Sipsey Fork drainage, including Lewis Smith Reservoir, Alabama, USA, and indirect sampling sites (i.e., fish collection locations). Site codes as in Table 1. Symbols for individual species reflect distributions of species whose origin is uncertain. “Other crayfish species” indicates that the site yielded at least one of the known native species: Cambarus obstipus , C. striatus , or Orconectes validus . Shaded area is the Bankhead National Forest. Inset shows location of the study area in Alabama. Rectangle indicates area of direct sampling shown in Figure 2. 

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... Sipsey Fork/Brushy Creek sampling, we collected crayfish from 60 sites ( Figure 2). Of the five crayfish species we found, three were expected [ Cambarus obstipus Hall, Cambarus striatus Hay, and Orconectes validus (Faxon)] and two were not [ O. lancifer (Hagen) and O. ( Procericambarus ) sp. nr ronaldi ]. We were aware that Orconectes virilis (Hagen) was invasive elsewhere in the Black Warrior River system but did not find it in the Sipsey Fork or Brushy Creek upstream of the reservoir. The three native species that we anticipated finding all occurred in perennially free-flowing stream segments upstream of any impoundment influences, as well as to varying extents in transition or impounded zones. Cambarus obstipus occurred frequently in the upper transition zones and upstream in both mainstem streams and their tributaries (Appendix 1). Orconectes validus had a similar distribution in Brushy Creek but also occurred in permanently impounded portions of the Sipsey Fork (Appendix 1). In the impounded and transition zones of the mainstems, C. striatus was found only in burrows near spring seeps; however, at one site in Brushy Creek nearly 10 km upstream of the transition zone, C. striatus was relatively abundant (Appendix 1). Cambarus striatus occurred in every tributary sampled, typically occurring upstream of the transition zones. Orconectes lancifer occurred in the impounded portions of the Sipsey Fork and Brushy Creek, as well as in the transition zones of two tributaries - one in each watershed (Figure 2, Appendix 1). One individual also was caught a short distance upstream of the transition zone in Grindstone Creek. We found O . sp. nr ronaldi only in the Brushy Creek watershed, where it occurred in the impounded and lower transition zones of the mainstem, as well as slightly upstream of the transition zones in both Brushy Creek tributaries that flowed into the impounded zone (Figure 2). We did not find it in collections farther upstream in those tributaries. We initially identified the species as O . juvenilis because, in addition to matching many other morphological features fairly closely, the incisor region of the mandible had a straight edge, an important diagnostic character distinguishing O . juvenilis from Orconectes ronaldi Taylor (Taylor 2000). Based on morphology, Dr. Chris Taylor (Illinois Natural History Survey) tentatively confirmed the identification. However, because the study area was far from the range of O. juvenilis , the possibility remained that the population represented a new species. Comparisons of COI sequences from our Alabama specimens to those in Kessler et al. (this issue) and in GenBank indicated that the two sequenced individuals were most similar to O. ronaldi . On average, sequences from the two Alabama specimens differed (uncorrected p-distance) from O. ronaldi by 3.2% and from O. juvenilis by 6.6%. Re-examination of the specimens indicated that the form I male gonopod was consistent with O. ronaldi , although the mandible was not; O. ronaldi typically has a serrated edge on the incisor region of the mandible (Taylor 2000). As is often the case with crayfish, morphological and genetic information were at odds, so for the present, we refer to the specimens as O . sp. nr ronaldi . We examined 18 crayfish from stomachs of 17 black bass collected in Lewis Smith Reservoir concurrently with the stream study. All 18 were Orconectes , representing four species; however, four individuals were not identified to species (Figure 1 and Table 2). Two of the species, O. lancifer and O. validus , also were found during our direct sampling, but the other two, Orconectes perfectus Walls and O. virilis , were not. Orconectes perfectus was found in fish stomachs from the Rock and Ryan creek arms of the reservoir as well as in the forebay of the dam and was collected over three months. We obtained a single O. virilis from a fish in the Ryan Creek arm of the reservoir. Given the generally high site fidelity and small home ranges documented for both largemouth bass and spotted bass in reservoirs (Warden and Lorio 1975; Winter 1977; Fish and Savitz 1983 ; Copeland and Noble 1994; Hunter and Maceina 2008 ), it is likely that most fish were collected within 2 km of where they fed on the crayfish. At least 44 crayfish occurred in the stomachs and intestines of 32 individual fishes collected from streams in the 1990’s. Nine of the crayfish could not be identified to genus, and 17 were identified to species with a high level of confidence. Because crayfish were removed from the intestines as well as the stomachs of these fishes, more small crayfish parts were encountered, relative to the samples from bass stomachs, making identifications more difficult. Both of the C. obstipus found in the fish guts came from fish captured in the Sipsey Fork mainstem, but six of the seven C. striatus came from fish collected in tributaries (Table 2). Four O. validus came from the Sipsey Fork mainstem, and the remaining 10 came from its tributaries. Because O. lancifer has the most distinctive rostrum, chelae, and gonopods of any crayfish in the study area, it is highly unlikely that it was overlooked in our evaluation of these ...

