2012 SOUTHEASTERN NATURALIST 11(2):219–238 Population Characteristics of the Mussel Villosa iris (Lea) (Rainbow Shell) in the Spring River Watershed, Arkansas Allison M. Asher1,2 and Alan D. Christian1,3,* Abstract - the goal of this study was to better understand population characteristics of Villosa iris (Lea) (Rainbow Shell) in the Spring River drainage in north-central Arkansas through documenting seasonal spatial patterns, movement behavior, population size, size-frequency distributions, sex ratios, and fecundity. We conducted monthly mark and recapture sampling between May and September 2007 (i.e., before, during, and after spawning) and documented the sex, size, fecundity, and spatial location of individual Rainbow Shell at 2 sites (SFSR1 and SR1). Population estimates were relatively high at both sites with 166 ± 32 (SE) and 381 ± 37 (SE) individuals at SFSR1 and SR1, respectively. Sex ratio was highly skewed toward males at SFSR1 with a ratio of 1.0:2.6, but only slightly skewed at SR1 with a ratio of 1.0:1.3. Mean fecundity was 27,849 ± 11,653 (SE) and 15,089 ± 11,710 (SE) glochidia at SFSR1 and SR1, respectively. Spatially, statistically more males were found upstream of non-gravid females during the spawning period. Mean movement for all sampling events was 1.6 ± 0.53 cm/day and 1.9 ± 0.58 cm/day for SFSR1 and SR1, respectively. Home range was 29.3 ± 27.7 cm2 and 43.0 ± 42.5 cm2 for SFSR1 and SR1, respectively. From our study, we conclude that Rainbow Shell exhibits traits, such as male-skewed sex ratio and non-uniform distribution of males and females, that may influence fertilization rates of females. Introduction Freshwater mussels (Bivalvia: Unioniformes) reach their greatest richness in North America with ≈300 taxa (Bogan and Roe 2008); however, starting in the early 1990s, it was documented that ≈70% of these taxa were imperiled (Williams et al. 1993, Lydeard et al. 2004). These apparent declines resulted in development of a national strategy for the conservation of native freshwater mussels (National Native Mussel Conservation Committee 1998) that spurred basic and applied biological and environmental research on freshwater mussels. However, for many species, there is still a paucity of information on basic biology, especially concerning reproductive behavior and biology. Freshwater mussels move both vertically and horizontally; however, the reasons for this behavior are not well understood but may be associated with reproduction. Vertical movement, burrowing into substrate and rising to the substrate surface, has been associated with day length (Perles et al. 2003, Schwalb and Pusch 2007), water temperature (Amyot and Downing 1997, 1Environmental Sciences Graduate Program, Arkansas State University, PO 847, State University, AR 7246. 2Current address - Department of Zoology, Southern Illinois University, 1125 Lincoln Drive, Carbondale, IL 62901. 3Current address- Department of Biology, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125. * Corresponding author - alan.christian@umb.edu. 220 Southeastern Naturalist Vol. 11, No. 2 Schwalb and Pusch 2007, Watters et al. 2001), discharge (Schwalb and Pusch 2007) and spawning (Watters et al. 2001). Vertical movement is believed to be an avoidance response to unfavorable conditions such as high water velocities (Di Maio and Corkum 1995) or predation (Amyot and Downing 1997). It is plausible that horizontal movement across the substrate surface could occur for similar reasons. For example, horizontal movement in freshwater mussels has been shown to increase spatial aggregation (Amyot and Downing 1998, Balfour and Smock 1995, Downing and Downing 1992, Downing et al. 1993) and is thought to occur in response to reproductive efforts to increase the chance for successful reproduction (Amyot and Downing 1998; Downing and Downing 1992; Downing et al. 1989, 1993). Information relating to population characteristics, especially reproductive characteristics, of mussels is more abundant, but still lacking for most species. Two characteristics that are of particular interest in studying reproduction are sex ratios and fecundity. Sex ratios in mussels vary by species, with femaleskewed (Garner et al. 1999), near equal, (Hanlon and Levine 2004, Rogers et al. 2001, Yeager and Neves 1986), and male-skewed sex ratios varying by drainages (Hagg and Staton 2003). Mean fecundity values range from lows of 9647 for Quadrula asperata (Lea) (Alabama Orb), 23,890 for Fusconaia cerina (Conrad) (Southern Pigtoe), and 25,767 for Obliquaria reflexa Rafinesque (Three-horn Wartyback), to higher values of 281,776 for Lampsilis ornata (Conrad) (Southern Pocketbook), 325,709 for Amblema plicata (Say) (Threeridge), and 566,000 for L. siliquoidea (Barnes) (Fatmucket) (Haag and Staton 2003, Perles et al. 2003). Furthermore, fecundity typically increases with length (Haag and Staton 