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Effects of selling on offspring survival and reproduction in a colonial simultaneous hermaphrodite (Bugula stolonifera, Bryozoa).

Introduction

For over a century, researchers have been interested in the potential for self-fertilization in simultaneous hermaphrodites (see reviews by Jain, 1976; Jarne and Charlesworth, 1993; Jarne and Auld, 2006). Specifically, studies have centered on the distinct benefits selfing can confer as compared to exclusive out-crossing (Maynard Smith, 1971; Charlesworth, 1980; Lively and Lloyd, 1990). Sexual reproduction requires the fusion of gametes, with each gamete containing 50% of its parent's genome. This reduction in parental legacy has been termed the cost of meiosis (Williams, 1975) and can be avoided by selfing via self-fertilization, the fusion of gametes from the same individual; by automixis, the activation of a meiotically divided cell (see Mogie, 1986); or by apomixis, a process particular to angiosperms and gymnosperms where seeds form without the need of meiosis and fertilization (see Bicknell and Koltunow, 2004). These processes can also alleviate some of the risks associated with sex, including finding an appropriate mate in copulating species and dilution of gametes in spawning species. Alternatively, it is thought that selfing can have deleterious effects such as decreased survival, growth rate, and fecundity, culminating in substantially decreased fitness or inbreeding depression (Lande and Schemske, 1985; Charlesworth and Charlesworth, 1987). This decrease in fitness could potentially stem from increased homozygosity, leading to retention of recessive deleterious alleles (Shields, 1982; Charlesworth and Charlesworth, 1987), or from the loss of genes in selfing populations, rendering the population as a whole incapable of adapting to changes in environmental conditions (Maynard Smith, 1978). These deleterious consequences associated with selfing were recently demonstrated empirically for the highly selfing nematode Caenorhabditis elegans (Morran et al., 2009). The authors documented that after 50 generations, increased mutation rate coupled with a change in environmental condition led to significantly reduced fitness in an obligate selfing strain of C. elegans, as compared to outcrossed controls. Other studies have shown that inbreeding depression can occur as rapidly as within the next generation (e.g., Charlesworth et al., 1994; Cohen, 1996; Escobar et al., 2007). Thus the evolution and maintenance of selfing is thought to depend on the trade-offs of various costs and benefits (Lande and Schemske, 1985).

Although selfing has historically been more heavily investigated in plants (Jain, 1976), and in particular in angiosperms (e.g., Darwin, 1876; Bicknell and Koltunow, 2004; Barringer, 2007), these processes in metazoans have recently received increased attention (Jarne and Auld, 2006). For dioecious organisms, automixis has been documented in some animals, including rotifers (Stelzer, 2008), bivalves (Foighil and Thiriot-Quievreux, 1991), beetles (Moore et al., 1956), grasshoppers (Atchley, 1978), wasps (Beuke-boom and Pijnacker, 2000), and vertebrates (Adams et al., 2003; Watts et al., 2006). Selfing is widely reported in simultaneous hermaphrodites, including cnidarians (Bucklin et al., 1984; Bassim et al., 2002; Sherman, 2008), molluscs (Meunier et al., 2004; Escobar et al, 2007; Smolensky et al., 2009), annelids (Finley et al., 2001; Mendez, 2006), flatworms (Christen and Milinski, 2003; Lagrue and Poulin, 2009), and ascidians (Ryland and Bishop, 1993; Cohen, 1996; Jiang and Smith, 2005; Manriquez and Castilla, 2005).

Conclusive evidence demonstrating the ability to self in simultaneous hermaphrodites comes from laboratory-based studies, wherein individuals are either maintained in isolation (e.g., Sabbadin, 1971) or gametes from a simultaneous hermaphrodite are procured (Bassim et al., 2002), and the effects of selfing examined. Alternatively, researchers have utilized genetic techniques to examine selfing in the field. Bucklin et al. (1984) collected 25 individuals of the brooding sea anemone Epiactis prolifera and, using allozyme electrophoresis, found that genotype frequencies of the brooded offspring were consistent with selfing. Dupont et al. (2007) utilized microsatellite markers to investigate selfing in two populations of the brooding ascidian Corella eumyota and found significant rates of selfing in both populations. It is thought that for species with limited dispersal potential, including those that brood embryos, inbreeding might be more prominent (Knowlton and Jackson, 1993). For these species, this could eventually lead to increased tolerance of inbreeding, culminating in populations that are capable of selfing with little to no inbreeding depression (Lande and Schemske, 1985; Uyenoyama, 1986).