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... the state’s crayfish. The number of formally recognized crayfish species in the state currently hovers around 86. However, many gaps still exist in our knowledge of crayfish distributions, especially for certain crayfish groups. Primary burrowing species and species occupying large waterbodies are particularly underrepresented in the historic data, and new species and species records continue to be found in Alabama (Schuster et al. 2008, 2015). Rapidly changing species distributions add to the difficulty of documenting distributions over large areas. Factors contributing to changing distributions can include land use changes, hydrologic changes (e.g., impoundment of rivers, hydrologic connection of previously discrete river systems, alteration of water tables), and species introductions, both intentional and unintentional. Species introductions can contribute to sudden and dramatic distributional changes due not only to the addition of the non-native species, but also to the extirpation or range contraction of native species (Arcella et al. 2014; Richman et al. 2015). We report on occurrences of crayfish species found in two mainstem streams that flow into Lewis Smith Reservoir, as well as in the reservoir itself, and compare those to previously known crayfish distributions from the streams and reservoir. The species were collected while working on a larger study examining changes in the aquatic faunal community composition along riverine-lacustrine transition zones (hereafter transition zones). The transition zones were stream segments that were inundated by the reservoir at high pool but flowing at low pool. The main objectives of this paper are to document recent occurrences of species previously unknown in the drainage and to discuss possible explanations for the new records. MATERIALS AND METHODS The study area in northwest Alabama, USA, lies within the Warrior Basin district of the Cumberland Plateau physiographic province, geologically dominated by Pottsville shale, sandstone, and coal (Boschung and Mayden 2004). Streams in the province typically flow through deeply entrenched valleys and have well- defined pool-riffle habitat sequences with substrate dominated by gravel, sand, slabrock, and bedrock ( Haag and Warren 2008), along with some cobble and boulders. The Sipsey Fork, a major tributary to the Mulberry Fork of the Black Warrior River, was impounded by the 91 m high Lewis Smith dam in 1961, creating the 8,580 ha Lewis Smith Reservoir (Boschung and Mayden 2004). We studied two mainstem streams flowing into the reservoir, Sipsey Fork and Brushy Creek, as well as three tributaries to each stream and several sites throughout the reservoir (Figures 1 and 2). Both streams originated in the Bankhead National Forest, with much of the Sipsey Fork headwaters protected in the Sipsey Wilderness and the Sipsey Wild and Scenic River corridor. We defined river- reservoir transition zones as the stream segments impounded when the reservoir was at the summer full-pool elevation targeted by managers (155.5 m above sea level) but flowing at the typical winter low-pool elevation (153.0 m). We determined the transition zones based on both elevation contours and examination of habitat during reservoir full- and low-pool periods. Both streams had potential transition zones of about 8 km. The tributaries were relatively small (wetted widths 3 – 7 m), rocky streams that typically had little flow in late summer. Because they had steeper channel slopes than the larger streams, the transition zones in the tributaries were much shorter (< 0.1 – 0.6 km). In the mainstem streams, we sampled every 1 – 3 km downstream of, in, and upstream of the transition zones. In three tributaries of each mainstem, we sampled from the impounded zone, if present, to well upstream of the transition zones. Sampling was most intensive and most effective for capturing crayfish during low pool in the autumn. We sampled the Sipsey Fork and its tributaries in September 2012, Brushy Creek and its tributaries and the Sipsey Fork upstream of the transition zone in September 2013, the lower transition zone mainstem and well upstream of the transition zones in tributaries in October 2014, and well upstream of the transition zones in mainstems and tributaries in April 2015. We used a variety of methods to capture crayfish. Although some methods were quantitative, the overall effort was a qualitative sampling approach aimed at documenting species occurrences in a variety of lotic and lentic habitats. Sampling methods in deep habitats included boat electrofishing, trawling, and trapping (minnow traps with 3.2 cm openings and baited initially with canned dog food and later with pieces of fresh fish). In wadeable habitats, we used backpack electrofishing, seining, visual searches (using mask and snorkel, view buckets, rock flipping, and searches for molted carapaces along banks), and occasionally digging of burrows along banks. Our