2003); however, this relationship is not always observed (Perles et al. 2003). Villosa iris (Lea) (Rainbow Shell) has a global conservation rank of G5Q (common, widespread, abundant, but taxonomic classification is a matter of conjecture among scientists) and a state conservation ranking in Arkansas of S2S3 (imperiled, very few populations; vulnerable, relatively few populations) (Harris et al. 2009). As its state status suggests, little is known about Rainbow Shell populations in Arkansas. Villosa iris is dioecious, has a maximum reported shell length of 75 mm, and is considered sexually dimorphic (Parmalee and Bogan 1998, Williams et al. 2008). Reproductively, Rainbow Shell is a long-term brooder (bradytictic) (Watters et al. 2001, Williams et al. 2008) and broods glochidia from May to July (Parmalee and Bogan 1998). It should be noted that the current taxonomic status of Rainbow Shell is uncertain as a recent phylogenetic analysis across the range revealed that the genus Villosa is polyphyletic with at least 9 clades that include species from 5 genera (Kuehnl 2009, Williams et al. 2008). The goal of this study was to document population characteristics of Rainbow Shell at 2 sites in the Spring River drainage, AR (Spring River and South Fork Spring River) and to compare these characteristics through time and between sites. The first objective was to determine population characteristics 2012 A.M. Asher and A.D. Christian 221 (i.e., population size, size frequency, fecundity, sex ratio) at each location and to compare these characteristics between sites and among sampling events. The second objective was to determine movement and spatial patterns (i.e., distance traveled between captures, total displacement, male and female spatial patterns) of Rainbow Shell for each sampling event at each site. To do so, we conducted a monthly mark and recapture study of Rainbow Shell from May to September 2007 and recorded the location, sex, and reproductive status (gravid or not gravid) of females. Methods Study area Two sampling sites were established in the 2 main streams of the Spring River drainage that is located in the Ozark Mountains of north-central Arkansas and southwestern Missouri. One site was located in the South Fork Spring River (SFSR1), and the other site was located in the Spring River (SR1) (Fig. 1). The SFSR begins in southeastern Missouri and flows southeast for 120 river km to its confluence with the Spring River just upstream of Hardy, AR. The underlying geology of the SFSR watershed is primarily limestone and land use is primarily pasture land (Martin 2008, Martin et al. 2009). Martin et al. (2009) reported Rainbow Shell from 11 locations within the SFSR, including the population we investigated for this study (SFSR1). Based on surface-exposed mussels, the mussel assemblage at SFSR1 was composed of 8 species, occupied an area approximately 70 m in length and 10 m in width, was located in a run habitat on the left descending bank upstream of the low-water bridge on Red Bud Road (County Road 26), and had substrate composed of primarily cobble and fines Figure 1. Villosa iris sampling sites on the South Fork Spring River (SFSR1; GPS coordinates: 15S 603340 4037709 [UTM]) and Spring River (SR1; GPS coordinates: 15S 633870 4022487 [UTM]) in Fulton County, AR. Map layers downloaded from GeoStor 5.0 (http://www.geostor.arkansas.gov). 222 Southeastern Naturalist Vol. 11, No. 2 (Martin 2008). The SR begins at Mammoth Spring, AR, which discharges ≈34 million liters of water/hour (Trauth et al. 2007), and flows south 92 river km until its confluence with the Black River. The geology and land use of the SR is similar to the SFSR, with underlying limestone and land use of primarily pasture land. Trauth et al. (2007) reported Rainbow Shell from 4 locations within the SR, including the population we studied (SR1; Fig. 1). Based on surface-exposed mussels, the mussel assemblage at SR1 was composed of 3 species, occupied an area approximately 73 m long and 13 m wide, was located in a run habitat on the left descending side channel, and had substrate composed of boulders with fines and cobble (Trauth et al. 2007). Mark and recapture sampling Mark and recapture sampling was conducted monthly at each site from May to September, 2007. However, we collected preliminary data in July 2006 at SR1 and included these data to estimate population size, calculate sex ratio, and compare shell length at SR1. During each sampling event, we surveyed mussels using a single snorkeling pass. Only mussels that could be visually observed at the substrate surface were located and marked with surveying flags after being completely removed from the substrate and being visually identified as Rainbow Shell; in other words, buried mussels were not excavated from below the substrate. Spatial locations of flagged Rainbow Shell were determined by