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Keywords:

  • fixed female preference;
  • frequency dependence;
  • rare male;
  • spermcast

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Negative frequency-dependent mating success – the rare male effect – is a potentially powerful evolutionary force, but disagreement exists as to whether previous work, focusing on copulating species, has robustly demonstrated this phenomenon. Noncopulating sessile organisms that release male gametes into the environment but retain their eggs for fertilization may routinely receive unequal mixtures of sperm. Although promiscuity seems unavoidable it does not follow that the resulting paternity obeys ‘fair raffle’ expectations. This study investigates frequency dependence in the mating of one such species, the colonial ascidian Diplosoma listerianum. In competition with an alternative sperm source males fathered more progeny if previously mated to a particular female than if no mating history existed. This suggests positive frequency-dependent selection, but may simply result from a mate order effect involving sperm storage. With fewer acclimation matings, separated by longer intervals, this pattern was not found. When, in a different experimental design, virgin females were given simultaneous mixtures of gametes at widely divergent concentrations, sperm at the lower frequency consistently achieved a greater than expected share of paternity – a rare male effect. A convincing argument as to why D. listerianum should favour rare sperm has not been identified, as sperm rarity is expected to correlate very poorly with ecological or genetic male characteristics in this pattern of mating. The existence of nongenetic female preferences at the level of colony modules, analogous in effect to fixed female preferences, is proposed. If visible to selection, indirect benefits from increasing the genetic diversity of a sibship appear the only likely explanation of the rare male effect in this system as the life history presents virtually no costs to multiple mating, and a near absence of direct (resource) benefits, whereas less controversial hypotheses of female promiscuity (e.g. trade up, genetic incompatibility) do not seem appropriate.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

If the reproductive success of a male varies with the proportion of its type in the population, mating is said to be frequency-dependent. Previous work has suggested that males of an unusual type often have a fitness advantage over their commoner rivals. This finding of negative frequency-dependent selection has been termed the rare male effect. Selection is balancing because the advantage holds only so long as the rare type is rare. When the rare type does so well as to become common the advantage disappears. Negative frequency dependence may help explain, without recourse to genetic load, how high levels of genetic variation are maintained within populations (e.g. Philosophical Transactions of the Royal Society, 1988, volume 319, pp. 457–640).

A rare male effect has been reported in several taxa including guppies, flour beetles, wasps and ladybirds, but the vast majority of studies have been performed on Drosophila. Here a wide variety of features – including geographical strains, inbred lines, enzyme variants, visible mutants, rearing media or temperature – from a number of Drosophila species, have been reported to produce rare male phenomena (see Knoppien, 1985 and Partridge, 1988 for reviews). However, by itself this range of studies may not be a robust demonstration of the effect. Some results have not been repeatable (e.g. Partridge & Gardner, 1983; Oberhauser, 1988), a publication bias may exist against studies where a rare male effect has not been found, and there may be problems with some of the methods and data analysis (Knoppien, 1985; Partridge, 1988). Doubt has been cast on the generality of rare male phenomena and important questions raised about the underlying mechanisms. Partridge (1988) summarized four possible causes of a rare male effect in copulatory species:

  • 1
    Disassortative mating preferences: a preference for mates with a dissimilar genotype. These may occur in the absence of active female choice and are usually included within discussions of sexual compatibility.
  • 2
    Female choice: females sample the male population, assess the frequencies of the different male types and bias their mating preferences towards the least common.
  • 3
    Male competition: males compete more with their own type than with an alternative type, or males from one type somehow facilitate matings by the other type.
  • 4
    Fixed female preferences: if the female population is composed of a mixture of morphs, each with different but fixed mating preferences, then a rare male effect can be generated (see O'Donald & Majerus, 1988 ).

Not all organisms mate by the direct transfer of numerous spermatozoa in an ejaculate or spermatophore. A phylogenetically diverse group of aquatic organisms mate at a distance by releasing male gametes into water for largely passive transfer between genetic individuals. Sperm fertilize eggs that have been retained by the acting female and embryos are often brooded. This largely sessile or sedentary group includes all bryozoans, most compound ascidians, plus many sponges, hydroids and algae as well as certain polychaete tube worms and bivalves. We refer to this reproductive strategy as ‘spermcast mating’. In such organisms ‘precopulatory’ screening of mates is severely constrained or absent (Bishop & Pemberton, 1997). It is of interest to incorporate this life history strategy into the empirical framework of frequency-dependent mate choice. Many of the evolutionary considerations are shared with wind pollinated plants in terrestrial environments where a small literature on the frequency dependence of reproductive success already exists (e.g. Elkassaby & Ritland, 1992; Cruzan & Barrett, 1996).