The phylum Bryozoa is dominated by species that brood embryos and release short-lived larvae that have low potential for dispersal (e.g., Strom, 1977; Zimmer and Woollacott, 1977). For example, larvae of Bugula spp. will usually initiate metamorphosis within 1-4 h after release (e.g., Woollacott et al., 1989; Wendt and Woollacott, 1999). At the level of the colony, bryozoans are simultaneous hermaphrodites; individual autozooids, however, generally express gender sequentially, first maturing male, then female, gonads. Alternatively, gonochoric zooids are known to occur in certain species (e.g., Dyrynda and Ryland, 1982; Hughes and Hughes, 1986). Because male and female reproductive structures occur within the same zooid, it was once thought that bryozoans were exclusive self-fertilizers (see Silen, 1966). Elegant work detailing bryozoan fertilization led researchers to conclude that these animals were, at least, capable of out-crossing. For instance, Silen (1966) showed that for two species of the genus Electra, sperm were released through the tips of certain tentacles of the lophophore. By "spermcasting" (Bishop and Pemberton, 2006), sperm can presumably be transferred to a different colony for fertilization, although the uptake of this sperm by another zooid of the same colony may also be possible. This manner of sperm release was later demonstrated in such a diverse array of other ctenostome and cheilostome bryozoans (Bullivant, 1967; Strom, 1969; Silen, 1972) that Silen (1972) concluded it was likely that cross-fertilization occurred in all groups of bryozoans.

Only a few studies have investigated selfing in bryozoans (see review by Ostrovsky, 2008) despite the more than 5500 extant species. For instance, Maturo (1991) found that colonies from six brooding species reared in isolation were able to release larvae, and Temkin (1991) found that isolated colonies of Membranipora membranacea were able to release embryos that developed into larvae. Working in natural populations, Yund and McCartney (1994) and McCartney (1997) used allozyme electrophoresis to provide evidence of selfing in populations of Celleporella hyalina from Walpole, Maine. These studies suggest that the ability to self is widespread in the gymnolaemate bryozoans, but the consequences of selfing to offspring fitness remain unclear. Interestingly, a series of studies examining reproduction in C. hyalina found that colonies from certain populations cultured in isolation released larvae that were unable to initiate metamorphosis (Hunter and Hughes, 1993a; Hoare and Hughes, 2001), whereas isolated colonies from other populations were able to self with no measured decrease in offspring survival (Hughes et al., 2002b, 2009). It is unknown from these latter studies if selfed progeny were able to reach reproductive maturity.

In this study, I report the results from experiments investigating the effects of selfing in the cheilostome Bugula stolonifera Ryland 1960. This species is cosmopolitan in temperate and tropical waters (Rodgers and Woollacott, 2006) and is suitable for this study for several reasons: (1) I am able to culture B. stolonifera from larvae through reproductively mature colonies in the laboratory, (2) brood chambers are distinct in this species, making it possible to determine onset of reproduction in cultured colonies, and (3) brood chambers are transparent, which allows for accurate counts of brooded embryos in gravid colonies. By culturing both solitary colonies and colonies growing adjacent to a conspecific (paired treatment), I examine the possibility of selfing and the subsequent effects on offspring survival and fecundity.

Materials and Methods

Experimental procedures

Sexually mature colonies of Bugula stolonifera were collected from the sides of floating docks in Eel Pond, Woods Hole, Massachusetts, in September 2008 and July 2009. Colonies were maintained overnight in the laboratory in complete darkness in a 38-1 glass aquarium equipped with a power filter providing water flow and aeration. To induce larval release, colonies were removed from the aquarium, placed in 1.5-1 glass bowls containing unfiltered Eel Pond seawater (UFSW), and exposed to fluorescent light. Larvae appeared about 15 min after exposure to light, and within an hour hundreds of larvae had aggregated on the illuminated side of the dish. Groups of 8 to 75 larvae were pipetted into 35-mm polystyrene dishes (BD Falcon #353001), which were then covered and transferred to the dark to facilitate larval attachment. No attempt was made to exclude closely related larvae from these experiments. To estimate the overall health of the collected colonies, the percentage of larvae initiating and completing metamorphosis was assessed. Percent metamorphic initiation was assessed after 4 h, and larvae not initiating metamorphosis were removed. Polystyrene dishes with attached metamorphs were then transferred to covered, 150-ml beakers containing 125 ml of UFSW and placed on a continuously oscillating orbital shaker to keep the water within the beaker mixed. The alga Rhodomonas sp. (Bigelow Laboratory CCMP757) was added the following morning, ensuring that ancestrulae could commence feeding immediately at completion of metamorphosis. Polystyrene dishes were maintained in a vertical position within the beaker to prevent the build-up of settled food and waste products adjacent to the growing individuals. Percent metamorphic completion was assessed about 72 h after metamorphic initiation, and individuals were then selected for the selfing experiment.