establishing a long-term X-axis on the first sampling date by inserting metal rods into the soil on the right and left banks. These rods remained in place throughout the sampling period and were documented by recording the GPS coordinates of both rods. During each sampling event, a temporary X-axis was established by attaching a meter tape to the right and left bank rods. A Y-axis was created by using another meter tape run parallel to the banks. The flagged mussel X and Y location was recorded to the nearest 0.01 meter. Variation in measurements of individual mussels, i.e. measurement error, was accounted for at each site by establishing a “dummy” marker within the study reach at each site. Dummy markers were measured exactly as flagged mussels. This allowed us to estimate the precision of our X and Y measurements at each site. Each previously unmarked Rainbow Shell collected was marked by etching a unique identification code on its right valve using a Dremel® tool. For each individual collected, we recorded the unique identification code, measured and recorded anterior-to-posterior shell length to the nearest 0.1 mm, and recorded the sex and reproductive status (gravid or not gravid) of females. The reproductive status of females was determined by gently prying open the valves and observing the gills for glochidia. Individuals were returned to their original location after being processed. Fecundity was estimated by counting glochidia collected from 5 gravid females at SR1 in July 2006 and 6 gravid females at SFSR1 in May 2007. Glochidia were harvested in two ways. At SR1, gravid females were collected, and pre2012 A.M. Asher and A.D. Christian 223 served in 100% ethanol, and glochidia were removed from the gills in the laboratory. At SFSR1, glochidia were collected from females in the field by prying open the valves, flushing de-ionized water over the gills, collecting the flushed glochidia, and preserving the glochidia in 100% ethanol. Females from SFSR1 were returned after glochidia harvest to the exact location they were collected. In the laboratory, the number of glochidia per female was determined by counting 5 glochidia subsamples from each female as follows. Each female’s glochidia were placed in a known volume of ethanol. The glochidia and ethanol were thoroughly mixed, a 1-mL subsample of the glochidia and ethanol mixture was removed and discharged into a gridded petri dish, and glochidia were counted under a dissection microscope. This was repeated 5 times per female. Total fecundity per female was estimated by multiplying the mean number of glochidia per/mL by the total volume of the glochidia and ethanol mixture. Calculations and statistical analyses Population estimates at each site were derived using mark and recapture methods outlined in Schumacher and Eschmeyer (1943) and only when a few individuals could be collected in each sampling event. Briefly, the population sizes (N) of Rainbow Shell were estimated using the formula: N = (Σk[n2(m + u)]) / Σk(nm), where N is the population estimate, k represents the total number of sampling events, n is the total number of marked individuals from prior sampling events, m is the number of marked individuals captured for the current sampling event, and u is the number of unmarked individuals captured for the current sampling event. The relative standard error of the population with a probability of 0.95 for each estimate was also estimated using the following formula where s2 is the sample variance. ______________________________________ √N 2[Ns2 / Σk(nm)] To determine if there were differences in male and female length among sampling events within a site and between sites on common sampling events, a Mann-Whitney U test was used (JMP IN® , 2001, SAS Institute Inc., Cary, NC). Locations of every male and female captured were mapped, distance traveled between captures calculated, and minimum convex polygons calculated using ArcMap 9.1 (ESRI, Inc. 2006). To convert X-Y meter coordinates to latitude and longitude coordinates of individual mussels, GPS coordinates of the X-axis were loaded into ArcMap and the X-Y coordinates (in meters) of all recaptured mussels were added to the data file. These data were edited to align the X-Y coordinates of the GPS coordinates recorded for the permanent X-axis rods. To calculate horizontal movement (i.e., distance traveled between captures), we used Arc GIS and the Hawth’s Tools (Beyer 2004) function of “convert locations to paths”. To calculate individual mussel home ranges for individuals captured 3 or more times, we created minimum convex polygons by calculating 224 Southeastern Naturalist Vol. 11, No. 2 the area of the polygon using the “create minimum convex polygons function” in Hawth’s Tools. To determine if males and females for each sampling event had uniform or non-uniform distribution patterns, we created