Conservation, Biology, and Natural History of Crayfishes from the Southern US 2010 Southeastern Naturalist 9(Special Issue 3):185–198 Phylogenetic Estimation of Species Limits in Dwarf Crayfishes from the Ozarks: Orconectes macrus and Orconectes nana (Decapoda: Cambaridae) Casey B. Dillman1,*, Brian K. Wagner2, and Robert M. Wood1 Abstract - Orconectes macrus (Neosho Midget Crayfish) and O. nana (Midget Crayfish) are a proposed sister-species pair with a small range on the western edge of the Ozark Highlands Physiographic province. Originally proposed as subspecies, O. macrus was elevated to full species status without explicit reason. This study utilized a single-locus mtDNA region (cytochrome oxidase I) to test: (1) monophyly of the species pair, (2) monophyly of the proposed species, and (3) examine if full species status is warranted for O. macrus. Phylogenetic reconstructions indicate that the two taxa indeed comprise a species pair and that each species is well supported as monophyletic. Additionally, there is significant phylogeographic structuring within each recognized species at fine spatial scales. Introduction Crayfishes are one of the most familiar aquatic invertebrates in eastern North America. This expansive area east of the Rocky Mountains is geologically and topographically diverse (Fenneman 1938), and supports one of the most diverse temperate aquatic fish faunas in the world (Burr and Mayden 1992, Warren et al. 2000). Crayfish biodiversity is also very high across this area (Hobbs 1988, Taylor et al. 2007). The diversity of aquatic organisms across the region is, at least in part, due to the varied habitats that occur throughout the landscape, particularly in areas of upland habitat that are characteristic of the Central Highlands Physiographic province (Central Highlands). The Central Highlands are the remnants of a once-continuous upland region that was altered by repeated advances and retreats of glacial ice (Mayden 1988, Pflieger 1971), with the maximum southern extent of glacial movement roughly equivalent to the Missouri and Ohio Rivers (Thornbury 1965). The Central Highlands are divided into the Eastern and Interior Highlands, with the Interior Highlands further subdivided into the Ozark and Ouachita Highlands. The Ozark Highlands is an isolated area in Arkansas, Kansas, Missouri, and Oklahoma (Thornbury 1965) that is approximately bordered by the Missouri River to the north, the Mississippi River to the east, and by the Arkansas River to the south. The aquatic fauna of the Ozark Highlands are a mixture of widespread and endemic taxa, with many of the latter occurring in both fishes (Pflieger 1997) and crayfishes (Pflieger 1996). Phylogenetic investigations into the 1Saint Louis University, Department of Biology, 3507 Laclede Avenue, St. Louis, MO. 63103. 2Arkansas Game and Fish Commission, 915 E. Sevier Street, Benton, AR 72015. *Corresponding author: dillmanc@slu.edu. 186 Southeastern Naturalist Vol. 9, Special Issue 3 fish fauna from the Ozark Highlands have shown that species exhibit complex interrelationships, with some species sister to taxa in adjacent drainages from the same province (e.g., Switzer and Wood 2002, 2009), while others are related to taxa from other isolated portions of the Central Highlands, i.e., Eastern Highlands or Ouachita Highlands, (e.g., Mayden 1988, Strange and Burr 1997, Wiley and Mayden 1985). The number of phylogenetic studies of fishes from the Ozark Highlands is far greater than for the invertebrate fauna; however, the first phylogenetic investigation into inter-relationships among crayfish species occurred in the Ozark Highlands for the genus Orconectes (Crandall and Fitzpatrick 1996). The scope of their study was genus-wide, but taxon sampling was greatest in the Ozark Highlands, where many endemic species of Orconectes occur (Pflieger 