ON THE REPRODUCTION OF PISIDIUM C . PFEIFFER , 1821 ( BIVALVIA : SPHAERIIDAE ) FROM SĄPOLNO ( NW . POLAND )

During a malacofaunistic survey in the environs of Sąpolno (Pomeranian Lakeland, NW. Poland) 16 species of Pisidium were found within an area of 2.5×2.0 km: P. amnicum (O. F. Müller), P. casertanum (Poli), P. crassum (Stelfox), P. globulare Clessin, P. henslowanum (Sheppard), P. hibernicum Westerlund, P. milium Held, P. moitessierianum Paladilhe, P. nitidum Jenyns, P. obtusale (Lamarck), P. personatum Malm, P. ponderosum (Stelfox), P. pseudosphaerium Schlesch, P. pulchellum Jenyns, P. subtruncatum Malm and P. supinum A. Schmidt. The number of offspring during the reproductive season depended on the species and on the parent’s size. The most fecund species was P. ponderosum, with up to 75 juveniles released at once. The offspring of P. amnicum were the largest (mean shell length 2.05 mm), but relatively small compared to the parent’s shell (mean 26.1%). The smallest juveniles were produced by P. moitessierianum (mean shell length 0.59 mm). The juvenile shells of the studied species were more or less elongated, with the height/length ratio ranging from 0.75 (P. amnicum, P. ponderosum, P. henslowanum) to 0.83 (P. subtruncatum, P. hibernicum). Distinct differences were observed in the reproduction of P. casertanum and P. ponderosum. However the differences between the reproductive parameters of P. ponderosum and the form described as P. casertanum var. humeriformis were rather small. key wordS: ovoviviparity, fecundity, infra-specific differences, shell of offspring


INTRODUCTION
Bivalves of the genus Pisidium C. Pfeiffer, 1821 are ovoviviparous hermaphrodites.Their embryos develop in brood pouches formed by ctenidia (only one pouch at a time) (Meier-Brook 1970, Holopainen & HanSki 1986, Piechocki 1991, KorniuShin 2007, piechocki & wawrzyniak-wydrowSka 2016).When the embryos are shell-covered, the pouch walls burst but the juveniles remain within the parent's body for some time and continue growing (Piechocki 1991, KorniuShin 2007).Only after they have been released, new pouches with new embryos can be formed.

MATERIAL AND METHODS
The material was collected in 1997-1999 in the environs of the village Sąpolno (Fig. 2).The bivalves were identified based on shell characters according to Piechocki & Dyduch-FalniowSka (1993).In order to avoid mistakes, individuals with atypical shells (e.g.misshapen or intermediate between species) were disregarded.The shells left as voucher material were additionally verified with the key of Glöer & Meier-Brook (2003).Based on anatomical differences some authors divided the genus Pisidium into an array of subgenera.Here I adopted the division used by Glöer & zettler (2005).
Adult bivalves (usually with shelled embryos visible through the shell) were kept in the laboratory (1) released offspring immediately after being placed in the culture, and the released juveniles were few (some may have been released in the field); (2) started releasing offspring only after a few months (unfavourable food conditions in the culture may have caused death of some embryos) were omitted from the fecundity data analysis.Small pieces of decaying tree leaves found in the water (mainly black alder, less often poplar or oak) and a small quantity of brown suspension from drainage ditches were placed in the containers as food.The bivalves were kept mostly singly, in containers of 25 mm diameter filled with water to 20 mm (rarely in groups, to collect a large number of juveniles).Only P. amnicum and the largest species: P. casertanum, P. ponderosum and P. hen slowanum (shell length more than 4.5 mm), were kept in larger containers of 60 mm diameter and water 30 mm deep.When the observations were concluded, both the juveniles and most of the adults were released in their original sites.Only a part of the largest individuals of all the species were retained in the laboratory.
The shells were measured with calibrated eyepiece to the nearest 0.01 mm (size below 1 mm) or 0.05 mm (range 2.3-5.0 mm).Adult shells more than 5 mm long were measured on graph paper, magnification 6× (accuracy ca.0.1 mm).Newly released juveniles were measured in a large drop of water; all the adult and juvenile P. amnicum were dried prior to measurements.
In the text "n" means the number of measured parents or offspring, r -Pearson's coefficient of linear correlation, SD -standard deviation.In the figures the numbering of water bodies (e.g.drainage ditches) is separate for each species.

