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J OURNAL OF C RUSTACEAN B IOLOGY, 32(6), 962-971, 2012 HYBRIDIZATION BETWEEN AN INVASIVE AND A NATIVE SPECIES OF THE CRAYFISH GENUS ORCONECTES IN NORTH-CENTRAL OHIO Sierra T. Zuber 1 , Katherine Muller 2 , Roger H. Laushman 1 , and Angela J. Roles 1,∗ 2 Department 1 Biology Department, Oberlin College, Oberlin, OH 44074, USA of Plant Biology and Conservation, Northwestern University, Evanston, IL 60208, USA ABSTRACT Hybridization between native and invasive species is an important but little-studied factor in crayfish invasions, with few documented natural cases. Here, we report genetic evidence of hybridization between invasive O. rusticus (Girard, 1852) and native O. sanbornii (Girard, 1852) in the Huron River in north-central Ohio based on a combination of molecular markers: nuclear DNA, mitochondrial DNA, and allozymes. Although we found no fixed differences between the species at nuclear DNA loci, fixed differences in mtDNA and allozyme loci confirmed the presence of individuals of hybrid ancestry. We also found preliminary evidence of possible mitochondrial recombination and biparental inheritance (though the existence of rare haplotypes cannot be excluded at the present time). We examined morphological features of both species in sympatry and allopatry and confirmed species-diagnostic morphological features including gonopod traits and shape of the annulus ventralis. This study is the second to detect hybridization between O. rusticus and a congener, which suggests that this may be an important mechanism of invasion by O. rusticus when closely related species are present. Further study of this system, including more extensive sampling and additional molecular markers, is necessary to understand the extent and implications of hybridization for the native species. K EY W ORDS: crayfish, hybridization, introgression, invasion, Orconectes DOI: 10.1163/1937240X-00002091 I NTRODUCTION A wide body of research has examined the ecological impacts of invasive crayfish, including displacement of native crayfishes (Capelli and Munjal, 1982; Hill and Lodge, 1999), reductions in prey taxa (Rosenthal et al., 2006; Pintor and Sih, 2011), and other community changes (Hamr, 2002; Roth et al., 2007; Bobeldyk and Lamberti, 2008). The displacement of native crayfishes is a particularly important consequence of invasion in North America, which holds the majority of the world’s crayfish biodiversity (Master et al., 1998; Perry et al., 2002; Crandall and Buhay, 2008). While the ecological impacts of crayfish invasions are well known, the evolutionary impacts have received little attention. In particular, hybridization between native and invader is an important evolutionary phenomenon in freshwater species invasions (Perry et al., 2002). Species that diverge in allopatry do not experience selective pressure to develop mating barriers, leading to the possibility of hybridization after secondary contact (Fisher, 1930; Dobzhansky, 1940; Kaneshiro, 1980; Dumont and Adriaens, 2009). Hybridization and reproductive interference can have a variety of impacts on an invasion, slowing or speeding the loss of a native species depending on the viability and fertility of hybrids and relative fitnesses of parental species and hybrid individuals (Ellstrand and Schierenbeck, 2000; Perry et al., 2001b). Few examples of crayfish hybridization exist in the literature. Induced hybridization has generally resulted in non-viable or sterile offspring or reduced brood sizes of ∗ Corresponding fertile offspring (Berrill, 1985; Lawrence and Morrissy, 2000; Reynolds, 2002; Lawrence, 2004). The relevance of these studies to natural populations is, however, unknown. Most reports of natural hybridization are supported solely by morphological evidence (Crocker, 1957; Crocker and Barr, 1968; Capelli and Capelli, 1980; Smith, 1981), which is not conclusive. Genetic evidence of natural crayfish hybridization has been found in two systems, both believed to be the result secondary contact (Procambarus spp.: Sbordoni et al., 1988; Cesaroni et al., 1992; Orconectes spp.: Perry et al., 2001b). While the studies of hybridization in Procambarus examined two native species, Perry et al. (2001b) studied hybridization between a native and an invasive species of Orconectes. Examining genetic markers (allozyme loci) and morphology in a hybrid zone between invasive Orconectes rusticus (Girard, 1852) and resident O. propinquus (Girard, 1852), Perry et al. (2001b) found evidence of introgression between species. While an earlier study of hybridization between these species suggested reproductive interference favoring the invasion of O. rusticus (Berrill, 1985), Perry et al. (2001b) demonstrated that hybrids outcompeted both parental species within the hybrid zone, thus slowing the advance of the O. rusticus invasion front. Orconectes rusticus is a notorious invader in North American waterways. Within the last 50 years O. rusticus has expanded its range from Indiana, Kentucky, and western Ohio to much of the Midwest and Northeast United States, author; e-mail: [email protected] © The Crustacean Society, 2012. Published by Brill NV, Leiden DOI:10.1163/1937240X-00002091 ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES plus the western states of Utah and Oregon, as well as north into Canada (Ontario), and south to Tennessee and North Carolina (United States Geological Survey, 2012). Use of O. rusticus as live bait is likely responsible for much of this spread (Berrill, 1978). We examined hybridization between introduced O. rusticus and native O. sanbornii (Faxon, 1884) in north-central Ohio. The native range of O. sanbornii extends from central Ohio to parts of Kentucky and West Virginia (Fitzpatrick, 1967; Thoma and Jezerinac, 2000). While O. sanbornii is not classified as endangered or threatened, populations in parts of Ohio and West Virginia are experiencing local decline with the introduction of O. rusticus through its use as live bait (Thoma and Jezerinac, 2000). Several studies have explored O. rusticus-O. sanbornii interactions, including one laboratory study demonstrating that O. sanbornii males prefer to copulate with O. rusticus females rather than with conspecifics (Butler and Stein, 1985). Butler and Stein (1985) also found that O. rusticus