1996). Orconectes (Procericambarus) nana Williams (Midget Crayfish), and O. (Procericambarus) macrus Williams (Neosho Midget Crayfish) comprise a species pair endemic to the Ozark Highlands. Both species are highly restricted in distribution and primarily found in the western-flowing rivers of the region (i.e., the Neosho and Illinois river basins and their tributaries; Fig. 1). Orconectes nana and O. macrus were originally distinguished from one another, and described as subspecies, based on differences in length of the terminal elements of the gonopods (i.e., central projection and mesial process) in relation to total length of the gonopod (Williams 1952). Orconectes macrus was later elevated to full species status by Hobbs (1972), though no explicit reason for elevation was given. Fitzpatrick (1987) reorganized Orconectes into subgenera based on gonopod morphology and placed both O. nana and O. macrus in subgenus Procericambarus. Orconectes nana is restricted to the Illinois River basin, and O. macrus is found in tributaries to the Neosho River north of the Illinois River. Both stream systems are direct tributaries of the Arkansas River. Outside of the core range of this proposed species pair, a single allopatrically distributed population of O. nana is found in Prairie Creek, a headwater tributary of the White River basin. Molecular phylogenetic and population genetic approaches were utilized to understand the history of the proposed sister-species pair, investigate potential lineage subdivision, and, as such, define the distribution and limits of the species from a historical (evolutionary) perspective. These data were used to test the following hypotheses: (1) monophyly of the species pair, (2) monophyly of the proposed species, and (3) full species status for O. macrus. Additionally, at the population level, these data were used to investigate the null hypothesis of no genetic subdivision within each species across their range. These data also allow inferences into the biogeographic history for these crayfishes from the extreme western edge of the Ozark Highlands. Methods Forty-five individuals of O. nana and 55 O. macrus were sampled from most of their range (Table 1). Additionally, sequence data from Taylor and Knouft (2006) was included to provide a robust test of monophyly of the focal taxa, and included an additional individual of O. macrus (Appendix A). 2010 C.B. Dillman, B.K. Wagner, and R.M. Wood 187 Total genomic DNA was isolated using the QIAGEN DNeasy® extraction kit (QIAGEN Inc., Valencia, CA) as described by the manufacturer for animal tissues. Modifications to the protocol included an overnight tissue digestion and a final elution volume of 200 μl in ddH20. Polymerase chain reaction (PCR) was used to amplify 642 base pairs (bp) of the Cytochrome Oxidase I (coxI) locus from the mitochondrial genome using primers H-COI 5'- TAAACTTCAGGGTGACCAAAAAATCA-3' and L-COI 5'- GGTCAACAAATCATAAAGATATTG- 3' (Folmer et al. 1994). PCR reactions (50 μL) Figure 1. Map showing the global distribution of Orconectes macrus (■) and Orconectes nana (●) in the western Ozark Highlands. The fine dashed line indicates the approximate species range for O. macrus, and the variable dashed line indicates the approximate species range for O. nana. 188 Southeastern Naturalist Vol. 9, Special Issue 3 Table 1. Sampling locality information for Orconectes macrus and O. nana. Collection Number Sample location Drainage State County Latitude, Longitude number Orconectes (Procericambarus) macrus 586–590 Spavinaw Creek Neosho AR Benton 36.39635°, -94.41488° CBD 06–32 617–619, 621 Beaty Creek Neosho AR Benton 36.41686°, -94.60036° CBD 06–35 627–631 Spavinaw Creek Neosho AR Benton 36.34262°, -94.58675° CBD 06–36 552–558 Spanker Creek Neosho AR Benton 36.43025°, -94.21000° CBD 06–29 565–570 Little Sugar Creek Neosho AR