Pisidium (Pisidium) amnicum (O. F. Müller, 1774)
In the studied area the species occurred in the rivers Brda and Lipczynka.The largest found empty shell was 11.2 mm long.Four distinct growth inhibition striae were usually visible on shells of more than 10 mm (less often 3 striae).The first stria was marked at the shell length of 3.7-8.2mm.
Adult individuals, collected in April-May and placed in the culture, released offspring in May.In the wild offspring release usually started at the beginning of May and finished in the second half of June.This conforms to the data on the populations from Germany and Great Britain (danneel & hinz 1976, Meier-Brook 1977, BaSS 1979).In Spain and France juveniles appeared already in April (araujo et al. 1999, Mouthon & dauFreSne 2008), and in Canada and Finland they were released as late as in June or July (Vincent et al. 1981, holopainen & hanSki 1986, rantanen et al. 1998).
The number of released offspring was correlated with the parent's shell length (r=0.73,n=14) (Fig. 3).In the culture the smallest offspring-releasing (7 young) individual had shell 5.2 mm long.The maximum number of offspring during one reproductive period was 46 (Table 1) and was similar to that reported by danneel & hinz (1976), bAss (1979) and korniuShin (2007).In the countries with warmer climate it was higher, with the maximum values: in Spain 73 (araujo et al. 1999), and in France even 163 juveniles produced by one parent (Mouthon & dauFreSne 2008).
Adults with shelled embryos were found from May till July (sporadically also in August).When placed in the laboratory, they usually released offspring in June and July (rarely as early as the end of May, or in August).The literature data on the timing of offspring release vary widely: April-June (Burky et al. 1981), June-July (haMill et al. 1979), June-November (Mackie 1979), July (heard 1965, holopainen 1979), or November-December (holopainen & jónaSSon 1983).
The number of released offspring was correlated with the parent's shell length (r=0.65,n=99) (Fig. 9).In the culture the smallest offspring-releasing (2 young) bivalve had shell 2.41 mm long.The maximum number of offspring during one reproductive season was 37 (Table 1), in another two populations it was 34 and 30, respectively.However, only three individuals were so fecund, the remaining ones produced not more than 22 juveniles.In the literature a similar number of offspring (42) was reported by heard (1965), while populations studied by other authors were less fecund (Mackie 1979, holopainen & jónaSSon 1983, piechocki & dyduch-FalniowSka 1993).

Pisidium (Euglesa) globulare Clessin, 1873
The species occurred mainly in shallow, initial sections of some drainage ditches (sometimes very abundantly).The bivalves spent dry periods in the layer of damp leaves or in the mud.However, at high water level (e.g. during local floods) they moved to the surrounding meadows.The shells were strongly convex, with wide umbones protruding above the upper margin (Fig. 6).The shell walls were relatively thin, with very numerous pores, which agrees with korniuShin's data (1999).
In the wild, throughout the year (including winter) a part of individuals contained shelled embryos.When placed in the culture, usually in a short time they released offspring.In the wild the time of release was limited by both the temperature and the water level (the temperature and water level usually permitted offspring release from the end of April till September, with irregular breaks).
The number of offspring was poorly correlated with the parent's shell length (r=0.52,n=134) (Fig. 11).In the culture the smallest offspring-releasing (2 young) bivalve had shell 2.30 mm long.The maximum number of offspring in one reproductive period was 23 (Table 1), in another two populations it was 21 and 17, respectively.

