Unusual Sperm Morphology in the Eurasian Bullfinch (Pyrrhula Pyrrhula)

Abstract

The sperm of the Eurasian Bullfinch (Pyrrhula pyrrhula) differs markedly in gross morphology from that of all other passerines examined to date. In other passerines, the sperm head is pointed and helical, and the midpiece comprises a mitochondrial helix extending along the flagellum; whereas in the Eurasian Bullfinch, the sperm acrosome is rounded, not helical, and the midpiece is extremely short. In a pairwise study, using principal component analysis (PCA), we combined quantitative and qualitative sperm morphology traits and conducted a phylogenetic correlation to compare the sperm morphology of Eurasian Bullfinch and Beavan's Bullfinch (P. erythaca) with nine other pairs of congeneric passerines. The analysis revealed that Eurasian Bullfinch was a dramatic outlier in sperm morphology and that Eurasian and Beavan's bullfinches are more different than any other pair of species. Excluding Eurasian Bullfinch from the analysis showed that most variation in sperm morphology in the other species was attributable to phylogeny. The Eurasian Bullfinch also has extremely small testes for its body size, which indicates that sperm competition is infrequent in this species; we discuss the possibility that relaxed selection, via lack of sperm competition, may have contributed to the species' unusual sperm morphology.

Morfología Espermática Inusual en Pyrrhula pyrrhula

Resumen

Los espermatozoides de Pyrrhula pyrrhula difieren marcadamente en su morfología de los de todas las demás especies de aves paseriformes examinadas hasta ahora. En otras aves paseriformes, la cabeza del espermatozoide es puntuda y helicoidal, y la parte media está formada por una hélice mitocondrial que se extiende a lo largo del flagelo, mientras que en P. pyrrhula el acrosoma del espermatozoide es redondeado, no es helicoidal y la parte media es extremadamente corta. En un estudio paralelo, utilizando análisis de componentes principales, combinamos caracteres cuantitativos y cualitativos de la morfología espermática y realizamos una correlación filogenética para comparar la morfología espermática de P. pyrrhula y de P. erythaca con la de otros nueve pares de especies paseriformes del mismo género. El análisis reveló que P. pyrrhula es una gran excepción en cuanto a la morfología espermática y que P. pyrrhula y P. erythaca son más distintos entre ellos que cualquier otro par de especies. Al excluir a P. pyrrhula del análisis, la mayor parte de la variación en la morfología espermática en las otras especies puede ser atribuida a la filogenia. Además, P. pyrrhula tiene testículos extremadamente pequeños con relación a su tamaño corporal, lo que indica que la competencia espermática es poco frecuente en esta especie. Discutimos la posibilidad de que un proceso de selección laxo debido a la ausencia de competencia espermática, podría haber contribuido a la morfología espermática inusual en esta especie.

Spermatozoa are among the most morphologically diverse of all cells (Cohen 1977). Three sets of factors are believed to account for inter-specific variation in sperm morphology: (1) phylogeny (Jamieson 1999), (2) mode of fertilization (internal vs. external; Franzén 1970), and (3) postcopulatory sexual selection and sexual conflict (Pitnick et al. 1999, Birkhead and Pizzari 2002, Pizzari and Snook 2003). However, the relative importance of these factors remains unknown. Interspecific variation in sperm phenotypes is considerable, and sperm morphology has been widely used as a taxonomic tool (e.g. Jamieson 1999). The two main groups of birds, the passerines and nonpasserines, differ fundamentally in sperm morphology. The spermatozoa of nonpasserines are relatively simple and similar to those of some reptiles; they are characterized by short length and a smooth, tapering head. By contrast, the sperm of passerines are typically longer, with a helical form and an elongated midpiece twisted around the flagellum (Ballowitz 1888, Retzius 1909, McFarlane 1963, Humphreys 1972, Lake 1981, Koehler 1995, Jamieson 1999). This basic sperm phenotype appears to be highly conserved in passerines (∼110 species examined; Ballowitz 1888, McFarlane 1963, Allen et al. 1968, Henley et al. 1978, Briskie and Montgomerie 1992, Koehler 1995, T. R. Birkhead pers. obs.), and most interspecific variation consists of changes in the relative size of different sperm components.

