HYBRID FITNESS SEEMS NOT TO BE AN EXPLANATION FOR THE ...

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between the parental morphs. The traditional ex- planation as to why assortative mating occurs is that reproductive isolation is reinforced due to hybrid unfitness.
J. Moll. Stud. (2000), 66, 149–156

© The Malacological Society of London 2000

HYBRID FITNESS SEEMS NOT TO BE AN EXPLANATION FOR THE PARTIAL REPRODUCTIVE ISOLATION BETWEEN ECOTYPES OF GALICIAN LITTORINA SAXATILIS KERSTIN JOHANNESSON 1 *, ANN LARSSON 1 , RACHEL CRUZ 2 , CARLOS GARCIA 2 and EMILIO ROLÁN-ALVAREZ 3 1

Department of Marine Ecology, Göteborg University, Tjärnö Marine Biological Laboratory, S-45296, Strömstad, Sweden. E-mail: K. [email protected] 2 Dtp de Biología Fundamental, Facultad de Biología, Universidad de Santiago de Compostela, 15706 Spain 3 Dto de Bioquímica, Genética e Inmunología, Facultad de Ciencias, Universidad de Vigo, 36200, Spain (Received 19 March 1999; accepted 7 July 1999)

ABSTRACT Partial reproductive isolation between closely related groups of organisms is suggested to be of central importance during speciation. Galician populations of Littorina saxatilis are phenotypically differentiated into an upper-shore and a lower-shore morph. These mate assortatively in the mid-shore zone of overlap, and genetic assessment shows an impeded gene flow between the parental morphs. The traditional explanation as to why assortative mating occurs is that reproductive isolation is reinforced due to hybrid unfitness. Earlier studies have, however, not found hybrids to be less viable. Likewise, growth and migratory behaviours are merely intermediate between those of the parental morphs. In the present study we compared male and female fertility components of the parental morphs and the hybrids to test hypotheses of decreased hybrid fertility. The results showed that hybrid males were as fertile as other males, and hybrid females did not produce fewer embryos, nor aborted embryos at a higher rate, than the parental morphs.

INTRODUCTION Hybrid zones are interbreeding areas between genetically distinct forms, which may be ecotypes of the same species, or taxa that are recognized as species between which some gene flow is possible (Barton & Hewitt, 1985). Selection usually contributes to hybrid zone formation, either in the form of endogenous selection (selection against mixing incompatible genomes), or as exogenous selection (gradient selection over zones of environmental transitions, e.g. ecotones). Endogenous selection in * to whom correspondence should be addressed.

particular, and possibly also exogenous selection, will produce hybrids with less fitness than the parental genotypes in the hybrid zone. Moreover, reproductive isolation due to prezygotic barriers may be an evolutionary response to low hybrid fitness, which is essentially the idea of the reinforcement hypothesis (Butlin, 1987). One of the most informative parameters for the dynamics of hybrid zones is the fitness of hybrids relative to that of pure forms (Arnold & Hodges, 1995). This parameter is, however, rarely estimated (reviewed by Barton & Hewitt, 1985; Arnold & Hodges, 1995), in part due to problems of estimating fitness in wild populations. One problem is that the fitness for a particular trait may vary over time, or over different life periods. Thus a complete picture of individual fitness can only be obtained from detailed longitudinal studies, or as a set of independent cross-sectional studies covering all parts of a life cycle (Arnold & Wade, 1984). On the rocky shores of Galicia, NW Spain, the upper shore is inhabited by one ecotype (sensu Turesson, 1933) of Littorina saxatilis, the ridged and banded (RB morph), while another ecotype, which is smooth and unbanded (the SU morph), is confined to the lower shore (Johannesson, Johannesson & Rolán-Alvarez, 1993). Snails hatched and raised in the laboratory develop the parental characters, and this indicates inherited differences (Johannesson et al., 1993). Also growth rate, estimated in natural populations, has a strong genetic component which differs among morphs (Johannesson, Rolán-Alvarez & Erlandsson, 1997). A number of additional traits, for example, embryo and radula characters, and characters