Pisidium (Euglesa) ponderosum (Stelfox, 1918) + var. humeriformis Stelfox
The taxonomic status of the bivalve described by StelFox (1918) as P. casertanum var.humeriformis and var.ponderosa raised controversies from the very beginning.Some authors regarded the last variety as a distinct species, others as a variety or subspecies of the conchologically variable P. casertanum.Studies on shell variation seemed to confirm that it was a form of P. casertanum (e. g. kuiper 1963, piechocki 1989, włoSik-Bieńczak 1992a).Ecological studies gave different results (Meier-Brook 1975, piechocki 1991, zettler & Glöer 2006).In recent publications P. ponderosum was treated as a distinct species, for example by Glöer & zettler (2005), piechocki & SulikowSka-drozd (2008), Glöer & dierckinG (2010), piechocki & wawrzyniak-wydrowSka (2016).Both forms occurred in the studied area (Brda River and Lake Sosnowe), syntopically or separately.The shells of ponderosa were strongly convex, in outline close to triangular (the short upper margin was usually invisible in lateral view) (Fig. 7).The shells of humeriformis were moderately convex (sometimes even flattened), trapezoidal in outline, with narrow umbones protruding above the upper margin (Fig. 8).The largest collected shells of the two forms were ca. 5 mm long.The hinge plate was very wide, with massive lateral teeth A1 and P1 as well as A2 and P2.The shell surface in ponderosa was feebly shiny, in humeriformis usually matt.In ponderosa and in humeri formis from a sandy bottom the shell walls were thick, without pores.Individuals of the form humeriformis living among plant roots or on a muddy bottom had thinner shell walls with pores.According to kuiper (1963) the boundary between the two forms was difficult to discern because of the existence of individuals with intermediate shell characters.In the study area only a few small specimens from the Brda River were difficult to assign to any form (those were disregarded in the analysis).
Adults of both forms collected in the spring, when placed in the culture, released offspring from half of May till half of June.In the wild, at the end of June only few adults contained shelled embryos and released them in July-August.The number of offspring was closely correlated with the parent's shell length: in ponderosa: r=0.83, n=98; in humeriformis: r=0.90, n=45 (Fig. 16).In the culture the smallest offspring-releasing bivalves had shells: ponderosa -2.62 mm long (5 young), humeriformis -2.35 mm long (7 young).The maximum number of offspring in both forms was 75 (Table 1).
The relationship between the two forms (conchological forms or closely related species) remains unclear.On the one hand they differ in shell structure which makes it possible to distinguish between them (Figs 7-8), on the other their reproductive parameters are similar .Assuming that The juvenile shell length in both typical P. pondero sum (=var.ponderosa) and var.humeriformis was usually smaller than in P. casertanum, but the variation ranges overlapped partly (Fig. 18).The shell proportions were very similar (Fig. 19).At the same length of parent's shell the number of released offspring in P. ponderosum and var.humeriformis was usually 2-3 times greater than in P. casertanum (e.g. at 4 mm 38, 43 and 13, respectively) (Fig. 20).The offspring release in P. ponderosum and var.humeriformis was usually earlier than in P. casertanum.

Pisidium (Euglesa) personatum Malm, 1855
The species occurred in a few drainage ditches and in some springs on wet meadows.Adults with shelled embryos were found from April to August.Placed in the culture, they released offspring from April till November.In some individuals the period between release of the first and the last juvenile was relatively long (even up to 3 months).In the studied sites the number of offspring was small and poorly correlated with the parent's shell length (r=0.46,n=33) (Fig. 21).In the culture the smallest offspring-releasing (5 young) bivalve had shell 2.28 mm long.The maximum number of offspring in one reproductive period was 10 (Table 1), in another population 8.According to heitkaMp (1980) and dyduch-FalniowSka (1983), P. personatum has two reproductive periods per year.
On release, the offspring had shells 0.80-1.43mm long (mean 1.08 mm, SD=0.12, n=298) (Fig. 22).The mean shell length in the studied two populations was 1.09 mm (ditch near Sąpolno) and 1.07 mm (ditch near Korne).The height/length ratio was 0.707-0.814(mean 0.758, SD=0.021, n=298) (mean values in the populations: ditch near Sąpolno -0.750, ditch near Korne -0.767).The offspring on average reached 39.4% of the parent's shell length (SD=4.9,n=167, range 25.0-52.1%).The species occurred in various kinds of water bodies: lakes, ponds, drainage ditches, and fens (on some wet meadows).In two sites with outflow of water from drains (the small fen on a slope of the Brda valley and concreted ditch at the railway) the shells were thick-walled, with wide poorly protruding umbones, slightly displaced posteriorly and with atypical hinge structure.
Adults with shelled embryos were found from early spring till the end of summer.When placed in the culture, they released offspring from April till October (rarely still in November).According to the literature data (MitropolSkij 1969, Meier-Brook 1970, heitkaMp 1980) the relatively long period of offspring release results from two consecutive reproductive periods.The number of offspring was correlated with the parent's shell length (r=0.76,n=142) (Fig. 23).In the culture the smallest offspring-releasing (4 young) individual had shell 1.79 mm long.The maximum number of offspring in one reproductive period was 26 (Table 1), and in the other studied populations it did not exceed 20.In one of the pop-ulations studied by Meier-Brook (1970) the maximum number of offspring was 26.