matrices in which the location of each male was compared to the location of each female. This allowed us to determine the upstream and downstream distribution of males in relation to females. In addition, females were coded as either gravid or non-gravid. A likelihood ratio test for goodness of fit was conducted to determine the significance of expected versus observed male and female spatial patterns (Sokal and Rohlf 1995). Results Population estimates, size, fecundity, and reproductive status At SFSR1, 111 captures representing 83 individuals and 28 recaptures from 7 sampling events (Table 1) resulted in a population estimate of 166 ± 32(SE) individuals. Both first time captures and recaptures were highest during the last sampling event (September) and lowest during the second sampling event (May). The female (n = 23) to male (n = 60) ratio at SFSR1 was 1.0:2.6. At SR1, 194 captures represented by 163 individuals and 31 recaptures resulted in a population estimate of 451 ± 43(SE) individuals (Table 1). Both first time captures and recaptures was highest during the last sampling event Table 1. Mark and recapture data for Villosa iris in the South Fork Spring River (SFSR1) and Spring River (SR1) sites for each sampling event. n = number of individuals previously marked for entire study period, m = number of individuals captured during a sampling event that were previously captured and marked, u = number of individuals captured during sampling event that were not previously captured and marked, N = population estimate, s2 = variance, and CI = confidence interval. Date n m u m + u n2 (m + u) nm m2/( m + u) N s2 95% CI SFSR1 May-1 0 0 9 9 0 0 0.00000 May-2 9 1 7 8 648 9 0.12500 May-3 16 7 18 25 6400 112 1.96000 June 34 3 7 10 11,560 102 0.90000 July 41 2 17 19 31,939 82 0.21053 Aug. 58 5 5 10 33,640 290 2.50000 Sept. 63 10 20 30 119,070 630 3.33333 Summary 83 28 83 111 203,257 1225 9.02886 166 0.274328 32 SR1 July 07 0 0 26 26 0 0 0.00000 May-1 26 0 2 2 1352 0 0.00000 May-2 28 1 24 25 19,600 28 0.04000 June 52 5 21 26 70,304 260 0.96154 July 73 2 24 26 138,554 146 0.15385 Aug. 96 8 32 40 368,640 768 1.60000 Sept. 129 15 34 49 815,409 1935 4.59184 Summary 163 31 163 194 1,413,859 3137 7.34722 451 0.064500 43 2012 A.M. Asher and A.D. Christian 225 (September) and lowest during the second sampling event (May). The female (n = 70) to male (n = 9 3) ratio at SR1 was 1.0:1.3. Male Rainbow Shell lengths at SFSR1 ranged from 35.9 to 59.1 mm throughout the sampling period with sampling event means ranging from 42.6 ± 6.7 (SE) mm to 48.7 ± 1.7 (SE) mm (Fig. 2, Table 2). Female lengths Figure 2. Length - frequency (number of individuals) distribution of male (black) and female (white) Villosa iris individuals per size class at the South Fork Spring River site (SFSR1) from May to September 2007. 226 Southeastern Naturalist Vol. 11, No. 2 at SFSR1 ranged from 29.4 to 54.6 mm throughout the sampling period, with individual sampling event means ranging from 39.4 ± 9.14 (SE) mm to 42.9 ± 3.6 (SE) mm (Fig. 2, Table 2). For SR1, mean male length ranged from 23.2 mm to 62.3 mm throughout the sampling period, and individual sampling event means ranged from 44.8 ± 3.8 (SE) mm to 50.3 ± 4.3 (SE) mm (Fig. 3, Table 2). Mean female length ranged from 23.5 to 54.9 mm throughout the sampling period, and individual sampling event means ranged from 39.8 ± 2.7 (SE) to 43.4 ± 2.3 (SE) mm (Fig. 3, Table 2). SFSR1 males were slightly significantly larger than SR1 males only during the May 2007 sampling period (S1: U = -1.97, P < 0.049). There were no signifi- cant differences in male lengths at all other sampling events nor were there any significant differences in female lengths between sites at any sampling event (S1: P > 0.05). Across all sampling events, 10 of 23 females at SFSR1 were gravid. The percentage of gravid females at SFSR1 declined from 77.8% in May to 0% in June, then gradually increased to 80% throughout the rest of the sampling period (Table 3). During the 2007 sampling period, 30 of 54 females at SR1 were gravid, and patterns of gravidity followed that of SFSR1, with the percentage of gravid females declining after May and slowly increasing through September (Table 3). Fecundity at SFSR1 (n = 6) ranged from 11,687 to 52,265 glochidia per female with a mean of 27,849 ± 11,653(SE). Fecundity at SR1 ranged from 3080 to 34,434 glochidia with a mean of 15,089 ± 11,710 (SE). Movement and spatial patterns Sample size was based on recaptures, and for some sampling periods was not large enough to perform statistical analysis. However, we did observe 3 Table 2. Mean lengths (mm) ± standard errors of South Fork Spring River site (SFSR1) and Spring River site (SR1) Villosa iris males and females for each sampling event from May to September 2007 (including the May 2006 sample event at SR). May 2006 May 2007 June July August September Males