Spermcast species are likely to encounter male gametes from different sources at dissimilar frequencies. A concentration gradient driven by diffusion processes is expected to form, such that sperm concentration decreases as a function of downstream distance and time (a pattern usually moderated by stochastic turbulence and advection). A female is likely to be subjected to large amounts of sperm from a few close males, with dilute sperm arriving infrequently from distant sources. Such considerations are of great importance as gene flow in sessile communities is largely restricted to the gamete and larval stages. Larval dispersal has traditionally been considered the dominant factor in the broadcast spawning groups with long lived, planktotrophic larvae that have been the classic subjects of marine research. However, spermcast species typically possess brooded larvae that disperse relatively short distances (Jackson, 1986), suggesting increased importance for sperm-mediated gene flow. Despite theoretical expectations of small-scale genetic structuring (Knowlton & Jackson, 1993), empirical evidence exists for relatively few sessile invertebrates. Where found, population subdivision may (e.g. Hellberg, 1995) or may not (e.g. Burnett et al., 1995) be attributable to philopatric larval behaviour.

Accumulating evidence suggests that the dynamics of gamete transfer in spermcast species differ greatly from previous assumptions based on broadcast spawners (Temkin, 1996; McCartney, 1997; Bishop, 1998; Yund, 2000). For example, Grosberg (1991) bred three homozygous lines for naturally occurring, rare, allozyme markers in the colonial ascidian Botryllus schlosseri, a spermcast species. These animals were used as focal males in field experiments. Progeny brooded by acting females along transects radiating out from the males were collected and screened for marker alleles. Although Grosberg concluded that sperm concentration must decrease rapidly within 0.5 m of a source colony he made two intriguing counterpoints. First, for two of the three alleles, even when background frequencies were subtracted, observed marker frequency probably exceeded that expected from a simple diffusion model. Secondly, although progeny frequencies reduced with distance, extrapolation of these values from one dimension (linear counts provided by transect data) to two dimensions (a series of concentric circles of potential sperm receivers) gave estimates of progeny numbers that possibly tended to increase with distance. A rare male effect could help explain departures from the predictions of diffusion models. Relatively few long-distance fertilizations can lead to extensive gene flow (Slatkin, 1985, 1987), and the likelihood of these minority sperm achieving successful fertilizations would be greatly increased by negative frequency-dependent selection.

Here we report investigations of frequency-dependent mating in the compound ascidian Diplosoma listerianum (Milne Edwards). Following the terminology of Waser (1993; p. 1), ‘mating’ is defined not by copulation or spawning but by the production of a zygote, i.e. by syngamy. Two groups of experiments were performed, which deal with alternative definitions of ‘rare’. The first considers variation of realized paternity with respect to mating history. Here a ‘rare’ male is one that is unfamiliar to a female that has been previously exposed to an alternative male type. For clarity the term ‘novel’ is adopted over ‘rare’, the female having a mating history to the alternative ‘acclimation’ type. This design mimics the chance transmission of gametes from relatively distant sources competing against sperm from local males. The second set of experiments considers a simpler interpretation of male frequency: that of two simultaneous sperm sources present at widely dissimilar concentrations. The genotype of sperm at the lower concentration can clearly be thought of as ‘rare’ and the alternative as ‘common’.

Biology of D. listerianum, laboratory culture and paternity analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

A colony of D. listerianum is founded by the settlement and metamorphosis of a sexually produced tadpole larva. Vegetative budding produces a colony of genetically identical zooids within an outer tunic. Colonies can move slowly across the substrate and fission and fusion are common. All zooids feed and, when mature, have both male and female gonads. Sperm are released by individual zooids in discrete emissions of a few thousand gametes, leave the colony via an exhalant opening in the tunic, and rapidly disperse (Bishop & Ryland, 1991; Bishop, 1998).

The movement of sperm is likely to be dominated by passive transport in currents. The substantial longevity of released sperm (half life of c. 8 h, Bishop, 1998) potentially allows viable male gametes to travel several kilometres from a source colony. Sperm are thought to enter another colony with the inhalant feeding current, although this has yet to be demonstrated. Fertilizing sperm pass up the fertilization duct (oviduct) of a zooid to the ovary (Burighel et al., 1986; Burighel & Martinucci, 1994a; Bishop & Sommerfeldt, 1996). Vitellogenic egg growth is triggered by the presence of sperm from a compatible colony (Bishop et al., 2000a), and the first ovulations take place at least 9 days later. A given zooid will then ovulate a single ovum at intervals of 7–12 days (Ryland & Bishop, 1990). Ovulation is not synchronized between zooids. Sperm are stored in the lumen of blind-ended peduncular extensions of the ovary that penetrate the follicle cell layers surrounding developing oocytes. Sperm do not pass through the ovarian epithelium into the oocyte cytoplasm until around the time of ovulation (Burighel & Martinucci, 1994b). The ovum/zygote is deposited directly into the colonial tunic, where the embryo develops to an advanced stage before being released, after about 13 days, as a free larva capable of swimming. In the laboratory the larval stage is brief (a few hours), before settlement and metamorphosis on the inside of culture tanks. Storage of compatible sperm enables D. listerianum to produce a lengthy series of progeny following a brief period of mating (Bishop & Ryland, 1991; Bishop, 1998). Self sperm re-entering the colony of their origin may pass into the oviduct of a zooid but are generally blocked and phagocytosed a fraction of the way along the duct (Bishop, 1996). Exogenous sperm from some sources may be blocked by a similar compatibility mechanism (Bishop, 1996).

