2013 SOUTHEASTERN NATURALIST 12(1):161–170 Growth Rates and Age Estimations of the Fuzzy Pigtoe, Pleurobema strodeanum: A Species Newly Listed under the Endangered Species Act Evelyn G. Reátegui-Zirena1,2, Paul M. Stewart1,*, and Jonathan M. Miller1 Abstract - Pleurobema strodeanum (Fuzzy Pigtoe), is listed as threatened under the Endangered Species Act. The purpose of this study was to estimate growth rate, morphometric ratios, and ages for this species. One hundred and sixty-one P. strodeanum were originally tagged and measured in 2004 at Eightmile Creek, Walton County, FL. In 2011, 28 of those 161 were recovered and re-measured. Growth-rate percentages were determined based on length, width, height, and volume. Age was estimated using the von Bertalanffy growth equation. Results indicate that over a period of seven years, P. strodeanum grew 0.48 mm/year (SD = 0.14) in length. The age estimations in this study ranged from 48.7 to 74.5 years. The growth constant (K) was 0.018 per year, and the asymptotic length (L∞) was 71.97 mm. Natural history data such as these will be useful in understanding the basic biology of P. strodeanum and may assist in successful mussel management. Introduction North American freshwater mussels (Unionidae) are one of the world’s most threatened groups of organisms. Mussel diversity is concentrated in the southeastern US, with Alabama being home to 183 species (Jeff Garner, Alabama Department of Conservation and Natural Resources [ADCNR], Florence, AL, 25 January 2012 pers. comm.), more than any other state (Williams et al. 2008). Longevity, growth, and age estimates are needed to better assess the risk of extinction of rare and endangered species, and add to an understanding of basic biology that may lead to successful mussel management (Williams et al. 1993). Pleurobema strodeanum, Walker (Fuzzy Pigtoe) is found in the Escambia, Yellow, and Choctawhatchee River drainages. Populations of this species are decreasing and it is currently listed as threatened under the Endangered Species Act (US Fish and Wildlife Service 2012). Management efforts are often based on accurate information regarding lifehistory traits such as age and growth, as such data can be used in modeling population dynamics (Haag 2009). Changes in length, width, and height of a shell over time are measurements commonly used for determining rates of growth in mussels (Dehnel 1956). Furthermore, the ratios between them are used to study the change in proportion of individuals as a consequence of growth (Lauzon- Guay et al. 2005), and these variations in shell morphology can be used as indicators of environmental change (Widarto 2007). 1Department of Biological and Environmental Sciences, Troy University, Troy, AL 36082. 2Current address - Institute of Environmental and Human Health, Department of Environmental Toxicology, Texas Tech Univesity, Box 41163, Lubbock, TX 79409. *Corresponding author - mstewart@troy.edu. 162 Southeastern Naturalist Vol. 12, No. 1 As a group, freshwater mussels are known to be long lived and slow growing. The lack of age and growth information for these organisms might be due in part to methodological issues (Haag and Rypel 2011). Growth rings occur on the external surface of mussel shells and have been used for studying growth and age. However, results are inconsistent among individuals and even different regions of a single shell. Because environmental disturbances, including handling during sampling, can cause the formation of external and internal false rings, age is often overestimated and growth rate can be underestimated (Haag 2009, Haukioja and Hakala 1978). Therefore, validation of annual ring counts is highly recommended to estimate age (Haag 2009). However, it is not the best approach for species of conservation concern that should not be sacrificed. Following tagged mussels under natural conditions is thought to be the most realistic method for estimating growth rate (Karatayev et al. 2006). With tag and recapture data, length can be used in the von Bertalanffy growth function (VBGF), which predicts length of an individual as a function of its age (Kesler and Downing 1997). Measurements of growth rate and estimation of K (growth constant), may lead to an understanding of whether or not a population is under stress and why or if they are in decline. The objectives of this study were to estimate P. strodeanum age, growth rate, and morphometric relationships over a seven-year period. Methods Pleurobema strodeanum was studied in Eightmile Creek, Walton County, FL. Mussels were collected in 2004 by handpicking using qualitative visual and tactile searches that covered 250 m downstream from a bridge crossing. Individuals were measured using digital calipers to the nearest 0.01 mm along three shell axes (length, width, and height). Mussels were tagged by removing a small portion of the periostracum on the shell and attaching a numbered Floy® shellfish tag using cyanoacrylate glue (Sickel et al. 1997). Voucher specimens were preserved in 70% ethanol and are curated in the Troy University collection. Seven years later, in 2011, 28 P. strodeanum, of the 161 originally tagged, were recaptured and measured. Growth rate percentages (G%) were calculated as follows: G% = 100 * (Mf - Mi) / Mi, where Mf and Mi are the final and initial measurements (length, width, height, and volume) in 2011 and 2004, respectively (Negishi and Kayaba 2009). Morphometric ratios were determined as follows, width to length (W:L), height to length (H:L), and height to width (H:W). Volume