To examine the effects of selfing on offspring survival, colonies were cultured either in isolation, the solitary treatment, or in the presence of one other conspecific, the paired treatment. A total of 58 colonies were cultured in 2008 (solitary n = 16, paired n = 10) and 2009 (solitary n = 22, paired n = 10). Cultured colonies were maintained at 24 [degrees]C in a constant temperature room with a 16 h:8 h light/dark cycle, and fed Rhodomonas sp. twice daily at a final concentration of at least 10,000 cells [ml.sup.-1] (Winston, 1976; Hunter and Hughes, 1993b). Colonies were maintained in UFSW on the orbital shakers and were cleaned and inspected daily. To prevent contamination from sperm contained within the field-collected water, water from the Eel Pond was aged for at least one week prior to use. Colonies were cleaned with a soft artist's brush daily to remove attached algal cells, and care was taken to prevent cross-contamination among beakers. Only one polystyrene dish was removed at a time. Dishes were removed with forceps when placed under the dissecting scope, and then transferred immediately after inspection and cleaning to a new beaker. Forceps and all surfaces were sterilized with 95% ETOH prior to examination of the next individual. Beakers remained covered the entire time to prevent contamination via splashing, and each beaker had a designated artist's brush for cleaning. Growth, as the number of bifurcations per colony, and onset of reproduction, as the presence of brood chambers, were assessed daily.

About one week after a filled brood chamber was observed, the total number of chambers and the number of brooded embryos per colony were recorded. To minimize larval release, reproductively mature colonies were moved to a dark constant temperature room also held at 24 [degrees]C. The feeding regime remained the same for these colonies. Colonies that had not reached reproductive maturity or had not brooded embryos remained on the original light/dark cycle until they became gravid. Larval releases were conducted every morning for 1 week as previously described, except that polystyrene dishes with attached colonies were placed in glass Stender dishes containing 20 ml UFSW. The smaller glass dish and volume of water facilitated larval collection. Larval release was allowed to continue for 2 h, after which colonies were cleaned, placed in new UFSW, and returned to the dark room. Collected larvae were transferred to clean polystyrene dishes and placed in the dark. Percent metamorphic initiation was determined after 4 h. The dishes were then flooded, ensuring that all settled metamorphs were submerged, and percent metamorphic completion was determined after 72 h.

Culturing experiments conducted in 2008 demonstrated that B. stolonifera colonies reared in isolation produced viable larvae that successfully completed metamorphosis. In summer 2009 these experiments were extended by transferring metamorphs from colonies reared in the solitary and paired treatments back to the field to examine the effects of selfing on offspring survival and reproductive fitness. Larvae from cultured individuals were allowed to settle on polystyrene weighing dishes (VWR #12577-005). After 4 h, percent metamorphic initiation was assessed, and unattached larvae were removed from the weighing dish. Metamorphs were marked by circling their position on the weigh boat to aid in identifying these individuals, thus preventing confusion with newly settled ancestrulae that might attach after dishes were transferred to the field. Weighing dishes were clipped into plastic binders, which were then affixed within a rectangular acrylic plastic chamber. The chambers (15.3 cm (H) X 7.6 cm (W) X 5.7 cm (D)) contained grooved sides, allowing the binders to slide into the grooves. A total of 5 weighing dishes were placed in each chamber, with 2.5 cm (vertical distance) between dishes. Previous tests showed that predation on newly settled individuals within these chambers was not prominent in Eel Pond, so protective screening was not used and the dishes were held in place simply by using several large rubber bands. Generally, B. stolonifera larvae will complete metamorphosis 24-48 h after initiation. Therefore, metamorphs (paired: n = 61; solitary: n = 59) were transferred to Eel Pond, Woods Hole, Massachusetts, within 24 h of settlement, enabling ancestrulae to feed immediately upon completion of metamorphosis. Chambers were suspended from the WHOI-MBL pier in Eel Pond at a site adjacent to numerous established B. stolonifera colonies. The chambers were weighted at the bottom to maintain vertical orientation within the water column and were suspended about 1 m below the surface. Within the chamber, colonies were oriented downward to prevent the build-up of sediment on the weigh boat. Colonies were inspected twice weekly for survival and onset of reproduction, and alien juveniles were removed from the weighing dish. About 2 weeks after the appearance of the first filled brood chamber, colonies were returned to the laboratory for larval collection. Larval release was conducted as previously described, except that weigh boats with attached colonies were placed in 250-ml bowls containing UFSW prior to exposure to light. The total number of larvae released and the total number of these individuals that initiated and completed metamorphosis were used to determine reproductive fitness.