Pisidium (Hiberneuglesa) hibernicum
Westerlund, 1894 In the studied area the species occurred mainly in the ditches draining lakes Bublinek (1st in the figures) and Sosnowe (2nd in the figures) (less often in lakes).Adults with shelled embryos were found from June till August.When placed in the culture they released offspring from the end of June till September (rarely longer, till October).The number of offspring was correlated with the parent's shell length (r=0.75, n=28) (Fig. 25).The fecundity of large individuals (shell length more than 2.6 mm) decreased gradually.The smallest bivalve in the culture which released offspring (3 young) had shell 1.75 mm long.The maximum number of offspring in one reproductive period was 19 (Table 1).odhner (1929) reported from Sweden the maximum of 20 juveniles at shell length of 2.6 mm.The fecundity in the population studied by Meier-Brook (1970) was much smaller (not exceeding 8).

Pisidium (Cingulipisidium) milium Held, 1836
In the studied area the species was found to inhabit various types of water bodies: lakes, some of the oxbows, ponds and drainage ditches.Adults with shelled embryos were found from May to July.In the culture offspring was released mainly in June and July (rarely as early as the end of May, or in August).The number of offspring was closely correlated with the parent's shell length (r=0.87,n=43) (Fig. 27).
In the culture the smallest offspring-releasing (3 young) bivalve had shell 1.93 mm long.The maxi-mum number of offspring in one reproductive period was 18 (Table 1), and in another two populations 17 in each.In the populations studied by heard (1965) and holopainen & jónaSSon (1989) the number of offspring was smaller, not exceeding 11.

Schlesch, 1947
In the studied area the species occurred in water bodies near the village of Korne (Lake Bublinek, its Adults with shelled embryos were found from May to October.When placed in the culture they released offspring from June till the end of October.The number of offspring was correlated with the parent's shell length (r=0.58,n=58) (Fig. 29).In the culture the smallest offspring-releasing (7 young) bivalve had shell 2.30 mm long.The maximum number of offspring in one reproductive period in the pond was 17 (Table 1), and in Lake Bublinek 10.

Pisidium (Cingulipisidium) nitidum Jenyns, 1832
The species occurred in a variety of water bodies: lakes, oxbows, drainage ditches.In the littoral of Lake Sosnowe (sandy bottom, depth 0.1-0.5 m) I found shells corresponding to the description of P. nitidum f. arenicola Stelfox.They were strongly striated, shiny (like P. crassum), but their hinge structure was typical of P. nitidum.
Adults with shelled embryos were found mainly from May to July (rarely in April or September).Usually shortly after placing them in laboratory they released offspring.The number of offspring was correlated with the parent's shell length (r=0.74,n=150) (Fig. 31).In the culture the smallest offspring-releasing (2 young) bivalve had shell 1.89 mm long.The maximum number of offspring in one reproductive period was 35 (Table 1), but in the other individuals from that population (Lake Sosnowe) it did not exceed 28.In another population (ditch draining Lake Bublinek) the number was even smaller, not exceeding 24.According to the literature two generations of young appear during the year, usually released from June till September (odhner 1929, heard 1965, ladle & Baron 1969, Meier-Brook 1970, holopainen & hanSki 1986, holopainen & jónaSSon 1989).The reported maximum numbers of offspring varied from 5 to 26 (Meier-Brook 1970, holopainen & hanSki 1986).
Adults collected in spring released offspring from April to June, those collected in autumn and winter reproduced from December to February.The number of offspring was closely correlated with the parent's shell length (r=0.89,n=19) (Fig. 33).In the culture the smallest offspring-releasing (2 young) bivalve had shell 2.02 mm long.The maximum number of offspring in one reproductive period was 30 (Table 1).
The newborn P. crassum were usually smaller than P. nitidum (Fig. 35), but their proportions were sim- ilar (Fig. 36).At the same parent's shell length the mean number of offspring in P. crassum was greater than in P. nitidum (Fig. 37).