Benton 36.47566°, -94.25027° CBD 06–30 576–580 Unnamed Creek Neosho AR Benton 36.48840°, -94.29850° CBD 06–31 598–601 Unnamed Creek Neosho AR Benton 36.48433°, -94.45797° CBD 06–33 608–611 Honey Creek Neosho AR Benton 36.48066°, -94.56296° CBD 06–34 800–804 Shoal Creek Neosho MO Newton 36.89594°, -94.36764° CBD 06–66 810–812, 814 Buffalo Creek Neosho MO McDonald 36.67116°, -94.60445° CBD 06–65 981, 983–986 Spring tributary to Spring River Neosho MO Lawrence 36.94581°, -93.79207° CBD07–23. Orconectes (Procericambarus) nana 478–482 Elk Horn Spring Br. Illinois AR Washington 36.06019°, -94.31139° CBD 06–22 488–490 Hamestring Creek Illinois AR Washington 36.09510°, -94.28716° CBD 06–23 503, 504, 506, 507 Spring 4 miles SE of Siloam Springs Illinois AR Benton 36.15478°, -94.49955° CBD 06–24 509–513 Creek at Siloam Springs Country Club Illinois AR Benton 36.19098°, -94.52324° CBD 06-25 519–523 Spring Fed Creek along Cornhoff Rd. Illinois AR Washington 36.15227°, -94.30472° CBD 06–26 529–535 Little Osage Creek Illinois AR Benton 36.25370°, -94.27140° CBD 06–27 637–641 Flint Creek Illinois AR Benton 36.24226°, -94.48721° CBD 06–37 649–653 Tributary of Flint Creek Illinois AR Benton 36.26191°, -94.42128° CBD 06–38 541–546 Prairie Creek at Atalanta Lake White AR Benton 36.33430°, -94.10320° CBD 06–28 2010 C.B. Dillman, B.K. Wagner, and R.M. Wood 189 consisted of 1–4 μL of total genomic DNA, 0.4 μM of each primer, 1U of Taq DNA polymerase (Promega Corporation, Madison, WI), 5 μL of Promega 10X DNA buffer, 2 mM MgCl2, 0.8 mM dNTPs, and ddH2O to volume. Thermal-cycling conditions consisted of an initial denaturation at 94 °C for 4 min followed by 45 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, and a final extension of 72 °C for 4 min. Amplified PCR fragments were purified of unincorporated dNTPs and primers using the QIAGEN MinElute® PCR purification kit (QIAGEN Inc., Valencia, CA). The purified products were subsequently used in cyclesequencing reactions with Applied Biosystems BigDye® terminated-cycle sequencing kits (Applied Biosystems, Foster City, CA). Thermal cycling for cycle sequencing was performed with PCR amplification primers and an initial denaturation step of 96 °C for 1 min followed by 45 cycles of 96 °C for 30 s, 50 °C for 15 s, and 60 °C for 4 min. DNA cycle-sequencing reactions for coxI were completed at Macrogen, Inc. sequencing facilities (World Meridian Venture Center 10F, Seoul, Korea). Sequence data were analyzed by eye for base calling using 4Peaks v1.7 (Griekspoor and Groothius 2005). Edited sequences were aligned using CLUSTAL X (Thompson et al. 1997), and the alignment was checked by eye. Phylogenetic hypotheses from the aligned sequences were reconstructed using maximum parsimony (MP) in PAUP*4.10b (Swofford 2003) with aid of the parsimony ratchet (Nixon 1999) as implemented in PAUPMacRat (Sikes and Lewis 2001). A heuristic search with TBR branch swapping was performed using 10 replicates of random sequence addition, holding 1 tree at each step with default MP settings and a maximum of 20,000 trees. The strict-consensus tree was constructed to determine nodes recovered in all topologies. Bootstrap support (Felsenstein 1985) with 10,000 pseudo-replications was completed using a full heuristic search, holding one tree at each step. Maximum likelihood topology searches were completed with GARLI v0.951 (Zwickl 2006), which implements a genetic algorithm to search tree space for the topology that gives the greatest -lnL score. GARLI v0.951 uses the GTR model of nucleotide evolution, a gamma distributed rate parameter, and an estimation of the proportion of invariable sites. Confidence in recovered nodes was assessed with 100 bootstrap replicates (Felsenstein 1985). Bayesian phylogenetic inference was performed with MrBayes v. 3.12b (Huelsenbeck and Ronquist 2001). Two independent