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of behaviour and physiology, also differ between the two ecotypes, but the genetic component of these traits is presently undefined (RolánAlvarez, Johannesson & Ekendahl, 1995, RolánAlvarez, Johannesson & Erlandsson, 1997). The two forms overlap in distribution at midshore levels and produce, at low rates, hybrids that are either smooth and banded, or ridged and unbanded. Over a scale of kilometres of shore, there is no correlation between genetic similarity (assessed by allozymes) and morphological similarity, and this suggests that the two forms are part of the same gene pool (Johannesson et al., 1993). In spite of this, at a scale of meters, gene flow between morphs is less than within morphs (Johannesson et al., 1993; RolánAlvarez et al., 1995). Strong assortative mating between parental forms in the sympatric midshore zone prevents a free flow of genes between parental forms (Johannesson, Rolán-Alvarez & Ekendahl, 1995; Rolán-Alvarez, Erlandsson, Johannesson & Cruz, 1999), and this may also explain part of the deficiency of hybrids. However, the main question remains unanswered: why has assortative mating developed at all? One possibility is that an assortative mating behaviour has been favoured by selection (i.e. ‘reinforced’) due to hybrids having less fitness than the parental forms in the hybrid zone. We have in an earlier study estimated the relative survival rates of the parental ecotypes and the hybrids, at different shore levels (Rolán-Alvarez, et al. 1997). Overall, each parental form survived best in its native habitat, while in the mid-shore the hybrids were recaptured at about equal rates to both the parental forms. Thus we found no suggestions of hybrid unfitness by measuring adult survival rate on the shore. Estimates of sexual selection suggest a disadvantage to hybrid females, in comparison with mating success of parental females (Johannesson et al., 1995; Rolán-Alvarez et al. 1995). However, this difference is far too small to explain the strong assortative mating present, and may indeed not at all decrease female fecundity as these females store sperm over several months (Johannesson, pers. obs.). In NE England, two other ecotypes of L. saxatilis are present, one in the upper and one in the mid-tidal zone (Hull, Grahame & Mill, 1996). Hybrids between the two forms are found at low rates (2%). The incidence of egg and embryo abortion in the hybrids is, furthermore, much higher than in the pure morphs (30–60%, and 1%, respectively, Hull et al. 1996). Thus in this area there is a barrier to gene flow between two ecotypes of L. saxatilis due to hybrid females

being less fertile than females of the parental morphs. Possibly, snail fertility may be critically different also among the Galician morphs. In an earlier study of hybrid fitness, female fecundity was estimated as the number of embryos carried by a female at a time. The results showed that number of carried embryos varied with size and morph of the female, but hybrid females seemed not to be less productive than the parental morphs (Cruz, Rolán-Alvarez & García, 1998). However, only embryos with a ‘normal appearance’ were considered. In the present study, we included assessments of male fertility and embryo quality. Littorina saxatilis has sex chromosomes and the male is the heterogametic sex (Rolán-Alvarez, Bruno & Gonzales, 1996). The heterogametic sex is more likely to be affected by postzygotis incompatibilities than the other sex (‘Haldane’s rule’ see e.g. Coyne 1992, Kelly & Noor, 1996). Trying to estimate male fertility thus seems to be important; likewise, embryo quality, estimated as the proportion of abortive embryos, may be critical, as found in the English population (Hull et al. 1996). Thus the overall aim was to test if any aspect of low hybrid fertility may, on the one hand, contribute to the structure of the hybrid zone and, on the other hand, promote reinforcement of the assortative mating behaviour.