Pisidium (Pseudeupera) subtruncatum Malm, 1855
In the study area the species was rather common and occurred in various kinds of water bodies: lakes, rivers, oxbows, drainage ditches, fish ponds.Adults collected in April-June released offspring from May till the beginning of July, and those collected later (July-September) reproduced soon after they were placed in the culture.The periods of offspring release reported in the literature vary (ladle & Baron 1969, dyduch-FalniowSka 1983, holopainen & jónaSSon 1983, Mouthon 2005, piechocki & wawrzyniak-wydrowSka 2016).The number of offspring was closely correlated with the parent's shell length (r=0.82,n=99) (Fig. 38).In the culture the smallest offspring-releasing (4 young) individual had shell 1.95 mm long.The maximum number of offspring in one reproductive period was: for Lake Sosnowe 45 (Table 1), for Brda River 44, in the remaining populations it did not exceed 30.According to the literature the bivalves started reproduction at shell length of 1.7-2.2mm and released at most 12-25 juveniles (ladle & Baron 1969, holopainen & jónaSSon 1983).According to Mouthon (2005) the number of young depended on the population density.On release the juveniles had shells 0.53-1.27mm long (mean 0.83 mm, SD=0.08, n=2,917) (Fig. 39).In the studied populations the shell length varied from 0.74 (Brda) to 1.00 mm (ditch draining Lake Sosnowe).The height/length ratio was mostly 0.82-0.84(mean 0.833, SD=0.016, n=2,095, range 0.781-0.879)(infra-population differences were rather small).The offspring reached on average 26.8% of the parent's shell length (SD=4.4,n=1,943,.The juvenile shells were close to trapezoidal with rounded angles, and the anterior, dorsal and posterior margins were of similar length. The bivalves from the Brda, under the bridge (road from Sąpolno to Czosnowo) corresponded to the description of P. subtruncatum var.tenuilineatiformis Feliksiak (shells with distinct, dense striation).One of them, found on 20.04.1999 (shell length 1.98 mm) released 6 young after 10 days.The juvenile shells were 0.63-0.66mm long and did not differ from the typical P. subtruncatum.

Pisidium (Pseudeupera) pulchellum Jenyns, 1832
In the studied area the species occurred in a short section (ca.30 m) of the ditch draining Lake Sosnowe.In 2008 the locality was destroyed by beaver.Adults with shelled embryos were found mainly in May and June.When placed in the culture they released offspring in June and July.Single adults with shelled embryos were still found at the end of August (offspring release in August or September).The number of offspring was closely correlated with the parent's shell length (r=0.91,n=22) (Fig. 40).
In the culture the smallest offspring-releasing (3 young) bivalve had shell 2.65 mm long.The maximum number of offspring in one reproductive period was 22 (Table 1).

Pisidium (Henslowiana) henslowanum (Sheppard, 1823)
The species occurred in lakes, rives, oxbows converted to fish ponds and in the ditches draining the ponds.Adults with shelled embryos were usually found from half of April till half of June.When placed in the culture they released offspring from May to July.The number of offspring varied, with the max- imum at parent's shell length of 4.6-4.9mm, and then decreased (Fig. 42).The correlation between the parent's shell length and the number of offspring was r=0.58, n=33.In the culture the smallest offspring-releasing (20 young) bivalve had shell 3.38 mm long (no smaller individuals could be collected).
The maximum number of offspring in one reproductive period was: for Lake Sosnowe -58 (Table 1), for Brda River -56.According to the literature data the species has one or two reproductive periods in a year (holopainen 1979, holopainen & jónaSSon 1989, pettinelli & Bicchierai 2009).In the populations studied by MitropolSkij (1970) and holopainen (1979) the maximum number of offspring was 40.
On release, the juveniles had shells 0.64-1.24mm long (mean 0.89 mm, SD=0.08, n=1,282) (Fig. 43).The mean shell length in the Brda was 0.85 mm, in Lake Sosnowe 0.90 mm.The height/length ratio was 0.674-0.813(mean 0.753, SD=0.028, n=234).On large shells there was a well-developed fold roughly at half height, on small shells it was close to the lower margin (arcuately bent in the way opposite to the shell margin).The offspring reached on average 20.2% of the parent's shell length (SD=2.4,n=1,062, range 14.0-29.8%).The newly released young were very active.

