Reproductive traits of nine freshwater mussel species (Mollusca: Unionidae) from Poyang Lake, China

Reproductive traits of nine freshwater mussel species (Mollusca: Unionidae) from Poyang Lake, China Abstract The Yangtze River basin has the highest species richness for freshwater mussels in East Asia, but little is known about the reproductive biology of most species. Here, the reproductive biology of nine freshwater mussel species (Unionidae) found in Poyang Lake, Jiangxi province, China was studied. Sex ratios were not significantly different from 1:1 in eight of the nine species examined. The minimum size and age of sexual maturity (0.2–5.6 yr for males and 0.3–5.9 yr for females) and of females first becoming gravid (0.6–8.9 yr) varied among species. Brooding seasons for glochidia varied among species, but seven of the nine species examined brooded glochidia through the spring and summer. In all species, high frequencies (mean 87.5%) were found to be gravid during their peak brooding period. Fecundity was positively correlated with shell length in the five species in which this was examined. Mean annual fecundity showed wide interspecific variation, ranging from 7,800 to 328,000 propagules/mussel. The findings of this study have implications for the ecology and conservation of unionid species in China and contributes to knowledge of the evolution of reproductive and life history characters of the Unionidae. INTRODUCTION Freshwater mussels (Unionidae) are a major component of food webs and play important roles in freshwater ecosystems (Howard & Cuffey, 2006; Vaughn, 2017). Unionids have a unique life cycle with an obligate parasitic larval stage (glochdia) that is dependent on a host fish (Wächtler, Drehermansur & Richter, 2001). Glochidia are brooded in a specialized marsupium formed by the interlamellar spaces (water-tubes) of the gills in female mussels. The portion of the gills used for brooding varies among species, ranging from the use of all four gills (tetragenous) to only a portion of the outer gills (ectobranchous) (Simpson, 1914; Davis & Fuller, 1981). Brooding time of glochidia in the gill marsupium also varies among species, with a continuum between short-term (tachytictic) and long-term (bradytictic) brooding strategies, as described in North American freshwater mussels (Sterki, 1895, 1898; Ortmann, 1911; Price & Eads, 2011). Short-term brooders brood glochidia for c. 2–6 weeks before release usually in spring or summer, while long-term brooders retain glochidia for up to 8 months, usually from late summer or autumn until release the following spring or summer (Haag, 2013). After release from the female, glochidia must find a suitable host fish, surviving outside of their mother for a few days at most (Kat, 2010). The larval stage is the most sensitive in the unionid life cycle (Hardison & Layzer, 2001). To a certain extent because of the disruption of the life cycles by anthropogenic stressors, mussels have become among the most imperiled animal groups on Earth (Regnier, Fontaine & Bouchet, 2009; Haag, 2012). Research on the reproductive traits of freshwater mussels can be traced back to the early 1900s that first revealed information on aspects such as sex ratios, fecundity and brooding periods (Coker et al., 1921; Bauer, 1987; Downing et al., 1989; Gordon & Smith, 1990; Haag & Staton, 2003). Brooding periods and arrangement of the gill marsupium have historically been important characteristics for the classification of the more than 290 North American freshwater mussel species (Ortmann, 1912; Heard & Guckert, 1971; Davis & Fuller, 1981; Lopes-Lima et al., 2017). Currently, in the context of the decline of freshwater mussel species and with the survival of many species seriously threatened, many scholars are actively researching the evolution of reproductive characters and life history strategies of unionids (Graf & Ó Foighil, 2000; Zanatta & Murphy, 2006; Barnhart, Haag & Roston, 2008; Haag, 2013; Pfeiffer & Graf, 2015; Lopes-Lima et al., 2017). Furthermore, information on the reproductive characters and life history is critically important for the conservation and recovery of many unionid species (Mcivor & Aldridge, 2007; Haag, 2012). The middle portion of the Yangtze River basin is one of the most species-rich regions for freshwater mussels on Earth (Zieritz et al., 2017), including many endemic species (He & Zhuang, 2013; Shu et al., 2014). However, research on the reproductive biology of unionids from this biodiverse region has largely been lacking. In recent decades, a series of anthropogenic stressors, including habitat destruction and degradation, water pollution and increasing commercial exploitation for human consumption and pearls, have resulted in the imperilment of many mussel species in the region (Shu et al., 2009; Xiong, Ouyang & Wu, 2012; Wu et al., 2017; Zieritz et al., 2017). Due to the lack of information on reproductive traits for most unionid species in the region, management and conservation efforts for unionids has been greatly hampered. It is thus critical that there is an improved understanding of the reproductive biology of Chinese unionid mussel species in order to provide bases for classification and conservation. This study is the first to examine the reproductive characteristics of nine native unionid mussel species found in Poyang Lake, the largest freshwater lake in the Yangtze River basin, in Jiangxi Province, China: Acuticosta chinensis (Lea, 1868), Anemina globosula (Heude, 1878), Arconaia lanceolata (Lea, 1856), Cristaria plicata (Leach, 1814), Lamprotula caveata (Heude, 1877), Lanceolaria eucylindrica Lin, 1962, Lanceolaria grayana (Lea, 1834), Schistodesmus lampreyanus (Baird & Adams, 1867) and Sinanodonta woodiana pacifica (Heude, 1878). Based on the information gathered on the reproductive biology of these species, some preliminary management strategies are proposed for unionid mussels in Poyang Lake and the Yangtze River drainage. This study not only begins to fill a major knowledge gap on the reproductive biology of Chinese freshwater mussels, but also lays a foundation for further study of the evolution of their reproductive and life history characters. MATERIAL AND METHODS Specimens were collected from Poyang Lake, located on the south side of the middle Yangtze River, China (Fig. 1). Nine mussel species were examined, selected because they were found to be widespread and abundant in Poyang Lake during annual surveys. A homemade bivalve harrow (width 65 cm, mesh 2 cm) was used to collect mussels qualitatively. Monthly sampling surveys were conducted from mid-August 2015 to mid-August 2016. During each monthly survey, at least ten individuals of each species were collected, and taken to the laboratory for dissection and analysis. Specimens were fixed in 10% buffered formalin then transferred to 70% ethanol. The numbers of each species collected for this study are shown in Table 1. Figure 1. View largeDownload slide Sampling sites of nine species of unoinid from Poyang Lake, China. Figure 1. View largeDownload slide Sampling sites of nine species of unoinid from Poyang Lake, China. Table 1. Sample size, size range, and sex ratios of nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 *Sex ratio significantly different from 1:1 (P < 0.05). View Large Table 1. Sample size, size range, and sex ratios of nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 *Sex ratio significantly different from 1:1 (P < 0.05). View Large A method of thin-sectioning of the shell was used to determine age accurately, as described by Hua, Neves & Jones (2001). For sexually mature individuals, a gonad-smear method (Wang et al., 2015) was used to determination the sex. Histological sections of the gonads were used to determine the sex of immature individuals, because when gametes were undeveloped the sex could not be determined using a smear (Galbraith & Vaughn, 2009). The gonadal tissue was fixed in Bonn’s solution, embedded in paraffin, sectioned at 5–7 μm and stained with haematoxylin-eosin (HE). The germ cells in sections were observed under a Nikon 80i microscope to determine the sex. Numbers of male and female individuals for each species were recorded and used to calculate sex ratios. A goodness-of-fit test was used to test if sex ratios were different from 1:1. Individuals with marsupia fully or partially charged with eggs or glochidia were considered gravid. Numbers of gravid females in each population every month was recorded and used for calculating gravidity. Fecundity for five of the mussel species (A. chinensis, A. lanceolata, L. caveata, L. grayana, S. lampreyanus) was estimated by counting the number of eggs, developing embryos and glochidia in all of the charged gills for each gravid female. For each specimen, a syringe with a 600-μm bore needle was used to dissect gills of gravid females and release the eggs, developing embryos and glochidia from each dissected gill into a Petri dish. Shell length (SL) and shell height (SH) of glochidia from each species examined were measured and glochidial index (i.e. SL × SH) was calculated (Wu et al., 1999). The eggs and glochidia of A. chinensis, A. lanceolata, L. grayana and S. lampreyanus were bound tightly to each other and formed a conglutination within the gills. For these species, we used a 5% NaOH solution to dissolve the conglutination until the embryos and glochidia were free; they were then diluted in 1000 ml of water, poured into a beaker and stirred well. Using a micropipettor, 1 ml of the suspension was subsampled and placed in a Petri dish. The number of suspended eggs and glochidia (propagules) was counted using a dissecting microscope. This fecundity estimate was extrapolated from the mean number of propagules in ten subsamples of the extracted suspension. The total fecundity (in units of propagules/mussel) was the average number of eggs and glochidia (10 × 1 ml subsamples) multiplied by 1000 ml (Chen et al., 2010). Regression analysis (SPSS®; IBM, Armonk, NY) was used to analyse SL vs age and SL vs fecundity relationships for A. chinensis, A. lanceolata, L. caveata, L. grayana and S. lampreyanus. RESULTS Eight of the nine mussel species examined were found to be completely dioecious. The numbers of specimens and sex ratios of the nine species examined are shown in Table 1. The sex ratios of eight of the species were consistent with an expected 1:1 sex ratio, with only Cristaria plicata showing a significant difference from a 1:1 ratio (χ2 = 4.618, df = 1, P < 0.05) and a skew towards males. During dissections, nearly 30% of Lamprotula caveata specimens examined showed signs of leech parasitism, with some individuals having as many of 26 parasitizing leeches. A single hermaphrodite was detected in Arconaia lanceolata (of 13 examined) and none in any other species examined. In this individual, both male and female follicles were found to coexist independently in the gonad, with the majority being female follicles. The female follicles of this individual were in the process of becoming fully mature, while male follicles were still in the process of proliferating and growing. The male follicles appeared to be slower-developing than female gonads in this individual. Shell growth models of nine species examined are presented in Figure 2. Two nonlinear growth models (i.e. exponential function and logarithmic function) were fitted to SL-at-age. The data had strong fit to the models for all the species examined with significant and high R2. Individuals less than 3 yr old were only found for A. lanceolata and Lanceolaria eucylindrica. For Lanceolaria grayana, all specimens collected were more than 5 yr old. Figure 2. View largeDownload slide Shell length–age relationships for nine species of freshwater mussels from Poyang Lake, China. All regressions are significant (P < 0.0001). Figure 2. View largeDownload slide Shell length–age relationships for nine species of freshwater mussels from Poyang Lake, China. All regressions are significant (P < 0.0001). Both minimum SL and minimum age for individuals at sexual maturity and gravidity varied among species (Table 2). Of the species examined, Acuticosta chinensis had the shortest time to maturity, with both sexual maturity and gravidity usually occurring in less than 1 yr. For Anemina globosula and Sinanodonta woodiana pacifica, although they reached sexual maturity in less than 1 yr, individuals becoming gravid took longer (i.e. >1 yr). Individuals of C. plicata, L. caveata and Schistodesmus lampreyanus had a narrow gap in ages of sexual maturity (between 1–2 yr old), but the age of minimum gravidity had a larger difference among the three species (Table 2). Age of both sexual maturity and minimum age for gravidity for A. lanceolata, L. eucylindrica and L. grayana was much older than the other species examined; for instance, the minimum gravidity of A. lanceolata was close to 9 yr. The absence of smaller sized mussels possibly resulted from sampling method, which may have failed to collect the mature mussels at all sizes and have been biased towards larger (and older) animals. Table 2. Observed minimum shell length (SLmin) and minimum age (Amin) values for sexual maturity and gravid individuals in nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Table 2. Observed minimum shell length (SLmin) and minimum age (Amin) values for sexual maturity and gravid individuals in nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Within species, sexual maturity in males usually occurred earlier than in females. Among the species examined, L. grayana had the largest difference for minimum sexual maturity between male and female (SL 64.2 mm vs 35.0 mm; age 5.4 yr vs 3.3 yr). The difference between sexual maturity and age of first gravidity was more than 2 yr in A. lanceolata, L. caveata and S. lampreyanus (Table 2). Brooding season varied among the species examined. Seven of the species examined were found to brood glochidia in spring and summer, with C. plicata and A. chinensis showing distinct differences. Cristaria plicata predominantly brooded in autumn and spring with two distinct peaks (Fig. 3). The brooding period for A. chinensis was unique among the species examined, lasting from February to October (Fig. 3). Figure 3. View largeDownload slide Proportion of female mussels that were gravid during each sampling month for nine unionid species from Poyang Lake, China. Figure 3. View largeDownload slide Proportion of female mussels that were gravid during each sampling month for nine unionid species from Poyang Lake, China. All of the species examined had a high proportion (>60%) of gravid females during their respective peak brooding periods. In A. chinensis, A. globosula, S. woodiana pacifica, A. lanceolata, L. eucylindrica and L. grayana, the percentage of gravid females during their peak brooding period was >90%, some reaching 100%. The other three species had a somewhat lower percentage of gravid females during the peak brooding period ranged from 67% to 78% (Fig. 3). The embryonic development of individuals within populations during the brooding season was desynchronized, as embryos and glochidia appeared in the marsupium simultaneously. For most species (S. woodiana pacifica, A. lanceolata, C. plicata, L. caveata, L. eucylindrica and L. grayana), only embryos were found in the marsupium during the early brooding season, with development proceeding in a nonsynchronous manner. A. chinensis and S. lampreyanus showed similar patterns of brooding, with embryos and glochidia co-occurring in the gill marsupia through the entire period of gravidity. A. globosula was distinct, with embryos and glochidia appearing in the marsupium early in the brooding period and all embryos developing into mature glochidia later in the period (Fig. 3). The size of the mature glochidia (as measured using the glochidial index, SL × SH; Wu et al., 1999) varied widely (0.026–0.090 mm2) among the species examined, with the largest glochidia in A. globosula and the smallest in A. chinensis (Fig. 4). Figure 4. View largeDownload slide Shell length-fecundity relationships for five unionid species from Poyang Lake, China. All regressions are significant (P < 0.0001). Figure 4. View largeDownload slide Shell length-fecundity relationships for five unionid species from Poyang Lake, China. All regressions are significant (P < 0.0001). Eight of the nine species examined were ectobranchous brooders of glochidia; only L. caveata was tetragenous. Fecundity varied widely among the species examined. L. caveata had the highest mean fecundity (mean fertility 175,417; mean SL 65.8 mm) and L. grayana had the lowest mean fecundity (mean fertility 36,867; mean SL 89.5 mm) (Table 3). Fecundity was positively and significantly correlated with length in all of the species examined, using a power function or a quadratic function (Fig. 4). Furthermore, fecundity continuously increased with SL in all of the species examined. Table 3. Shell length (SL) and fecundity for five species of freshwater mussels from Poyang Lake, Yangtze River basin, China. Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) Table 3. Shell length (SL) and fecundity for five species of freshwater mussels from Poyang Lake, Yangtze River basin, China. Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) DISCUSSION The characterization of the nine species examined here adds to the knowledge of reproductive biology of Chinese freshwater mussel species, which previously had received little attention, and contributes to discussion of the evolution of life history traits of Chinese unionid species more generally. Comparison of reproductive traits In unionid species, sex ratios reported in most studies were consistently close to 1:1 (Table 4). Biased sex ratios were only found in two of the 19 species examined to date (i.e. Nodularia douglasiae and Cristaria plicata). In the nine mussel species examined in this study, incidence of hermaphroditism was low and only a single hermaphrodite was found in Arconaia lanceolata. The single hermaphrodite found in A. lanceolata was likely accidental, as both male and female follicles coexisted independently in the gonad (Coe, 1943). Heard (1975) classified functional hermaphrodites as female or male hermaphrodites according to the proportions of male and female tissue. Thus, the single hermaphrodite found was a female hermaphrodite as the gametes present were predominantly female. Table 4. Summary of reproductive traits of 19 unionid species in China. Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) *Sex ratios significantly different from 1:1 (P < 0.05). Table 4. Summary of reproductive traits of 19 unionid species in China. Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) *Sex ratios significantly different from 1:1 (P < 0.05). Skewed sex ratios and hermaphrodites in freshwater mussels had been reported in many studies. When the population densities are too low, some unionid species are known to switch genders or become hermaphroditic (Heard, 1975). However, in most unionid populations, hermaphrodites only represent a small percentage of the total (Bauer, 1987; Çek & Sereflişan, 2011). Some mussels demonstrate protandry, with small mussels being mostly male with only a portion of the gonad being converted to the female form during growth (Tudorancea, 1972; Kat, 1983; Downing et al., 1989). Nearly all mature females examined were gravid during the peak of the brooding season in Acuticosta chinensis, Anemina globosula, Arconaia lanceolata, Lanceolaria eucylindrica and L. grayana (Table 4), while in Aculamprotula fibrosa, Solenaia oleivora and Lamprotula leai, the percentage of gravid females were less than 50% (Table 4). In some species of Lamprotula and Aculamprotula abortion of glochidia appeared to occur frequently as a result of environmental disturbance (Wu et al., 2017). Species with low rates of gravidity and high rates of aborted progeny are known to be among the most imperiled Chinese freshwater mussel species (Wu et al., 2017). A number of studies have reported high gravidity rates (>85%) in females of unionid species (Jansen & Hanson, 1991; Bruenderman & Neves, 1993; Woody & Holland-Bartels, 1993), although some species have been reported with lower percentages, ranging from 64% to 75% (Haggerty & Garner, 2000; Garner, Haggerty & Modlin, 2015). For most mussel species and populations, it appears that the majority of sexually mature females become gravid during the peak of the breeding season (Haag & Staton, 2003). Among Chinese mussel species, the fecundity of Sinanodonta woodiana woodiana, A. chinensis, Lamprotula caveata, L. leai and S. oleivora was uniformly high (>100,000). All of these species were tetragenous and also had high rates of fecundity. Solenaia oleivora has the highest fecundity of the 19 Chinese mussels examined to date, but also the lowest glochidial index (Table 4). These data are too limited to determine if fecundity in Chinese mussels is related to marsupial type or glochidial size and more data from more species and populations are needed to examine this further. Fecundity is an important reproductive trait for freshwater mussels as survivorship for glochidia and juvenile mussels is normally very low (typically <1/10,000 glochidia produced will reach sexual maturity) (Bauer & Wächtler, 2001). Fecundities for unionids vary widely among species (Wächtler et al. 2001). For North American mussel species, fecundities span nearly four orders of magnitude, ranging from 2,000 to 100,000,000 progeny per female, but most species are considerably lower than the maximum reported (about 200,000; Haag, 2013). Fecundity also varied widely among Chinese mussel species, from 7,800 in Anemina arcaeformis to 1,715,000 in S. oleivora, but the fecundity of most species was <100,000 (Table 4). Some researchers have attempted to explain the differences in fecundity among unionid species. The size of the brooding area on the gills is variable among species, and has been used to account for fecundity differences among species (Haggerty et al., 1995), yet Haag & Staton (2003) found that fecundity did not relate to reproductive success. Bauer (1987) showed an inverse relationship between glochidial size and fecundity when analysing seven species. However, Haag (2013) argued that fecundity for North American freshwater mussels may be determined primarily by physical and energetic constraints, rather than life-history traits including glochidial size, lifespan, brooding strategies or host-use strategies (e.g. host attraction and host specialization). The brooding period of freshwater mussels varies among species. Chinese freshwater mussels studied so far have mainly been reported to brood in spring and summer, but the brooding periods were often much longer than the typical tachytictic definition of 2–6 weeks, e.g. 6 months or more (A. chinensis, S. woodiana pacifica, A. lanceolata, Sinohyriopsis cumingii, Lepidodesma languilati; Table 4). A smaller number of species brood over a shorter period (<4 months) in the spring from February to May (A. globosula, S. oleivora, Cuneopsis pisciculus, L. leai, L. caveata; Table 4). Other species show unique brooding periods. The brooding period of A. fibrosa was in autumn and winter (October to January), consistent with the long-term brooding reported for North American mussel species with glochidial release occurring in the spring or summer (Graf & Ó Foighil, 2000; Haag, 2013). Cristaria plicata demonstrates a unique pattern with two brooding peaks—in the autumn from September to November and the spring from March to April. The spring brooding period in C. plicata appeared to be a continuation of the autumn one; after November, with the water temperature dropping, egg development proceeded slowly or even stopped, with embryos and developing glochidia overwintering in the gill marsupia. As water temperature rose in March, glochidia matured quickly and were released, but the rate of gravidity was lower than that in autumn. Anemina arcaeformis had its brooding season from October to March. This species also appears to be capable of