exhibits faster juvenile growth and lower susceptibility to predation than O. sanbornii, giving the invader additional competitive ecological advantages. However, Butler and Stein (1985) did not observe the outcome of mating attempts, thus the possibility of hybridization between these species in natural populations remained speculation. In the course of our field research on the Huron River, where these two species exist in sympatry, species identification on the basis of morphology is not always clear. We sought to identify species diagnostic genetic markers by genotyping nuclear DNA loci, mitochondrial DNA loci, and allozyme loci in allopatric populations, then genotyping the sympatric population to determine whether hybridization is occurring. We also explored morphological differences between the species in allopatry as we discovered that some morphological traits frequently used to identify species in the field exhibit more within-species variation than we had expected. 963 M ATERIALS AND M ETHODS Sample Collection Crayfish were collected in fall 2009 from the Portage River, Vermilion River, and Huron River, and in summer 2010 from a second site on the Huron River (Fig. 1). The Portage and Vermilion rivers were selected as allopatric sites for O. rusticus and O. sanbornii, respectively, whereas the species are sympatric in the Huron River. Crayfish were caught in seine and hand nets, by eye and by disturbing rocks in riffle zones. We collected 21 O. rusticus (9 male, 12 female) from the Portage River, 24 O. sanbornii (12 male, 12 female) from the Vermilion River, 36 sympatric crayfish (21 male, 15 female) from the Huron River Monroeville site (HRM) and 12 sympatric crayfish (9 male, 3 female) from the Huron River Lover’s Lane Bridge site (LLB). Crayfish were transported back to the lab and maintained in 75.7 liter tanks (20 gallon) until frozen and preserved whole at −80°C. DNA Analysis DNA Isolation.—DNA was isolated from pleopod tissue, collected before we sacrificed crayfish, using the Qiagen Puregene DNA Purification kit (Qiagen, Germantown, MD). For each individual, one or more pleopods were removed for DNA isolation and stored in 300 μl Cell Lysis Buffer (Qiagen). Tissue was disrupted with a mortar and pestle and maintained at room temperature. Proteins were digested with 1.5 μl Proteinase K (20 mg/ml) at 55°C for 1 h, then precipitated with 100 μl Puregene Protein Precipitation Solution and pelleted by centrifugation; this step was repeated if the supernatant was not clear. DNA was precipitated from the supernatant with 300 μl 100% isopropanol and pelleted by centrifugation, then washed with 300 μl 70% ethanol and dried overnight at room temperature. DNA was rehydrated in 50 μl Puregene DNA Hydration Solution and incubated either for one hour at 65°C with occasional agitation or overnight at room temperature. Purified DNA was stored at −20°C. Loci and Amplification.—We amplified four nuclear DNA regions: glyceraldehyde-3-phosphate dehydrogenase Fig. 1. Map of crayfish sampling sites in north-central Ohio on the Portage River (allopatric for Orconectes rusticus), Vermilion River (allopatric for O. sanbornii), and Huron River (sympatric for both species), with GPS coordinates for each site. Crayfish were collected fall 2009 and summer 2010. 964 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012 (GAPDH), 28S ribosomal RNA (28S), histone H3 (H3) and phosphoenolpyruvate carboxykinase (PEPCK). We amplified two mitochondrial DNA regions: 16S ribosomal RNA (16S) and cytochrome oxidase II (COII). Primer sequences, amplification conditions, and sequencing details are reported in the Supplementary Material in the online edition of this journal, which can be accessed via http://booksandjournals. brillonline.com/content/1937240x/32/6. RFLP.—Restriction digests were performed on 16S and COII amplified from allopatric samples to identify diagnostic differences in cut sites. For 16S, we used the restriction enzyme AseI, which cuts in O. sanbornii but not in O. rusticus. For COII, we used PvuII, which cuts in O. rusticus but not in O. sanbornii. Each 30 μl reaction contained 12 μl of undiluted PCR product, 3 μl of 10× restriction enzyme buffer (NEB), 20 units of restriction enzyme, and water to volume. Reactions were incubated at 37°C for 1-2 h before being stopped with 1 μl EDTA (0.5 M). The resulting fragments were run on a 2% agarose gel and stained with ethidium bromide. Species identity was assigned by visual inspection of fragment number and length. Allozyme Analysis We extracted enzymes from frozen tail muscle tissue ground with a mortar and pestle in liquid nitrogen and extracted enzymes using three extraction buffers (recipes in Supplementary Material). Extracted enzymes were filtered through Miracloth and absorbed onto nitrocellulose wicks (Whatman 3mm), then stored at −80°C. Enzymes were separated using horizontal electrophoresis on 11% or 12% starch gels (StarchArt Lot #W670) for 6-8 h with various electrode/gel buffer systems at 4°C (Table 1 and Supplementary Material). We stained for 12 enzymes (Table 1) using stains given in Murphy et al. (1990). Gels were examined by eye after staining and scored by hand relative to a standard run on each gel (crayfish HL1; see Supplementary Material). For Idh, 18 allopatric O. rusticus and 24 allopatric O. sanbornii were genotyped; for all other enzymes 12 allopatric individuals of each species were genotyped to assess variability. Morphology Analysis For sympatric samples from the Huron River, each crayfish was field identified to species based on coloration. A reddish-brown lateral carapace “rusty spot” and orange and black claw tips characterize O. rusticus; a mottled brown tail and orange or orange and dark claw tips characterize O. sanbornii. For the Huron River sites, we field-identified 23 O. rusticus, 10 O. sanbornii, and 3 putative hybrids at HRM in 2009 and 12 O. rusticus at LLB in 2010. Preserved allopatric (9 male, 12 female O. rusticus; 11 male, 7 female O. sanbornii) and sympatric (19 male, 18 female) crayfish were measured for 15 quantitative morphological characteristics (Fig. 2). Because overall size is highly variable in crayfish and many traits covary strongly with size, we analyzed 15 size-adjusted characters to account for this source of variation, e.g., chela length divided by carapace length, plus carapace length itself. For each character, differences between the species in allopatry were analyzed by ANOVA using JMP (SAS Institute). Traits that 1) exhibited significant differences in the mean in allopatry, and 2) had non-overlapping distributions were designated as species diagnostic; these characters were then examined in sympatric crayfish to observe similarities and differences between allopatric and sympatric populations. Rostral margins and the annulus ventralis were photographed with a Canon EOS Rebel digital camera mounted on a dissecting microscope (Westover Scientific, Mill Creek, WA, USA). Photographs of allopatric individuals were examined by eye for qualitative differences in shape for these two traits. We then assessed which species the photographs of sympatric individuals resembled most closely. R ESULTS DNA Analysis Sequence Comparison of Allopatric Crayfish.