During the course of a comparative study of passerine sperm morphology, we noticed that the sperm morphology of the Eurasian Bullfinch (Pyrrhula pyrrhula) differed dramatically from that of other passerines. The aim here is to describe the sperm morphology of the Eurasian Bullfinch and compare it quantitatively with the sperm of other passerines.

Materials and Methods

We obtained sperm from eight captive-bred and three wild, sexually mature male Eurasian Bullfinch during the breeding season. For two wild birds, we obtained sperm from fecal samples (Immler and Birkhead 2005); for the remainder, we obtained sperm from the seminal glomera following dissection (under license). We measured the linear dimensions of the testes using a vernier calliper (accurate to 0.1 mm) and by their mass; we recorded the mass of the seminal glomera (to the nearest milligram). One seminal glomerus from each of two captive-bred males was macerated and fixed in 5% formaldehyde solution, and sperm were counted using a neubauer counting chamber, as described in our previous study (Birkhead and Fletcher 1995). The total number of sperm in the seminal glomerus was multiplied by two to estimate the total number of sperm stored by the male. In the Zebra Finch (Taeniopygia guttata), the mass and number of sperm in the two seminal glomera did not differ significantly (T. R. Birkhead unpubl. data). None of the captive-bred Eurasian Bullfinches had access to females in the previous two months; hence, none had copulated. Because they were “rested,” the numbers of sperm stored in their seminal glomera was likely maximal (Birkhead 1991, Birkhead and Fletcher 1995). The other seminal glomerus was used to provide sperm for velocity analysis using a Hobson sperm tracker (Hobson Tracker, Sheffield, United Kingdom; see Birkhead et al. 1995). We diluted sperm from the cloacal end of the seminal glomerus in Dulbecco's sperm extender at 35°C and video-recorded sperm movement to obtain an estimate of their forward path velocity (VAP) and other aspects of motility. Although passerine body temperature is generally ∼40°C, we had found previously that sperm velocity measurements made just below body temperature were more consistent. Having measured the sperm velocity of several passerine species previously at 35°C (T. R. Birkhead unpubl. data), we also measured the velocity of Eurasian Bullfinch sperm at this temperature. Sperm for morphology measurements were fixed in 5% formaldehyde and examined using a conventional light microscope at magnifications up to 1,000×. Sperm was stained with fluorescent stains: Mitotracker Green FM (Invitrogen Corporation, Carlsbad, California), a fluorescent dye specific to mitochondria to identify and measure the midpiece; and Hoechst 33342 (Sigma Chemical Company, St. Louis, Missouri) to identify the sperm nucleus. We measured 15 morphologically normal spermatozoa per male. We also fixed some sperm in glutaraldehyde and used scanning and transmission electron microscopy to examine different sperm components (i.e. acrosome, head, midpiece, and flagellum). We compared the morphology of the Eurasian Bullfinch sperm with that of several other passerines (see below), including a sister species, Beavan's Bullfinch (P. erythaca; two wild-caught males purchased from a U.K. dealer), and the closest relative to Pyrrhula, the Pine Grosbeak (Pinicola enucleator; a single wild-caught male) (for a molecular phylogeny of Pyrrhula, see Arnaiz-Villena et al. 2001).

To assess how distinctive the bullfinch sperm was in size and design, we compared it with that of Beavan's Bullfinch and nine other pairs of congeneric passerines. Sperm from the other species had been collected opportunistically (e.g. from road kills) and the nine species-pairs used were the only ones available in an avian sperm data bank held by T.R.B. and, hence, constitute a relatively random sample. These species-pairs include (1) Moustached Warbler and Sedge Warbler, (2) Common Linnet and European Goldfinch, (3) Carrion Crow and Rook, (4) Corn Bunting and Yellowhammer, (5) Willow Tit and Great Tit, (6) House Sparrow and Cape Sparrow, (7) Common Chiffchaff and Willow Warbler, (8) Blackcap and Lesser Whitethroat, and (9) Eurasian Blackbird and Song Thrush. See Appendix for scientific names of birds.