Statistics

The effect of treatment (solitary or paired) on the ability of cultured colonies to reach reproductive maturity was investigated using Fisher's Exact Test. Significant differences in all other measured variables between the experimental treatments within each year were investigated using a one-way ANOVA with the statistical package Minitab ver. 15. The measured variables were as follows: time to reach reproductive maturity, colony size at reproductive maturity, total number of brood chambers per colony, percentage of filled brood chambers per colony, number of larvae released per treatment, number of larvae initiating metamorphosis per treatment, and number of individuals completing metamorphosis per treatment. Because there were two individuals in each beaker in the paired treatment and one individual in each beaker in the solitary treatment, the number of larvae released and the number of individuals initiating and completing metamorphosis from the paired treatments were halved prior to analyses to correct for this discrepancy. Prior to conducting the ANOVA, all data sets were initially examined for normality and equal distributions. Data sets failing these tests were transformed to meet these assumptions using either the squared, square root, or 4th root transformation, depending on the data set. The time to reach reproductive maturity from 2009 continued to fail these tests, so the untransformed data were subjected to the non-parametric Kruskal-Wallis analysis, with condition as the factor. These data are presented as medians; all other data are presented as untransformed means [+ or -] 1 standard error.

Results

Fitness of field-collected parental colonies

In 2008, 375 larvae were sampled from field-collected colonies. Of these, 342 (91.2%) initiated metamorphosis, and 330 (96.5%) of those completed metamorphosis. In 2009, 950 larvae were collected, 884 (93.1%) initiated metamorphosis, and 871 (98.5%) of those completed metamorphosis. Due to the high rates of metamorphic initiation and completion, parental colonies were considered healthy, and individuals successfully completing metamorphosis were selected for the selling experiments.

Colony growth in culture

Bugula stolonifera colonies were amenable to culturing, as there was no mortality in either year. For both years, growth rates varied between the treatments such that, on average, solitary colonies were significantly larger at reproductive maturity than colonies cultured in pairs (Fig. 1; Table 1). Although solitary colonies were significantly larger at the time of assessment, there were no significant differences in time to reach reproduction between the conditions in either year (Fig. 1; Table 1).

[FIGURE 1 OMITTED]

Effect of selfing on brooding and larval fitness

In 2008, 12 of 16 solitary colonies and 10 of 10 colonies cultured in pairs reached reproductive maturity, demonstrated by the production of brood chambers (P = 0.122). On average, there was no significant difference between treatments in the total number of brood chambers developed per colony (Tables 1 and 2). In contrast, colonies cultured in pairs had a significantly higher percentage of filled brood chambers at the time of assessment (Tables 1 and 2), as well as a significantly higher total number of larvae released (P = 0.030) and total number of offspring initiating (P = 0.016) and completing (P = 0.042) metamorphosis compared to the solitary treatment (Fig. 2). Additionally, larvae released from solitary colonies experienced reduced rates of metamorphic initiation (33.1%) compared to larvae from the paired treatment (43.7%). Of those that initiated, offspring from solitary colonies also experienced reduced rates of metamorphic completion (81.1%) compared to the paired treatment (97.7%) (Fig. 2).