—We obtained sequences from both species for all nuclear and mitochondrial loci except the nuclear locus H3 (only one sequence, from O. rusticus). The nuclear loci for which we had multiple sequences proved largely uninformative for species identification due to either lack of variation or lack of fixed dif- Table 1. Allozymes extracted from Orconectes rusticus and O. sanbornii tail muscle tissue, separated by gel electrophoresis, and stained. R = O. rusticus, S = O. sanbornii. * See Supplementary Material; + Multiple, resolution was not sufficient to determine exact number of alleles present. Enzyme Abbreviation IUBMBNC number Running buffer system∗ Alleles observed Aspartate aminotransferase Alkaline phosphatase Esterase Fructose-bisphosphatase Aat Alp Est Fbp 2.6.1.1 3.1.3.1 3.1.1.3.1.3.11 2a 7 10 5(1), 5(2), 5(3) Glucose-6-phosphate dehydrogenase Glutamate dehydrogenase Isocitrate dehydrogenase G6ph Gdh Idh 1.1.1.49 1.4.1.2 1.1.1.42 2a 7, 8(-) 5(1), 5(2), 5(3), P Lactose dehydrogenase Malate dehydrogenase Malic enzyme Phosphoglucoisomerase Phosphoglucomutase Ldh Mdh Me Pgi Pgm 1.1.1.27 1.1.1.37 1.1.1.40 5.3.1.9 5.4.2.2 MC 6.9 MC 6.9, 1a, 2a MC 6.9 10 7, 8(-) 1 1 + R−2 S−1 1 1 R−1 S−1 1 1 1 + 1 965 ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES Table 2. Summary of sequence variation within and between species. R = Orconectes rusticus, S = O. sanbornii. GenBank Accession numbers JQ397605-JQ397631. Locus GAPDH 28S PEPCK H3 16S COII No. sequences R S Base pairs aligned 1 3 3 1 3 3 3 3 1 0 3 3 763 698 544 – 543 556 No. shared No. fixed polymorphisms differences 3 0 2 – 1 3 1 0 0 – 17 28 two mitochondrial loci (HL17; Supplementary Material). Of sympatric crayfish from the LLB site collected in 2010, 83% (10 of 12) had the O. rusticus mitochondrial haplotype for both loci (Table 3). The other two individuals had conflicting banding patterns for the two loci (Supplementary Material). In total, three individuals were found to have conflicting banding patterns at the two mitochondrial loci. We repeated the amplification and digestion for all three DNA samples and obtained the same result in all cases. For two cases (HL17, SLL12) the individuals displayed the pattern characteristic of O. sanbornii for the 16S locus and the pattern of O. rusticus for the COII locus. The remaining individual, SLL10, had the O. rusticus pattern for COII but for 16S displayed three bands – with sizes matching the two digested bands characteristic of O. sanbornii and a single undigested band as in O. rusticus. We discuss possible interpretations of these results below. Allozyme Analysis Fig. 2. Quantitative characters measured on preserved allopatric Orconectes rusticus and O. sanbornii, and sympatric crayfish of both species and putative hybrids. Measurements were made to the nearest hundredth of a millimeter with digital calipers (Mitutoyo Absolute Digimatic CD-6 CX, Mitutoyo America) and a dissecting microscope (Westover Scientific, Mill Creek, WA, USA). Diagram modified with permission from Perry et al. (2001b). Body measurements: 1, carapace length; 2, areola length; 3, areola width; 4, carapace width; 5, acumen length; 6, rostrum width. Chela measurements: 7, dactyl length; 8, chela length; 9, palm width; 10, palm length; 11, chela gap width; 12, number of carpus spines. Gonopod measurements (males only): 13, gonopod total length; 14, central projection length; 15, mesial projection length. ferences (Table 2). The mitochondrial loci, 16S and COII, amplified consistently and contained both shared and fixed differences (Table 2). Thus, we were able to use differences in restriction sites of mitochondrial loci to identify maternal lineage in sympatric crayfish, but could not gain additional information on the ancestry of sympatric crayfish from any of the nuclear loci that we sequenced. GenBank Accession numbers are reported in Table 2. RFLP Analysis of Sympatric Crayfish.—Of the sympatric crayfish from the HRM site collected in 2009, 28% (9 of 32) had the O. sanbornii banding pattern and 69% (22 of 32) had the O. rusticus banding pattern for both loci (Table 3). One individual had conflicting banding patterns for the Of the 12 enzymes screened in allopatric individuals, eight were monomorphic across both species and four polymorphic (Table 1). Three of the polymorphic loci were not species diagnostic. The last polymorphic marker, Idh, was monomorphic within each species but variable between the species, thus Idh displays species-specific variability and was informative in sympatric individuals (Table 3). Following Perry et al. (2001a), we scored the O. rusticus allele as 110. The O. sanbornii allele moved only 85% as far from the cathode as the O. rusticus allele, thus we scored it as 94. We genotyped 43 sympatric crayfish (31 from HRM and 12 from LLB) for the Idh allozyme locus. Of these, 69.8% were homozygous for the O. rusticus allele, 18.6% were homozygous for the O. sanbornii allele and 11.6% were heterozygotes (Table 3). Two individuals exhibited disagreement between the allozyme banding pattern and mtDNA identity, with one individual having the O. rusticus genotype for Idh (HL16; on-line Supplementary Material) and the other having the O. sanbornii genotype for Idh (HL12; on-line Supplementary Material), suggesting the presence of backcrossed individuals. Morphological Analysis Annulus ventralis shape was qualitatively different for the