Pisidium (Henslowiana) supinum A. Schmidt, 1851
In the studied area the species occurred in the rivers Brda and Lipczynka.Adults collected from the end of April till the beginning of June released offspring from May to August, those collected later (e.g.half of August) released their offspring just after being placed in the culture.Those collected too early (around half of April) often started releasing young only as late as 3-4 months after being placed in the culture, and the number of offspring was rather small (omitted in Fig. 44).The number of offspring was correlated with the parent's shell length (r=0.74,n=27) (Fig. 44).In the culture the smallest offspring-releasing (3 young) individual had shell 2.51 mm long.The maximum number of offspring in one reproductive period was 46 (Table 1) and was close to that reported by Mouthon (2011).That author observed offspring release in France in two periods: May-June and August, and the smallest adults with shelled embryos had shells 1.68 mm long.
On release, the juveniles had shells 0.52-1.29 mm long (mean 0.80 mm, SD=0.10, n=658) (Fig. 45).The height/length ratio was 0.734-0.833(mean Pisidium (Odhneripisidium) moitessierianum Paladilhe, 1866 In the studied area the species occurred in the Brda River.Adults with shelled embryos were found mainly in April and May (rarely as early as in March), and offspring was released shortly after placing the bivalves in the culture.Also at the end of summer some individuals reproduced; they released offspring in September and October.The number of offspring varied and was rather poorly correlated with the parent's shell length (r=0.57,n=53) (Fig. 46).In the culture the smallest offspring-releasing (7 young) individual had shell 1.50 mm long.The maximum number of offspring released in one reproductive period was 21 (Table 1).In Finland the species had one reproductive period in a year and released offspring in July (holopainen 1979).In France offspring was released in two periods: May-June and August-September (Mouthon 2011).In France the maximum number of offspring in one reproductive period was 17 (Mouthon 2011), according to other authors it was much smaller (holopainen 1979(holopainen , GriGoroVich et al. 2000)).

COMPARATIVE REMARKS
The occurrence of as many as 16 species of Pisidium offered a unique opportunity to compare their reproductive parameters without the need to consider the effect of climate conditions.The variation in the shell length of juveniles and the shell proportions is presented in Figures 48 and 49. Figure 50 presents the variation of relative size of the offspring (ratio of juvenile to parent shell length).Species of the same subgenus were placed next to each other (as in the text).Particular reproductive parameters differed more or less distinctly not only between the species, but also between conspecific populations from differ-ent habitats.The anatomical structure (division into subgenera) had no greater effect on the differences in the studied parameters.
The number of offspring was positively correlated with the parent's shell length, and the regression curves were best described by exponential equations (different between species and even populations).For P. pulchellum the exponent was the highest and amounted to 5.3, for P. supinum 4.6, for P. crassum 4.2, and for P. nitidum and P. ponderosum 4.0 (it was smaller for the remaining species).This resulted from the fact that the mean fecundity of the pisids depended mainly on their size.However, also other factors (e. g. mortality of embryos) had an effect on the parent's actual fecundity.Too great a number of released offspring in relation to the parent size resulted in a small size of the released young (limited space for incubation).When most individuals in a population were very fecund, the size of the offspring decreased (and vice versa).

Figs
Figs 1-2.Location of the study area: 1 -general view, 2 -map of the environs of Sąpolno

Fig. 37 .
Fig. 37. Comparison of Pisidium crassum and P. nitidum: parent size and number of offspring

Table 1 .
Genus Pisidium: maximum number of offspring released in one reproductive period