The evolution of sperm size in birds.
Recent empirical studies of sperm-size variation in a variety of taxa have now shown that such a simple size-number tradeoff is unlikely (Gomendio and Roldan 1991, 1993; Briskie and Montgomerie 1992; Dixson 1993; Gage 1994). Instead, sperm tend to be larger in species subject to more intense sperm competition. For example, Gomendio and Roldan (1991) found that polyandrous species of both rodents and primates have longer sperm than their monandrous counterparts (see also Dixson 1993). As there appears to be a positive correlation between swimming velocity and sperm length (Gomendio and Roldan 1991), longer sperm may have evolved as an adaptation to outdistance sperm from rival males in the race to fertilize ova. A similar pattern of sperm-size variation among mating systems was found in butterflies (Gage 1994), a taxon in which both fertile eupyrene and nonfertile apyrene sperm morphs are produced. Across a variety of species, eupyrene sperm length correlated with the intensity of sperm competition, but no such pattern was evident with apyrene sperm. Gage (1994) concluded that selection for faster swimming speed was probably the main factor that led to the increased length of eupyrene sperm.
In birds the situation is more complex, as females store sperm prior to fertilizations in specialized sperm storage tubules (SSTs) at the junction of the uterus and vagina (e.g., Fujii 1963; Shugart 1988; Birkhead et al. 1990a; Briskie and Montgomerie 1993). SSTs are typically long, narrow tubular structures with a single opening into the lumen of the oviduct. The number of SSTs varies from species to species, ranging from 300 to 20,000 per female, depending at least in part on female body size (Birkhead and Moller 1992a; Briskie and Montgomerie 1993). Sperm enter the SSTs within a few hours of insemination, where they remain until they are used to fertilize the eggs (Bobr et al. 1964; Verma and Cherms 1965; Bakst 1981). Sperm that are not stored within SSTs are unlikely to survive for more than a short period in the oviduct and may become entrapped in albumin by the passage of an egg (Lorenz 1966). Thus, most sperm that eventually fertilize ova have probably come from SSTs and not directly from an insemination. This certainly must be true for the many passerine bird species (Passeriformes) that do not copulate during the egg-laying period (Birkhead and Moller 1993).
To examine how sperm storage might have shaped the evolution of sperm size in birds, Briskie and Montgomerie (1992) analyzed interspecific variation in sperm length in relation to both the size and number of SSTs in females. They observed a fivefold difference in sperm length across 20 passerine bird species. As in other taxa, most of this variation was due to variation in tail length: long sperm are long because they have long tails (see also Allen et al. 1968; Dybas and Dybas 1981; Pitnick and Markow 1994). Unlike mammals and butterflies, however, neither social mating system nor relative testis size (both used as measures of the intensity of sperm competition) were related to variation in sperm length in birds. Instead, Briskie and Montgomerie (1992) found, across species, a negative correlation between sperm length and SST number, and a positive correlation between sperm length and SST length (also see Birkhead and Moller 1992a for a similar result). They suggested that competition for access to a limited number of SSTs could explain the negative relationship between sperm length and the number of SSTs: if SSTs are limiting, selection will favor males that produce longer-tailed sperm (and hence faster sperm; Gomendio and Roldan 1991) that can outdistance sperm from rival males in the race to reach the SSTs. Furthermore, it would also pay males to produce sperm long enough to fill an SST, thus excluding rival sperm (Briskie and Montgomerie 1992). Females, on the other hand, might undergo evolutionary changes in SST size to control how sperm are subsequently allocated in fertilizations (Briskie and Montgomerie 1992, 1993). For example, by evolving longer SSTs, females may induce sperm competition by providing space to store sperm from several males concurrently, or to force sperm to stratify within SSTs, a possible mechanism to ensure last-male precedence (Compton et al. 1978; Briskie and Montgomerie 1992).