MATERIALS AND METHODS Sampling wild populations Samples of L. saxatilis were from two localities (Barcelos and Silleiro Cape) on the Baiona coast of Galicia (NW Spain). At each locality 23–89 snails (3 mm) were collected at upper-, mid- and lowershore levels, in November and December 1996. The upper-shore samples were taken from the top of the barnacle (Chthamalus stellatus) belt and included only the RB-morph, although a minority of hybrids was present (7% at Barcelos and 8% at Silleiro). The low-shore samples were from the blue mussel Mytilus galloprovincialis) belt below, and included only the SU-morph, while here a few hybrids were present (0–2%). The mid-shore samples were from the patchy zone where the barnacle belt and the mussel belt fuse. These samples included both the parental forms (RB and SU), and hybrids (HY, 70% at Barcelos and 32% at Silleiro). Defining both morph and zone of sampling we use the following designations RBu (RB from upper shore), RBm, HYm and SUm (morphs from mid shore) and SUl (SU from low shore). Each individual was classified using previously described characters, that is, absence or presence of

HYBRID FITNESS OF LITTORINA shell ornamentation, and absence or presence of dark bands on the shell (Johannesson et al., 1993). This time, however, we classified all individuals that were not unquestionably RB or SU morph as hybrids, which tended to increase the proportion of hybrids. In Silleiro, for example, we earlier defined 11–18% of the snails in the mid shore as hybrids (Johannesson et al. 1993), while in this study we found 32% of one sample to be hybrids. The group of hybrids most likely consists of true F1 hybrids mixed with snails which are the result of introgression and backcrossing, and thus there is no definitive border between hybrid and parental groups.

Male fertility Male gonad quality was assessed using relative gonad area and colour of the gonad. In a pilot study these characters were shown to be different between sexually active and non-active snails. That is, relative gonad area was significantly larger in mating compared to non-mating individuals) (2  25.86, df  1, n  40, P  0.001). Likewise, actively mating males had a stronger coloured gonad than had inactive males (2  26.03, df  2, n  40, P  0.001). We assumed that male activity also reflected male fertility and used these indirect estimates as indices for male fertility. In the present study, animals were dissected under seawater and the colour of the male gonad was assessed using a scale where ‘1’ was white or near white, ‘2’ was slightly coloured, and ‘3’ strongly coloured in yellowish or green. To quantify the size of the gonad, this tissue was stained by acid fuchsine (0.250 g fuchsine and 12.5 ml HC1 in 150 ml distilled water) for 10 s, on a glass plate. Thereafter a glass cover was placed over the gonad and the tissue was gently squeezed. The area of the squeezed gonad was recorded with a binocular microscope equipped with a video camera, and the perimeter of the gonad was manually painted on the monitor screen. A digital image analyser measured the area of the gonad with an accuracy of 0.001 mm2. We compared male fertility among male morphs and between localities, focusing on hybrid fertility compared to parental morph fertility, using the estimates described above. Thus, the effect of the two factors Morph (fixed with five levels) and Locality (random with two levels) on gonad area and colour were analysed using an orthogonal ANOVA. To get a balanced design (n  11), we randomly deleted up to 43 observations in each sample. In one sample (RBm from Barcelos), however, we only had 5 observations, and in this group each observation was used twice and one (random) was used three times. Balancing a design in this way increases the risk of a Type II error (a true difference will not be revealed), while a strongly unbalanced design increases the risk of a type I error (rejecting a true null hypothesis) (Underwood, 1997). We repeated the tests using the unbalanced data sets to be able to compare how the different designs affected the results.