SFSR1 - 48.7 ± 4.4 42.6 ± 6.9 48.2 ± 3.2 46.4 ± 2.1 48.6 ± 1.9 SR1 50.3 ± 4.3 44.8 ± 3.8 44.8 ± 4.5 45.3 ± 4.0 47.1 ± 2.1 47.0 ± 2.2 Females SFSR1 - 42.9 ± 3.6 39.4 ± 9.1 40.7 ± 3.6 39.8 ± 5.5 42.3 ± 2.2 SR1 43.4 ± 2.3 40.3 ± 2.8 41.1 ± 3 42.0 ± 6.8 42.2 ± 3.7 39.8 ± 2.7 Table 3. The percentage and sample size (n) of Villosa iris females gravid for each sampling period (May–September) at the South Fork Spring River (SFSR1) and Spring River (SR1) sites from May to September 2007. May June July August September SFSR1 77.8 (9) 0.0 (5) 25.0 (4) 50.0 (2) 80.0 (5) SR1 47.1 (17) 21.4 (14) 50.0 (6) 90.0 (10) 88.2 (17) 2012 A.M. Asher and A.D. Christian 227 interesting patterns (Table 4). First, overall movement for males and females combined for the entire sampling period was greater at SR1 (1.89 ± 0.58 cm/ day) than at SFSR1 (1.64 ± 0.53 cm/day). Second, overall female movement was greater than male movement at both sites. Third, male movement over the entire study period was greater at SR1, while female movement over the entire study period was slightly greater at SFSR1. Figure 3. Length-frequency (number of individuals) distribution of male (black) and female (white) Villosa iris individuals per size class at the Spring River site (SR1) in May 2006 and from May to September 2007. 228 Southeastern Naturalist Vol. 11, No. 2 For those individuals with at least 3 observations (a capture and at least 2 recaptures), movement during the sampling period resulted in an overall mean home range of 35.1 ± 22.6 cm for both sites combined. The home range of Rainbow Shell was larger at SR1 (43.0 ± 42.5 cm; n = 3) than at SFSR1 (29.3 ± 27.7 cm; n = 4). Spatial distributions of males and females at SFSR1 were significantly different from a uniform distribution for the May, July, August, and September sampling periods (Table 5, Fig. 4). In May, more males were observed upstream of all females (G1 = 5.8, P < 0.016) and gravid females (G3 = 18.58, P < 0.001) than expected from a uniform distribution. During July (G3 = 22.57, P < 0.001) and August (G3 = 13.18, P < 0.004), more males were observed upstream of non-gravid females than expected from a uniform distribution. During September, more males were observed downstream of all females (G1 = 23.19, P < 0.001) and gravid females (G3 = 81.79, P < 0.001) than expected from a uniform distribution. Spatial distributions of males and females at SR1 were significantly different than a uniform distribution for the June, August, and September sampling dates (Table 6, Fig. 5). In June, significantly more males were observed upstream of non-gravid females (G3 = 60.08, P < 0.001) than expected from a uniform Table 4. Sample size of recaptures (n) and mean movement in cm/day (x̅) and standard error (SE) for recaptured male (M) and female (F) Villosa iris from the South Fork Spring River (SFSR1) and Spring River (SR1) sites. Recaptured individuals were not encountered during all sampling events and values are for each sampling period between May to September 2007. O = overall. May– May– May– May– June– June– June– July– July– Aug.– June July Aug. Sept. July Aug. Sept. August Sept. Sept. O SFSR1 M n 3 2 4 5 - - 2 3 3 1 23 x̅ 0.26 3.35 2.17 1.44 - - 3.24 0.61 1.08 0.72 1.55 SE 0.25 1.27 1.06 0.53 - - 3.04 1.19 0.41 - 0.52 F n - - 1 - - - 1 - - - 2 x̅ - - 1.11 - - - 4.22 - - - 2.67 SE - - - - - - - - - - 3.05 M+F n 3 2 5 5 - - 3 3 3 1 25 x̅ 0.26 3.35 1.96 1.44 - - 3.57 0.61 1.08 0.72 1.64 SE 0.25 1.27 0.92 0.53 - - 1.87 1.19 0.41 - 0.53 SR1 M n 1 - 2 2 1 1 3 2 2 6 20 x̅ 5.66 - 1.46 1.12 2.39 0 1.00 0.07 1.19 2.33 1.63 SE - - 2.28 1.68 - - 0.99 0.13 1.75 1.47 0.74 F n 3 1 - 1 - 3 1 - 1 - 10 x̅ 2.98 0.56 - 2.46 - 3.08 1.26 - 1.48 - 2.39 SE 2.00 - - - - 1.89 - - - - 0.92 M+F n 4 1 2 3 1 4 4 2 3 6 30 x̅ 3.65 0.56 1.46 1.57 2.39 2.31 1.06 0.07 1.29 2.33 1.89 SE 1.93 - 2.28 1.31 - 2.02 0.71 0.13 1.03 1.47 0.58 2012 A.M. Asher and A.D. Christian 229 distribution. Interestingly, significantly more males were observed downstream of all females (G3 = 155.43, P < 0.001) and gravid females (G3 = 377.16, P less than 0.001) in August. However, the pattern changed in September, with significantly Table 5. The number of observed and expected (based on uniform distribution) observations of each individual Villosa iris male location compared to each individual female location with the calculated G and P-values (bold indicates significance at P ≤ 0.05) for each comparison for each month of the study period at the South Fork Spring River site (SFSR1). For the strictly male versus female comparison: MUF = males upstream of females; MDF = males downstream of females. For the males upstream or downstream of gravid females and non-gravid females: MUGF = males upstream of gravid females, MDGF = males downstream of gravid females, MUNGF = males upstream of non-gravid females, and MDNGF = males downstream of non-gravid females. NA = no gravid females observed for June. Month Treatment Observed Expected G P-value May MUF 82 68.00 MDF 54 68.00 5.80614 0.0160 MUGF 44 25.50 MDGF 24 25.50 MUNGF 14 25.50 MDNGF 20 25.50 18.58776 0.0003 June MUF 16 12.50 MDF 9 12.50 1.98645 0.1587 MUGF NA NA MDGF NA NA MUNGF NA NA MDNGF NA NA July MUF 29 30.00 MDF 31 30.00 0.06668 0.7962 MUGF 3 15.00 MDGF 12 15.00 MUNGF 26 15.00 MDNGF 19 15.00 22.57311 0.0000 Aug. MUF 10 8.00 MDF 6 8.00 1.01069 0.3147 MUGF 2 4.00 MDGF 6 4.00 MUNGF 8 4.00 MDNGF 0 4.00 13.18334 0.0043 Sept. MUF 36 62.50 MDF 89 62.50 23.19900 0.0000 MUGF 22 31.25 MDGF 78 31.25 MUNGF 14 31.25 MDNGF 11 31.25 81.79490 0.0000 230 Southeastern Naturalist Vol. 11, No. 2 more males observed upstream of all females (G1 = 11.22, P < 0.001) and gravid females (G3 = 388.76, P < 0.001). Discussion Population estimates and characteristics Our population estimates for both sites were higher than previous reports at both SFSR1 and SR1. In a study just 1 year prior to this study, Martin (2008) used a stratified random sampling protocol and estimated the population size at SFSR1 to be 16 ± 14 Rainbow Shell individuals. This estimate is considerably lower than our estimate of 166 ± 32 individuals. At the same time, our population estimate at SR1 (451 ± 43) was over 100 individuals higher than Trauth et al. (2007), which estimated the Rainbow Shell population there to be 273 ± 109 individuals just 2 years prior to our sampling. Both studies (Martin 2008, Trauth et al. 2007) used the same stratified random sampling design of a defined assemblage that estimates populations using equations described by Christian and Harris (2005). This protocol is ideal for large-scale surveys that sample all species encountered; however, the stratified random sampling method tends to both over and under Figure 4. Observed and expected occurrences of Villosa iris male locations compared to female locations for monthly sampling period at the South Fork Spring River site (SFSR1). MDNGF = males downstream of non-gravid females, MUNGF = males upstream of non-gravid females, MDGF = males downstream of gravid females, MUGF = males upstream of gravid females, MDF = males downstream of females, and MUF = males upstream of females. 2012 A.M. Asher and A.D. Christian 231 estimate population size under certain conditions. For example, because the Christian and Harris (2005) method only samples the assemblage once during a year, it does not account for seasonal vertical and horizontal movements or Table 6. The number of observed and expected (based on uniform distribution) observations of each individual Villosa iris male location compared to each individual female location with the calculated G and P-values (bold indicates significance at P ≤ 0.05) for each comparison for each month of the study period at the Spring River site (SR1). For the strictly male versus female comparison: MUF = males upstream of females; MDF = males downstream of females. For the males upstream or downstream of gravid females and non-gravid females: MUGF = males upstream of gravid females, MDGF = males downstream of gravid females, MUNGF = males upstream of non-gravid females, and MDNGF = males downstream of non-gravid females. Month Treatment Observed Expected G P-value May MUF 73 68 MDF 63 68 0.7359 0.3910 MUGF 41 34 MDGF 23 34 MUNGF 32 34 MDNGF 40 34 6.4930 0.0108 June MUF 86 84 MDF 82 84 0.0952 0.7576 MUGF 15 42 MDGF 21 42 MUNGF 71 42 MDNGF 61 42 60.0816 0.0000 July MUF 60 60 MDF 60 60 0.0000 0.0000 MUGF 22 30 MDGF 38 30 MUNGF 38 30 MDNGF 22 30 8.7375 0.0033 Aug. MUF 47 150 MDF 253 150 155.4278 0.0000 MUGF 44 75 MDGF 226 75 MUNGF 3 75 MDNGF 27 75 377.1645 0.0000 Sept. MUF 311 272 MDF 233 272 11.2225 0.0008 MUGF 290 136 MDGF 190 136 MUNGF 21 136 MDNGF 43 136 388.7647 0.0000 232 Southeastern Naturalist Vol. 11, No. 2 underrepresented species like mark and recapture techniques have the ability to do. Nevertheless, we believe our population estimates are also an underestimate of the true population because we were not able to capture all of the individuals in the population. For example, Rogers et al. (2001) concluded that the methods of Schumacher and Eschmeyer (1943) underestimate the true population size when only surfaced individuals are sampled. Additionally, Berg et al. (2008) recognized true abundances of smaller individuals are often underestimated due to sampling techniques. Freshwater mussels remain burrowed through the juvenile stage, often not surfacing until reproductively mature (Balfour and Smock 1995, Yeager et al. 1994). Since only surfaced individuals were sampled, juveniles were underrepresented in our study. This bias can be seen in the size-frequency graphs of each site, in which none of the 246 individuals were less than 20 mm in length. The sex ratio in our study was skewed toward males in both populations. While SR1 had a slight male bias of 1.0:1.3, the SFSR1 population had 2.6 males per female. Sex ratios can be a response to overcome limitations of reproductive mechanisms (van Ekrom Schurink and Griffiths 1991) or an indicator of a Figure 5. Observed and expected occurrences of Villosa iris male locations compared to female locations for monthly sampling period at the Spring River site (SR1). MDNGF = males downstream of non-gravid females, MUNGF= males upstream of non-gravid females, MDGF = males downstream of gravid females, MUGF = males upstream of gravid females, MDF = males downstream of females, and MUF = males upstream of females. 