[FIGURE 2 OMITTED]

In 2009, 20 of 22 solitary and 10 of 10 paired colonies reached reproductive maturity (P = 0.466). Here, colonies cultured in the paired treatment on average had significantly more brood chambers per colony as well as a significantly higher percentage of filled brood chambers compared to solitary colonies (Tables 1 and 2). As in 2008, paired colonies also had a significantly higher number of larvae released (P < 0.001) and a higher number of individuals initiating (P < 0.001) and completing metamorphosis (P < 0.001) compared to colonies cultured in the solitary treatment (Fig. 2). Additionally, as in 2008, larvae released from solitary colonies in 2009 experienced reduced rates of metamorphic initiation (71.3%) and completion (65.2%) compared to those cultured in pairs (rate of initiation = 86.7%; rate of completion = 95.4%) (Fig. 2).

Effect of selfing on offspring reproductive fitness

A total of 61 metamorphs from the paired treatments were transferred to Eel Pond the morning after larval attachment, 59 (96.7%) of which were found to complete metamorphosis when examined 3 days later. As colonies from the paired treatments routinely released numerous larvae, only one release event was required to collect sufficient larvae (> 50) for this experiment. The larvae from the five cultured pairs were allowed to settle on five polystyrene weighing dishes (n = 4-17 individuals per dish), and the dishes were affixed within one acrylic plastic chamber. These colonies were inspected twice weekly; they grew well and appeared healthy. Neither mortality nor predation was observed on any weighing dish while the colonies could be tracked individually ([approximately equal to]15 days post-settlement). About 12 days post-settlement the colonies on 4 of 5 dishes had grown to sufficient size to begin overgrowing each other, and by 15 days were so intertwined that distinguishing individual colonies became impossible. The remaining dish had only three individuals attached, and these could be followed directly. By day 12 post-settlement, individuals on all dishes had developed brood chambers, and by day 15 had commenced brooding embryos. Although the colonies could not be tracked individually, the dishes were monitored over the next 2 weeks as previously stated, and any newly settled ancestrulae were removed. Sixteen days after brooded embryos were observed, colonies were transferred back to the laboratory for larval release, which was conducted over the next 4 days. Colonies were found to release numerous larvae, with metamorphic initiation and completion rates approximating the rates found in field-collected colonies (Table 3). Colonies were then removed from the weighing dishes and assessed for survival and reproductive maturity. Of the 59 colonies, 56 (94.9%) were recovered, and all retained brooded embryos at the time of removal.

For the solitary treatments, 58 metamorphs were transferred to Eel Pond, 37 (63.8%) of which had completed metamorphosis when examined 3 days later. In contrast to colonies from the paired treatments, few larvae were collected from solitary colonies during a single release event. Therefore, four release events were required to collect approximately the number of larvae collected from the paired treatment colonies. The larvae from 14 different solitary colonies were settled on 19 weighing dishes (n = 1-8 individuals per dish), which were affixed in four acrylic plastic chambers. Due to the fewer number of offspring per dish as compared to the paired treatment, all individuals could be followed directly. Colonies were inspected twice weekly, and growth was found to vary greatly. By day 12 post-settlement, growth varied from ancestrulae with no buds to colonies approximating the size of those from the paired conditions. Additionally, by this time, 17 of the 37 individuals had been lost. Prior to their dislodgement from the weighing dish, all of these individuals were observed to be either simply ancestrulae with a regressed polypide or ancestrulae with but a few budded zooids. Of the 20 surviving colonies, 13 reached reproductive maturity. By day 23 post-settlement, all reproductively mature colonies brooded embryos. After an additional 14 days, these colonies were transferred back to the laboratory for larval release, which was conducted over the next 3 days. These 13 colonies released a total of three larvae, none of which initiated metamorphosis (Table 4). In addition, during larval release, several aborted embryos were found at the base of the weighing dish, underneath the attached colonies. At the completion of the larval release period, only 2 of the 13 colonies still contained brooded embryos. For these colonies, 3 out of 75 and 2 out of 92 brood chambers were filled.

Discussion

The ability to self could convey distinct advantages compared to dependence on a mate for outcrossing (Maynard Smith, 1971; Charlesworth, 1980; Lively and Lloyd, 1990). Previous work directly examining selfing in bryozoans has shown that selfing is possible in several different species (Maturo, 1991; Temkin, 1991; Hughes et al., 2009), but the consequences for survival and reproductive fitness have rarely been investigated. Here I show that selfing in Bugula stolonifera results in the release of viable offspring, but these individuals experience a significant reduction in fitness. As compared to outcrossed controls, this is expressed as significantly fewer larvae released, reduced rates of metamorphic initiation and completion, and decreased survival and fecundity. Indeed, no viable larvae were collected from reproductively mature selfed colonies.