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Female fertility In the females we estimated the numbers and sizes of normally as well as abnormally developing embryos. Embryos were grouped in four categories of different developmental stages, as suggested by Janson (1985): 1—eggs and embryos without distinct shells; 2— shelled embryos with vela (veligers); 3—shelled embryos with reduced vela (post-veligers); and 4— hatched embryos, i.e. those which had left the egg capsula. Within each category the numbers of normal and abnormal embryos were counted. Shell diameter of the female ( 0.1 mm), and embryo diameter (as in Rolán-Alvarez et al. 1996) of 2–3 of the hatched embryos ( 0.02 mm) were measured. The variation in embryo number and embryo size over morph and locality was analysed using a balanced (n  8) orthogonal ANOVA with Morph as a fixed factor with five levels (RBu, RBm, HYm, SUm and SUl), and Locality as a random factor with two levels (Silleiro and Barcelos). The total number of embryos (all developmental categories), but not embryo size, was log-transformed to get insignificant heterogeneity of variances (P  0.05 in Cochran’s test). In cases where no hatched embryos were present, the largest of the stage 3 embryos were measured instead. A few females had fewer than ten eggs and embryos. These were mostly RB (25 individuals), but three were SU morph and five were hybrids. Lack of, or few, embryos may be due either to these females being more or less immature, sterile due to parasitic infections, or infertile for other reasons. These females were excluded from the analysis, since for natural reasons they contributed poor estimates of the frequency of abnormal embryos.

RESULTS Male fecundity Gonad area was positively correlated with size of snail (r  0.60, n  66, P  0.001), but using gonad area/size of snail as an index of gonad area in the ANOVA removed the correlation (r  0.06, P  0.10). Gonad colour, on the other hand was uncorrelated with size of snail (r  0.02, n  66, P  0.10) and was used directly. Gonad colour and gonad area did not in the balanced design appear to be significantly affected by snail morph, locality, or by the morph and locality interaction (Table 1). Repeating the ANOVA with an unbalanced design gave essentially the same results except for a significant difference between localities in gonad area with SIL snails having a large gonad index on average than the BAR snails (P  0.02). Hybrid males, however, seemed definitely not to be less fecund than males of the parental morphs at both localities (Fig. 1).

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Table 1. Effects of morph (fixed factor with five levels: RBu, RBm, HYm, SUm, and SUl), locality (random factor with two levels: Silleiro and Barcelos), and the interaction, on an index for male gonad colour (see text) and an index for gonad area (gonad area/snail size). Variances were homogeneous (P  0.05 in both, Cochran’s test). P-values in brackets are the corresponding values of an unbalanced ANOVA analysis. Factor

SS

df

MS

F-ratio versus

F

P

Index of male gonad colour Morph, M 2.87 Locality, L 0.036 Morph  Locality 0.69 Residual 27.8

4 1 4 100

0.718 0.036 0.173 0.278

ML Residual Residual

4.15 0.13 0.62

0.10 (0.13) 0.72 (0.90) 0.65 (0.21)

Index of male gonad area Morph, M Locality, L Morph  Locality Residual

4 1 4 100

0.366 1.41 0.296 0.492

ML Residual Residual

1.24 2.87 0.60

0.42 (0.74) 0.09 (0.02) 0.66 (0.11)

1.46 1.41 1.18 49.2

Table 2. Effects of morph (fixed factor with five levels: RBu, RBm, HYm, SUm, and SUl), locality (random factor with two levels: Silleiro and Barcelos), and the interaction on the number of embryos and their hatching size. Log embryo number was used to avoid heterogeneous variances, while the variances of average size were homogeneous (P  0.05 in both; Cochran’s test). Factor

SS

df

MS

F-ratio versus

F

Log embryo number Morph, M Locality, L Morph  Locality Residual

2.77 0.027 1.36 2.56

4 1 4 70

0.692 0.027 0.340 0.037

ML Residual Residual

2.04 0.73 9.29

0.25 0.39 0.0001

Embryo size Morph, M Locality, L Morph  Locality Residual

0.049 0.0003 0.0067 0.156

4 1 4 70

0.0124 0.0003 0.0017 0.0022

ML Residual Residual

7.40 0.14 0.75

0.039 0.70 0.56

Female fecundity Embryo number varied over morph, but in a slightly different way in the two localities, producing a significant interaction between the factors Morph and Locality (Fig. 2A, Table 2). The reason for this was most likely a larger average size of the RBu females at Silleiro, compared to the RBu females at Barcelos (8.1 mm at Silleiro and 5.9 mm at Barcelos, which differ according to a one-factor ANOVA, n  13, P  0.001). Average sizes of the other morphs did not differ between localities (Barcelos and Silleiro) (P  0.05). Embryo size, on the other hand, differed among morphs but not between localities (Fig. 2B, Table 2). At both localities, both RBu and RBm tended to have higher numbers of embryos than SUm and SUl, but these were also of a smaller size than the