2012 A.M. Asher and A.D. Christian 233 changing population (Heard 1975). Male-skewed sex ratios have been reported for Quadrula asperata (1.0:3.8) and Lampsilis ornata (1.0:5.5) in the Sipsey River, AL (Haag and Staton 2003). However, male-biased sex ratios are not observed in all freshwater mussel populations (Berg et al. 2008, Haag and Staton 2003, Rogers et al. 2001, Yeager and Neves 1986), and some populations exhibit female-skewed sex ratios (Garner et al. 1999). Downing et al. (1989) suggested that a male-skewed sex ratio emphasizes the importance of female fertilization. However, current evolutionary theory for dioecious species is that sex ratios at conception commonly should be 1:1 (Charnov 1982). One class of factors that have been shown to influence sex ratios post conception is environmental factors. Martin (2008) found that SFSR1 had a low index of biotic integrity (IBI) score (i.e., impaired), but higher habitat assessment score (marginal) and microinvertebrate index score (i.e., very good). The lower IBI score, indicative of altered fish composition compared to a less altered site, could suggest reproductive barriers for freshwater mussels either due to poor water quality, as indicated by low IBI scores, or lack of host fish or host-fish migration. Fecundity is a measure of reproductive potential and is linked with female size, as larger females generally have higher fecundity (Downing et al. 1993, Haag and Staton 2003), although exceptions have been reported (Perles et al. 2003). Fecundity was much lower at SR1 than SFSR1, even though we did not detect any differences in female size between the two sites. Villosa iris is considered bradytictic (Watters et al. 2001), releasing glochidia in late spring to early summer (Parmalee and Bogan 1998). Because glochidia were collected from SR1 females in July, compared to May collection at SFSR1, seasonal differences in fecundity may be the reason for the differences we observed. For example, it is likely the females at SR1 had already discharged glochidia and were only partially recharged from the current spawning period. Thus, we believe that fecundity estimates from SFSR1 are likely to be more representative measurement of pre-glochidia release fecundity. Both populations had size distributions similar to those often reported in the literature showing a conspicuous absence of individuals under 20 mm (Christian et al. 2005, Payne and Miler 1989). When lengths of females and males were compared between rivers, no differences were observed except during May 2007, in which SFSR1 males were longer than SR1. At first glance, both populations would appear to lack recruitment. However, as previously mentioned, juvenile mussels burrow into the substrate (Balfour and Smock 1995, Yeager et al. 1994). Sediments were not extensively sampled; therefore, we believe that juveniles were not effectively sampled. Thus, population size structure and recruitment are likely underestimated and reflective of individuals over 20 mm in length. Movement Our observed horizontal movement rates were higher than movement rates of an Elliptio complanata (Lightfoot) (Eastern Elliptio) assemblage at Buzzards 234 Southeastern Naturalist Vol. 11, No. 2 Branch, VA, a headwater stream consisting of primarily sandy substrate with silt and gravel (Balfour and Smock 1995). However, our movement rates are much lower than those of Peck et al. (2007), in which native and relocated Potamilus capax (Green) (Fat Pocketbook) had combined displacement values ranging up to ≈225 cm/day (27 m over a 3-month period). Displacement values, measures of distance between first and last known location, are not reflective of the overall movement and would be equal to or less than actual movement distances. Horizontal movement rates in this study were more comparable to those observed by Schwalb and Pusch (2007) in the River Spree, Germany, a stream with sandy substrate. Schwalb and Pusch (2007) observed mean movement of 1.9 cm/day for Unio pictorum (L.) (Painter’s Mussel), 1.4 cm/day for U. tumidus (Philipsson) (Swollen River Mussel), and 2.1 cm/day for Anodonta anatina (L.) (Duck Mussel). Neither study determined the exact position of an individual, but instead used the relative movement between quadrats. Our Rainbow