two species in allopatry (Fig. 3; O. rusticus N = 12, O. sanbornii N = 10). No sympatric crayfish showed an intermediate morphology for the annulus ventralis; every 966 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012 Table 3. Summary of genotypes, haplotypes, and qualitative morphological characters of crayfish sampled from the sympatric Huron River. R = Orconectes rusticus; S = O. sanbornii. Shape of the female’s annulus ventralis was identified as described in Fig. 3. Note that for the allozyme locus Idh, the O. rusticus allele is scored as 110 (Perry et al., 2001a) and the O. sanbornii allele as 94 based on relative migration distances. Site Idh genotype RR RS SS N 16s haplotype R S R/S N COII haplotype R S N Annulus ventralis shape R S N HRM 2009 LLB 2010 Overall sympatric Allopatric O. rusticus Allopatric O. sanbornii 20 3 8 31 10 2 0 12 30 5 8 43 18 0 0 18 0 0 24 24 23 9 0 32 10 1 1 12 33 10 1 44 7 0 0 7 0 7 0 7 24 10 34 12 0 12 36 10 46 7 0 7 0 7 7 7 5 12 3 0 3 10 5 15 12 0 12 0 10 10 sympatric female crayfish (N = 15) was confidently assigned to one species’ shape or the other without reference to additional features or genetic data (Table 3; 10 O. rusticus shape, 5 O. sanbornii shape). In all but one case, annulus ventralis shape corresponded to mtDNA identity. The exception was HL16, which had O. sanbornii mtDNA and the O. rusticus Idh genotype: this putative hybrid individual had the O. rusticus annulus ventralis shape (online Supplementary Material). Rostral margins were variable within and between species, contrary to our expectations of concave margins in O. rus- ticus and straight margins in O. sanbornii (on-line Supplementary Material; Jezerinac et al., 1995). Both species exhibited approximately straight rostral margins in allopatry (except for two allopatric O. rusticus with concave rostral margins), thus rostral margin shape was not species diagnostic. All sympatric individuals photographed (N = 43) except HL1 had straight rostral margins. Thus, while only O. rusticus exhibited concave rostral margins, more often they displayed straight margins, as in O. sanbornii. For quantitative characters, species in allopatry exhibited significantly different means for 10 size-corrected traits (Ta- Fig. 3. Typical annulus ventralis shapes. A, Oroconectes rusticus (left); B, O. sanbornii (right). The annulus ventralis of O. rusticus females has a fairly straight medial suture on a raised ridge in the caudal half of the annulus ventralis, with two protrusions in the rostral quadrants, which slightly overhang the central region to create a small pocket. The annulus ventralis of O. sanbornii females is nearly flat, with a three-segment zig-zag medial suture that is strongly asymmetric. We observed a bias in our sample as to which direction the first suture segment slanted: of the 10 allopatric O. sanbornii individuals photographed, nine had a first suture slant from the animal’s rostral right to caudal left, one had a first suture slant from the animal’s rostral left to caudal right. 967 ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES Table 4. Mean (s.e.) and range of ratios of body, chela, and gonopod measurements for allopatric Orconectes rusticus (N = 21 total; 9 males) and O. sanbornii (N = 18 total; 11 males). A difference between the species means was tested by one-way ANOVA. L = length, W = width. Measurement Body ratios Carapace W/L Areola L/Carapace L Areola W/L Areola W/Carapace L Rostrum W/ Carapace L Acumen L/Carapace L Rostral W/Acumen L Chela ratios Dactyl L/Chela L Dactyl L/Palm L Palm W/Chela L Palm L/Chela L Palm W/Palm L Chela gap W/Chela L Gonopod ratios Central projection L/Gonopod L (Central projection L – Mesial projection L)/ Gonopod L O. rusticus O. sanbornii F -ratio P -value Mean (s.d.) Min-Max Mean (s.d.) Min-Max 0.49 (0.005) 0.34 (0.003) 0.15 (0.007) 0.05 (0.002) 0.10 (0.002) 0.09 (0.003) 1.16 (0.06) 0.44-0.55 0.32-0.37 0.09-0.22 0.03-0.08 0.09-0.13 0.05-0.12 0.92-1.88 0.47 (0.005) 0.33 (0.003) 0.18 (0.007) 0.06 (0.002) 0.10 (0.002) 0.09 (0.003) 1.09 (0.06) 0.44-0.51 0.31-0.36 0.14-0.22 0.05-0.07 0.07-0.12 0.05-0.12 0.76-2.01 F1,40 F1,40 F1,40 F1,40 F1,40 F1,39 F1,39 = 8.452 = 6.273 = 6.068 = 4.178 = 0.7035 = 0.4305 = 0.8496 0.006 0.016 0.018 0.048 0.407 0.516 0.362 0.61 (0.01) 1.66 (0.07) 0.39 (0.008) 0.38 (0.01) 1.06 (0.04) 0.04 (0.004) 0.52-0.69 1.20-3.17 0.28-0.47 0.21-0.46 0.81-1.82 0.01-0.08 0.56 (0.01) 1.35 (0.08) 0.40 (0.008) 0.42 (0.01) 0.95 (0.04) 0.04 (0.004) 0.53-0.61 1.08-1.62 0.33-0.46 0.33-0.52 0.80-1.12 0.02-0.06 F1,37 F1,37 F1,37 F1,37 F1,37 F1,37 = 16.05 = 8.220 = 0.0964 = 5.162 = 4.447 = 0.3273 0.0003 0.007 0.758 0.029 0.042 0.571 0.31 (0.02) 0.05 (0.006) 0.16-0.44 0.00-0.18 0.08 (0.02) 0.01 (0.006) 0.05-0.11 0.00-0.03 F1,38 = 105.9 F1,38 = 23.93 ble 4). While significant differences in the mean establish an average difference between the species, only traits which exhibit non-overlapping distributions are useful for species identification. Of these 10 traits, only the ratio of gonopod central projection length to total gonopod length displayed both significantly different means and non-overlapping distributions for the two species in allopatry (Table 4; Fig. 4). In all gonopod measurements and ratios, the range of values for allopatric individuals was far greater for O. rusticus (N = 9) than for O. sanbornii (N = 11). When these gonopod measurements were assessed in sympatric crayfish (N = 28), values fell primarily above the range for allopatric O. sanbornii – though a few in each case fell below the range of allopatric O. rusticus. In no case did a sympatric crayfish have a value for a gonopod length ratio that fell above the range of values for allopatric O. rusticus. Thus, the sympatric population displays morphology somewhat intermediate to the two species considered in allopatry for the species diagnostic gonopod length ratio. D ISCUSSION Our observations of nine sympatric individuals that exhibit mixed genotypes for nuclear and mitochondrial loci confirms that O. rusticus and O. sanbornii are hybridizing in the Huron River (Table 3). We identified five individuals heterozygous for the allozyme locus (Idh), two individuals with the allozyme genotype of one species and the mitochondrial genotype of the other, and two individuals with differ- <0.0001 <0.0001 ent haplotypes