Appendix  Species mean dimensions for sperm components and other attributes used in the PCA (see Fig. 1D)

For all species, we measured five spermatozoa from two males per species using light microscopy at 400× magnification; intramale variation in sperm morphology is generally small and considerably less than interspecific differences (Morrow and Gage 2001, Birkhead et al. 2005). We found the within-species repeatability values for different sperm traits to be high (head length = 0.85, midpiece length = 0.94, total length = 0.99, number of midpiece curves = 0.98, and number of head curves = 0.75). We photographed sperm with a digital video camera and measured them using LEICA IM50 Image Manager (Leica Microsystems Imaging Solutions, Cambridge, United Kingdom). We measured length of the head, length of the mid-piece, length of the flagellum, and total length (TL) to the nearest 0.01 μm. We calculated the straightened helix length of the midpiece using the formula in Birkhead et al. (2005). We also counted the number of head curves (see Fig. 1C) and number of curves along the helical midpiece (Fig. 1C), and scored the presence or absence of a head membrane (see Humphreys 1972) and a pointed or rounded acrosome (see Fig. 1A; also see Appendix). All measurements were made three times and were highly repeatable within an individual (r > 0.90, P < 0.001; Lessells and Boag 1987). Using principal component analysis (PCA), we combined sperm components and compared the 10 species-pairs using Freckleton et al.'s (2002) phylogenetic correlation (where a lambda [λ] value of 1 indicates complete phylogenetic dependence and a value of 0 indicates no phylogenetic dependence) to estimate the relative importance of phylogeny in explaining the variance in sperm morphology between and within genera. All values are reported as mean ± SE, unless otherwise stated.

Fig. 1.

(A) Scanning electron micrograph of the head of a House Sparrow sperm. Typical of most passerines, it has a helical head and pointed acrosome; arrow indicates the junction of the head and the flagellum, which is also the start of the mitochondrial helix (a single curve is shown—see text and [D]). (B) Scanning electron micrograph of Eurasian Bullfinch sperm showing the cylindrical head, no elongated mitochondrial helix, and rounded acrosome. (C) Fluorescent-stained sperm heads of Eurasian Bullfinch (left), Common Linnet (middle), and Beavan's Bullfinch (right). Nucleus is stained blue; the midpiece, which is stained green, lies posterior to the nucleus, and its size is indicated by the red bar. The Eurasian Bullfinch sperm has no head curve, the Common Linnet has three, and Beavan's Bullfinch has two. (D) Relationship between sperm-morphology principal components 1 and 2 for 10 pairs of congeneric passerines. Species pairs are coded with the same color. The Eurasian Bullfinch is a clear outlier (see text)

Results and Discussion

Sperm morphology.

Compared with those of other passerines (see Briskie and Montgomerie 1992), the Eurasian Bullfinch's sperm is relatively short: 46.87 ± 3.83 μm (n = 11) (captive-bred males: 47.19 ± 4.28 μm, n = 8; wild males: 46.01 ± 2.75 μm, n = 3). The sperm has a rounded acrosome and head and no obvious mitochondrial helix (Fig. 1B, C). Like that of the Eurasian Bullfinch, the sperm of Beavan's Bullfinch (Fig. 1C) was short (length = 49.11 μm); but, instead of being rounded, its head was more pointed and spiral-shaped. The spermatozoa of both Pyrrhula species had an extremely small mid-piece, in contrast to the elongated mitochondrial helix that forms the midpiece in almost all other passerines' sperm (McFarlane 1963, Humphreys 1972), including that of the Pine Grosbeak, whose sperm was similar to other passerines in length (162.7 ± 9.63 μm, n = 30 sperm, n = 1 male) and structure.