Shell movement rates also were close to those of E. complanata in Lac de l’Achigan (Amyot and Downing 1997). Although movement rates were similar among the unionids, the habitats were contrasting, as Lac de l’Achigan is a lentic system. Numerous factors, such as substrate type, stream order, discharge (Schwalb and Pusch 2007), temperature (Perles et al. 2003, Schwalb and Pusch 2007), day length (Amyot and Downing 1997, Perles et al. 2003, Schwalb and Pusch 2007), spawning period (Amyot and Downing 1997, 1998), and density and position of other individuals (Downing and Downing 1992, Huang et al. 2007) have been shown to influence movements of unionids and could have influenced the horizontal movement of the Rainbow Shell in our study. Since females brood glochidia, we hypothesized females would have lower movement rates than males, allocating energy toward brooding rather than movement (Amyot and Downing 1998). Observations in our study were the same as Amyot and Downing (1998), with females having higher mean movement rates than males. However, due to low sample size per sampling event, statistical tests were not feasible. Comparisons of male and female unionid movement rates have been made for lentic systems; however, they have not been investigated for lotic systems. Amyot and Downing (1998) reported the mean distance travelled by females in the lentic study area was slightly greater than that of males, but differences in mean distances were not significant. Spatial patterns In our study, the male and female distribution patterns were associated with the spawning period of Rainbow Shell. In May, when locations of surfaced males were compared to surfaced gravid and non-gravid females, more males were located upstream of gravid females than compared to a uniform distribution. In July and August, more males were located upstream of non-gravid females than expected from a uniform distribution (but not at SR1 for August). This finding is consistent with the observation that female Rainbow Shell released glochidia in spring and early summer, resulting in a spawning period during summer to 2012 A.M. Asher and A.D. Christian 235 early fall (Parmalee and Bogan 1998). It was also consistent with our findings in which the percentage of gravid females peaked in May, was lowest in June, and increased in each subsequent month until 80% of the females were observed gravid in September. Male and female spatial distributions at SR1 were similar to that of SFSR1, with 78.6% of the females non-gravid in June. Our observation of more males upstream (see comment about SR1) of non-gravid females (July and August) at both sites corresponds to the spawning period and might be interpreted as a behavior to increase fertilization success (Downing et al. 1989). Meanwhile, our observation of more males downstream of females in September corresponded with a cessation of spawning and spatial position being less relevant. Thus, the observation that more males are located upstream of females supports the hypothesis that during spawning events, more males would be located upstream of females than other times of the year. Information on the population characteristics and spatial patterns of freshwater mussels has been generally lacking. Our study resulted in 2 findings. First, population estimates at both study sites were larger than previously reported, and we conclude that mark and recapture methods tend to provide more accurate and precise estimates than quadrat-based sampling. Quadrat-based sampling surveys a portion of the mussel assemblage during 1 sampling event, whereas mark and recapture surveys the entire assemblage during multiple sampling events. Second, based on a male-skewed sex ratio, movement, and spatial patterns observed in our study, we conclude that Rainbow Shell exhibits behaviors that may lead to increased fertilization rates of females. For example, although mussels are considered sedentary animals with small home ranges, movement potentially increases the chance for fertilization and may aid mussels in avoiding harmful conditions or locating more suitable microhabitat. Furthermore, males and females exhibited a non-uniform distribution, at least in association with spawning events, with males being spatially located upstream of females. Thus, due to external fertilization of freshwater mussels, this spatial positioning of males should increase fertilization rates and ultimately reproductive success. Acknowledgments Funding for this project was provided by the Arkansas State University Environmental Sciences Graduate program and the Arkansas Biosciences Institute at ASU. We thank M.N. Asher, E.A. Daniells, K. Inoue, D.M. Hayes, R.L. Lawson, H.C. Martin, and A.J. Peck for field and lab assistance. We thank N. Young and A.J. Peck for assistance with ArcGIS. We thank J. 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