runs, each consisting of five million generations, were completed with the Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) search algorithm, and MrModelTest (Nylander 2004) was used to select the best evolutionary parameters for the data as partitioned by codon position (i.e., 1st, 2nd, 3rd codon). The recovered log likelihood scores at each sampling interval (1000 generations) were plotted against the generation number to establish when stationarity was reached. Those trees that were part of the burn-in (i.e., pre-stationarity) were removed, and all of the remaining trees from each independent run were compiled into a single tree file. The resultant posterior probability 190 Southeastern Naturalist Vol. 9, Special Issue 3 Figure 2 (opposite page). Strict consensus maximum parsimony topology from the 20,000 most parsimonious phylogenetic reconstructions. Numbers associated with nodes are maximum parsimony bootstrap/maximum likelihood bootstrap/Bayesian PPS. In cases where a phylogenetic reconstruction methodology did not provide support for a recovered node, support values are given in order. Three clades of O. macrus and two groups of O. nana are recovered. Orconectes nana “B” is not a clade, but was considered separate for population estimations. scores (PPS) were used to infer support for the nodes in a 50% majority rule consensus tree. We consider bootstrap values of 70 or higher in either MP or ML and PPS of 0.95 or higher to be strong support for recovered nodes. Given the low levels of divergence sometimes observed in investigations of closely related interspecific taxa, standard intraspecific population genetic methodologies were utilized. Average pairwise genetic distances using Kimura 2-parameter estimates for within and between group averages were calculated using MEGA 3.0 (Kumar et al. 2004). Pairwise ΦST estimates for the mitochondrial loci (an FST analog for haploid data) for an a priori grouping scheme that mirrored phylogenetic recovery, i.e., three clades of O. macrus and two groups of O. nana, were computed in ARLEQUIN v2.0 (Schneider et al. 2000). Hierarchical population subdivision was investigated with an analysis of molecular variance (AMOVA; Excoffier et al. 1992) also based on recovered clades. Sequential Bonferroni correction was used (Rice 1989) to correct for multiple pairwise comparisons. Results A total of 642 nucleotides of cox-I sequence data was generated for 101 individuals from the O. nana and O. macrus species pair. The majority of nucleotide characters were constant (393), 32 characters were variable but uninformative for parsimony reconstruction, and the remaining 217 characters were parsimony informative. Parsimony searches recovered 20,000 most-parsimonious trees of 1827 steps each. The strict consensus of all 20,000 parsimony trees is shown in Fig. 2 (MP bootstrap support, ML bootstrap support, and PPS respectively shown above each node). The maximum parsimony hypothesis recovered 100 of 101 individual O. nana and O. macrus as monophyletic, and the two taxa are strongly supported as sister species. Each recognized species is further subdivided into smaller groups of individuals: clades A, B, and C for O. macrus, clade A for O. nana. Orconectes nana “B” was not a clade, but was recovered in some MP reconstructions, and it is used here only to ease discussion of the results. One individual tentatively identified as O. macrus (607) is recovered outside the larger clade containing all remaining O. nana and O. macrus, but given that O. macrus (607) is not a form I male, identification cannot be 100% positive. Monophyly for the species pair, exclusive of O. macrus (607) is highly supported, as is monophyly for each species by bootstrapping in MP searches (Fig. 2). 