Effect of a conspecific on reproduction

For B. stolonifera the presence of a conspecific did not induce an earlier onset of reproduction, nor did solitary colonies delay reproduction (Fig. 1; Table 1). These results demonstrate that this species is capable of reaching reproductive maturity without extrinsic cues. Previous studies examining the onset of reproduction in bryozoans have found that embryonic effects (genetic control or maternal contributions) and external cues can be responsible for reproductive timing, depending on the species. Working with the encrusting species Membranipora membranacea, Harvell and Grosberg (1988) found that conspecific crowding and colony damage resulted in an earlier onset of reproduction. Additionally, Harvell and Helling (1993) found that damage to one side of a colony of M. membranacea induced earlier onset of reproduction in adjacent zooids than in zooids on the undamaged side of the same colony, showing within-colony variation in reproductive timing. In contrast, Keough (1989a) collected offspring from B. neritina colonies growing in two areas, one with early reproductive onset and one with late, and found through a common garden growth experiment that reproductive patterns follow the patterns of the parental populations. That there was no significant difference in time to reach reproduction between treatments for my study supports Keough's findings, suggesting that for Bugula spp. the time to reach reproduction is determined prior to larval release. This pattern is not uniform within Bryozoa, however; onset of reproduction, as well as potential within-colony variation in reproductive timing, needs further investigation for other genera.

Although the presence of a conspecific did not significantly affect reproductive timing in B. stolonifera, it does appear to have had an effect on energy directed toward reproduction. In 2009, paired colonies were significantly smaller than solitary colonies, yet they had significantly more brood chambers (Tables 1 and 2). Although not significant, the data from 2008 trended similarly. This increase in brood chamber development in paired colonies suggests increased resource allocation toward the production of female zooids. Hence, the potential for outcrossing could lead to greater female investment. Sex allocation theory for simultaneous hermaphrodites predicts that selfing and mating groups of two should result in decreased production of males and increased production of females (Charnov, 1982; Fischer, 1984). As the number of individuals in the mating group increases, male allocation should increase due to sperm competition. That the solitary colonies produced fewer brood chambers suggests that the paired colonies were responding to some conspecific cue that resulted in greater female investment. Bishop et al. (2000) found that allosperm induced vitellogenic egg growth not only in the bryozoan Celleporella hyalina, but also in the ascidian Diplosoma listerianum. Further, Hughes et al. (2002a) found that colonies of C. hyalina exposed to allosperm produced significantly more female zooids compared to non-exposed controls. Hunter and Hughes (1995), however, found that the number of male and female zooids in cultured C. hyalina colonies varied with differing food and temperature combinations. It appears that sex allocation in bryozoans is complex, and that allocation can vary in response to numerous environmental signals.
Table 1
Effect of culturing treatment on the time in days to reach
reproduction, number of bifurcations at onset of reproduction, number
of brood chambers developed per colony, and percentage of filled brood
chambers per colony

Year  Measurement     Source   df      MS      [F.sub.stat]  P value

2008  Time          Treatment   1     12.20         1.68       0.209
                    Error      20      7.26

      Bifurcations  Treatment   1   1037.2         23.99      <0.001
                    Error      20     43.2

      Total brood   Treatment   1      3.01         0.30       0.587
      chambers      Error      20      9.88

      Filled brood  Treatment   1      0.6247      11.08       0.003
      chambers      Error      20      0.0564

2009  Time          Treatment   1      -             -         0.358

      Bifurcations  Treatment   1    756.1         11.23       0.002
                    Error      28     67.3

      Total brood   Treatment   1  23721           12.48       0.001
      chambers      Error      28   1901

      Filled brood  Treatment   1      0.3449       6.67       0.015
      chambers      Error      28      0.0517

Time to reach reproduction in 2009 analyzed by the Kruskal-Wallis
nonparametric test (H = 0.84; MS and [F.sub.stat] not calculated); all
others analyzed by a one-way ANOVA.