The remote transfer of individual spermatozoa allows a female to be simultaneously inseminated by multiple partners. Simultaneous mating with two males can produce broods of mixed paternity (Bishop et al., 1996). Sequential mating to two sperm sources provides a consistent pattern of sperm precedence, with initial bias in paternity towards the first of two acting males. The proportion of second-male paternity subsequently increases with time until it dominates in late progeny (Bishop et al., 2000b).

All laboratory clones were founded from individual larvae of wild-mated parents. In addition to spontaneous colony fission, virgin colonies can be cut up and the pieces (ramets) reattached to separate substrates, allowing a single genotype to be replicated within and between experimental treatments. General protocols for laboratory culture are provided in Bishop et al. (2001). Ramets are grown and crossed in small volumes (c. 850 mL) of filtered, UV treated and aged sea water (FSW). Culture rooms are maintained at a constant temperature and with a set light : dark cycle. Great care is taken to prevent the accidental transfer between genetic individuals of water droplets or aerosols that may contain sperm.

Four laboratory clones, designated E, F, 35 and 36, were used in the experiments reported here. Clones E and F were the acting females whose progeny were collected and analysed. For clone E as a female, clone F and 35 acted as the males. For clone F as a female, clones E and 36 acted as the males. This deployment of clones 35 and 36 was necessitated by the incompatibility of the reverse pairings. These pairings of fully compatible clones are the same as used to examine sperm precedence by Bishop et al. (2000b), and further details are provided in that paper.

Acting male ramets released sperm into fresh FSW. The concentration of sperm could be calculated (30 mL subsamples filtered onto a membrane, stained and counted by fluorescent microscopy as described by Bishop, 1998), and diluted with FSW to give a standardized concentration. Sperm from two donor clones could be mixed to give known concentrations. Crosses were carried out by placing the acting female ramet into the sperm suspension.

Progeny were subjected to the same protocols and paternity markers for randomly amplified polymorphic DNA (RAPD) analysis as described by Bishop et al. (2000b). The behaviour of marker bands is highly repeatable. Primer OPY-04 gives a 560-bp fragment homozygous in clone F but absent in clones E, 35 and 36. Primer OPR-13 gives a 500-bp amplification fragment homozygous in clone E, but absent in clones F, 35 and 36. A previously unpublished homozygous band at c. 310 bp accompanies the c. 500 bp fragment in clone E, and was used to confirm clone E paternity.

Part A: Many acclimation matings separated by short intervals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Mating sequences and numbers of replicates are shown in Table 1. All acting female ramets were virgins at the start of the experiment. Treatment females were acclimated to one male genotype over 13 single-male matings, each separated by 5 ± 1 days. Acting males released sperm for 24 h. Treatment females were immersed in this sperm suspension for a further 24 h to allow sperm to be taken up. Acting females in control groups were water-changed for FSW (no sperm). Five days after the final acclimation cross, a mixed mating was performed on all acting females. For this, sperm suspensions from both males, for each of the two mating groups, were diluted to give an equal, known sperm concentration. Thus both treatments had a mating history with one of the two males. Controls had experienced neither sperm source before. Hence the treatments were ‘acclimated’ to one of the genotypes of sperm in the mixed mating, whereas the other sperm type was ‘novel’.

Table 1.  Mating sequences and numbers of replicates for all experiments. In experiment 1 – represents control II which was added to the experiment late from standard culture stocks, and so did not have a strictly similar history to the other tanks.
Experiment 1
Acting femaleAcclimation matingsMixed mating (day 0)Number of replicates
Experiment 1A(×13, once every c. 5 days)  
 EFF and 353
35F and 353
none (FSW)F and 353
 FEE and 363
36E and 363
none (FSW)E and 363
Experiment 1B(115, 78, 38 days before mixed mating)  
 FEE and 363
36E and 363
none (FSW)E and 362
E and 362
Experiment 2
Acting femaleCommon sperm sourceRare sperm sourceNumber of replicates
EF354
 35F4
FE364
 36E4

Before the mixed mating any released progeny were removed at weekly intervals. After the mixed mating progeny were collected every 3–8 days until production of progeny ceased. Paternity analysis was spread between replicates.