Principal component analysis of sperm morphology.

Using PCA to combine the various sperm-morphology traits, we were able to define sperm morphology along two principal axes. The first and second principal components (PC1 and PC2) explained 58% and 29% of the variance in sperm traits, respectively (87% in total). The traits that had strong positive loading on PC1 were total sperm length, straight helix length, length of flagellum, and number of midpiece curves around the flagellum; those that had strong loadings on PC2 were the number of head turns and the shape of the acrosome. Most of the variance derives from size differences of various sperm traits between species.

Plotting the two principal components against each other clearly shows (Fig. 1D) that the two Pyrrhula species are more different from each other than any other species-pair and that the Eurasian Bullfinch is an extreme outlier in its sperm morphology. This is quantitatively confirmed by the index of phylogenetic dependence. In an analysis that includes all data, λ = 0.58 (approximate 95% CI: 0.14 to 0.93); but when the Eurasian Bullfinch data is excluded, the estimate of lambda increases to 0.93 (CI: 0.54 to 0.96). This suggests that when Pyrrhula is excluded, there is strong phylogenetic dependence in sperm morphology. In other words, congeneric species are more similar to each other in sperm morphology than expected by chance. We confirmed this result by systematically removing species other than Pyrrhula in turn and recalculating lambda: in all cases, lambda varied only between 0.51 and 0.60.

Excluding the Eurasian Bullfinch from analyses and controlling for phylogeny (using independent contrasts; Purvis and Rambaut 1995) also shows that a negative correlation (r = −0.730, P < 0.001) exists between contrasts in PC1 and PC2, indicating broadly that longer sperm (PC1) tend to have a more rounded acrosome with fewer head curves (PC2) (Fig. 1D). The biological significance of this pattern is unknown.

Interspecific comparisons of other reproductive traits.

Our quantitative results confirm that the sperm morphology of the Eurasian Bullfinch is extremely unusual. It is unusual compared with the sperm of most other passerines, but also unusual in how much it differs from the sperm of Beavan's Bullfinch. It is also obvious from Figure 1 that the sperm morphology of the two Pyrrhula species is more different than that of any other species-pair. In one sense, our analysis is preliminary, in that we have compared only a small number of species. When more data become available, it will be possible to conduct more robust analyses using both larger numbers of genera but also more species per genus. Nonetheless, our data suggest that (excluding Pyrrhula) phylogeny explains much of the variation in sperm phenotype in passerine birds. This has been noted previously (McFarlane 1963), but not quantified. In other taxa, sperm-morphology traits, and sperm ultrastructure in particular, have been used as phylogenetic tools (Jamieson 1999); it is clear that if sperm morphology had been used to infer phylogeny from the data presented here, Beavan's Bullfinch would be closer to corvids and the Eurasian Bullfinch might not have been classified as a passerine at all.

The outstanding question is: what evolutionary forces have brought about such a profound difference in the phenotype of the Eurasian Bullfinch sperm, compared with other passerines? Assuming that the phylogenetic relationship between Pyrrhula bullfinches and their close relatives are correct and that Pyrrhula does not occupy a basal position in the passerine phylogeny—and there is no evidence that it does (Arnaiz-Villena et al. 1998, 1999, 2001)-some other factor(s) must be involved. One possibility is postcopulatory sexual selection, mediated through sperm competition or cryptic female choice or both. There is increasing evidence that postcopulatory sexual selection shapes a wide range of behavioral, physiological, and anatomical reproductive traits in both sexes (Birkhead and Pizzari 2002). In birds, interspecific differences in both female and male reproductive anatomy (including sperm morphology) are related to the intensity of sperm competition in birds (and other taxa), and there is evidence that male and female reproductive traits have coevolved (Briskie and Montgomerie 1992, Briskie et al. 1997, Johnson and Briskie 1999). For example, across species, the size of the female's sperm-storage tubules is positively correlated with the total length of sperm (Briskie and Montgomerie 1992, Briskie et al. 1997).