for the two mitochondrial loci. The existence of individuals with different nuclear and mitochondrial haplotypes suggests that hybrids are fertile and are backcrossing with one or both of the parent species. The mixed maternal origins of mitochondrial haplotypes in individuals of hybrid ancestry suggests that hybridization does not occur exclusively through males of one species mating with females of the other; instead, males of each species must have mated with females of the other or with hybrid individuals. As we have small samples sizes and few loci, we cannot at present speculate on directional trends in hybridization nor upon rates of hybridization. In fact, while we can establish a minimum of nine individuals of likely hybrid ancestry, our limited set of molecular markers lacks the resolution to identify all individuals of hybrid ancestry. We observed three individuals with conflicting data for the two, mtDNA loci. These individuals could represent rare haplotypes that exist in the allopatric populations but were not present in our samples. Alternatively, these unusual haplotypes could represent biparental inheritance followed by recombination in the mitochondrial genome, which is most commonly observed in cases of hybridization (Rokas et al., 2003; Barr et al., 2005) and is known to occur in animals (Sutovsky et al., 2000). Hybridization events may be especially prone to exhibiting evidence of recombination due to the breakdown of species-specific mechanisms that prevent paternal mtDNA inheritance (Barr et al., 2005). Our results are the first to report evidence of possible paternal leakage in Fig. 4. Boxplots of ratios exhibiting statistically significant differences (see Table 1) between allopatric Orconectes rusticus (N = 21 total; 9 males) and allopatric O. sanbornii (N = 18 total; 11 males). Sympatric individuals from the Huron River are included for comparison (N = 37 total; 19 males). Three Form II individuals are included (1 O. sanbornii and 2 sympatric individuals) as their values do not differ from the Form I individuals. A, carapace length; B, carapace width/length; C, areola length/carapace length; D, areola width/length; E, dactyl length/chela length; F, dactyl length/palm length; G, palm length/chela length; H, palm width/length; I, central projection length/total gonopod length; J, central minus mesial projection length/total gonopod length. 968 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012 ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES crayfish. Inter-population crosses in one crayfish species detected no evidence of mitochondrial recombination (Nguyen and Austin, 2004), however, if the mechanisms preventing paternal transmission are species-specific, then recombination would not be expected in intraspecific crosses. Our evidence stems from proposed interspecific crosses, where species-specific mechanisms may fail. In addition, we report evidence for recombination between two mitochondrial loci. As the frequency of crossing-over is positively correlated with distance, within-locus recombination is less likely than between-locus recombination. We caution that our results are suggestive but inconclusive: further, more extensive, sampling is necessary to confirm or refute the possibility of biparental inheritance and recombination in the mitochondrial genomes of these crayfish. We also lack the resolution in terms of sample sizes and number of loci to determine the rate at which hybridization may occur. However, we observe that the percentage of putative hybrids in our sample was fairly consistent between sites approximately eight miles of river distance apart (3/12 = 25% for LLB, sampled in 2010 and 6/32 = 19% for HRM, sampled in 2009) with an estimate of 20% overall, similar to the 23.4% reported for O. rusticus and O. propinquus in Trout Lake (Perry et al., 2001b). However, the addition of more molecular markers and larger sample sizes is necessary to make a reliable estimate of the rates of hybridization and backcrossing in the Huron River (Perry et al. used two diagnostic nuclear loci and sampled 781 individuals). The ability to identify hybrids by sight in the field is invaluable. We found many morphological traits that exhibit significantly different means between the two species in allopatry, though few appear promising for field identification of the two pure species and the hybrid due to overlapping ranges (Table 4). For example, the two ratios used by Perry et al. (2001b) to distinguish male O. rusticus, O. propinquus, and O. virilis (areola width/carapace length, (central-mesial projection length)/gonopod total length) had significantly different means in our sample, but were not truly diagnostic due to overlapping ranges. Only two, nonsize-corrected gonopod traits (central projection length and mesial projection length; Supplementary Material), one sizecorrected gonopod trait (central projection length/gonopod total length), and the shape of the annulus ventralis exhibited non-overlapping distributions. These differences are unsurprising as characteristics of gonopods and the annulus ventralis are commonly used to distinguish and classify crayfish species (Fitzpatrick, 1987). However, the lack of difference in the size-corrected values is surprising because differences in size-corrected characters have been used to identify crayfish species. Unexpectedly, rostral margin shape did not show fixed differences between species; concave rostral margins are a distinguishing characteristic of O. rusticus in dichotomous keys (Jezerinac et al., 1995). However, it is unknown whether O. rusticus in the Portage River may have experienced hybridization with another species of Orconectes in the past, leading to higher variance in these morphological measures. Additional sampling of O. rusticus populations throughout their native range is necessary to determine the generality of this result. 