was calculated using the formula: V = (Length x Width x Height) / C, where the constant (C) was determined with the method used by Martins et al. (2011). Briefly, 18 P. strodeanum shells between 31.6 mm and 60.4 mm in length 2013 E.G. Reátegui-Zirena, P.M. Stewart, and J.M. Miller 163 were sealed with Parafilm®. Mussels were immersed in water, and the volume was determined by displacement to the nearest 0.1 ml in a graduated cylinder. These numbers were plotted against the volume obtained by multiplying the length, width, and height. The regression line was calculated, and the slope (C) was 2.51 (R2 = 0.975, P < 0.001). The von Bertalanffy growth equation was used to determine the age of the individuals. The equation is expressed as: Lt = L∞ (1 - e - K [t - t0)]) It is usually applied when age and body size are known; however, it can be inverted to find age as follows: t = ln[(L∞ - Lt) / (L∞ - L0)] / (-K), where L∞ (asymptotic length) is the theoretical maximum length at infinite age, Lt is the length of the organism at time t (age), L0 is the length of the organism at time 0 since age is not known, and K is Brody’s growth constant. Using mark and recapture growth data, L∞ and K can be estimated using a linear regression of the Ford-Walford relationship (Ricker 1975): L∞ = [a / (1 – β)] K = -ln β, where a is the y intercept and β is the slope (Anthony et al. 2001, Haag 2009). Assuming constant growth, the Brody’s growth constant (K) was divided by seven to be used in the inverted von Bertalanffy growth equation since the estimated K was based on a seven-year period. Data were plotted using Excel 2007 for Windows, and linear regressions between growth rate (%) and all the measurements taken were performed using SPSS® version 11.0. Data were tested for normality, and a t-test was applied to measure differences between smaller and larger individuals’ growth-rate percentages, and differences between morphometric ratios in 2004 and 2011. If data were not normal, a Mann Whitney (U) or Wilcoxon (Z) test was used. A t-test was used for width, width:length, and height:length; Mann-Whitney (U) tests were used for length, height, and volume; and Wilcoxon (Z) test for height:width. In addition, ratios in 2004 and 2011 were plotted against length to determine if samples were biased by the size of the individuals. Results In the present study in 2004, maturity for P. strodeanum was determined at 37.65 mm based on reproductive activity. Juveniles were not sampled, and all (n = 161) but seven individuals were larger than 40 mm. The theoretical maximum length (L∞) found for this species was 71.97 mm, a close approximation of the 75 mm reported by Williams et al. (2008). During 2011 resampling, a presumed juvenile individual (providing limited evidence for recruitment but not considered for analysis) was collected (length = 21.58 mm, width = 14.25, and height = 8.55). 164 Southeastern Naturalist Vol. 12, No. 1 In 2004, 161 P. strodeanum were marked, and 28 were recaptured in 2011 (for a recapture rate of 17.4%). Of the 28 P. strodeanum recaptured, 27 were used for analysis (one individual displayed negative growth and was not used in any analysis). Of the tagged mussels, the smallest P. strodeanum was 37.65 mm and the largest was 51.62 mm in length in 2004, while the individuals ranged from 41.89 mm to 53.04 mm in 2011. From 2004 to 2011, individuals grew an average of 3.37 mm in length, and showed a mean increase in width of 14.08% in seven years. This width increase was almost double the percentage of growth in length (7.84%) or height (6.87%). The mean volume for this species increased 31.44%. For purposes of analysis, it was assumed that growth was constant each year, and results were divided by seven to estimate the annual growth rate (Table 1). Individuals grew an average of 0.48 mm in length per year. Pleurobema strodeanum growth rate percentage in length (r2 = 0.273, P = 0.005), width (r2 = 0.609, P < 0.001), and volume (r2 = 0.490, P < 0.001) decreased with increased initial size (Fig. 1). When growth rate percentages of the nine smaller and the nine larger individuals were tested, there was a significant difference in length (U = 10.0, P = 0.006), width (t = 7.33, P < 0.001), and volume (U = 15.0, P = 0.024). From 2004 to 2011, the ratio of width to length increased and the height-to-width ratio decreased, while relative height (height:length) remained the same (Fig. 2). When morphometric ratios were tested, there was a significant change in width-to-length ratio (Z = -2.600, P = 0.009) and height-to-width ratio (t = 4.094, P < 0.001). Also, when ratios were plotted against length, linear regressions were not significant, suggesting that samples were not biased by the size of the individuals. The Brody’s growth constant (K) was 0.125 in a seven-year period (0.018 per year). The estimated ages ranged from 41.3 to 70.5 years in 2004, with a mean of 51.9 (SD = 6.16), and 48.7 to 74.5 years in 2011, with a mean of 58.9 (SD = 6.48) (Fig. 3). Discussion In comparable studies using different species, Kesler et al. (2007) compared the growth rates of Elliptio complanata Lightfoot (Eastern Elliptio) among three sites in Rhode Island over 14 years. Individuals at one site grew 4.3 mm, while the other two sites had growth rates that were 3 to 8 times Table 1. Pleurobema strodeanum growth (length, width, height and volume) over seven years and per year. Growth (mm) Growth rate (percentage) Measurements Over 7 years Per year Over 7 years Per year Length 3.37 0.48 7.84 1.12 Width 3.57 0.51 14.08 2.01 Height 1.21 0.17 6.87 0.98 Volume (mm3) 2.47 0.35 31.44 4.50 2013 E.G. Reátegui-Zirena, P.M. Stewart, and J.M. Miller 165 Figure 1. Regression of growth rate (%) increase f o r P l e u - robema strodeanum and (A) init ial length (mm), (B) initial width (mm), and (C) initial volume (mm3). 