Until recently, it has been difficult to measure directly the intensity of sperm competition in any species. Generally, indirect indices of sperm competition have been used, such as social mating system, copulation rates, or relative testis size (Gomendio and Roldan 1991; Briskie and Montgomerie 1992; Dixson 1993; Gage 1994). With the advent of molecular methods for parentage analysis (see Burke 1989), it has become clear that the intensity of sperm competition varies dramatically from one species to the next. Thus, data on social mating systems or copulation behavior are often a poor guide to the underlying genetic mating system (Birkhead and Moller 1992b), and therefore to the real intensity of sperm competition. Even direct observations of mating behavior in wild animals can give misleading impressions of the frequency of sperm competition (e.g., Westneat 1987, 1990). In this study, we reexamine the relationship between sperm size and the intensity of sperm competition in birds, using species for which parentage has been measured with molecular techniques. Our results suggest that sperm competition has indeed influenced the evolution of sperm morphology in birds, but that this has occurred primarily as an indirect result of female strategies to control and manipulate the patterns and processes of sperm storage, rather than through a simple process of increasing sperm swimming speed.
MATERIALS AND METHODS
We obtained information on rates of extrapair paternity in birds from the literature, through personal communications with researchers, and from our own unpublished studies (all data and sources given in the appendix). All of these studies used at least one molecular method (e.g., single and multilocus DNA fingerprinting, protein gel electrophoresis) for assigning parentage. The rate of extrapair paternity (defined as the number of extrapair offspring divided by the total number of offspring sampled) was used as a measure of the intensity of sperm competition. Where more than one study had been done on a species, we used the arithmetic mean in our analyses.
Sperm length, SST length, and SST number were measured directly from birds collected by us. Some of these data have been published previously (see the appendix). Details of dissection methods and measurement protocol can be found in Birkhead and Hunter (1990), Briskie and Birkhead (1993), and Briskie and Montgomerie (1993). Briefly, we collected sperm either by gently massaging the cloacal protuberance of live males, salvaging sperm from the seminal glomera of freshly killed males (see Wolfson 1952), or retrieving ejaculates from dummy females fitted with a false cloaca (Pellatt and Birkhead 1994). Semen samples were spread onto a glass slide and then examined as unstained mounts at 400x magnification under a phase-contrast or light microscope. Total sperm length was measured for at least 10 haphazardly chosen sperm per sample. Means were calculated for each species by averaging the means from each individual. The number of males examined per species varied from one to five (mean 2.1). Although sample sizes were small for some species, most variation ([greater than]99%) in sperm length in birds is found among species, rather than within species or individuals (Briskie and Montgomerie 1992).
To obtain data on the size and number of SSTs, we collected, preserved and dissected the utero-vaginal region of the oviducts of one to five females (mean 2.5) for each of the species for which we had sperm sizes. For each female, three to five haphazardly chosen oviductal folds were removed and examined as a wet mount at 100x and 400x using either phase-contrast or bright-field light microscopy (Briskie and Birkhead 1993). SSTs were counted and the length of a haphazardly chosen subsample (15 to 50 SSTs) measured using a calibrated ocular micrometer. The mean number of SSTs per fold was then multiplied by the total number of folds to estimate the total number of SSTs per female. As for males, mean values of each measure were calculated for each species studied, and only these means were used in further analyses. All birds (males and females) were collected under permit. In total, we obtained information on sperm size, SST anatomy, and rate of extrapair paternity for 21 species of passerine birds (representing seven families) from Europe, North America and Australia (see the appendix).
To assess the relative importance of different variables that might influence sperm length, we performed path analysis (see Sokal and Rohlf 1995), a statistical method using multiple regression analysis (and related techniques), to help interpret causal relations in a system of correlated variables. The method can be used to test explicit causal hypotheses or as a means of exploratory data analysis to suggest possible causal relations that can later be subjected to experimental testing. Here we use path analysis as a means of exploring the causal factors that might influence the evolution of sperm length.