2010 C.B. Dillman, B.K. Wagner, and R.M. Wood 191 Maximum likelihood reconstruction using the GTR+I+Γ model resulted in a similar topology to MP, and bootstrap support, when present, from 192 Southeastern Naturalist Vol. 9, Special Issue 3 ML is given as the middle number in the string of support metrics at each node (Fig. 2). Monophyly of the species pair is strongly supported, as is monophyly of O. nana. Interestingly, bootstrap support for monophyly of O. macrus with ML is weak (bootstrap = 64), but the node is recovered and clades inside of the larger O. macrus clade exhibit support metrics similar to MP bootstrapping. Orconectes macrus 607 again is not recovered inside of the clades that comprise the remainder of the species pair. Models of nucleotide evolution for the 1st, 2nd, and 3rd codon positions were determined to be GTR+Γ, HKY85+I, and GTR+I, respectively. The two independent Bayesian searches each resulted in a total of 5001 sampled trees. Burn-in was completed by 50,000 generations (i.e., 50 sampled trees), and the first 100,000 generations were removed, leaving 4900 trees for reconstructing the majority-rule consensus. The PPS for each node is shown on the recovered maximum parsimony hypothesis (Fig. 2). The recovered topologies were similar to the MP hypothesis, and monophyly of the species pair and of each species, again exclusive of O. macrus 607, is strongly supported. Interestingly, support for O. macrus clade A was weak in PPS when compared to bootstrap support. Average pairwise inter- and intraclade divergence values are given above the diagonal and along the diagonal, respectively, in Table 2. Intraclade divergence estimates for O. nana and O. macrus ranged from a low of 0.01% to a high of 1.1% divergence within their respective clades. Interclade divergence (i.e., between species) ranged from 1.4 to 9.7%. Orconectes macrus (clade B) showed an average of 1.1% divergence within the clade, indicating a substantial amount of nucleotide sequence variation within this recovered group. Intraspecific ΦST values (0 = complete mixing, 1 = complete subdivision) ranged from 0.354 to 0.686 between clades of O. macrus to 0.818 between groups of O. nana (Table 2). Between O. nana and O. macrus, the values ranged from 0.859 to 0.976. All pairwise comparisons showed significant differentiation at P < 0.00005, except between clades B and C of O. macrus, which was significant at P < 0.001. The hierarchical distribution of genetic variance as recovered from AMOVA (Table 3) was significant in two of the three categories. Not surprisingly, the majority of variation (62.9%) was explained among groups, i.e., between the recognized species O. macrus and O. nana. However, this group was not statistically differentiated. The Table 2. Average pairwise interclade divergence (above the diagonal) and intraclade divergence (along the diagonal, in italics) for recovered clades of Orconectes nana and O. macrus. Below the diagonal are inter- and intraspecific ΦST values. An asterisk for ΦST estimates indicates significant differentiation with a P-value less than 0.0005. O. nana O. nana O. macrus O. macrus O. macrus Species A “B” A B C O. nana A 0.002 0.014 0.097 0.091 0.089 O. nana “B” 0.818* 0.003 0.097 0.086 0.085 O. macrus A 0.876* 0.951* 0.007 0.046 0.045 O. macrus B 0.899* 0.976* 0.686* 0.011 0.045 O. macrus C 0.911* 0.859* 0.666* 0.354 0.001 2010 C.B. Dillman, B.K. Wagner, and R.M. Wood 193 remaining variation of 28.12% and 8.88% was explained by the categories of among populations within groups and within populations, respectively, and were statistically significant. Interestingly, when all of O. nana was grouped as one population, i.e., clade A and group “B,” statistically significant differentiation between these species was demonstrated (data not shown). All statistical comparisons remained significant after sequential Bonferroni correction (Rice 1989). Discussion The sampling provided by this