initial crayfish results led us to ask about the distributions of crayfish species in the continuously impounded portion of the reservoir. We obtained crayfish from an independent, concurrent study of black bass ( Micropterus henshalli Baker et al. and Micropterus salmoides (Lacepède)) diets in the reservoir. Black bass were captured via boat electrofishing at seven sites in the reservoir (Figure 1; Table 1) from July 2013 to September 2014 (Table 2). Bass were placed on ice and taken to the laboratory, where stomachs were dissected and contents stored in 95% ethanol. We attempted to identify all crayfish remains to species. In an attempt to determine whether certain crayfish taxa were new to the study area, we also examined gut contents of potentially predaceous fishes that were collected in the Sipsey Fork, Brushy Creek, and their tributaries from October 1993 to July 1995, preserved in 5% formalin, and stored in 70% ethanol (in part, Haag and Warren 1998). These sites were farther upstream in the drainages than were most of our recently sampled sites (Figure 1). We dissected all fish deemed capable of eating adult crayfish (assessed subjectively based on fish size and gape size) and removed all crayfish and parts of undigested crayfish exoskeletons from stomachs and intestines. We attempted to identify whole crayfish specimens, as well as the parts, to species. We used a DNA bar-coding approach to assist in the identification of the Orconectes ( Procericambarus ) sp. collected. Initial morphological identifications suggested that these individuals were Orconectes juvenilis (Hagen). We extracted DNA from two specimens and amplified a region of the mitochondrial cytochrome oxidase subunit I gene (COI), a standard bar-coding region of the mitochondrial genome useful for diagnosing problematic or unknown specimens. M ethods of DNA isolation, amplification, and sequencing are described in Taylor et al. (2014). Following methods described in Kessler et al. (this issue), we compared the COI sequences from our specimens to sequences from GenBank and from the Kessler et al. dataset, including each of the members of the O. juvenilis complex as well as other North American crayfish (see Table 2 in Kessler et al. in this issue, for details, including GenBank accession numbers). ...

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... the state’s crayfish. The number of formally recognized crayfish species in the state currently hovers around 86. However, many gaps still exist in our knowledge of crayfish distributions, especially for certain crayfish groups. Primary burrowing species and species occupying large waterbodies are particularly underrepresented in the historic data, and new species and species records continue to be found in Alabama (Schuster et al. 2008, 2015). Rapidly changing species distributions add to the difficulty of documenting distributions over large areas. Factors contributing to changing distributions can include land use changes, hydrologic changes (e.g., impoundment of rivers, hydrologic connection of previously discrete river systems, alteration of water tables), and species introductions, both intentional and unintentional. Species introductions can contribute to sudden and dramatic distributional changes due not only to the addition of the non-native species, but also to the extirpation or range contraction of native species (Arcella et al. 2014; Richman et al. 2015). We report on occurrences of crayfish species found in two mainstem streams that flow into Lewis Smith Reservoir, as well as in the reservoir itself, and compare those to previously known crayfish distributions from the streams and reservoir. The species were collected while working on a larger study examining changes in the aquatic faunal community composition along riverine-lacustrine transition zones (hereafter transition zones). The transition zones were stream segments that were inundated by the reservoir at high pool but flowing at low pool. The main objectives of this paper are to document recent occurrences of species previously unknown in the drainage and to discuss possible explanations for the new records. MATERIALS AND METHODS The study area in northwest Alabama, USA, lies within the Warrior Basin district of the Cumberland Plateau physiographic province, geologically dominated by Pottsville shale, sandstone, and coal (Boschung and Mayden 2004). Streams in the province typically flow through deeply entrenched valleys and have well- defined pool-riffle habitat sequences with substrate dominated by gravel, sand, slabrock, and bedrock ( Haag and Warren 2008), along with some cobble and boulders. The Sipsey Fork, a major tributary to the Mulberry Fork of the Black Warrior River, was impounded by the 91 m high Lewis Smith dam in 1961, creating the 8,580 ha Lewis Smith Reservoir (Boschung and Mayden 2004). We studied two mainstem streams flowing into the reservoir, Sipsey Fork and Brushy Creek, as well as three tributaries to each stream and several