166 Southeastern Naturalist Vol. 12, No. 1 greater. Kesler et al. (2007) suggested that the great difference in growth rates could be due to food limitation/availability at the site with less growth. In P. strodeanum, there was a significant difference in growth rate percentages Figure 2. Morphometric relationship means (± 1 SE) for Pleurobema strodeanum measured in 2004 and 2011. Note: W = width, L = length, and H = height. Figure 3. Mussel estimated ages from the inverted von Bertalanffy growth equation using tag and recapture growth rates for Pleurobema strodeanum in 2004 and 2011. 2013 E.G. Reátegui-Zirena, P.M. Stewart, and J.M. Miller 167 when considering length, width, and volume between smaller and larger individuals. The smaller P. strodeanum had a higher growth rate than did larger individuals. This result was expected since mussels grow more rapidly during their first few years, and the growth rate decreases over time (San Miguel et al. 2004). For instance, annual growth rates of Pronodularia japanensis (Lea) individuals greater than 40 mm (adults) were less than 2%, while juveniles (10 mm) had an annual growth rate of ≈100% (Negishi and Kayaba 2009), suggesting a strong size-dependent relationship. Variations in allometric relationships are thought to be due to site-specific factors, reproductive seasons, sex, and are species-specific (Gimin et al. 2004). Shell length and obesity (width:length) increased with age in Pleurobema strodeanum, while relative height (height:length) remained the same. Similar results have been observed in Mytilus edulis L. (Blue Mussel), which has been widely studied and used as a reference for variations in shell morphology. In M. edulis, the shell width-to-length ratio increased with age, though shell height-to-width ratio decreased with age and increased shell length (Seed 1968). These observations were confirmed by Lauzon-Guay et al. (2005), who additionally found that the height-to-length ratio decreased as M. edulis grew. This ratio change is supported by the observation that shell morphology is greatly influenced by mussel density, and more elongate shells (lower height:length) are expected in the field if mussels grow in crowded conditions (Seed 1968). Moreover, relative height might decrease with an increase in shear stress exposure (Bailey and Green 1988). Similar results were found for Velesunio ambiguous (Philippi) (The Flood Plain Mussel), which suggested that the decrease in relative shell height is due to the progressively dorsal arching of the shell (Widarto 2007). The growth constant (K) is a major determinant for life span and maximum shell length. The constant declines as life span and maximum shell length increases, and it describes the shape of an individual’s growth curve (Bauer 1992). Growth constants (K) vary widely among mussel species, and reported values range from 0.063 mm for Pinna nobilis L. (Noble Pen Shell) (Galinou-Mitsoudi et al. 2006) to 0.655 for Megapitaria squalida (Sowerby) (Schweers et al. 2006). Members of the tribe Pleurobemini, to which Pleurobema strodeanum belong, are long-lived and slow-growing, having low K values. Fusconaia cerina (Conrad), F. ebena (Lea), and P. decisum (I. Lea) (Southern Clubshell) are reported to reach ages of 45–51 years. The K value was determined for F. cerina (0.173), P. coccineum (0.134), and P. decisum (0.145), and it was considered that a K = 0.05 is extremely slow growth (Haag and Rypel 2011). However, none of these species’ growth constants were as low as the K for P. strodeanum in the present study, which was only 0.018 per year. The growth constant (K) is an indicator of the energy allocated for growth compared to other functions such as reproduction (Haag and Rypel 2011). The very low K found for P. strodeanum in this study might be a response to environmental factors such as temperature, trophic conditions, and stress. Pilarczyk et al. (2006) 168 Southeastern Naturalist Vol. 12, No. 1 showed a decline from the 1990s to 2004 in the number of sites at which candidate species were reported in the Choctawhatchee drainage, of which Eightmile is a tributary. Within this watershed and its tributaries, P. strodeanum showed the greatest decrease in the number of sites in which it was found. The extremely low K that we found suggested that this population may be under stress. The decrease in distribution along with a slow growth rate supports a need for conservation efforts to identify the limiting factors for growth. Only measuring mature individuals dictates that K and growth rate are lower, therefore, more work needs to be done including juveniles so that a more accurate set of growth variables can be measured. This study has provided some important insights into the ecology of this Federally threatened mussel species, showing that tag-recapture methods can be used to obtain estimates of important life-history and growth parameters. As a future recommendation, additional studies are necessary to assess population demographics such as recruitment and survival to better understand population trends over time. Our team is currently doing such work at this site for these and other candidate species through occupancy and detection studies. 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