To perform path analysis we first constructed a structural model [ILLUSTRATION FOR FIGURE 1A OMITTED] using the following variables: sperm length, rate of extrapair paternity, SST size, and SST number. This structural model is explicitly constructed to focus on factors influencing sperm length and is based on the following assumptions about the relations among these four variables:
(1) Extrapair paternity (EPP) directly influences sperm length, and both the number and size of SSTs. We assume here that extrapair copulations (EPCs) are controlled by females (e.g., Smith 1988) and that increased EPC rate and careful timing of EPCs are means by which females can increase sperm competition. The result of these two female tactics is an increase in EPP. The relation between sperm length and EPP rate (path E in [ILLUSTRATION FOR FIGURE IA OMITTED]) would be positive if EPP selects for faster swimming sperm to increase access to unfertilized ova (Gomendio and Roldan 1991) or SSTs (Briskie and Montgomerie 1992). Alternatively, the relation would be negative if increased EPP selects for the production of more and smaller sperm (Parker 1982). To increase sperm competition, selection should also favor females with longer SSTs (path B), which can simultaneously accommodate the sperm from multiple males, and to decrease the number of SSTs (path C), which will limit the availability of safe storage sites for sperm (Briskie and Montgomerie 1992).
(2) The number of SSTs directly influences sperm length (path F). This relation is expected to be negative if reduced numbers of SSTs select for longer, faster-swimming sperm due to increased competition for limited storage sites (Briskie and Montgomerie 1992).
(3) The size of SSTs directly influences sperm length (path D). This relation is expected to be positive if longer sperm gain an advantage by filling SSTs and thereby exclude sperm from rival males (Briskie and Montgomerie 1992).
(4) There is a trade-off (negative correlation) between size and number of SSTs (path A). This is unlikely to be a result of energetic limitations between SST size and number, but rather a function of the relatively limited space for SSTs. That is, the surface area for SSTs at the utero-vaginal junction is limited, and for purely physical reasons it may not be possible to keep increasing SST size without having to reduce their number to free up space. We have modeled this trade-off as a direct influence of SST length on the number of SSTs, assuming that size is the causal variable, but our conclusions are unaffected by reversing the direction of causation or by making this a simple correlation. A negative correlation between the size and number of SSTs in birds has been reported by Briskie and Montgomerie (1993).
Based on this structural model [ILLUSTRATION FOR FIGURE 1A OMITTED], we calculated path coefficients (using standardized, partial regression coefficients; see Li 1975; Sokal and Rohlf 1995). Path coefficients estimate the strength and sign of each causal relationship in the structural model and allow us to produce a path diagram where the relative strength of each relation is indicated [ILLUSTRATION FOR FIGURE 1B OMITTED]. The statistical significance of these relations was determined from the multiple regression analyses. We also determined [R.sup.2], the proportion of variation in sperm length explained by the model, using normal equations (Li 1975; Mitchell 1993; Sokal and Rohlf 1995). Unexplained variation is then simply [square roof of 1 - [R.sup.2]].
Before performing path analyses, each variable was tested for normality (using the Shapiro-Wilk W-test), and each pair-wise relation for linearity, and appropriately transformed, if necessary. We performed the path analysis both on the (transformed) raw data and on (transformed) contrast scores controlling for the effects of phylogeny (Harvey and Pagel 1991). Contrast scores were calculated using the Comparative Analysis by Independent Contrasts program (Purvis and Rambaut 1994). We generated contrast scores from two different phylogenies: one based on DNA-DNA hybridization (Sibley and Ahlquist 1990) and the other on more traditional methods using morphological, biogeographic, and behavioral traits (Howard and Moore 1991). Regressions on contrast scores were forced through the origin as recommended by Harvey and Pagel (1991). Examples of the contrast method and its rationale can be found in Briskie and Montgomerie (1992), Birkhead et al. (1993a) and Moller and Briskie (1995).