sites throughout the reservoir (Figures 1 and 2). Both streams originated in the Bankhead National Forest, with much of the Sipsey Fork headwaters protected in the Sipsey Wilderness and the Sipsey Wild and Scenic River corridor. We defined river- reservoir transition zones as the stream segments impounded when the reservoir was at the summer full-pool elevation targeted by managers (155.5 m above sea level) but flowing at the typical winter low-pool elevation (153.0 m). We determined the transition zones based on both elevation contours and examination of habitat during reservoir full- and low-pool periods. Both streams had potential transition zones of about 8 km. The tributaries were relatively small (wetted widths 3 – 7 m), rocky streams that typically had little flow in late summer. Because they had steeper channel slopes than the larger streams, the transition zones in the tributaries were much shorter (< 0.1 – 0.6 km). In the mainstem streams, we sampled every 1 – 3 km downstream of, in, and upstream of the transition zones. In three tributaries of each mainstem, we sampled from the impounded zone, if present, to well upstream of the transition zones. Sampling was most intensive and most effective for capturing crayfish during low pool in the autumn. We sampled the Sipsey Fork and its tributaries in September 2012, Brushy Creek and its tributaries and the Sipsey Fork upstream of the transition zone in September 2013, the lower transition zone mainstem and well upstream of the transition zones in tributaries in October 2014, and well upstream of the transition zones in mainstems and tributaries in April 2015. We used a variety of methods to capture crayfish. Although some methods were quantitative, the overall effort was a qualitative sampling approach aimed at documenting species occurrences in a variety of lotic and lentic habitats. Sampling methods in deep habitats included boat electrofishing, trawling, and trapping (minnow traps with 3.2 cm openings and baited initially with canned dog food and later with pieces of fresh fish). In wadeable habitats, we used backpack electrofishing, seining, visual searches (using mask and snorkel, view buckets, rock flipping, and searches for molted carapaces along banks), and occasionally digging of burrows along banks. Our initial crayfish results led us to ask about the distributions of crayfish species in the continuously impounded portion of the reservoir. We obtained crayfish from an independent, concurrent study of black bass ( Micropterus henshalli Baker et al. and Micropterus salmoides (Lacepède)) diets in the reservoir. Black bass were captured via boat electrofishing at seven sites in the reservoir (Figure 1; Table 1) from July 2013 to September 2014 (Table 2). Bass were placed on ice and taken to the laboratory, where stomachs were dissected and contents stored in 95% ethanol. We attempted to identify all crayfish remains to species. In an attempt to determine whether certain crayfish taxa were new to the study area, we also examined gut contents of potentially predaceous fishes that were collected in the Sipsey Fork, Brushy Creek, and their tributaries from October 1993 to July 1995, preserved in 5% formalin, and stored in 70% ethanol (in part, Haag and Warren 1998). These sites were farther upstream in the drainages than were most of our recently sampled sites (Figure 1). We dissected all fish deemed capable of eating adult crayfish (assessed subjectively based on fish size and gape size) and removed all crayfish and parts of undigested crayfish exoskeletons from stomachs and intestines. We attempted to identify whole crayfish specimens, as well as the parts, to species. We used a DNA bar-coding approach to assist in the identification of the Orconectes ( Procericambarus ) sp. collected. Initial morphological identifications suggested that these individuals were Orconectes juvenilis (Hagen). We extracted DNA from two specimens and amplified a region of the mitochondrial cytochrome oxidase subunit I gene (COI), a standard bar-coding region of the mitochondrial genome useful for diagnosing problematic or unknown specimens. M ethods of DNA isolation, amplification, and sequencing are described in Taylor et al. (2014). Following methods described in Kessler et al. (this issue), we compared the COI sequences from our specimens to sequences from GenBank and from the Kessler et al. dataset, including each of the members of the O. juvenilis complex as well as other North American crayfish (see Table 2 in Kessler et al. in this issue, for details, including GenBank accession numbers). ...

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... 2. Direct sampling sites from which we collected crayfish in the Sipsey Fork, Brushy Creek, and their tributaries. “Other” represents sites where only known native species (as in Figure 1) were collected. Sites where no crayfish were collected are not shown. Site numbers coincide with those in Appendix 1, where collection details are provided.  ...