study indicates that these two species have not been in contact for an extensive period of time (as indicated by the deep divergence between recognized species). Additionally, these data indicate that there is sub-division within each of the recognized species (i.e., recovered clades in each species) and that the subdivision within each clade is geographically structured, as is the divergence between each of these species (≈9.5% divergence). Orconectes nana clade A, as recognized by the moderately supported mitochondrial lineage data recovered in this study, is confined to the Illinois River proper, Osage Creek (a tributary to the Illinois River), and the disjunct population located in Prairie Creek (White River drainage). A previous stream connection between the Neosho River and the White River has been hypothesized based on faunal distribution patterns in fishes (Branson 1967), and data presented here from O. nana support this hypothesized former connection. The second group (“B”) of O. nana is restricted to Flint Creek, a tributary to the Illinois River that is very close geographically to clade A. There is, however, a 1.4% sequence divergence between clade A and what is termed “B” here. Thus, there appears to be fine-scale subdivision in O. nana across its very small range. Three clades of O. macrus (A, B, and C) were recovered in these analyses. Individuals of O. macrus clade A were found as far south as Spavinaw Creek and its tributary, Beaty Creek, in Arkansas. They were also found in sampled tributaries of Little Sugar Creek and Elk River in Arkansas. Orconectes macrus can be found throughout the Spring River and its tributaries, exclusive of North Fork Spring River and portions of Shoal Creek in Missouri (Pflieger 1996). Sampling for the more northern localities was less dense. Despite the less-dense sampling of these more northerly distributed individuals, the Table 3. Hierarchical analysis of molecular variance (AMOVA) based on the recovered phylogenetic hypotheses. Orconectes macrus and O. nana were each separated into distinct groups with three and two populations respectively. Orconectes nana “B” was considered a separate group from O. nana A despite it not being a distinct clade. Sum of Percentage Source of variation d.f. squares Variance of variation P-value Among groups 1 1103.34 17.41 62.99 0.10 Among populations within groups 3 297.13 7.77 28.12 0.00 Within populations 95 233.3 2.46 8.88 0.00 Total 99 1633.77 27.64 99.99 194 Southeastern Naturalist Vol. 9, Special Issue 3 molecular data suggest they comprise two distinct evolutionary groups, reported here as clades B and C, that are ≈4.5% divergent from O. macrus clade A. Clade B (O. macrus) contains five individuals that were sampled from Shoal Creek. This clade also contains one individual sampled from Sycamore Creek in Ottawa County, OK by Taylor and Knouft (2006), located downstream from the mouth of Shoal Creek. Clade B also contained the largest average sequence divergence estimate within any of the clades. Clade C (O. macrus) was sampled from the headwaters of the Spring River. It was one of the most geographically isolated populations to be included in this study, and exhibited ≈4.5% divergence from clade B O. macrus, indicating a lack of maternal gene flow among these clades. Interestingly, Clade C had the lowest ΦST value among all pairwise comparisons, suggesting the more northerly distributed populations (i.e., Shoal Creek, Spring River, and their tributaries) may have shared more recent contact than the populations found in the southern part of their range. Orconectes macrus is also found in a small portion of the Spring River in Cherokee County, KS (Ghedotti 1998), and inclusion of samples from this area could provide increased resolution concerning the northern and southern distributions of O. macrus. Additionally, it