969 The lack of species-specific variation in the nuclear DNA and allozyme loci we tested suggests very little phylogenetic distance between O. rusticus and O. sanbornii, contrary to their current classification in separate subgenera: Procericambarus and Crockerinus, respectively. While the current classification scheme is based on morphology (Fitzpatrick, 1987), recent studies have addressed phylogenetic relationships within Orconectes using molecular markers (Crandall and Fitzpatrick, 1996; Fetzner, 1996; Taylor and Hardman, 2002; Wetzel et al., 2004; Taylor and Knouft, 2006). Of these studies, only Taylor and Knouft (2006) included both O. rusticus and O. sanbornii. In their parsimony analysis of COI sequences in most of the 82 species of Orconectes, O. rusticus and O. sanbornii were quite closely related, in a clade with only five other species, with O. rusticus in the group sister to O. sanbornii, the most basal member of the clade (Crockerinus and Procericambarus contain nearly 40 species in Fitzpatrick’s classification). Thus, the available molecular data seems to support a closer relationship between O. rusticus and O. sanbornii than previously determined. It is noteworthy that while O. propinquus is not part of the clade that includes O. rusticus and O. sanbornii, it does share a common ancestor with them one node back from the clade for O. sanbornii and O. rusticus; thus O. rusticus is known to hybridize with two relatively closely related species within the genus. Our data confirm hybridization between O. rusticus and O. sanbornii in the Huron River of north-central Ohio. The genotypes of putative hybrids also indicate that backcrossing is occurring; thus hybrids are viable and fertile. We are not presently able to estimate the rate at which F1 hybrids or backcrosses are formed; additional nuclear loci must be developed to further understand the dynamics and importance of hybridization in this invasion. More generally, assays for hybridization in additional O. sanbornii rivers that have been invaded by O. rusticus will allow us to establish whether this is a common or important mechanism of invasion for O. rusticus in this region. We also report the first evidence in crayfish of possible mitochondrial recombination and paternal leakage, resulting from interspecific mating; more extensive sampling is necessary to confirm or reject this hypothesis (alternatively, these results could represent rare haplotypes). We suggest that the use of multiple mitochondrial loci may increase the power to detect rare events of recombination and paternal inheritance in mitochondrial genomes. Finally, phylogenies based on additional markers would aid in understanding the true evolutionary relationships within the genus as well as provide information on the potential for hybridization of the invasive O. rusticus with other members of the genus. ACKNOWLEDGEMENTS The work described above comes from the undergraduate Honors Theses of KM and STZ. We would like to thank the Oberlin Biology Department and the Oberlin College Office of Sponsored Programs for funding this study. We would also like to thank Michael J. Moore for guidance with the lab work and two anonymous reviewers for their insightful comments on the manuscript. STZ thanks Katie A., Anthony B., Marta R. and Robin T. for lab and field assistance, and Joshua T. G. for help in creating maps and figures. 970 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012 R EFERENCES Agerberg, A. 1990. Genetic variation in three species of freshwater crayfish; Astacus astacus L., Astacus leptodactylus Aesch., and Pacifastacus leniusculus (Dana), revealed by isozyme electrophoresis. Hereditas 113: 101-108. Barr, C. M., M. Neiman, and D. R. Taylor. 2005. Inheritance and recombination of mitochondrial genomes in plants, fungi and animals. New Phytologist 168: 39-50. Berrill, M. 1978. Distribution and ecology of crayfish in the Kawartha Lakes region of southern Ontario. Canadian Journal of Zoology 56: 166177. . 1985. Laboratory induced hybridization of two crayfish species, Orconectes rusticus and O. propinquus. Journal of Crustacean Biology 5: 347-349. Bobeldyk, A. M., and G. A. Lamberti. 2008. A decade after invasion: evaluating the continuing effects of rusty crayfish on a Michigan river. Journal of Great Lakes Research 34: 265-275. Buhay, J. E., G. Moni, N. Mann, and K. A. Crandall. 2007. Molecular taxonomy in the dark: evolutionary history, phylogeography, and diversity of the cave crayfish in the subgenus Aviticambarus, genus Cambarus. Molecular Phylogenetics and Evolution 42: 435-448. Butler IV, M. J., and R. A. Stein. 1985. An analysis of the mechanisms governing species replacements in crayfish. Oecologia (Berlin) 66: 168177. Capelli, G. M., and J. F. Capelli. 1980. Hybridization between crayfish of the genus Orconectes: morphological evidence. Crustaceana 39: 121132. , and B. L. Munjal. 1982. Aggressive interactions and resource competition in relation to species displacement among crayfish of the genus Orconectes. Journal of Crustacean Biology 2: 486-492. Cesaroni, D., G. Allegrucci, and V. Sbordoni. 1992. A narrow hybrid zone between two crayfish species from a Mexican cave. Journal of Evolutionary Biology 5: 643-659. Crandall, K. A., and J. E. Buhay. 2008. Global diversity of crayfish (Astacidae, Cambaridae, and Parastacidae – Decapoda) in freshwater. Hydrobiologia 595: 295-301. , and J. F. Fitzpatrick Jr. 1996. Molecular systematics: using a combination of procedures to estimate phylogeny. Systematic Biology 45: 1-26. Crocker, D. W. 1957. The crayfishes of New York State. N.Y. Museum of Science Service Bulletin 335: 1-97. , and D. W. Barr. 1968. Handbook of the Crayfishes of Ontario. Life Sciences Miscellaneous Publications, Royal Ontario Museum, University of Toronto, 158 pages, 87 figures. Dobzhansky, T. 1940. Speciation as a stage in evolutionary divergence. The American Naturalist 74: 312-321. Dumont, H. J., and E. Adriaens. 2009. Experimental hybridization of two African Streptocephatus species (Crustacea, Branchiopodia: Anostraca). Current Science 96: 88-90. Ellstrand, N. C., and K. A. Schierenbeck. 2000. Hybridization as a stimulus for the evolution of invasiveness in plants? Proceedings of the National Academy of Sciences 97: 7043-7050. Faxon, W. 1884. Descriptions of new species of Cambarus; to which is added a synonymical list of the known species of Cambarus and Astacus. Proceedings of the American Academy of Arts and Sciences 20: 107158. Fetzner Jr., J. W. 1996. Biochemical systematics and evolution of the crayfish genus Orconectes (Decapoda: Cambaridae). Journal of Crustacean Biology 16: 111-141. , and K. A. Crandall. 