Part B: Few acclimation matings separated by long intervals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Three acclimation matings to single sperm donors were performed at intervals of 37–40 days. At these times a procedural control (control I) was water-changed for FSW. Thirty-eight days after the final acclimation cross, a mixed mating was performed on all females. This consisted of 400 mL of sperm suspensions from both males (total 800 mL). ‘Control II’ was added to the experiment at the time of the mixed mating and consisted of stock animals previously maintained in reproductive isolation under standard culture conditions. The sequences of mating and numbers of replicates are shown in Table 1.

Collections of progeny were made 71, 41 and 1 day before the mixed mating, then on days 4, 12, 17 and subsequently every 2–3 days after the mixed mating until the release of progeny ceased. Paternity analysis was performed on six well-preserved progeny from each replicate at each collection interval, where available.

Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

All females were virgins before a single, simultaneous mating to two sperm sources at widely dissimilar concentrations. Acting males released sperm for 6 h. Sperm suspensions from various subcolonies of the same clone (ramets) were pooled and used to generate alternative ‘rare’ and ‘common’ treatments within a mating group. Sperm concentration in each stock was initially assessed from 3 × 30 mL samples. Based on these counts, dilutions were performed in an attempt to produce 15 : 1 ratios of common : rare sperm, in 800 mL. This process took c. 2½ h. Sperm suspensions were then transferred to acting female colonies for 9 h. Mating was stopped by moving the females into FSW. Mating sequences and numbers of replicates are shown in Table 1.

Progeny were collected every 2–3 days until all progeny had been released. For offspring brooded by clone E, paternity analysis was performed on six progeny from each replicate at each collection interval, where available. All well-preserved progeny from acting female F were analysed (this cross gave fewer offspring).

Recounts of sperm concentration were performed on the preserved filters several weeks after the matings. Without the time pressures of the preliminary counts filters could be carefully observed. Sperm staining also appears to strengthen slightly with time. Recounts thus provided a more accurate measure of sperm concentration.

Experiment 1A

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Figure 1 shows the results of molecular paternity analysis for this experiment. As no progeny were produced by the controls during the first two (acting female clone E) or three (female clone F) collection intervals, and all analysed offspring from treatment ramets were fathered by the acclimation male during these periods, it is likely that these initial fertilizations used stored sperm from the earlier single-male matings. These fertilizations took place in the absence of intermale competition/choice and so are not included in further calculations. The control tanks provide a ratio of paternity in the absence of a mating history. The treatment tanks give a ratio of paternity following a mating history (F : 35 paternity in female clone E mating group: control= 119 : 1; F acclimation, 35 novel=30 : 0; 35 acclimation, F novel=2.5 : 1. Ratios of E : 36 paternity in the female clone F mating group: control=3.2 : 1; E acclimation, 36 novel=10.5 : 1; 36 acclimation, E novel=2.8 : 1). ‘Expected’ paternities for acclimated (treatment) animals can be constructed from the paternity ratios in the absence of mating history, provided by the nonacclimated control tanks. These values can be compared with ‘observed’ values to assess the impact of a mating history on realized paternity. This data is shown in Fig. 2 . A consistent and, where testable, highly significant difference can be seen with the acclimation genotype achieving higher observed paternity than expected.

image

Figure 1. Paternity with time for both mating groups in experiment 1A. All well-preserved progeny of the replicates shown were analysed except where marked with an asterisk ‘*’. ‘#’ represents replicates where only alternate progeny were analysed.

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image

Figure 2. Pooled data from all replicates of ‘observed’ vs. ‘expected’ paternity of the acclimated animals based on paternity ratios of the previously unmated controls in experiment 1A. Within individual groups the data are organized by the different acclimation males. The left hand pair of bars always represent the acclimation male's paternity and the right hand bars the paternity of the novel male. Values are G -tests of observed against expected frequencies. In ‘#’=values are Gtotal ( Sokal & Rohlf, 1995 ; box 17.4). ‘##’=replicates are heterogeneous, values are Gadj employing Williams' correction ( Sokal & Rohlf, 1995 ; box 17.1). For female clone E, clone F=acclimated, clone 35=novel the analysis is not possible due to zero values of clone 35 paternity.

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Experiment 1B

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

After the mixed mating a delay was expected before progeny were released. This was found in all control replicates and replicates acclimated to clone 36. However, females acclimated to clone E produced progeny during the first two collection intervals after the mixed mating. These are too early to have been fertilized by sperm from the mixed mating. A more likely explanation, supported by the molecular paternity analysis, is that these used stored sperm from the single-male acclimation matings. Subsequent calculations exclude these individuals.