The quantitative results from path analyses are known to be sensitive to small sample size and collinearity among predictor variables, as well as to the departures from linearity, normality, homoscedasticity, and measurement error that can influence the results of any multiple regression (Petraitis et al. 1996). We took the necessary precautions to minimize the effects of the latter four problems (see above), but our sample size is small relative to the number of paths in the structural model, and two of our predictor (independent) variables, SST length and SST number, are significantly negatively correlated when we do not control for phylogeny (Table 1).
TABLE 1. Path coefficients for the relations between extrapair paternity rate, sperm length, sperm storage tubule (SST) length, and SST number for 21 species of passerine birds [ILLUSTRATION FOR FIGURE 1 OMITTED]. For each path, coefficients were calculated using the normalized raw data (not controlled for phylogeny) and using data controlling for the phylogenies of Sibley and Ahlquist (1990) and Howard and Moore (1991). Sample sizes (n) are the number of species (raw data) or the number of independent contrasts (when controlling for phylogeny). [R.sup.2] is the proportion of variance in sperm length explained by the path model (see Sokal and Rohlf 1995). ** [less than] 0.01, *** [less than or equal to] 0.001. Path Raw data Sibley and Ahlquist Howard and Moore A -0.49 -0.36 -0.48 B 0.69(***) 0.45(***) 0.64(***) C -0.41 -0.19 -0.21 D 0.85(***) 0.85(***) 0.93(**) E -0.14 -0.06 -0.18 F -0.20 -0.23 -0.24 G 0.47 0.37 0.25 H 0.50 0.86(**) 0.69 I 0.75(**) 0.95(**) 0.91(**) [R.sup.2] 0.78 0.87 0.94 n 21 19 10
Petraitis et al. (1996) suggest that sample size should be at least five times larger than the number of paths to ensure stable parameter estimates. With six paths, our sample size (n = 21) is smaller than the minimum recommended, so we examined the effect of sample size on our conclusions using a bootstrapping procedure (Sokal and Rohlf 1995). To do this we ran path analyses 1000 times on our dataset, separately for sample sizes of 10, 15, and 20. For each iteration we sampled with replacement using the Resampling Statistics program (Macintosh version 4.0.1; see Simon 1992). For each of the tested sample sizes, the relative magnitude (and sign) of the path coefficients remained the same as did their statistical significance. Thus, our conclusions appear to be unaffected by the relatively small sample size.
The main effects of collinearity are to reduce the ability to detect statistical significance and to increase the magnitude of path coefficients (Petraitis et al. 1996). While these effects are important, they are minimized by controlling for phylogeny (because predictor variables are no longer correlated), and should not influence the qualitative conclusions of our study. Thus, caution is required mainly in interpreting the absolute magnitude, and not the direction, of the path coefficients that we report.
Sperm length varied more than sixfold over our sample of 21 species of passerine birds (appendix), from 42.7 [[micro]meter] in the Red-Backed Shrike to 277.5 [[micro]meter] in the Yellow Warbler. An analysis of sperm length in relation to the rate of extrapair paternity suggests that the intensity of sperm competition has a substantial effect on sperm length [ILLUSTRATION FOR FIGURE 2 OMITTED]. In other words, species with high rates of extrapair paternity (and therefore sperm competition) had longer sperm than species with lower rates of extrapair paternity. This result agrees with that of earlier studies of mammals, birds, and insects showing a positive relationship between sperm size and the intensity of sperm competition (Gomendio and Roldan 1991, 1993; Briskie and Montgomerie 1992; Dixson 1993; Gage 1994).
The path analyses confirmed that sperm competition has a large influence on sperm length, but that it does so only indirectly ([ILLUSTRATION FOR FIGURE 1B OMITTED]; Table 1). Most variation in sperm length is accounted for by variation in SST length (path D in [ILLUSTRATION FOR FIGURE 1B OMITTED]), while most variation in SST length is explained by the incidence of extrapair paternity (path B). Both of these relationships are positive and significant (Table 1); thus, those species with the highest rates of extrapair paternity are also those with the longest SSTs and the longest sperm. The relationship between sperm length and both SST number and the rate of extrapair paternity were both negative and nonsignificant (Table 1). All of the patterns revealed by path analyses were the same whether we used the raw data or either set of contrast scores controlling for phylogeny (Table 1).