metamorphosis from glochidia to juvenile without parasitizing a fish (Wu, 1998; Xu, 2013). Nonparasitic larval metamorphosis in Unionidae has also been reported in a few North American species and may in part explain the large geographic distribution of unionid species demonstrating this ability (Lefever & Gurtis, 1911; Howard, 1914; Barfield & Watters, 1998; Dickinson & Sietman, 2008). How metamorphosis without a host has evolved in some Unionidae is a phenomenon deserving further study. Brooding period has historically been considered an important trait in the classification of unionid mussels. Coker et al. (1921) first described the brooding period of 54 North American unionid species, of which 36 species were tachytictic (short-term brooders) and 18 were bradytictic (long-term brooders). For Japanese unionid mussels, Kondo (1987) classified them into the following four categories; winter breeder with glochidial growth, summer breeder with glochidial growth, winter breeders without glochidial growth and summer breeders without glochidial growth. Haag (2012) has pointed out that tachytictic and brachytictic are simplistic categories and that brooding period among unionid species is much more varied, as Kondo (1987) suggested, and may be a continuum rather than discrete types. To what degree evolutionary history (phylogeny and common ancestry) and/or environment (temperature, host availability, ecophenotypic plasticity) controls the period of gravidity is unclear. Additional evolutionary (phylogenetic) and ecological studies related to the timing of gravidity in the Unionida are needed. The gill marsupium is a critical reproductive feature of unionid mussels, being the site of fertilization and of development of glochidia. The type and morphology of the gill marsupia have also been an important trait in the classification of unionids (e.g. Lopes-Lima et al., 2017). Two types of marsupium have been observed in the Chinese freshwater mussels examined in this study and in the literature (Table 4). Members of the subfamilies Anodontinae and Unioninae were all ectobranchous. The only tetragenous species examined were from the genera Lamprotula and Solenaia, both from the subfamily Gonideinae (Table 4; Lopes-Lima et al., 2017). In N. douglasiae, A. lanceolata, L. eucylindrica, L. grayana, Schistodesmus lampreyanus and Schistodesmus spinosus, the glochidia were always aggregated in the marsupium as a conglutinate mass (Table 4). The remaining species had diffuse glochidia within the marsupium (A. fibrosa, C. pisciculus, S. cumingii, L. languilati, A. lanceolata and Sinanodonta). In some North American unionids, species use elaborate conglutinates (packages of glochidia) that resemble fish fry, worms or the pupae of aquatic insects to actively attract a host fish (Zanatta & Murphy, 2006; Barnhart et al. 2008; Haag, 2012). For Chinese freshwater mussels, host attraction behaviours have not been reported, but we speculate that the arrangement of the glochidia in the gill marsupium (conglutinate vs diffuse) may be related to host use and host attraction. Conservation implications Poyang Lake has the highest species richness and densities of unionid mussels reported from the Yangtze River drainage. Recent human-induced habitat changes have made conditions less favourable for freshwater mussels, with many populations and species showing signs of serious declines (Wu et al., 2017). Additionally, many unionids are harvested for human consumption and pearls; unregulated mussel fishery practices and nonscientific management styles are likely contributing to the dramatic decline in freshwater mussel populations in the region (Wu et al., 2017). The new information provided by this study on the reproductive traits of nine species in Poyang Lake provides critical life-history data that can be incorporated into management strategies for the mussel fishery. It is recommended that commercial mussel harvest be carried out during the non-gravid period, to ensure that the maximum number of mussels are able to complete their reproductive cycle. Some harvested species that were examined in this study have a very long brooding period (>6 months) for their glochidia (e.g. A. chinensis, C. plicata) and it is therefore recommended that harvest of these species be targeted within certain size or age classes on a year-to-year basis, in order to avoid large disruptions of the reproductive cycle for the entire population. The length-fecundity relationships detailed in this study (Fig. 4) demonstrate that fecundity increases throughout the life of all of the species examined, indicating that larger (and older) individuals are capable of producing more offspring. This means that efforts should be made to avoid the harvest of the largest animals in a population in order to maximize their potential to reproduce and provide new recruits to the population. These data can also be used to help create life tables to construct population models (e.g. Hassal et al., 2017) that can help to determine acceptable and sustainable levels of harvest and mortality. To complete their development, unionid mussels must (in almost all cases) parasitize a suitable host fish species, but investigations of mussel-fish parasite-host relationships remains a critical knowledge gap for most Chinese unionid species. The condition (e.g. size, age and health) of the host fish may affect the development of glochidia while encysted and their ability to successfully metamorphose into a free-living juvenile mussel (Barnhart et al. 2008; Douda et al., 2016; Modesto et al., 2018). It is recommended that regulations on fish harvest should take the life cycle of mussels into consideration. A suggestion would be to limit the harvest of known host fish species during the months of March to July in Poyang Lake in order to allow for glochidia to successfully complete their parasitic phase. Information on the reproductive patterns of unionids in combination with host testing will greatly enhance management and conservation of the diverse (yet increasingly imperiled) mussel assemblage in the Poyang Lake and Yangtze River drainage. 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Journal of Experimental Zoology Part A: Ecological Genetics & Physiology , 315A : 30 – 40 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molluscan Studies Oxford University Press

Reproductive traits of nine freshwater mussel species (Mollusca: Unionidae) from Poyang Lake, China

Journal of Molluscan Studies , Volume Advance Article (3) – May 23, 2018

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Abstract

Abstract The Yangtze River basin has the highest species richness for freshwater mussels in East Asia, but little is known about the reproductive biology of most species. Here, the reproductive biology of nine freshwater mussel species (Unionidae) found in Poyang Lake, Jiangxi province, China was studied. Sex ratios were not significantly different from 1:1 in eight of the nine species examined. The minimum size and age of sexual maturity (0.2–5.6 yr for males and 0.3–5.9 yr for females) and of females first becoming gravid (0.6–8.9 yr) varied among species. Brooding seasons for glochidia varied among species, but seven of the nine species examined brooded glochidia through the spring and summer. In all species, high frequencies (mean 87.5%) were found to be gravid during their peak brooding period. Fecundity was positively correlated with shell length in the five species in which this was examined. Mean annual fecundity showed wide interspecific variation, ranging from 7,800 to 328,000 propagules/mussel. The findings of this study have implications for the ecology and conservation of unionid species in China and contributes to knowledge of the evolution of reproductive and life history characters of the Unionidae. INTRODUCTION Freshwater mussels (Unionidae) are a major component of food webs and play important roles in freshwater ecosystems (Howard & Cuffey, 2006; Vaughn, 2017). Unionids have a unique life cycle with