P

SU embryos. Hybrid females, being of intermediate average size to the SU and RB morphs, revealed intermediate numbers of embryos and intermediate embryo sizes (Fig. 2). Thus none of the estimates of female fecundity suggested hybrid unfitness. Incidence of abnormal embryos Hybrid females showed no tendency to have higher numbers of abnormal eggs and embryos than either of the two parental morphs. This was true for both localities samples. For some reason, however, there were large differences between the proportion of abnormal embryos of the SU females in Barcelos and Silleiro, producing a significant interaction between the two factors morph and locality (Table 3, Fig. 3). A

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Figure 1. Male fertility, assessed as indices for gonad colour and gonad area, in different morphs of Littorina saxatilis. A high index suggests greater fertility than a low index (see text). The morphs were ridged and banded from the upper shore (RBu), ridged and banded from the mid shore (RBm), hybrids from mid shore (HYm), smooth and unbanded from mid shore (SUm), and smooth and unbanded from low shore (SUl). The estimates are from Barcelos (black bars) and Silleiro (white bars) on the Galician coast of Spain.

Table 3. Effects of morph (levels: RBu, RBm, HYm, SUm, and SUl), locality (levels: Silleiro and Barcelos), and the interaction on proportion of abnormal embryos in the female brood pouch. Log proportion of abnormal embryos was used, and variances were homogeneous (P  0.05, Cochran’s test). Factor Morph, M Locality, L Morph  Locality Residual

SS

df

MS

F-ratio versus

F

P

0.162 0.016 2.42 13.6

4 1 4 70

0.041 0.016 0.604 0.195

ML Residual Residual

0.067 0.083 3.10

0.99 0.77 0.021

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Figure 2. Female fertility, estimated as log embryo numbers and size of hatching embryos, in different morphs of Galician Littorina saxatilis. Morph and sample definitions as in Fig. 1.

general result was that the proportion of abnormally developed embryos in the female brood pouches decreased significantly from earlier to later stages of development. However, also in this respect hybrid females did not show any aberrant pattern (Fig. 4). DISCUSSION The mid-shore zone, where the two main morphs of the Galican L. saxatilis overlap, shows

several characteristics of a classical hybrid zone, for example, being narrow and with a partial barrier to gene flow. More surprising is that the hybrids seem to survive as well as the parental morphs (Rolán-Alvarez et al. 1997), and that among the hybrid females production of embryos and the reproductive status of the males seem not to be impeded by hybrid incompatibilities (Cruz et al., 1998 and this study). In their classic paper Barton & Hewitt (1985) suggest that most hybrid zones are tension zones maintained by endogenous selection

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Figure 3. Female fertility, estimated as the proportion of abnormally developing (abortive) embryos, in different morphs of Galician Littorina saxatilis. Morph and sample definitions as in Fig. 1.

Figure 4. Percent abortive embryos in different developmental categories of embryos, assessed in different morphs of Galician Littorina saxatilis from two localities (Balcaros and Silleiro). Morph definitions as in Fig. 1.

against hybrids. Probably, this is the case in the British population of L. saxatilis where hybrid infertility causes a high incidence of embryo abortion (Hull et al., 1996). Also, in Sweden hybrid populations between different ecotypes tend to have more inviable embryos than popu-

lations of parental morphs (Janson, 1985). Hybrid unfitness is, however, not always found when hybrid viability is compared to parental morph viability. Indeed, in a recent review of 44 hybrid zones only 13 (30%) had hybrids with decreased viability compared to both parental