Table 2
Number of brood chambers and percentage of filled brood chambers per
colony for each treatment

Year  Treatment     Brood chambers       P      Filled brood        P
                                       value     chambers (%)     value

2008  Paired      42.6 ([+ or -]12.0)  0.587  72.2 ([+ or -]3.0)  0.003
      Solitary    34.7 ([+ or -]12.1)         38.4 ([+ or -]6.4)

2009  Paired     111.9 ([+ or -]16.7)  0.001  78.3 ([+ or -]3.3)  0.015
      Solitary    52.3 ([+ or -] 8.6)         59.2 ([+ or -]4.9)

The total number of brood chambers and percentage of filled brood
chambers were assessed for each colony about one week after observing
the first brooded embryo. Values are means [+ or -] 1 S.E. Differences
were analyzed with a one-way ANOVA.


Effect of selfing on embryo production

Compared to colonies cultured in pairs, solitary colonies were found to brood significantly fewer embryos, measured as the percentage of filled brood chambers per colony (Tables 1 and 2). This significant difference could be a consequence of various reproductive barriers minimizing selfing. Pre-zygotic barriers to self-fertilization have not been directly investigated in these animals; if rigid mechanisms do exist, embryos could still be produced via automixis. Robertson (1903) suggested that this could occur in the steno-laemate cyclostome Crista spp., but this has not been further investigated. For many species of bryozoans, sperm enter the maternal zooid via an opening at the base of the lophophore--a supraneural pore or the intertentacular organ (ITO) depending on the species (Temkin, 1994). This entry site could serve as a barrier to self-fertilization through selective uptake of non-self sperm. Dyrynda and King (1982), however, found spermatozoa in the coelomic cavity of a female zooid on a colony that had been maintained in isolation for several days. Additionally, Temkin (1994) found that in isolated colonies of M. membranacea, the ITO allowed spermatozeugmata to enter the coelomic cavity of maternal zooids. These studies suggest that there is little regulation of sperm entry into female zooids, but rather that any conspecific sperm contacting a lophophore is transferred to the maternal coelom. Once inside the maternal zooid, genetically based barriers such as those described for Ciona intestianilis (Harada et al., 2008) could prevent self-fertilization. Temkin (1991), however, found that some coelomic oocytes extracted from isolated colonies of M. membranacea contained sperm nuclei. It seems likely, therefore, that the significant difference in brooded embryos between treatments documented in my study was a result of post-zygotic inbreeding depression--specifically, increased embryonic abortion. Although it was not quantified due to the large amount of debris from the UFSW and introduced algal cells that would accumulate each day, aborted embryos were routinely found at the base of solitary colonies. Large decreases in numbers of brooded embryos did not necessarily correlate with increased larval output in this treatment. For instance, in 2008 one solitary colony was found to contain 123 brood chambers, 108 ([approximately equal to]87%) of which were filled. Larval releases conducted over the next four days resulted in a total of five larvae released, but the number of brooded embryos decreased from 108 to 37. The majority of these embryos were most likely aborted, and these rates of decrease without increased larval output were not observed in the paired treatment colonies.

Effect of selfing on offspring fitness

Offspring from solitary colonies experienced reduced fitness compared to offspring from colonies in the paired treatment. Solitary colonies released significantly fewer larvae and had significantly fewer individuals initiating and completing metamorphosis (Fig. 2; Table 3). Not only was there reduced larval output from these colonies, but rates of metamorphic initiation and completion were also reduced. For example, in 2009 paired colonies released a total of 646 larvae. Of these, 87% initiated metamorphosis and 95% of these completed metamorphosis. Solitary colonies released a total of 129 larvae; 71% initiated metamorphosis and 65% completed metamorphosis. Hence, not only were there fewer larvae released via selfing, but the fitness of these selfed larvae was also significantly compromised. By transplanting metamorphs back to the field in 2009, I was also able to show that for those offspring that were able to complete metamorphosis, selfing resulted in decreased survival and reproductive fitness compared to outcrossed controls (Table 4). These results demonstrate that, although selfing can occur in B. stolonifera, there are significant deleterious effects manifested at every stage of growth from embryos to reproductively mature colonies. Further, they suggest that selfing is rare in this population. If selfing were prominent, it would be expected that deleterious alleles would have been purged over time and these animals would be able to self with little to no inbreeding depression (Crnokrak and Barrett, 2002). For instance, Swindell and Bouzat (2006) provided evidence that the purging of deleterious recessive alleles in Drosophilia melanogster led to significantly reduced inbreeding depression in certain lineages. The results from my study suggest that the Eel Pond population of B. stolonifera is neither selfing nor maintaining a mixed-mating system, but is routinely outcrossing.
Table 3
Results of one-way ANOVA examining the effect of culturing treatment on
total larvae released, total number of individuals initiating
metamorphosis, and total number completing metamorphosis