Figure 3 shows the results of paternity analysis. For both treatments and control I, clone E appears to have fathered slightly more of the progeny than clone 36. This pattern is reversed for control II tanks (Ratios of E : 36 paternity in this female clone F mating group: control I=1.3 : 1; control II=1 : 1.5; E acclimation, 36 novel=1.3 : 1; 36 acclimation, E novel=1.2 : 1).

image

Figure 3. Results of paternity analysis for experiment 1B. RAPD PCR was performed on six well preserved progeny from each replicate at each collection interval where available. Early progeny, left of the dotted line, are thought fertilized by stored sperm from the acclimation matings.

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As with Experiment 1A the control tanks provide a ratio of paternity in the absence of a mating history. These form ‘expected’ paternities to compare against ‘observed’ values. Results are skewed depending on which control data are used to construct the ‘expected’ values. In both cases no consistent difference in mating history could be seen. All differences are nonsignificant (G-test of observed against expected frequencies employing Williams' correction).

It was not possible to allocate paternity for one of the offspring from the treatment acclimated to clone E (43–45 days collection interval, left-hand replicate of Fig. 3). Repeated amplifications produced the same result of a disagreement between the c. 500 bp and c. 310 bp bands. Paternity could therefore not be assigned and the individual was excluded from further analysis.

Experiment 2

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Table 2 shows the sperm concentrations and dilutions to produce the common : rare ratios. Dilutions were based on preliminary sperm counts [column (a) in Table 2 ]. Subsequent recounts improved these estimates [column (b)]. Common : rare ratios [column (c)] were subsequently recalculated [column (d)]. No attempt was made to standardize absolute sperm concentration between treatments [column (e)]. The mating group of acting female E, mated to males F and 35, showed the greatest variation in all parameters.

Table 2.  Experiment 2. Estimated sperm concentrations (left hand block) and subsequent dilutions to produce common : rare ratios (central block). Letters in parentheses refer to the text. All concentrations are in sperm mL −1 . Right hand block shows G -test of observed against expected frequencies of paternity employing Williams' correction [f]( Sokal & Rohlf, 1995 ; box 17.1). Data from replicates are pooled due to small expected rare male paternities of some individual replicates. P -values are exact [g], allowing a test of combined probabilities from independent tests of significance [h] ( Sokal & Rohlf, 1995 ; part 18.1).
Acting femaleSperm donorOriginal estimation of undiluted conc. [a]Recounted undiluted [b]Sperm mixOriginal estimation of diluted ratio. [c]Recalculated Ratio [d]Overall sperm conc. [e]Gadj pooled by Treatment [f] P [g] Combined P[h]
  EF107166F common, 35 rare15 : 130.7 : 11463.670.05530.0002
35403135 common, F rare15 : 1 7.3 : 13413.340.0003 
  FE157141E common, 36 rare15 : 116.2 : 11380.280.59530.0178
3613010836 common, E rare15 : 113.8 : 11108.150.0043 

Figure 4 shows total numbers of released progeny and paternity for the alternative common : rare treatments in both mating groups (all replicates combined). Figure 5 shows the central result of this experiment. Ratios of common : rare sperm concentrations [column (d) Table 2 ] have been used to calculate expected paternities based on fair-raffle expectations that can be compared with observed values from molecular paternity analysis. Observed paternity ratios track expected values. However, all four rare sperm types achieved higher observed paternity than expected. It follows that all four common sperm types achieved lower paternity than expected. The trend is consistent but the magnitude of the difference and sample size varies. This translates into different probability values for each pooled set of replicates for the null hypothesis that observed frequencies differ from expected values by sampling error alone [column (g) Table 2 ]. Importantly, overall probabilities for each mating group are significant [column (h)].

image

Figure 4. Experiment 2. Summed data from all replicates of numbers of progeny (lines, right hand axes) and RAPD paternity (bars, left hand axes) with time for both mating groups. Note different scales.

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image

Figure 5. Experiment 2. Pooled data of ‘observed’ vs. ‘expected’ paternity based on measured ratios of the sperm concentrations. Within individual groups the data are organized by the alternative treatments of different common/rare male roles. The left hand pair of bars always represent the rare male's paternity and the right hand bars the paternity of the common male. Frequencies are shown on top of the relevant bar; note scales differ. Table 2 columns (g) and (h) show probabilities.

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Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Several (13) acclimation matings, separated by short intervals (c. 5 days) imposed a detectable effect on realized paternity following a single mixed mating in the colonial ascidian D. listerianum (experiment 1A). When in competition with an alternative sperm source, males fathered more progeny if previously mated to a particular female than if no mating history existed. This suggests positive frequency-dependent selection under these conditions, the opposite of the rare male effect. When the number of acclimation matings was reduced (to three) and the time between them increased (to c. 5 weeks), paternity from a final competitive mixed mating was not significantly different from virgin animals (experiment 1B). So under these conditions reproduction was independent of previous mating history.