an obligate parasitic larval stage (glochdia) that is dependent on a host fish (Wächtler, Drehermansur & Richter, 2001). Glochidia are brooded in a specialized marsupium formed by the interlamellar spaces (water-tubes) of the gills in female mussels. The portion of the gills used for brooding varies among species, ranging from the use of all four gills (tetragenous) to only a portion of the outer gills (ectobranchous) (Simpson, 1914; Davis & Fuller, 1981). Brooding time of glochidia in the gill marsupium also varies among species, with a continuum between short-term (tachytictic) and long-term (bradytictic) brooding strategies, as described in North American freshwater mussels (Sterki, 1895, 1898; Ortmann, 1911; Price & Eads, 2011). Short-term brooders brood glochidia for c. 2–6 weeks before release usually in spring or summer, while long-term brooders retain glochidia for up to 8 months, usually from late summer or autumn until release the following spring or summer (Haag, 2013). After release from the female, glochidia must find a suitable host fish, surviving outside of their mother for a few days at most (Kat, 2010). The larval stage is the most sensitive in the unionid life cycle (Hardison & Layzer, 2001). To a certain extent because of the disruption of the life cycles by anthropogenic stressors, mussels have become among the most imperiled animal groups on Earth (Regnier, Fontaine & Bouchet, 2009; Haag, 2012). Research on the reproductive traits of freshwater mussels can be traced back to the early 1900s that first revealed information on aspects such as sex ratios, fecundity and brooding periods (Coker et al., 1921; Bauer, 1987; Downing et al., 1989; Gordon & Smith, 1990; Haag & Staton, 2003). Brooding periods and arrangement of the gill marsupium have historically been important characteristics for the classification of the more than 290 North American freshwater mussel species (Ortmann, 1912; Heard & Guckert, 1971; Davis & Fuller, 1981; Lopes-Lima et al., 2017). Currently, in the context of the decline of freshwater mussel species and with the survival of many species seriously threatened, many scholars are actively researching the evolution of reproductive characters and life history strategies of unionids (Graf & Ó Foighil, 2000; Zanatta & Murphy, 2006; Barnhart, Haag & Roston, 2008; Haag, 2013; Pfeiffer & Graf, 2015; Lopes-Lima et al., 2017). Furthermore, information on the reproductive characters and life history is critically important for the conservation and recovery of many unionid species (Mcivor & Aldridge, 2007; Haag, 2012). The middle portion of the Yangtze River basin is one of the most species-rich regions for freshwater mussels on Earth (Zieritz et al., 2017), including many endemic species (He & Zhuang, 2013; Shu et al., 2014). However, research on the reproductive biology of unionids from this biodiverse region has largely been lacking. In recent decades, a series of anthropogenic stressors, including habitat destruction and degradation, water pollution and increasing commercial exploitation for human consumption and pearls, have resulted in the imperilment of many mussel species in the region (Shu et al., 2009; Xiong, Ouyang & Wu, 2012; Wu et al., 2017; Zieritz et al., 2017). Due to the lack of information on reproductive traits for most unionid species in the region, management and conservation efforts for unionids has been greatly hampered. It is thus critical that there is an improved understanding of the reproductive biology of Chinese unionid mussel species in order to provide bases for classification and conservation. This study is the first to examine the reproductive characteristics of nine native unionid mussel species found in Poyang Lake, the largest freshwater lake in the Yangtze River basin, in Jiangxi Province, China: Acuticosta chinensis (Lea, 1868), Anemina globosula (Heude, 1878), Arconaia lanceolata (Lea, 1856), Cristaria plicata (Leach, 1814), Lamprotula caveata (Heude, 1877), Lanceolaria eucylindrica Lin, 1962, Lanceolaria grayana (Lea, 1834), Schistodesmus lampreyanus (Baird & Adams, 1867) and Sinanodonta woodiana pacifica (Heude, 1878). Based on the information gathered on the reproductive biology of these species, some preliminary management strategies are proposed for unionid mussels in Poyang Lake and the Yangtze River drainage. This study not only begins to fill a major knowledge gap on the reproductive biology of Chinese freshwater mussels, but also lays a foundation for further study of the evolution of their reproductive and life history characters. MATERIAL AND METHODS Specimens were collected from Poyang Lake, located on the south side of the middle Yangtze River, China (Fig. 1). Nine mussel species were examined, selected because they were found to be widespread and abundant in Poyang Lake during annual surveys. A homemade bivalve harrow (width 65 cm, mesh 2 cm) was used to collect mussels qualitatively. Monthly sampling surveys were conducted from mid-August 2015 to mid-August 2016. During each monthly survey, at least ten individuals of each species were collected, and taken to the laboratory for dissection and analysis. Specimens were fixed in 10% buffered formalin then transferred to 70% ethanol. The numbers of each species collected for this study are shown in Table 1. Figure 1. View largeDownload slide Sampling sites of nine species of unoinid from Poyang Lake, China. Figure 1. View largeDownload slide Sampling sites of nine species of unoinid from Poyang Lake, China. Table 1. Sample size, size range, and sex ratios of nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 *Sex ratio significantly different from 1:1 (P < 0.05). View Large Table 1. Sample size, size range, and sex ratios of nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 Species n Length (mm) Percent female Percent male Acuticosta chinensis 1,324 16.4–59.0 50 50 Anemina globosula 198 33.0–81.6 51 49 Sinanodonta woodiana pacifica 130 39.4–140 38 62 Arconaia lanceolata 138 54.9–125.0 50 50 Cristaria plicata* 157 56.1–245.0 34 66 Lamprotula caveata 1,712 21.6–94.1 49 51 Lanceolaria eucylindrica 211 64.8–118.7 44 56 Lanceolaria grayana 279 35.0–115.8 50 50 Schistodesmus lampreyanus 325 23.0–57.3 45 55 *Sex ratio significantly different from 1:1 (P < 0.05). View Large A method of thin-sectioning of the shell was used to determine age accurately, as described by Hua, Neves & Jones (2001). For sexually mature individuals, a gonad-smear method (Wang et al., 2015) was used to determination the sex. Histological sections of the gonads were used to determine the sex of immature individuals, because when gametes were undeveloped the sex could not be determined using a smear (Galbraith & Vaughn, 2009). The gonadal tissue was fixed in Bonn’s solution, embedded in paraffin, sectioned at 5–7 μm and stained with haematoxylin-eosin (HE). The germ cells in sections were observed under a Nikon 80i microscope to determine the sex. Numbers of male and female individuals for each species were recorded and used to calculate sex ratios. A goodness-of-fit test was used to test if sex ratios were different from 1:1. Individuals with marsupia fully or partially charged with eggs or glochidia were considered gravid. Numbers of gravid females in each population every month was recorded and used for calculating gravidity. Fecundity for five of the mussel species (A. chinensis, A. lanceolata, L. caveata, L. grayana, S. lampreyanus) was estimated by counting the number of eggs, developing embryos and glochidia in all of the charged gills for each gravid female. For each specimen, a syringe with a 600-μm bore needle was used to dissect gills of gravid females and release the eggs, developing embryos and glochidia from each dissected gill into a Petri dish. Shell length (SL) and shell height (SH) of glochidia from each species examined were measured and glochidial index (i.e. SL × SH) was calculated (Wu et al., 1999). The eggs and glochidia of A. chinensis, A. lanceolata, L. grayana and S. lampreyanus were bound tightly to each