is possible that within the broad categories of longer versus shorter terminal elements of the gonopods used to diagnose this species pair, detailed intraspecific investigations may reveal fixed geographic variation in the length of these terminal elements as documented in the recovered haplotype tree. Both O. macrus and O. nana exhibited population subdivision. While this investigation utilized a single mtDNA locus, the subdivision noted provides an important first step in understanding the evolutionary history of these highly localized dwarf crayfish species. Based on results from this study it is evident that biodiversity, in terms of evolutionary lineages, is greater than currently recognized taxonomically. In addition, historical intra-drainage complexity is indicative of relationships within this group. Specifically, there is biogeographic structuring within each species in the Illinois and Neosho Rivers and their tributaries, and there was also likely a historical headwater connection with the White River. These data indicate that anthropogenic movement of these taxa could be detrimental to understanding the full history of this species group, as well as our full understanding of the nature of these species. Efforts should be made to discourage and eliminate movement of individuals between areas, thus maintaining the natural evolutionary history of dwarf Orconectes from the Ozark Highlands. Acknowledgments We thank Mark Kottmyer for extensive field collection help, and Stuart Welsh for publication support. The publication of this manuscript was supported, in part, by the US Geological Survey Cooperative Research Unit Program, including the West Virginia Cooperative Fish and Wildlife Research Unit. We would also like to thank the US Fish and Wildlife Service and Arkansas Game and Fish Commission for funding. For help in the field, C.B. Dillman and R.M. Wood also thank Justin Baker, Jeff Ray, Nick Lang, and the summer 2007 Aquatic Ecology class at Saint Louis University. 2010 C.B. Dillman, B.K. Wagner, and R.M. Wood 195 Literature Cited Branson, B.A. 1967. Fishes of the Neosho river system in Oklahoma. American Midland Naturalist 78:126–154. Burr, B.M., and R.L. Mayden. 1992. Phylogenetics and North American freshwater fishes. Pp. 18–75, In R.L. Mayden (Ed.). Systematics, Historical Ecology, and North American Freshwater Fishes. Stanford University Press, Stanford, CA. 969 pp. Crandall, K.A., and J.F. Fitzpatrick, Jr. 1996. Crayfish molecular systematics: Using a combination of procedures to estimate phylogeny. Systematic Biology 45:1–26. Excoffier, L., P.E. Smouse, and J.M Quattro. 1992. 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Robison, S.T. Ross, and W.C. Starnes. 2000. Diversity, distribution, and conservation status of the native freshwater fishes of the Southern United States. Fisheries 25:7–31. Wiley, E.O., and R.L. Mayden. 1985. Species and speciation in phylogenetic systematics, with examples from the North American fish fauna. Annals of the Missouri Botanical Garden 72:596–635. Williams, A.B. 1952. Six new crayfishes of the Genus Orconectes (Decapoda: Astacidae) from Arkansas, Missouri and Oklahoma. Transactions of the Kansas Academy of Science 55:330–351. Zwickl, D. 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph.D. Dissertation. University of Texas, Austin, TX. Program available online at http:// www.zo.utexas.edu/faculty/antisense/Garli.html. 2010 C.B. Dillman, B.K. Wagner, and R.M. Wood 197 Appendix A. Orconectes and outgroup taxa from the dataset of Taylor and Knouft (2006). GenBank Genus (subgenus) and species access no. Cambarus (Cambarus) C. bartonii cavatus Hay (Appalachian Brook Crayfish) AY701190 Cambarus (Depressicambarus) C. graysoni Faxon (Twospot Crayfish) AY701192 Cambarus (Lacunicambarus) C. diogenes Girard (Devil Crawfish) AY701191 Hobbseus