2002. Genetic variation, pp. 291-326. In, D. M. Holdich (ed.), Biology of Freshwater Crayfish. Blackwell Science, Oxford. Fisher, R. A. 1930. Chapter VI: sexual reproduction and sexual selection, pp. 121-131. In, The Genetical Theory of Natural Selection. Clarendon Press, Oxford. Fitzpatrick Jr., J. F. 1967. The Propinquus group of the crawfish genus Orconectes (Decapoda: Astacidae). Ohio Journal of Science 67: 129172. . 1987. The subgenera of the crawfish genus Orconectes (Decapoda: Cambaridae). Proceedings of the Biological Society of Washington 100: 44-74. Girard, C. 1852. A revision of the North American Astaci, with observations on their habits and geographic distribution. Proceedings of Academy of Natural Sciences of Philadelphia 6: 87-91. Hamr, P. 2002. Orconectes, pp. 585-608. In, D. M. Holdich (ed.), Biology of Freshwater Crayfish. Blackwell Science, Oxford. Hill, A. M., and D. M. Lodge. 1999. Replacement of resident crayfishes by an exotic crayfish: the roles of competition and predation. Ecological Applications 9: 678-690. Jezerinac, R. F., G. W. Stocker, and D. C. Tarter. 1995. The crayfishes (Decapoda: Cambaridae) of West Virginia. Bulletin of the Ohio Biological Survey New Series, Columbus, OH. Kaneshiro, K. 1980. Sexual isolation, speciation and the direction of evolution. Evolution 34: 437-444. Lawrence, C. S. 2004. All-male hybrid (Cherax albidus × Cherax rotundus) yabbies grow faster than mixed-sex (C. albidus × C. albidus) yabbies. Aquaculture 236: 211-220. , and N. M. Morrissy. 2000. Genetic improvement of marron Cherax tenuimanus Smith and yabbies Cherax spp. in Western Australia. Aquaculture Research 31: 69-82. Master, L. L., S. R. Flack, and B. A. Stein. 1998. Rivers of Life: Critical Watersheds for Protecting Freshwater Diversity. Nature Conservancy, Arlington, VA. Murphy, R. W., J. W. Sites Jr., D. G. Buth, and C. H. Haulier. 1990. Proteins I: isozyme electrophoresis, pp. 45-126. In, D. M. Hillis and C. Moritz (eds.), Molecular Systematics. Sinauer Associates, Sunderland, MA. Nguyen, T. T. T., and C. M. Austin. 2004. Inheritance of molecular markers and sex in the Australian freshwater crayfish, Cherax destructor Clark. Aquaculture Research 35: 1328-1338. Perry, W. L., J. L. Feder, and D. M. Lodge. 2001b. Implications of hybridization between introduced and resident Orconectes crayfishes. Conservation Biology 15: 1656-1666. , D. M. Lodge, and J. L. Feder. 2002. Importance of hybridization between indigenous and nonindigenous freshwater species: an overlooked threat to North American biodiversity. Systematic Biology 51: 255-275. , J. L. Feder, G. Dwyer, and D. M. Lodge. 2001a. Hybrid zone dynamics and species replacement between Orconectes crayfishes in a Northern Wisconsin lake. Evolution 55: 1153-1166. Pintor, L. M., and A. Sih. 2011. Scale dependent effects of native prey diversity, prey biomass and natural disturbance on the invasion success of an exotic predator. Biological Invasions 13: 1357-1366. Reynolds, J. D. 2002. Growth and Reproduction, pp. 152-191. In, D. M. Holdich (ed.), Biology of Freshwater Crayfish. Blackwell Science, Oxford. Rokas, A., E. Ladoukakis, and E. Zouros. 2003. Animal mitochondrial DNA recombination revisited. Trends in Ecology and Evolution 18: 411417. Rosenthal, S. K., S. S. Stevens, and D. M. Lodge. 2006. Whole-lake effects of invasive crayfish (Orconectes spp.) and the potential for restoration. Canadian Journal of Fisheries and Aquatic Sciences 63: 1276-1285. Roth, B. M., J. C. Tetzlaff, M. L. Alexander, and J. F. Kitchell. 2007. Reciprocal relationships between exotic rusty crayfish, macrophytes, and Lepomis species in northern Wisconsin lakes. Ecosystems 10: 74-85. Sbordoni, V., G. Allegrucci, F. Baldari, and D. Cesaroni. 1988. Evolutionary genetics and morphometrics of a cave crayfish population from Chiapas (Mexico). International Journal of Speleology 17: 65-80. Smith, D. G. 1981. Evidence for hybridization between two crayfish species (Decapoda: Cambaridae: Orconectes) with a comment on the phenomenon in cambarid crayfish. American Midland Naturalist 105: 405-407. Sutovsky, P., R. D. Moreno, J. Ramalho-Santos, T. Dominko, C. Simerly, and G. Schatten. 2000. Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biology of Reproduction 63: 582-590. Taylor, C. A., and M. Hardman. 2002. Phylogenetics of the crayfish subgenus Crockerinus, genus Orconectes (Decapoda: Cambaridae), based on cytochrome oxidase I. Journal of Crustacean Biology 22: 874-881. , and J. H. Knouft. 2006. Historical influences on genital morphology among sympatric species: gonopod evolution and reproductive isolation in the crayfish genus Orconectes (Cambaridae). Biological Journal of the Linnaean Society 89:1-12. Thoma, R. F., and R. F. Jezerinac. 2000. Ohio Crayfish and Shrimp Atlas. Ohio Biological Survey, Columbus, OH. ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES Tsang, L. M., K. Y. Ma, S. T. Ahyong, T. Y. Chan, and K. H. Chu. 2008. Phylogeny of Decapoda using two nuclear protein-coding genes: origin and evolution of the Reptantia. Molecular Phylogenetics and Evolution 48: 359-368. United States Geological Survey. 2012. Orconectes rusticus. USGS Nonindigenous Aquatic Species Database, Gainesville, FL. http://nas.er.usgs. gov/queries/FactSheet.aspx?speciesID=214 Revision Date: 1/30/2008. Wetzel, J. E., W. J. Poly, and J. W. Fetzner Jr. 2004. Morphological and genetic comparisons of Golden Crayfish, Orconectes luteus, and Rusty Crayfish, O. rusticus, with range corrections in Iowa and Minnesota. Journal of Crustacean Biology 24: 603-617. R ECEIVED: 14 February 2012. ACCEPTED: 16 June 2012. S UPPLEMENTARY M ATERIAL Nuclear and Mitochondrial Loci.—Isolated DNA was amplified using PCR with a final reaction volume of 15 μl: 0.6 μl DNA (0.02× of stock; 0.5× for PEPCK), 0.15 μl of dNTPs (10 mM), 1.5 μl of 10× Sigma RedTaq PCR reaction buffer, 0.6 μl (0.225 μl for 16S and COII) of forward and reverse primers (10 μM), 0.6 μl RedTaq DNA polymerase (1 unit/μl; Sigma), and water to volume. Primer sequences, sources, and amplification conditions are reported in Table S1. PCR products were purified by combining 10 μl of PCR product with 4 μl of ExoSAP-IT (USB) and incubating at 37°C for 15 min to degrade remaining primers and nucleotides. A second incubation at 80°C for 15 min deactivated the enzyme. Samples were stored at −20°C until shipped on dry ice for sequencing at SeqWright (Houston, TX, USA) with an ABI Prism 3730xL DNA sequencer. Sequence Analysis.