The two sets of experiments were designed to complement each other. The total period of acclimation in experiment 1A was chosen to be slightly greater than the c. 5 week interval used in experiment 1B. The c. 5 days interval between matings was similar to the gap between first and second males used in sperm precedence experiments (Bishop et al., 2000b). The short (5 days) gap between the final acclimation mating and mixed mating in experiment 1A made it likely that a proportion of offspring released after the mixed mating would be fertilized by sperm from the acclimation crosses (a biologically realistic situation as a spermatozoon is likely to arrive at an ovary where several viable spermatozoa are already in storage). The appearance of even a few novel male progeny after so many acclimation matings probably represents a mate order advantage to the final batch of sperm in the absence of competition from later-arriving gametes. However, overall, the paternity was biased in favour of the acclimation male. Such a result may be caused by matings from previously stored sperm.

Experiment 1B failed to show a frequency-dependent effect on realized paternity. The null hypothesis that mating history has no effect on future mating pattern may be accepted. The c. 5 week mating interval was designed to avoid the carry-over of fertilizations from the acclimation crosses into the cohort of progeny from the mixed mating as experienced in experiment 1A. By increasing mating interval, influences of ‘defensive’ behaviour (sensuRice & Holland, 1997) of stored acclimation sperm could have been reduced or eliminated. An alternative explanation of the results is that insufficient acclimation matings were performed for an effect to be displayed. A further possibility arises from the modular, colonial nature of the study organism: zooidal turnover (individual zooids have a life span of c. 6 months), with zooids dying and others reaching sexual maturity over the course of the experiment, could have eroded the effect of earlier acclimation matings.

Relatively few studies have examined the effects of female acclimation or prior mating experience on male sexual fitness. Pruzan (1976) used three strains of Drosophila pseudoobscura to study mating frequencies after various acclimation procedures. In common with our results a mating advantage to the acclimation type was found when the competing male types were equal in number in the mixed mating. McLain (1991) reported the opposite trend for female stink bugs, Nezara viridula. Females that remated soon (<2 days) after an initial mating preferred a different male type. Likewise some D. melanogaster strains discriminate against certain genotypes of acclimation sperm (Childress & Hartl, 1972). Hughes et al. (1999) found mature female guppies (Poecilia reticulata) had significantly lower probability of mating with an acclimation than novel male type and produced significantly fewer offspring when they did. Two studies using immature guppies had failed to demonstrate negative frequency-dependent selection and had sometimes shown positive frequency-dependent selection (Breden et al., 1995; Rosenqvist & Houde, 1997).

Pruzan (1976 ) suggested that ‘Considering the important implications that frequency-dependent mating selection contains for evolutionary theories, equilibria between the gene frequencies of rare and common types would be reached much more rapidly if sexual selection is altered by prior mating experiences.’ The results of this study suggest that this factor may not be important for D. listerianum . From other studies it does not seem possible to generalize as to the direction of frequency-dependent selection following prior mating experience and/or conditioning.

Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

When virgin colonies of D. listerianum were given mixtures of compatible sperm at widely divergent concentrations, offspring were shared between the two sperm sources in approximately the ratio of each mixture. However, there existed a small but statistically significant deviation from the fair-raffle model, in that sperm at the lower concentration consistently achieved a greater than expected share of paternity. This is negative frequency-dependent selection – the rare male effect.

Various features of the experimental design are worth further consideration to back up claims of a two-sided effect, although the magnitude of frequency dependence varied between opposing halves of both mating groups (a stronger rare male effect existed in the two mating groups when sperm of clones E or F were in the rare male role). Experiment 2 hinged on the reciprocal nature of the crosses, whereby each batch of sperm was used in both rare and common roles. Any error in the estimation of sperm concentration that altered the results for one treatment would have had the opposite effect in the alternative treatment – a kind of self-controlling and stabilizing design. Thus any one half of a mating group is inconclusive, but when the two halves are considered in combination, errors balance out, allowing overall deviations from fair-raffle expectations to be detected. Such a deviation was apparent in the present results.

It is worth considering if there are any further methodological explanations for the patterns found? Several possible areas are considered:

Problems with the molecular markers

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

As previously described RAPD has proved an extremely reliable method for assigning paternity in D. listerianum. We have considered the effects of errors in mistaking adult zooids for progeny, of chimeric fusions of metamorphs, of errors in the markers themselves and we cannot think of a realistic mechanism by which any bias could significantly modify the results within the counter-balanced design of the experiment (this is more thoroughly considered by Pemberton, 2000).

The pattern of mating, via the remote transfer of individual spermatozoa, facilitated the manipulation of sperm concentrations. Simultaneous mating to two sperm donors avoided mate-order effects. A comparable process with copulatory species is only really possible through mixed artificial insemination (see Dziuk, 1996), limiting its biological relevance outside domestic breeds. In D. listerianum a form of simultaneous mixed mating is expected to be normal in the wild.