Unlike the significant positive effect of extrapair paternity on SST length (path B), we found only a weak, negative, and nonsignificant relationship between extrapair paternity and the number of SSTs (path C). Thus selection on females apparently does not change the number of SSTs in direct response to varying levels of sperm competition. Instead, the number of SSTs was negatively (though not significantly) related to SST length (path A) and was significantly related only to a large amount ([R.sup.2] = 0.56-0.90 depending upon model; see Table 1) of unexplained variation (path I). We know from other studies (Briskie and Montgomerie 1993) that body size alone can account for a substantial proportion of this unexplained variation ([R.sup.2] = 0.62 for body size for the Sibley/Ahlquist model), but it was not included in the present path analysis because of the need to minimize the number of paths (see materials and methods). This suggests that the number of SSTs may largely be a function of allometry (larger birds require more SSTs to compensate for the larger size of the oviduct) and perhaps space constraints (physical space for increases in SST length can come about only at the expense of SST number). As before, analyses using either the raw data or the contrast scores calculated using the two different phylogenies resulted in the same patterns (Table 1).
Although the rate of extrapair paternity was the best predictor of SST length, there was considerable unexplained variation in SST length (path H), and that was significant for the Sibley/Ahlquist model (Table 1). Unexplained variation ([R.sup.2]) ranged from 0.25-0.74 of the total variation, depending upon the model, and could not be accounted for by variation in female body mass (which accounted for only 6% of the variation in SST length in the Sibley/Ahlquist model). It is possible that some of this unexplained variation in SST length arises from measurement error, although it also seems likely that at least some of this variation must be due to one or more as yet unidentified variables.
Sperm length varies widely from species to species and we found that, in passerine birds, much of this variation was related to differences in the intensity of sperm competition. Unlike previous studies, which have proposed that longer sperm are a direct adaptation to sperm competition through increased swimming speed, our results suggest that sperm length in passerine birds is most dependent on the morphology of the female sperm storage sites. It is these sperm storage structures, in turn, that seem most influenced by variation in the intensity of sperm competition. In our path analyses, length of SSTs was significantly positively correlated with increasing rates of extrapair paternity, and thus sperm length was only indirectly correlated with the intensity of sperm competition. Indeed, the direct relation between sperm length and the rate of EPP was slightly negative and far from significant. Why should females vary the size of their sperm storage organs in response to variation in the intensity of sperm competition? And why should males produce sperm to track these changes?
One advantage to females in manipulating SST size is that it could give them control over which sperm are subsequently used for fertilizations. For a given sperm length, increasing SST length could allow females to increase the number of sperm stored per SST. A greater sperm load per SST might then increase a female's ability to control paternity in two different ways. First, if sperm from rival males do not mix within an SST, but instead form discrete stratified layers with the last male's sperm forming a layer over the previous male's sperm, females could control the paternity of their offspring simply by copulating last with the male they prefer as the father. Under this hypothesis, females increase the length of their SSTs as a way to keep sperm from rival males segregated and in a position to ensure paternity from a specific male. However, this mechanism of paternity control seems unlikely. Although there is stratification of sperm within SSTs in several species (Briskie and Montgomerie 1993), there is now some evidence that stratification is not the primary mechanism of last-male sperm precedence in birds (Birkhead et al. 1995; Colegrave et al. 1995; Birkhead and Biggins, unpubl.). Instead, last-male precedence appears to result from the relative numbers of sperm from each male entering the SSTs and the time interval between successive inseminations by different males (Birkhead et al. 1995; Colegrave et al. 1995).