One possible explanation for the unusual sperm morphology of the Eurasian Bullfinch is that it has evolved in response to changes in the female reproductive tract. We examined the reproductive tract of a single captive female Eurasian Bullfinch that died from a prolapse during egg laying. However, the gross anatomy of the oviduct of this female was not noticeably different from that of many other passerines we have examined (T. R. Birkhead unpubl. data). The only difference we noted was that the sperm-storage tubules were relatively long (mean = 300.2 ± 102.84 μm, n = 38), in relation to the total length of the Eurasian Bullfinch's sperm, compared with the species reported by Briskie and Montgomerie (1992). It is not obvious how relatively long sperm-storage tubules could be responsible for the unusual shape of the Eurasian Bullfinch's sperm. Of course, we cannot exclude the possibility that other, more subtle aspects of the female oviduct may have influenced sperm morphology.

The other possibility is that a low level of sperm competition in the Eurasian Bullfinch may have played a role in its unusual sperm morphology. No estimate of extrapair paternity exists for the Eurasian Bullfinch, but because the levels of extrapair paternity and relative testes mass are positively correlated across species (Møller and Briskie 1995, S. Calhim and T. R. Birkhead unpubl. data), relative testes mass provides an index of the intensity of sperm competition. The same is true for the dimensions of the cloacal protuberance and the male's extragonadal sperm stores, the seminal glomera (Birkhead et al. 1993). Three comparisons showed the testes of the Eurasian Bullfinch to be extremely small in relation to its body size: (1) The observed testes mass of the Eurasian Bullfinch (0.063 ± 0.022 g, n = 16) is nearly half the mass predicted (i.e. 0.124 g) from Møller's (1991) equation on the basis of the relationship between body mass and testes mass of 247 bird species. (2) Considering the relationship between log-transformed body mass and log-transformed testes mass for only the 10 pairs of species in the present study, the Eurasian Bullfinch had the lowest residual testes mass (−0.76 vs. −0.51 to +0.34 for other species). (3) The same analysis restricted to 14 members of the Fringillidae (range of body mass: 11–29 g) also confirmed that the Eurasian Bullfinch has relatively small testes (0.29% of body mass), compared with 0.65–2.2% for the other species (testes data from Møller 1991; supplemented with data from Sheldon and Birkhead 1994, T. R. Birkhead unpubl. data).

The cloacal protuberance of the Eurasian Bullfinch also appeared to be relatively small. No systematic interspecific comparison was possible, but the height of the cloacal protuberance in four captive-bred birds was 3–4 mm, which is small compared with other finches. For example, in the Common Chaffinch (Fringilla coelebs), it is ∼7 mm (n = 7) (T. R. Birkhead unpubl. data). The small size of the cloacal protuberance in the Eurasian Bullfinch reflects the small size of the paired seminal glomera, which reside in the cloacal protuberance (Wolfson 1954, Birkhead et al. 1993). The estimated number of sperm in the seminal glomera was 1.12 × 10⁶ (bird A: 0.52 × 10⁶ and bird B: 1.72 × 10⁶). These values are very small compared with the data available for a few other species. For example, in the much smaller domesticated Zebra Finch (∼15 g), the mean number of stored sperm in rested males was 4.8 × 10⁶; and in the Bengalese Finch (Lonchura striata; body mass: 15–17 g), it was 7.7 × 10⁶ (Birkhead 1991). Numbers were larger in the Common Chaffinch (32.9 × 10⁶; Sheldon and Birkhead 1994), and even larger in the polygynandrous Smith's Longspur (Calcarius pictus; 217 × 10⁶; Briskie 1993) and the Dunnock (Prunella modularis; 1,060 × 10⁶; Birkhead et al. 1991). The socially monogamous Zebra Finch and polygynandrous Dunnock probably represent opposite ends of the spectrum in terms of intensity of sperm competition, so the fact that the Eurasian Bullfinch had fewer sperm than even the Zebra Finch, together with their relatively small testes mass, strongly suggests that sperm competition is unlikely in the Eurasian Bullfinch.