H. valleculus (Fitzpatrick) (Choctaw Riverlet Crayfish) AY701193 Orconectes (Billecambarus) O. harrisonii (Faxon) (Belted Crayfish) AY701189 Orconectes (Buannulifictus) O. hobbsi Penn (Ponchartrain Painted Crawfish) AY701211 O. meeki brevis (= O. meeki) Williams AY701212 (Meek's Short Pointed Crayfish) O. meeki meeki (= m. meeki) (Faxon) (Meek's Crayfish) AY701213 O. palmeri longimanus (= O. palmeri)(Faxon) AY701214 (Western Painted Crayfish) Orconectes (Crockerinus) O. eupunctus Williams (Coldwater Crayfish) AF474349 O. illinoiensis Brown (Shawnee Crayfish) AY701226 O. jeffersoni Rhoades (Louisville Crayfish) AF474351 O. marchandi Hobbs (Mammoth Spring Crayfish) AF474353 Orconectes (Faxonius) O. indianensis (Hay) (Indiana Crayfish) AY701198 O. limosus (Rafinesque) (Spinycheek crayfish) AY701199 O. wrighti Hobbs (Hardin Crayfish) AY701200 Orconectes (Gremicambarus) O. compressus (Faxon) (Slender Crayfish) AY701217 O. jonesi Fitzpatrick (Sucarnoochee River Crayfish) AY701221 O. nais (Faxon) (Water Nymph Crayfish) AY701223 Orconectes (Hespericambarus) O. deanae Reimer and Jester (Conchas Crayfish) AY701205 O. difficilis (Faxon) (Painted Crayfish) AY701206 O. hartfieldi Fitzpatrick and Suttkus (Yazoo Crayfish) AY701207 O. maletae Walls (Kisatchie Painted Crayfish) AY701208 O. perfectus Walls (Complete Crayfish) AY701210 O. perfectus (= O. perfectus2) Walls (Complete Crayfish) AY701209 Orconectes (Orconectes) O. inermis inermis (= O. inermis) Cope (Ghost Crayfish) AY701201 O. pagei Taylor and Sabaj (Mottled Crayfish) AY701202 O. pellucidus (Tellkampf) (Mammoth Cave Crayfish) AY701203 198 Southeastern Naturalist Vol. 9, Special Issue 3 GenBank Genus (subgenus) and species access no. Orconectes (Procericambarus) O. acares Fitzpatrick (Redspotted Stream Crayfish) AY701227 O. barrenensis Rhoades (Barren River Crayfish) AY701228 O. carolinensis Cooper and Cooper (North Carolina Spiny Crayfish) AY701229 O. cristavarius Taylor (Spiny Stream Crayfish) AY701230 O. forceps (Faxon) (Surgeon Crayfish) AY701231 O. hylas (Faxon) (Woodland Crayfish) AY701232 O. juvenilis (Hagen) (Shrimp Crayfish) AF474352 O. longidigitus (Faxon) (Longpincered Crayfish) AY701234 O. luteus (Creaser) (Golden Crayfish) AY701235 O. macrus Williams (Neosho Midget Crayfish) AY701236 O. medius (Faxon) (Saddlebacked Crayfish) AY701237 O. menae (Creaser) (Mena Crayfish) AY701238 O. mirus (Ortmann) (Wonderful Crayfish) AY701239 O. neglectus (= O. n. chaenodactylus) Williams (Gap Ringed Crayfish) AY701240 O. neglectus (= O. n. neglectus) (Faxon) (Ringed Crayfish) AY701241 O. ozarkae Williams (Ozark Crayfish) AY701242 O. peruncus (Creaser) (Big Creek Crayfish) AY701243 O. punctimanus (Creaser) (Spothanded Crayfish) AY701244 O. putnami (Faxon) (Phallic Crayfish) AY701245 O. quadruncus (Creaser) (St. Francis River Crayfish) AY701246 O. ronaldi Taylor (Mud River Crayfish) AY701247 O. rusticus (= O. rusticus) (Girard) (Rusty Crayfish) AY701248 O. rusticus (= O. rusticus2) (Girard) (Rusty Crayfish) AY701249 O. saxatilis Bouchard and Bouchard (Kiamichi Crayfish) AY701250 O. spinosus (Bundy) (Coosa River Spiny Crayfish) AY701251 O. williamsi Fitzpatrick (Williams’ Crayfish) AY701252 Orconectes (Rhoadesius) O. kentuckiensis Rhoades (Kentucky Crayfish) AF474369 O. sloanii (Bundy) (Sloan Crayfish) AY701197 Orconectes (Trisellescens) O. alabamensis (Faxon) (Alabama Crayfish) AY701215 O. chickasawae Cooper and Hobbs (Chickasaw Crayfish) AY701216 O. cooperi Cooper and Hobbs (Flint River Crayfish) AY701218 O. etnieri Bouchard and Bouchard (Ets Crayfish) AY701219 O. immunis (Hagen) (Calico Crayfish) AY701220 O. mississippiensis (Faxon) (Mississippi Crayfish) AY701222 O. rhoadesi Hobbs (Fishhook Crayfish) AY701224 Procambarus (Ortmannicus) P. acutus (Girard) (White River Crawfish) AF474366 Procambarus (Scapulicambarus) P. clarkii (Girard) (Red Swamp Crawfish) AY701195