Alternatively, if sperm from rival males mix within SSTs, then females could use increased SST length as a mechanism to force sperm to compete directly with each other. In this way, only those sperm that are the highest quality or most successful at reaching the eggs would be used in fertilization. Keller and Reeve (1995) have called this process the "sperm sexual selection hypothesis" and see females as accepting inseminations from multiple male partners as a way of choosing sperm characteristics in much the same way as females may choose other secondary sexual characteristics of males. Thus, by increasing SST length, females may provide increased opportunities for greater competition among sperm from different males and thereby increase the probability that their sons will also have high fertilization success (Keller and Reeve 1995). The degree to which sperm within SSTs mix is not known, but a positive correlation between SST length and the extent of sperm mixing would lend support to the hypothesis that increased SST length functions to incite sperm competition and facilitate female choice.
Any change in female strategy to control paternity through manipulation of their SST length is bound to place selection on males to compensate. Our path analyses show that sperm length has indeed tracked changes in SST length: those species of birds with the longest SSTs also had the longest sperm. A positive correlation between sperm size and female sperm storage organs has also been found in a variety of other groups. For example, Dybas and Dybas (1981) found a strong positive correlation between sperm size and the size of the spermathecae in featherwing beetles (Bambaria spp.). A similar relationship was reported by Pitnick and Markow (1994) for fruit flies (Drosophila spp.) and by Rothschild (1991) for fleas (Siphonoptera). Several hypotheses have been proposed to account for this pattern, two of which seem relevant to the causal relations we have postulated in passerine birds.
First, males may increase sperm length as a way to fill the female's sperm storage sites and thereby prevent sperm from rival males from being stored (Dybas and Dybas 1981; Briskie and Montgomerie 1992; Pitnick and Markow 1994). Ladle and Foster (1992) called this the "blocking hypothesis," and suggested that such a function could explain the evolution of giant sperm in both ostracod crustaceans and ptiliid beetles. In these groups, a single sperm is so large that it occupies most of the female's reproductive tract and may block inseminations from any rival male. However, such a function does not appear to account for the evolution of giant sperm in some Drosophila species, as sperm from multiple males are not excluded from female sperm storage areas by the presence of previous inseminations (Pitnick and Markow 1994). Although excluding rival sperm from SSTs may partly account for an increase in sperm length in birds, it cannot be the whole picture for the simple reason that bird sperm are not long enough to fill the length of the average SST (Briskie and Montgomerie 1992, 1993). Thus, unless the first male to mate with a female is able to fill an SST to capacity, his sperm are liable to be covered or mixed with sperm from succeeding males. The potential for first males to inseminate enough sperm to fill all SSTs does seem readily achievable as the total sperm-storage capacity of a female is often less than 1% of a single ejaculate (Birkhead et al. 1993b). However, even if a male were able to fill all SSTs at a given time, the fact that SSTs continue to grow and elongate over the copulation period (Briskie 1994) may mean that a male is unable to permanently fill an SST and prevent his sperm from subsequently being stored with rival sperm. Continued growth of SSTs after insemination may even be a female strategy to promote sperm mixing or layering (see above).
Second, if increased sperm length does not function as a mechanism to block access to SSTs, it may act to resist displacement (Dybas and Dybas 1981; Pitnick and Markow 1994). It is possible that sperm evolved a greater length because such sperm are either more resistant to displacement, or because they are able to displace other sperm. As SSTs are long and narrow tubular structures, it may be particularly difficult to displace sperm with long tails in such a confined space. Sperm with longer tails are thought to generate greater forces than short-tailed sperm (Katz and Drobnis 1990), which would enable long-tailed sperm to both resist displacement and to help displace other sperm. Whether sperm actively displace each other within SSTs is unknown, but on the basis of current knowledge of the mechanisms of sperm competition in birds it seems unlikely (Birkhead et al. 1995; Colegrave et al. 1995). Nonetheless, the displacement hypothesis could be tested by examining how sperm interact within the SSTs and seeing if this differs between species with differing relative sperm lengths.