The mean velocity of sperm from two captive-bred Eurasian Bullfinch males was 21.65 μm s⁻¹, which was relatively slow compared with those of 10 other passerine species for which data exist (range: 21–43 μm s⁻¹; mean of two males per species); only one species, the Magpie (Pica pica), had slower sperm (T. R. Birkhead unpubl. data). It was also noted that Eurasian Bullfinch sperm moved in a distinctive manner. During forward motility, the sperm of other passerines rotated tightly along their long axes, exhibiting a dextral helical waveform (Vernon and Woolley 1999); Eurasian Bullfinch sperm exhibited a much larger roll than the sperm of other passerines (D. Woolley pers. comm.).

Our results suggest that sperm competition may be absent or rare in the Eurasian Bullfinch. If that is the case, it is possible that relaxed selection may have resulted in the evolution of the unusual sperm morphology. A similar pattern exists in rodents, where a large number of species has been examined. Breed (2002) has shown that species with unusually shaped sperm heads also tend to have relatively small testes.

However, if a low incidence of sperm competition accounts for the unusual sperm morphology in the Eurasian Bullfinch, we can predict that sperm competition or relative testes mass, or both, will be higher in Beavan's Bullfinch. It is difficult to test this prediction, because Beavan's Bullfinch is poorly known; it is assumed to be monogamous, but no estimate for extrapair paternity exists. After an exhaustive search, we obtained breeding-season testes dimensions for three individual males from museum skins. The mean value of 0.0523 g (n = 3), with a mean body mass of 19.5 g (Clement et al. 1993), indicates that, like the Eurasian Bullfinch (and contrary to the prediction), Beavan's Bullfinch also has relatively small testes. As a genus, Pyrrhula spp. differ in a number of ways from other fringillids (Newton 1972, Clement et al. 1993), including a lack of territoriality, a very quiet song, and a more frugivorous diet. It is possible that the ecological shift to a more frugivorous diet in Pyrrhula may have reduced the opportunities or evolutionary benefits of extrapair copulations and, hence, resulted in relaxed selection on sperm morphology.

A formal analysis of the relationship between testes dimensions and the two sperm principal components, controlling for both body mass and phylogeny, revealed no significant relationships (analysis not shown). However, with just 10 species-pairs, the statistical power of this test is low. We are currently collecting data from more species to test the hypothesis that sperm morphology is shaped by the intensity of sperm competition (see also Birkhead et al. 2005).

Without more data, it is probably useless to speculate about the cause of the Eurasian Bullfinch's unusual sperm morphology. We are currently constructing a new Pyrrhula phylogeny to compare with that of Arnaiz-Villena et al. (2001); we are also trying to obtain sperm from other Pyrrhula species and are collecting data on sperm morphology and testes size from other passerine species for a more rigorous comparative analysis.

In conclusion, our results show that PCA provides a useful method for combining sperm traits and that phylogeny accounts for much of the variation in passerine sperm morphology, with the Eurasian Bullfinch as a striking exception. The sperm of this species differs from that of any other passerine examined so far and from that of its closest relative, Beavan's Bullfinch.

Acknowledgments

We are especially grateful to S. Fitzpatrick for his assistance with this study. We also thank R. Dallai and P. Luppetti for the SEM pictures; I. Newton for allowing us to use his unpublished data; H. Moore and N. Jenkins for use of their sperm tracker; J. Proctor and H. H. Chen for their help; A. Gamuf (Natural History Museum, Vienna), P. Berthold, and C. Spottiswoode for providing data; and S. Pitnick and D. Woolley for valuable discussion. The authors' roles were as follows: T.R.B. designed the study and wrote the paper; S.I. made all sperm measurements, ran statistical analyses, and contributed to the writing; E.J.P. provided detailed technical support and maintained the sperm library; R.F. provided statistical advice and devised and executed the phylogenetic analyses. The research was supported by a grant from the Natural Environment Research Council to T.R.B.