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populations (Arnold & Hodges, 1995). Indeed, Moore (1977) suggested that hybrids are even more fit than the parental forms in some environments, and that this is the explanation for seemingly stable hybrid zones. Accepting the results of no obvious hybrid unfitness leaves us with the important question of why, after all, strong assortative mating has evolved in the Galician populations. If hybrids are not particularly unfit we have indeed no evolutionary force that could promote the reinforcement of prezygotic reproductive barriers. Alternatively, the assortative mating may have evolved as a secondary consequence of selection on other traits. One such trait is related to microhabitat distribution. Indeed, it seems as if a non-random microdistribution of the parental morphs in the mid-shore zone may explain roughly half the assortative mating (Rolán-Alvarez et al., 1999). However, there is also a component of assortative mating other than microhabitat selection, and there is some suggestion that anatomical differences in penis morphology may contribute to the isolation through mechanical incompatibility (Cruz, Carvía & Rolán-Alvarez, in prep).

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isolation in Littorina saxatilis (Olivi) in northeast England. Journal of Molluscan Studies, 62: 89-99. JANSON, K. 1985. Variation in the occurrence of abnormal embryos in females of the intertidal gastropod Littorina saxatilis Olivi. Journal of Molluscan Studies, 51: 64-68. JOHANNESSON, J., JOHANNESSON, B. & ROLÁNALVAREZ, E. 1993. Morphological differentiation and genetic cohesiveness over a microenvironmental gradient in the marine snail Littorina saxatilis. Evolution, 47: 1660-1787. JOHANNESSON, K., ROLÁN-ALVAREZ, E. & EKENDAHL, A. 1995. Incipient reproductive isolation between two sympatric morphs of the intertidal snail Littorina saxatilis. Evolution, 49: 1180-1190. JOHANNESSON, K., ROLÁN-ALVAREZ, E. & ERLANDSSON, J. 1997. Growth rate differences between upper and lower shore ecotypes of the marine snail Littorina saxatilis (Olivi) (Gastropoda). Biological Journal of the Linnean Society, 61: 267-279. KELLY, J.K. & NOOR, M.A.F. 1996. Speciation by reinforcement: a model derived from studies of Drosophilia. Genetics, 143: 1485-1497. MOORE, W.S. 1977. An evaluation of narrow hybrid zones in vertebrates. The Quarterly Review of Biology, 52: 263-277. ROLÁN-ALVAREZ, E., ROLÁN, E. & JOHANNESSON, K. 1996. Differentiation in radular and embryonic characters, and further comments on gene flow, between two sympatric morphs of Littorina saxatilis (Olivi). Ophelia, 45: 1-15. ROLÁN-ALVAREZ, E., BUNO, I. & GONSALVEZ, J. 1996. Sex is determined by sex chromosomes in Littorina saxatilis (Olivi) (Gastropoda, Prosobranchia). Hereditas, 124: 261-267. ROLÁN-ALVAREZ, E., JOHANNESSON, K. & EKENDAHL, A. 1995. Frequency- and density-dependent sexual selection in natural populations of Galician Littorina saxatilis Olivi. Hydrobiologia, 309: 167-172. ROLÁN-ALVAREZ, E., JOHANNESSON, K. & ERLANDSSON, J. 1997. The maintenance of a cline in the marine snail Littorina saxatilis: the role of home site advantage and hybrid fitness. Evolution, 51: 1838-1847. ROLÁN-ALVAREZ, E., ERLANDSSON, J., JOHANNESSON, K. & CRUZ, R. 1999. Mechanisms of incomplete prezygotic reproductive isolation in an intertidal snail: testing behavioural models in wild populations. Journal of Evolutionary Biology (in press). TURESSON, G. 1922. The species and the variety as ecological units. Hereditas, 3: 100-113. UNDERWOOD, A.J. 1997. Experiments in ecology. Cambridge University Press, Cambridge.