other and formed a conglutination within the gills. For these species, we used a 5% NaOH solution to dissolve the conglutination until the embryos and glochidia were free; they were then diluted in 1000 ml of water, poured into a beaker and stirred well. Using a micropipettor, 1 ml of the suspension was subsampled and placed in a Petri dish. The number of suspended eggs and glochidia (propagules) was counted using a dissecting microscope. This fecundity estimate was extrapolated from the mean number of propagules in ten subsamples of the extracted suspension. The total fecundity (in units of propagules/mussel) was the average number of eggs and glochidia (10 × 1 ml subsamples) multiplied by 1000 ml (Chen et al., 2010). Regression analysis (SPSS®; IBM, Armonk, NY) was used to analyse SL vs age and SL vs fecundity relationships for A. chinensis, A. lanceolata, L. caveata, L. grayana and S. lampreyanus. RESULTS Eight of the nine mussel species examined were found to be completely dioecious. The numbers of specimens and sex ratios of the nine species examined are shown in Table 1. The sex ratios of eight of the species were consistent with an expected 1:1 sex ratio, with only Cristaria plicata showing a significant difference from a 1:1 ratio (χ2 = 4.618, df = 1, P < 0.05) and a skew towards males. During dissections, nearly 30% of Lamprotula caveata specimens examined showed signs of leech parasitism, with some individuals having as many of 26 parasitizing leeches. A single hermaphrodite was detected in Arconaia lanceolata (of 13 examined) and none in any other species examined. In this individual, both male and female follicles were found to coexist independently in the gonad, with the majority being female follicles. The female follicles of this individual were in the process of becoming fully mature, while male follicles were still in the process of proliferating and growing. The male follicles appeared to be slower-developing than female gonads in this individual. Shell growth models of nine species examined are presented in Figure 2. Two nonlinear growth models (i.e. exponential function and logarithmic function) were fitted to SL-at-age. The data had strong fit to the models for all the species examined with significant and high R2. Individuals less than 3 yr old were only found for A. lanceolata and Lanceolaria eucylindrica. For Lanceolaria grayana, all specimens collected were more than 5 yr old. Figure 2. View largeDownload slide Shell length–age relationships for nine species of freshwater mussels from Poyang Lake, China. All regressions are significant (P < 0.0001). Figure 2. View largeDownload slide Shell length–age relationships for nine species of freshwater mussels from Poyang Lake, China. All regressions are significant (P < 0.0001). Both minimum SL and minimum age for individuals at sexual maturity and gravidity varied among species (Table 2). Of the species examined, Acuticosta chinensis had the shortest time to maturity, with both sexual maturity and gravidity usually occurring in less than 1 yr. For Anemina globosula and Sinanodonta woodiana pacifica, although they reached sexual maturity in less than 1 yr, individuals becoming gravid took longer (i.e. >1 yr). Individuals of C. plicata, L. caveata and Schistodesmus lampreyanus had a narrow gap in ages of sexual maturity (between 1–2 yr old), but the age of minimum gravidity had a larger difference among the three species (Table 2). Age of both sexual maturity and minimum age for gravidity for A. lanceolata, L. eucylindrica and L. grayana was much older than the other species examined; for instance, the minimum gravidity of A. lanceolata was close to 9 yr. The absence of smaller sized mussels possibly resulted from sampling method, which may have failed to collect the mature mussels at all sizes and have been biased towards larger (and older) animals. Table 2. Observed minimum shell length (SLmin) and minimum age (Amin) values for sexual maturity and gravid individuals in nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Table 2. Observed minimum shell length (SLmin) and minimum age (Amin) values for sexual maturity and gravid individuals in nine freshwater mussel species from Poyang Lake, Yangtze River drainage, China. Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Species SLmin (mm) and Amin (yr) of sexually mature female (F) and male (M) individuals SLmin (mm) and Amin (yr) of gravid females SL (F) Age (F) SL (M) Age (M) SL Age Acuticosta chinensis 18.9 0.3 16.4 0.2 23.4 0.6 Anemina globosula 40.0 0.6 33.0 0.4 49.2 1.0 Sinanodonta woodiana pacifica 41.2 0.7 39.4 0.6 66.0 1.1 Arconaia lanceolata 66.6 4.8 54.9 3.4 92.4 8.9 Cristaria plicata 68.0 1.2 56.1 1.0 98.2 1.9 Lamprotula caveata 22.1 1.6 21.6 1.5 42.3 4.6 Lanceolaria eucylindrica 68.1 5.9 64.8 5.6 77.3 7.1 Lanceolaria grayana 64.2 5.4 35.0 3.3 69.0 5.8 Schistodesmus lampreyanus 25.4 1.6 23.0 1.3 38.1 3.4 Within species, sexual maturity in males usually occurred earlier than in females. Among the species examined, L. grayana had the largest difference for minimum sexual maturity between male and female (SL 64.2 mm vs 35.0 mm; age 5.4 yr vs 3.3 yr). The difference between sexual maturity and age of first gravidity was more than 2 yr in A. lanceolata, L. caveata and S. lampreyanus (Table 2). Brooding season varied among the species examined. Seven of the species examined were found to brood glochidia in spring and summer, with C. plicata and A. chinensis showing distinct differences. Cristaria plicata predominantly brooded in autumn and spring with two distinct peaks (Fig. 3). The brooding period for A. chinensis was unique among the species examined, lasting from February to October (Fig. 3). Figure 3. View largeDownload slide Proportion of female mussels that were gravid during each sampling month for nine unionid species from Poyang Lake, China. Figure 3. View largeDownload slide Proportion of female mussels that were gravid during each sampling month for nine unionid species from Poyang Lake, China. All of the species examined had a high proportion (>60%) of gravid females during their respective peak brooding periods. In A. chinensis, A. globosula, S. woodiana pacifica, A. lanceolata, L. eucylindrica and L. grayana, the percentage of gravid females during their peak brooding period was >90%, some reaching 100%. The other three species had a somewhat lower percentage of gravid females during the peak brooding period ranged from 67% to 78% (Fig. 3). The embryonic development of individuals within populations during the brooding season was desynchronized, as embryos and glochidia appeared in the marsupium simultaneously. For most species (S. woodiana pacifica, A. lanceolata, C. plicata, L. caveata, L. eucylindrica and L. grayana), only embryos were found in the marsupium during the early brooding season, with development proceeding in a nonsynchronous manner. A. chinensis and S. lampreyanus showed similar patterns of brooding, with embryos and glochidia co-occurring in the gill marsupia through the entire period of gravidity. A. globosula was distinct, with embryos and glochidia appearing in the marsupium early in the brooding period and all embryos developing into mature glochidia later in the period (Fig. 3). The size of the mature glochidia (as measured using the glochidial index, SL × SH; Wu et al., 1999) varied widely (0.026–0.090 mm2) among the species examined, with the largest glochidia in A. globosula and the smallest in A. chinensis (Fig. 4). Figure 4. View largeDownload slide Shell length-fecundity relationships for five unionid species from Poyang Lake, China. All regressions are significant (P < 0.0001). Figure 4. View largeDownload slide Shell length-fecundity relationships for five unionid species from Poyang Lake, China. All regressions are significant (P < 0.0001). Eight of the nine species examined were ectobranchous brooders of glochidia; only L. caveata was tetragenous. Fecundity varied widely among the species examined. L. caveata had the highest mean fecundity (mean fertility 175,417; mean SL 65.8 mm) and L. grayana had the lowest mean fecundity (mean fertility 36,867; mean SL 89.5 mm) (Table 3). Fecundity was positively and significantly correlated with length in all of the species examined, using a power function or a quadratic function (Fig. 4). Furthermore, fecundity continuously increased with SL in all of the species examined. Table 3. Shell length (SL) and fecundity for five species of freshwater mussels from Poyang Lake, Yangtze River basin, China. Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) Table 3. Shell length (SL) and fecundity for five species of freshwater mussels from Poyang Lake, Yangtze River basin, China. Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) Species Mean SL (mm) Mean fecundity ± SD (propagules/mussel) Acuticosta chinensis 39.4 83,407 ± 22,999 (41,900–113,200) Arconaia lanceolata 108.5 102,625 ± 45,890 (41,600–169,200) Lamprotula caveata 65.8 175,417 ± 66,807 (96,200–309,000) Lanceolaria grayana 89.5 36,867 ± 17,838 (12,600–67,400) Schistodesmus lampreyanus 44.9 46,133 ± 28,967 (4,600–98,600) DISCUSSION The characterization of the nine species examined here adds to the knowledge of reproductive biology of Chinese freshwater mussel species, which previously had received little attention, and contributes to discussion of the evolution of life history traits of Chinese unionid species more generally. Comparison of reproductive traits In unionid species, sex ratios reported in most studies were consistently close to 1:1 (Table 4). Biased sex ratios were only found in two of the 19 species examined to date (i.e. Nodularia douglasiae and Cristaria plicata). In the nine mussel species examined in this study, incidence of hermaphroditism was low and only a single hermaphrodite was found in Arconaia lanceolata. The single hermaphrodite found in A. lanceolata was likely accidental, as both male and female follicles coexisted independently in the gonad (Coe, 1943). Heard (1975) classified functional hermaphrodites as female or male hermaphrodites according to the proportions of male and female tissue. Thus, the single hermaphrodite found was a female hermaphrodite as the gametes present were predominantly female. Table 4. Summary of reproductive traits of 19 unionid species in China. Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) *Sex ratios significantly different from 1:1 (P < 0.05). Table 4. Summary of reproductive traits of 19 unionid species in China. Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) Subfamily Taxon Brooding period Brooding location for glochidia Aggregation of glochidia within marsupium Gravidity rate (%) Mean fecundity (propagules/mussel) Glochidial index Sex ratio (M:F) Ref. Anodontinae Acuticosta chinensis Feb-Oct Ectobranchous Conglutinate 93 83,000 0.026 1.02 This study Anemina arcaeformis Dec-Mar Ectobranchous Diffuse – 7,800 – – Liu (2008) Anemina globosula Feb-Apr Ectobranchous Diffuse 100 91,000 0.090 0.98 This study Sinanodonta woodiana pacifica Apr-Aug Ectobranchous Diffuse 80 68,000 0.088 –; 1.60 Zheng & Wei (2000); this study Lanceolaria eucylindrica Mar-Aug Ectobranchous Conglutinate 100 – 0.040 1.27 This study Lanceolaria grayana Mar-Aug Ectobranchous Conglutinate 100 37,000 0.040 0.99 This study Sinanodonta woodiana woodiana Mar-Jun Ectobranchous Diffuse – 610,000 0.080 – Liu (2008) Arconaia lanceolata Apr-Sep Ectobranchous Diffuse 100 102,000 – 1.00 This study Cristaria plicata Sep-Apr Ectobranchous Diffuse 71;100 – 0.081 1.03; 1.91* Tian (1993); this study Unioninae Aculamprotula fibrosa Nov-Jan Ectobranchous Diffuse 55 – 0.059 1.19 Zhu et al. (1997) Cuneopsis pisciculus Apr-Jun Ectobranchous Diffuse – 10,000 – 1.00 Liu (2008) Schistodesmus lampreyanus Feb-May Ectobranchous Conglutinate 68 46,000 – 1.24 This study Schistodesmus spinosus May-Sep Ectobranchous Conglutinate – 24,000 – – Liu (2008) Lepidodesma languilati Feb-Aug Ectobranchous Diffuse – 37,000 0.131 – Liu (2008) Nodularia douglasiae Feb-Jul Ectobranchous Conglutinate 84.21 184,000 0.020 0.83; 0.61* Liu (2008); Chen et al. (2010) Gonideninae Sinohyriopsis cumingii Apr-Oct Ectobranchous Diffuse – – 0.054 – Wu et al. (2000) Lamprotula caveata Apr-Aug Tetragenous Diffuse 79 175,000 0.036 1.03 This study Lamprotula leai Feb-May Tetragenous Diffuse 36 328,000 – 1.10 Xu et al. (2013) Solenaia oleivora Feb-May Tetragenous Diffuse 35.00 1,715,000 0.010 1.02 Wang et al. (2015) *Sex ratios significantly different from 1:1 (P < 0.05). Skewed sex ratios and hermaphrodites in freshwater mussels had been reported in many studies. When the population densities are too low, some unionid species are known to switch genders or become hermaphroditic (Heard, 1975). However, in most unionid populations, hermaphrodites only represent a small percentage of the total (Bauer, 1987; Çek & Sereflişan, 2011). Some mussels demonstrate protandry, with small mussels being mostly male with only a portion of the gonad being converted to the female form during growth (Tudorancea, 1972; Kat, 1983; Downing et al., 1989). Nearly all mature females examined were gravid during the peak of the brooding season in Acuticosta chinensis, Anemina globosula, Arconaia lanceolata, Lanceolaria eucylindrica and L. grayana (Table 4), while in Aculamprotula fibrosa, Solenaia oleivora and Lamprotula leai, the percentage of gravid females were less than 50% (Table 4). In some species of Lamprotula and Aculamprotula abortion of glochidia appeared to occur frequently as a result of environmental disturbance (Wu et al., 2017). Species with low rates of gravidity and high rates of aborted progeny are known to be among the most imperiled Chinese freshwater mussel species (Wu et al., 2017). A number of studies have reported high gravidity rates (>85%) in females of unionid species (Jansen & Hanson, 1991; Bruenderman & Neves, 1993; Woody & Holland-Bartels, 1993), although some species have been reported with lower percentages, ranging from 64% to 75% (Haggerty & Garner, 2000; Garner, Haggerty & Modlin, 2015). For most mussel species and populations, it appears that the majority of sexually mature females become gravid during the peak of the breeding season (Haag & Staton, 2003). Among Chinese mussel species, the fecundity of Sinanodonta woodiana woodiana, A. chinensis, Lamprotula caveata, L. leai and S. oleivora was uniformly high (>100,000). All of these species were tetragenous and also had high rates of fecundity. Solenaia oleivora has the highest fecundity of the 19 Chinese mussels examined to date, but also the lowest glochidial index (Table 4). These data are too limited to determine if fecundity in Chinese mussels is related to marsupial type or glochidial size and more data from more species and populations are needed to examine this further. Fecundity is an important reproductive trait for freshwater mussels as survivorship for glochidia and juvenile mussels is normally very low (typically <1/10,000 glochidia produced will reach sexual maturity) (Bauer & Wächtler, 2001). Fecundities for unionids vary widely among species (Wächtler et al. 2001). For North American mussel species, fecundities span nearly four orders of magnitude, ranging from 2,000 to 100,000,000 progeny per female, but most species are considerably lower than the maximum reported (about 200,000; Haag, 2013). Fecundity also varied widely among Chinese mussel species, from 7,800 in Anemina arcaeformis to 1,715,000 in S. oleivora, but the fecundity of most species was <100,000 (Table 4). Some researchers have attempted to explain the differences in fecundity among unionid species. The size of the brooding area on the gills is variable among species, and has been used to account for fecundity differences among species (Haggerty et al., 1995), yet Haag & Staton (2003) found that fecundity did not relate to reproductive success. Bauer (1987) showed an inverse relationship between glochidial size and fecundity when analysing seven species. However, Haag (2013) argued that fecundity for North American freshwater mussels may be determined primarily by physical