Sperm precedence work (Bishop et al., 2000b) suggested that ‘differential fertilizing capacity’ (DFC) (Dziuk, 1996) exists between the clones used in these experiments. In Experiment 1A DFC, along with a relatively small sample size, is likely to be responsible for the complete absence of clone 35 paternity in progeny of female E acclimated to male F. With the other mating group of female clone F and males E and 36, experiment 1A and sperm precedence experiments showed greater symmetry between the reciprocal pairings. DFC probably contributes to the results of experiment 2. It can be seen that the most significant over-representation of the rare male is found when the ‘dominant’ clones of previous work (F and E) are in that role. Knoppien (1985; p. 90) warns of confounding frequency dependence of mating success with differential mating success (analogous to DFC). However, as with inaccuracies in the estimation of sperm concentration, any effect of DFC would balance out between the two halves of a mating group, allowing a rare male effect to be assessed.

Reasons why D. listerianum might favour rare sperm sources are not obvious. A colonial, sessile life history combined with spermcast mating produces a suite of interconnected factors that may be more or less correlated with the frequency of sperm reaching a recipient. These may include:

Distance: a rare sperm source may be one that is located far away.

Relatedness: in highly structured sessile populations (e.g. for the colonial ascidian B. schlosseri,Grosberg, 1987, 1991; the bryozoan Celleporella hyalina, Goldson et al., 2001) a distant sperm source may be less related to the acting female than a close, common donor.

Size: sperm output would be expected to increase with donor colony size but with a life history that includes colony fission and movement an acting female has no information as to whether the whole genetic individual (genet), or just an upstream subcolony (ramet) is being sampled.

Age: a rare donor colony may produce fewer sperm because it is young or because it is senescing and old (e.g. Rinkevich et al., 1992; Gardner & Mangel, 1997).

Reproductive synchrony: a source of rare sperm may be a recent recruit or may have matured late.

Sexual allocation: low sperm output may be due to preferential investment in female or somatic functions (Ghiselin, 1969; Heath, 1977; Charnov, 1979).

It is only possible to conclude that relative abundance of sperm probably provides an acting female zooid with very little information about heritable fitness. This would tend to favour explanations for frequency-dependent mating that do not invoke an evolved adaptive response.

Negative frequency dependence may affect the ‘degree’ of polyandry through an increase in the evenness of paternity shared between males and possibly maximizes the number of fathers under certain circumstances (very rare sperm sources may achieve a few fertilizations where they would probabilistically achieve none in the absence of a rare male effect). The net result is that the genetic diversity of a brood increases. As discussed by Bishop & Pemberton (1997) spermcast organisms potentially represent a relatively uncluttered terrain for the study of polyandry as most of the costs (e.g. time and energy, predation, female injury, reduced paternal care and transfer of disease) and many of the nongenetic benefits (e.g. nutritional benefits, additional paternal care) in copulating species can be largely discounted. Genetic benefit hypotheses that invoke a ‘trade-up’ to higher quality males or that relate to socially monogamous species (polyandry compensating for low fertility or poor quality of the social mate) seem applicable to neither the life history strategy nor the two-sided nature of the present results. Also the hypothesis that multiple mating minimizes intragenomic conflict (Zeh & Zeh, 1997) is not valid here as embryonic development occurs out of reach of female control, preventing the reallocation of resources from genetically incompatible to compatible offspring. This leaves the somewhat controversial (e.g. Arnqvist, 1989) offspring diversity hypotheses that rely on benefits through either sib competition (Ridley, 1993), inbreeding avoidance (which may also require sib competition, Stockley et al., 1993 p. 177) or uncertainties in future environmental conditions to justify the production of diverse broods. Unfortunately insufficient basic biology of the study organism is known to favour any particular explanation.

It is of interest to consider how this experiment fits into the summary of suggested mechanisms of the rare male effect provided by Partridge (1988):

Disassortative mating preferences

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Grosberg (1991) noted that the functioning of an incompatibility system geared to restrict inbreeding could act to decrease local fertilization success in populations where a degree of kin structure was found. Thus sperm-mediated gene flow would be greater than expected, with maximum male fertilization success at an intermediate distance from a source colony. D. listerianum does have a compatibility system ( Bishop et al., 1996 ), but the present study avoids this complicating factor by choosing fully compatible mating partners (see Methods section) and utilizing both males in both rare and common roles.