The somewhat unexpected results of our analyses thus reveal several areas worthy of further study. First, it is clear that early models on the evolution of sperm size, suggesting that intense sperm competition will select for smaller sperm (Parker 1982), are incorrect. Certainly, interspecific variation in sperm size in birds (Briskie and Montgomerie 1992; Birkhead and Moller 1992b; this study) and mammals (Gomendio and Roldan 1991) seems largely due to a positive relation between size and the intensity of sperm competition. Recent models have already begun to address some of these newly discovered patterns (e.g., Parker 1993), but the ways in which females use and manipulate ejaculates after insemination clearly need to be considered more thoroughly in any future model of sperm-size evolution.
Although we found that EPP has an important influence on the evolution of SST length in birds, a large amount of unexplained variation in SST length remained in our models (ILLUSTRATION FOR FIGURE 1B OMITTED], path H). This suggests that there may still be a number of as yet unidentified variables influencing the evolution of SST morphology. For example, some of the unexplained variation in SST length may be due to the obvious layering of sperm in SSTs in some species: species with high levels of sperm layering may be using different strategies of sperm storage and/or sperm precedence than species with little or no layering. These differences might then favor varying changes in SST length, depending on the sort of male-female "arms race" that may develop (see Briskie and Montgomerie 1992, 1993). Alternatively, it is possible that the rate of EPP is not always a good index of sperm competition at the gametic level. Females, for example, who carefully control copulation frequency and timing could conceivably increase EPP without increasing the competition incurred by sperm. Such a decoupling of EPP and sperm competition and EPP seems unlikely, but the precise link between these two variables is worthy of further study.
Our study also suggests that the correlation between sperm size and sperm competition need not be the result of a positive relation between size and swimming speed, as has been suggested previously (Gomendio and Roldan 1991; Briskie and Montgomerie 1992). Indeed, the only published data on this speed/size relation across species are based on data from five species of mammals (Gomendio and Roldan 1991). Moreover, there does not appear to be any simple relationship between sperm length and swimming speed in passerine birds (T. R. Birkhead and F. Fletcher, unpubl. data). More data are needed from both birds and mammals to further assess this relationship, but does this mean that the swimming ability of sperm is unimportant? Briskie and Montgomerie (1992) previously reported a significant negative correlation between sperm length and the number of SSTs, suggesting that sperm competition for access to a limited number of SSTs was responsible for selection on sperm length via increased swimming speed. It is now clear that this negative correlation may be due to the negative relationship between the length and number of SSTs, most likely the result of space constraints on SST size - space for increases in size of SSTs are possible only by reducing their number. This still means that SSTs may be limiting in situations of intense sperm competition, as females seem to increase SST size under such conditions, with the concomitant reduction in SST number. Thus, the seemingly inevitable reduction of SST number may further increase the intensity of sperm competition and reinforce any advantage to swimming ability afforded by longer sperm.
Finally, we are obviously only beginning to understand some of the complexities of the relation between male sperm and female sperm storage, a relation that has a profound effect on sperm competition and mating strategies in birds. Our analyses confirm that sperm competition has played an important role in the evolution of both sperm size and the morphology of the female reproductive tract. However, the indirect pathways by which many of these changes have occurred suggest that the patterns of coevolution at the gamete level are as complex and diverse as any behavioral strategy observed to date. The strong relationship between the degree of extrapair mating by females and the size of their SSTs suggests that female choice at the gametic level may also have played a greater part in the fertilization process than previously imagined. Determining exactly how these differences in SST and sperm morphology ultimately translate into fitness differences may provide the key to understanding why sperm size varies so much.
We thank D. Hasselquist, B. Kempenaers, E. D. Ketterson, A. Langefors, P. M. Moore, V. Nolan, P. G. Parker, S. A. Raouf, T. von Schantz, M. Wellbourn, and C. Ziegenfus for letting us use results from their unpublished parentage studies. A. Dixon, J. T. Lifjeld, T. E. Martin, and T. Sztkely helped provide some of the birds used in this study. C. Potvin provided useful feedback on the path analyses. Funding was provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to RM. TRB was supported by a Biotechnology and Biological Research Council grant and a Leverhulme Research Fellowship.
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