparisons between future sampling results and the crayfish densities we reported.
The O. n. neglectus invasion in Jolliff Spring Branch poses an increased threat to the conservation status of the imperiled O. eupunctus. Previous studies indicate that crayfish invasions in upland streams progress fairly rapidly downstream (Peay et al. 1999;Bubb et al. 2005;Kerby et al. 2005); those studies as well as the O. n. chaenodactylus invasion in the nearby Spring River (Magoulick and DiStefano 2007) suggest that O. n. neglectus in Jolliff Spring Branch could expand its range and enter the reach of Barren Fork occupied by O. eupunctus within a decade. The Jolliff Spring Branch O. n. neglectus invasion should be monitored closely and the data reported here should be considered in future conservation status assessments for the imperiled O. eupunctus.