and energetic constraints, rather than life-history traits including glochidial size, lifespan, brooding strategies or host-use strategies (e.g. host attraction and host specialization). The brooding period of freshwater mussels varies among species. Chinese freshwater mussels studied so far have mainly been reported to brood in spring and summer, but the brooding periods were often much longer than the typical tachytictic definition of 2–6 weeks, e.g. 6 months or more (A. chinensis, S. woodiana pacifica, A. lanceolata, Sinohyriopsis cumingii, Lepidodesma languilati; Table 4). A smaller number of species brood over a shorter period (<4 months) in the spring from February to May (A. globosula, S. oleivora, Cuneopsis pisciculus, L. leai, L. caveata; Table 4). Other species show unique brooding periods. The brooding period of A. fibrosa was in autumn and winter (October to January), consistent with the long-term brooding reported for North American mussel species with glochidial release occurring in the spring or summer (Graf & Ó Foighil, 2000; Haag, 2013). Cristaria plicata demonstrates a unique pattern with two brooding peaks—in the autumn from September to November and the spring from March to April. The spring brooding period in C. plicata appeared to be a continuation of the autumn one; after November, with the water temperature dropping, egg development proceeded slowly or even stopped, with embryos and developing glochidia overwintering in the gill marsupia. As water temperature rose in March, glochidia matured quickly and were released, but the rate of gravidity was lower than that in autumn. Anemina arcaeformis had its brooding season from October to March. This species also appears to be capable of metamorphosis from glochidia to juvenile without parasitizing a fish (Wu, 1998; Xu, 2013). Nonparasitic larval metamorphosis in Unionidae has also been reported in a few North American species and may in part explain the large geographic distribution of unionid species demonstrating this ability (Lefever & Gurtis, 1911; Howard, 1914; Barfield & Watters, 1998; Dickinson & Sietman, 2008). How metamorphosis without a host has evolved in some Unionidae is a phenomenon deserving further study. Brooding period has historically been considered an important trait in the classification of unionid mussels. Coker et al. (1921) first described the brooding period of 54 North American unionid species, of which 36 species were tachytictic (short-term brooders) and 18 were bradytictic (long-term brooders). For Japanese unionid mussels, Kondo (1987) classified them into the following four categories; winter breeder with glochidial growth, summer breeder with glochidial growth, winter breeders without glochidial growth and summer breeders without glochidial growth. Haag (2012) has pointed out that tachytictic and brachytictic are simplistic categories and that brooding period among unionid species is much more varied, as Kondo (1987) suggested, and may be a continuum rather than discrete types. To what degree evolutionary history (phylogeny and common ancestry) and/or environment (temperature, host availability, ecophenotypic plasticity) controls the period of gravidity is unclear. Additional evolutionary (phylogenetic) and ecological studies related to the timing of gravidity in the Unionida are needed. The gill marsupium is a critical reproductive feature of unionid mussels, being the site of fertilization and of development of glochidia. The type and morphology of the gill marsupia have also been an important trait in the classification of unionids (e.g. Lopes-Lima et al., 2017). Two types of marsupium have been observed in the Chinese freshwater mussels examined in this study and in the literature (Table 4). Members of the subfamilies Anodontinae and Unioninae were all ectobranchous. The only tetragenous species examined were from the genera Lamprotula and Solenaia, both from the subfamily Gonideinae (Table 4; Lopes-Lima et al., 2017). In N. douglasiae, A. lanceolata, L. eucylindrica, L. grayana, Schistodesmus lampreyanus and Schistodesmus spinosus, the glochidia were always aggregated in the marsupium as a conglutinate mass (Table 4). The remaining species had diffuse glochidia within the marsupium (A. fibrosa, C. pisciculus, S. cumingii, L. languilati, A. lanceolata and Sinanodonta). In some North American unionids, species use elaborate conglutinates (packages of glochidia) that resemble fish fry, worms or the pupae of aquatic insects to actively attract a host fish (Zanatta & Murphy, 2006; Barnhart et al. 2008; Haag, 2012). For Chinese freshwater mussels, host attraction behaviours have not been reported, but we speculate that the arrangement of the glochidia in the gill marsupium (conglutinate vs diffuse) may be related to host use and host attraction. Conservation implications Poyang Lake has the highest species richness and densities of unionid mussels reported from the Yangtze River drainage. Recent human-induced habitat changes have made conditions less favourable for freshwater mussels, with many populations and species showing signs of serious declines (Wu et al., 2017). Additionally, many unionids are harvested for human consumption and pearls; unregulated mussel fishery practices and nonscientific management styles are likely contributing to the dramatic decline in freshwater mussel populations in the region (Wu et al., 2017). The new information provided by this study on the reproductive traits of nine species in Poyang Lake provides critical life-history data that can be incorporated into management strategies for the mussel fishery. It is recommended that commercial mussel harvest be carried out during the non-gravid period, to ensure that the maximum number of mussels are able to complete their reproductive cycle. Some harvested species that were examined in this study have a very long brooding period (>6 months) for their glochidia (e.g. A. chinensis, C. plicata) and it is therefore recommended that harvest of these species be targeted within certain size or age classes on a year-to-year basis, in order to avoid large disruptions of the reproductive cycle for the entire population. The length-fecundity relationships detailed in this study (Fig. 4) demonstrate that fecundity increases throughout the life of all of the species examined, indicating that larger (and older) individuals are capable of producing more offspring. This means that efforts should be made to avoid the harvest of the largest animals in a population in order to maximize their potential to reproduce and provide new recruits to the population. These data can also be used to help create life tables to construct population models (e.g. Hassal et al., 2017) that can help to determine acceptable and sustainable levels of harvest and mortality. To complete their development, unionid mussels must (in almost all cases) parasitize a suitable host fish species, but investigations of mussel-fish parasite-host relationships remains a critical knowledge gap for most Chinese unionid species. The condition (e.g. size, age and health) of the host fish may affect the development of glochidia while encysted and their ability to successfully metamorphose into a free-living juvenile mussel (Barnhart et al. 2008; Douda et al., 2016; Modesto et al., 2018). It is recommended that regulations on fish harvest should take the life cycle of mussels into consideration. A suggestion would be to limit the harvest of known host fish species during the months of March to July in Poyang Lake in order to allow for glochidia to successfully complete their parasitic phase. Information on the reproductive patterns of unionids in combination with host testing will greatly enhance management and conservation of the diverse (yet increasingly imperiled) mussel assemblage in the Poyang Lake and Yangtze River drainage. 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Journal of Molluscan StudiesOxford University Press

Published: May 23, 2018

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