Female choice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

It seems unrealistic to suppose that individual zooids have the cognitive ability to assess, rank and actively choose a male on grounds of relative sperm concentration. Mechanisms of female choice by individual zooids of D. listerianum are likely to be basic. One possibility may involve a habituation to the common sperm type, perhaps based on an immune-like response of the female (e.g. chickens –Steele & Wishart, 1992 in Birkhead, 1995). Sperm uptake from a single source has been reported to fall dramatically during continued availability (Bishop & Sommerfeldt, 1996). Phagocytes in the lumen of the fertilization duct of D. listerianum and heterophagic vacuoles in cells of the oviductal epithelium (Burighel & Martinucci, 1994a,b) are thought likely candidates for selection and removal of certain sperm types (Bishop, 1996). Negative frequency dependence could be a by-product of such mechanisms. If the female response (via immune-like phagocytotic processes) were specific to a particular type (or haplotype subset) of sperm, then it could result in the selective removal of common over rare gametes. The influence need only be mild to account for the results of experiment 2 and fit in with the conclusions from experiments 1A and 1B. In experiment 1A the acclimation male outperformed the novel male. If sperm of a common type were severely rejected then the opposite result would be predicted.

Male competition

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

By itself male competition does not seem to offer any obvious explanation for the results (for a possible exception see below). One factor that can be discounted here is the potential for a rare male effect to be generated by differences in male mating behaviour. Knoppien (1985; p. 101) suggests that typical rare male experiments cannot discern between whether females prefer to actively choose the rare male or whether the rare male compensates for its rarity by becoming more sexually active. The sperm suspensions used during these experiments gave no possibility for the males to be aware of the presence of the female or other males and adjust sperm output or quality accordingly.

Fixed female preferences

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References

Preferences are considered fixed or frequency independent if the likelihood of mating upon encounter is unaffected by male frequency. If the female population is composed of a mixture of morphs each with different but fixed mating preferences for different male morphs then a rare male effect can be generated (O'Donald & Majerus, 1988). The D. listerianum experiments used a single female clone within and between treatments, so variation in genotype was eliminated leaving only variation through ontogeny or environmental effects. Nongenetic properties have been previously reported to give a rare male effect (see Knoppien, 1985; p. 97). In the present experiments zooidal demography may have had such an influence as colonies always contain zooids of various ages. If the relative ability of sperm from different sources to obtain fertilizations varied with acting-female zooid age or condition, a rare male effect could be generated. Female ageing has been associated with a change in frequency-dependent selection in Drosophila (Pruzan, 1976; Klobutcher, 1977).

The three experiments presented here give three different patterns, with positive (experiment 1A), neutral (experiment 1B) and negative (experiment 2) frequency dependence. It is of interest to consider the effect of these patterns on gene flow in wild populations. Negative frequency dependence following exposure to sperm mixtures (experiment 2) should ‘lift’ the long tail of a leptokurtic (e.g. Levin & Kerster, 1974) gamete dispersal curve: in a continuous population although the majority of fertilizing gametes come from nearby males (Engel et al., 1999), rare sperm arriving from relatively distant sources would have an enhanced chance of success. Likewise, migrants attached to ships' hulls or rafting on natural or man-made debris (e.g. Barnes, 2002) would be more likely to leave a genetic legacy along their route if the few sperm that did reach sedentary populations were favoured. However, the finding of positive frequency dependence with respect to mating history (experiment 1A) suggests caution – precedence of stored sperm could impart resistance to genetic invasion. We are a long way from fully understanding the reproductive attributes that determine a species' susceptibility to genetic introgression via natural or anthropogenic processes. The possibility exists that mechanisms promoting genotypic diversity within broods have an indirect effect on patterns of gene flow in these sessile, remotely mating organisms.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Biology of D. listerianum, laboratory culture and paternity analysis
  6. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  7. Part A: Many acclimation matings separated by short intervals
  8. Part B: Few acclimation matings separated by long intervals
  9. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  10. Results
  11. Experiment 1A
  12. Experiment 1B
  13. Experiment 2
  14. Discussion
  15. Experiment 1 – Variation of paternity with respect to mating history: ‘novel’ vs. ‘acclimation’ males
  16. Experiment 2 – Mating success of two simultaneous sperm sources at widely dissimilar concentrations: ‘rare’ vs. ‘common’ males
  17. Dilution protocols
  18. Differential ageing
  19. Problems with the molecular markers
  20. Disassortative mating preferences
  21. Female choice
  22. Male competition
  23. Fixed female preferences
  24. Acknowledgments
  25. References
  • Arnqvist, G. 1989. On multiple mating and female fitness: comments on Loman et al . (1988). OIKOS 54: 248250.
  • Barnes, D.K.A. 2002. Invasions by marine life on plastic debris. Nature 416: 808809.
  • Birkhead, T.R. 1995. Sperm competition: evolutionary causes and consequences. Reprod. Fertil. Dev. 7: 755775.
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  • Burnett, W.J., Benzie, J.A.H., Beardmore, J.A. & Ryland, J.S. 1995. Patterns of genetic subdivision in populations of a clonal cnidarian, Zoanthus coppingeri, from the great-barrier-reef. Mar. Biol. 122: 665673.
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Received: 29 May 2002;revised 5 September 2002;accepted 10 October 2002