Year  Measurement   Source    df    MS    [F.sub.stat]  P value

2008  Released     Treatment   1  31.03        5.82      0.030
                       Error  14   5.33

      Initiated    Treatment   1  18.57        7.51      0.016
                       Error  14   2.47

      Completed    Treatment   1  10.18        5.27      0.042
                       Error  11   1.93

2009  Released     Treatment   1   6.479      49.98     <0.00l
                       Error  22   0.130

      Initiated    Treatment   1   7.999      29.87     <0.001
                       Error  22   0.268

      Completed    Treatment   1  10.933      28.70     <0.001
                       Error  22   0.381


As previously discussed, it does not appear that there is strict discrimination between self and non-self sperm by maternal zooids. Hence, how these animals minimize selfing in a natural population remains unclear. One mechanism could be a consequence of the distribution pattern of bryozoans within a given locale (see Ryland, 1973). Bryozoans often form patchy distributions, whereby high numbers of adults are contained within a small spatial scale. Further, bryozoan larvae are known to settle on adult colonies, such that a bryozoan colonial mass could be made up of multiple, genetically distinct individuals. Keough (1989b) found no consistent deleterious effects on growth and survival of B. neritina juveniles growing adjacent to mature colonies. It could be that this settling behavior and patchy distribution, without resulting in intra-specific competition, allow for ample opportunities to outcross, as well as minimize the chances of taking up own-self sperm. Investigations into the genetic relatedness of individuals within these patchy distributions are currently being conducted.
Table 4
Results from investigating survival and fecundity of offspring from
colonies cultured in each treatment

Treatment  Transferred  Recovered colonies  Reproductive colonies
           metamorphs

Paired         61               56                    56
Solitary       58               20                    13

Treatment  Larvae released    Initiated    Completed metamorphosis
                            metamorphosis

Paired           1030        1017 (98.7%)         986 (97.0%)
Solitary            3           0                      -

Sexually mature colonies were collected from the field (Eel Pond, Woods
Hole, MA) for larval release 14 d after brooded embryos were observed.
Percent initiated are of total released; percent completed are of total
initiated.


Selfing in natural populations

The results from my experiments suggest that although the Eel Pond population of B. stolonifera is capable of selfing, this is not routinely occurring, as evidenced by the significantly reduced fitness of selfed offspring compared to outcrossed controls. If this decreased fitness were due to deleterious alleles, then successful selfing events could rapidly purge these alleles, particularly when selection favors selfing over outcrossing. Long-distance dispersal events can lead to the introduction of small numbers of individuals into a new area, and theoretically a single self-compatible individual could colonize an area following this type of introduction (Baker, 1955). Results from investigations with Celleporella hyalina showed a differential ability to self among geographically distinct populations (Hughes et al., 2009). These findings suggest that the ability for a population as a whole to self might be traced back to the colonizing individuals. For instance, if the population within a given locale were founded by few individuals, the selection for selfing would be greater and could result in the establishment and subsequent propagation of self-compatible individuals. In my study, solitary colonies were indeed able to reach reproductive maturity, self, and release offspring. Selfed offspring were themselves shown to reach reproductive maturity and release larvae. Therefore, it remains a possibility that under different circumstances, selfing in B. stolonifera could lead to the production of viable, self-compatible offspring. Results from this study, however, establish that B. stolonifera colonies in Eel Pond are not selfing, but rather are routinely outcrossing, and that any potential selfing events would result in the production of inviable offspring.

Acknowledgments

I am grateful to Ed Enos (Marine Biological Laboratory, Woods Hole) for his assistance in animal procurement. I thank Robert Woollacott, David Haig, and James McCarthy (all of Harvard University) for their thoughtful comments that improved this study. I also thank Helene Ferranti (Harvard University), the associate journal editor and two anonymous reviewers for providing helpful comments. This research was supported by the Robert G. Goelet Research Grant awarded to Collin H. Johnson (2008, 2009) and by funds from Robert M. Woollacott.

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Received 5 April 2009; accepted 21 June 2010.

Address for correspondence: E-mail: cjohnson@oeb.harvard.edu

COLLIN H. JOHNSON

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138
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