—Sequences were edited and aligned with either Sequencher 4.10.1 (Gene Codes) or Geneious (Biomatters). For the nuclear loci GAPDH, 28S, H3 and PEPCK, we examined the sequences for fixed differences between the species. For the mitochondrial loci 16S and 971 COII, we identified species-specific differences in restriction enzyme cut sites using Enzymex3.1 (Mekentosj). Buffers used for allozyme extraction from crayfish muscle tissue and gel electrophoresis. Extraction buffers Agerberg (1990): 21.40 g sucrose, 41.75 ml 0.1 M KH2 PO4 , 20.75 ml 1.0 M K2 HPO4 , 3.75 ml phenoxyethanol, in distilled water to 250 ml total volume Fetzner (1996): Saturated pyridoxal 5 -phosphate in distilled water W. L. Perry (pers. commun. to RHL): 0.04 g EDTA, 0.01 g NADP, 0.01 g NAD, 0.25 ml 2-mercaptoethanol, in 100 ml 0.1 M Tris-HCl buffer, pH 7.0 Running buffer systems 1a – Citric acid electrode buffer, 0.010 Histidine-HCl gel buffer, pH 7.0 2a – Tris-citric acid electrode buffer, 0.067 × (electrode buffer) gel buffer, pH 7.0 5(1) – Tris-citric acid electrode buffer, 0.7 × (electrode buffer) gel buffer, pH 7.2 5(2) – Tris-citric acid electrode buffer, pH 7.2, 0.7 × (electrode buffer) gel buffer, pH 7.4 5(3) – Tris-citric acid electrode buffer, pH 7.2, 0.035 × (electrode buffer) gel buffer, pH 7.4 7 – Lithium hydroxide-boric acid electrode buffer, Triscitric acid gel buffer, pH 8.3 8(-) – Lithium hydroxide-boric acid electrode buffer, pH 8.1, Tris-citric acid gel buffer, pH 8.5 10 – Tris-Borate-EDTA electrode buffer, 0.25 × (electrode buffer) gel buffer, pH 8.6 MC 6.9 – Morpholine-citric acid electrode buffer, 0.05 × (electrode buffer) gel buffer, pH 6.9 P – Tris-citric acid electrode buffer, pH 6.3, Tris-citric acid gel buffer pH 6.7 Table S1. Primers and amplification cycles for mitochondrial and nuclear loci amplified in Orconectes rusticus and O. sanbornii. 1 Perry et al., 2001b; 2 Buhay et al., 2007; 3 Fetzner and Crandall, 2002; 4 Tsang et al., 2008. Genome mtDNA Nuclear Primer sequence (5 → 3 ) Amplification profile f: AGATAGAAACCAACCTGG r: CCTGTTTAACAAAAACAT 36 cycles of: 30 s at 94°C, 2 min at 50°C, 2 min at 72°C COII 1 f: AGCGCCTCTCCTTTAATAGAACA r: CCACAAATTTCTGAACATTGACCA 36 cycles of: 30 s at 94°C, 2 min at 50°C, 2 min at 72°C GAPDH 2 f: TGACCCCTTCATTGCTCTTGACTA r: ATTACACGGGTAGAATAGCCAAACTC Initial 2 min at 95°C; 40 cycles of: 30 s at 95°C, 30 s at 56°C, 1 min at 72°C; final 10 min at 72°C 28S rDNA3 f: CCCSCGTAAYTTAAGCATAT r: CCTTGGTCCGTGTTTCAAGAC Initial 2 min at 94°C; 35 cycles of: 30 s at 94°C, 30 s at 50°C, 1 min at 72°C; final 5 min at 72°C H34 f: ATGGCTCGTACCAAGCAGACVGC r: ATATCCTTRGGCATRGTGAC Initial 5 min at 94°C; 35 cycles of: 30 s at 94°C, 30 s at 40°C, 1 min at 72°C; final 5 min at 72°C PEPCK 4 f: GCAAGACCAACCTGGCCATGATGAC r: CGGCYCTCCATGCTSAGCCARTG Initial 3 min at 94°C; 35 cycles of: 30 s at 94°C, 30 s at 60°C, 1.5 min at 72°C; final 10 min at 72°C Locus 16S rDNA1 S1 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012 Table S2. Sympatric Orconectes rusticus, O. sanbornii, and putative hybrid crayfish from two sites on the Huron River with allozyme (Idh), mtDNA (16s and COII), and morphology data (Field ID, rostral margin, and annulus ventralis for females). The Field ID is based on coloration (see Materials and Methods). R = O. rusticus, S = O. sanbornii, PH = putative hybrid (mix of O. rusticus and O. sanbornii coloration), – = no band or did not assay, ) ( = concave, | | = approximately parallel, / \ = wedge-shaped. For Idh, the O. rusticus allele was scored as 110 and the O. sanbornii allele as 94. ∗ Putative hybrid ancestry. Individual MONROEVILLE HL01 HL02 HL03 HL04 HL05 HL06 HL07 HL08 HL09 HL10 HL11 HL12∗ HL13 HL14 HL15 HL16∗ HL17∗ HL18 HR01 HR02 HR03 HR04∗ HR05 HR06 HR07 HR08 HR09 HR10 HR11 HR12∗ HR13∗ HR14 HR15 HR16 HR17 HR18 Field ID Idh 16S COII Rostral margin Annulus ventralis R S PH R R R R R S R S S R S S PH R R R R R S R R R R R R R PH R R S R S S RR SS RR RR RR RR RR RR SS – SS SS – SS SS RR RR RR RR RR RR RS RR RR RR RR RR – RR RS RS RR SS – – SS R S R – – R R R S R S R R – – S S R R R R S R R R R R R R R R R S R S S R S R R R R R R S R S R R S S S R R – R R S R R R R R R – R R R S R S S )( || || || || || /\ || /\ – || || – || || || || || || || || /\ || || || || /\ – || || || /\ || – – /\ R RR RR RR RR RR RR RR RS RR RS RR RR R R R R R R R R R R/S R S R R R R R R R R R R R R || || || || || || /\ || || || || || LOVER’S LANE BRIDGE SLL01 R SLL02 R SLL03 R SLL04 R SLL05 R SLL06 R SLL07 R SLL08∗ R SLL09 R SLL10∗ R SLL11 R R SLL12∗ R S S R R S R R R S S R R R S2 ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES Table S3. Mean (s.e.) and range of gonopod and body measurements taken on allopatric Orconectes rusticus and O. sanbornii. Measurements in mm except number of carpus spines. L = length, W = width. Numbers in front of character name refer to Fig. 3. A difference between the species means was tested with one-way ANOVA. Character Body measurements 1. Carapace L 2. Areola L 3. Areola W 4. Carapace W 5. Acumen L 6. Rostrum W Chela measurements 7. Dactyl L 8. Chela L 9. Palm W 10. Palm L 11. Chela gap W 12. No. carpus spines Gonopod measurements 13. Total L 14. Central projection L 15. Mesial projection L O. rusticus O. sanbornii F -ratio P -value Mean (s.e.) Min-Max Mean (s.e.) Min-Max 28.09 (1.16) 9.69 (0.44) 1.45 (0.08) 13.99 (0.64) 2.43 (0.11) 2.74 (0.12) 16.07-41.47 5.45-15.51 0.72-2.42 7.67-21.55 1.53-3.44 1.68-4.30 24.19 (1.16) 8.07 (0.44) 1.42 (0.08) 11.47 (0.64) 2.19 (0.11) 2.30 (0.12) 16.30-34.26 5.45-11.63 1.00-1.92 7.39-16.07 0.96-3.06 1.49-3.28 F1,40 F1,40 F1,40 F1,40 F1,39 F1,40 = 5.64 = 6.74 = 0.05 = 7.70 = 2.33 = 6.88 0.023 0.013 0.832 0.008 0.135 0.012 13.03 (0.87) 21.15 (1.33) 8.40 (0.57) 8.07 (0.57) 1.00 (0.13) 1.33 (0.11) 5.97-21.26 9.61-34.35 3.22-14.00 3.96-12.98 0.23-2.48 1-2 10.06 (0.94) 17.82 (1.44) 7.08 (0.61) 7.42 (0.62) 0.74 (0.13) 1.39 (0.12) 5.12-17.42 9.33-29.36 3.95-11.73 4.46-11.54 0.21-1.27 1-2 F1,37 F1,37 F1,37 F1,37 F1,37 F1,37 = 5.35 = 2.88 = 2.52 = 0.60 = 2.05 = 0.12 0.026 0.098 0.121 0.442 0.161 0.727 10.52 (0.41) 3.35 (0.28) 2.78 (0.30) 7.42-12.82 1.39-5.03 1.10-4.99 8.11 (0.37) 0.68 (0.25) 0.60 (0.27) 6.54-10.51 0.37-0.97 0.35-0.84 F1,18 = 18.89 F1,18 = 49.53 F1,18 = 29.55 0.0004 <0.0001 <0.0001