Maintenance of two genetic entities by habitat selection - CiteSeerX

Evolutionary Ecology, 1995, 9, 131-138

Maintenance of two genetic entities by habitat selection THIERRY DE MEEÛS1, MICHAEL E. HOCHBERG2 and FRANCOIS RENAUD 1 1

Laboratoire de Parasitologie Comparée, CNRS-URA 698, Université Montpellier II, Case 105, Place E. Bataillon, 34095 Montpellier Cedex 05, France 2 Ecole Normale Supérieure, CNRS-URA 258, Laboratoire d'Ecologie, 46 Rue d'Ulm, 75230 Paris Cedex 05, France

Summary In the laboratory, the two species of copepods Lepeophtheirus thompsoni and Lepeophtheirus europaensis, ectoparasites of flatfishes, can meet and mate on at least one host species. In the wild however, these two species are found isolated on their sympatric hosts. Habitat selection theoretically represents a powerful enough mechanism to explain the maintenance of genetic heterogeneity in the wide sense. In this paper, the host colonization process is studied for both parasite species. It is shown that each parasite can develop and reach adult age on each host species. However, L. thompsoni is highly selective; it almost totally refuses to colonize hosts other than its natural one. Lepeophtheirus europaensis, on the contrary, readily infests turbot and brill in single-host experiments, but strongly prefers the brill when it has a choice. It appears that these two genetic entities are sympatrically maintained due to strong habitat selection. Such a pattern could theoretically only occur in a soft-selection context (density dependence). This point is discussed with respect to the different patterns in host use found in the geographical distribution of these parasites. Keywords: Selected polymorphism; habitat selection; genetic isolation; habitat specialization; soft selection; competition; parasitic copepods; specificity Introduction

In the Western Mediterranean (France), it has been shown that there is total reproductive isolation among two congeneric species of caligid copepods Lepeophtheirus thompsoni and Lepeophtheirus europaensis, ectoparasites of flatfishes (Zeddam et al., 1988). Lepeophtheirus thompsoni is specific to turbot (Psetta maxima), a marine scophthalmid and L. europaensis parasitizes two hosts: brill (Scophthalmus rhombus) a marine scophthalmid (sympatric with turbot) and flounder (Platichthys flesus), a pleuronectid inhabiting lagoons. In the laboratory, L. thompsoni and L. europaensis can meet and mate on the turbot, producing viable and fertile hybrids (De Meeûs et al., 1990). In nature, although turbot and brill are sympatric, the two parasite species are completely genetically isolated (Zeddam et al., 1988). Selective maintenance of a genetic polymorphism in a variable environment is possible only when the populations are regulated by density dependence in each habitat (Levene, 1953; Dempster, 1955; Maynard Smith and Hoekstra, 1980) (i.e. soft selection) and requires highly restrictive conditions (Maynard-Smith, 1962, 1966; Maynard Smith and Hoekstra, 1980). It has nevertheless been demonstrated that habitat selection broadens these conditions (Maynard Smith, 1966), especially if there is a correlation between adaptation and the chosen habitat (i.e. through pleiotropy) (Rausher, 1984; Garcia-Dorado, 1986; Hedrick, 1990). Furthermore, recent * To whom correspondence should be addressed. 0269-7653

© 1995 Chapman & Hall

132

De Meeûs et al.

work (De Meeûs et al., 1993) has shown that when this correlation is achieved by epistasis, habitat selection following a Markovian trial and error process like that in Doyle's (1975) model always results in the maintenance of different genetic entities in their respective habitats. On the other hand, if density dependence is absent from these habitats (i.e. hard selection) and habitat selection is possible, only the most favourable habitats are exploited by the most efficient specialist. The parasites studied in the present work have a direct life cycle, in accordance with the Caligidae cycle described by Kabata (1972; De Meeûs et al., 1990). After mating on the body surface of the host, females colonize the gill chamber. Eggs are laid in two ovigerous sacks. Hatching gives rise to a free swimming larva called 'nauplius 1'. A first moult produces a n' auplius 2' followed by the infective stage (copepodid). At 15°C, this free-swimming phase may last more than 4 days (Johannessen, 1978). After attachment to its host, the copepodid becomes mucophagous. Four chalimus, two pre-adult stages and the mature adults then follow. In this paper, the different capabilities of the two parasite species to infest turbot and brill (their marine hosts) are experimentally investigated. Choice behaviour of infective stages, when faced with the two different host species, is then analysed. This study provides a demonstration that the two copepod species are maintained and isolated on their respective sympatric hosts by the existence of strong habitat selection, suspected to be of Doyle's type. According to the theory, this pattern requires soft selection and confirms the importance of habitat selection in maintaining biological diversity. Material and methods Hosts and parasites Ovigerous copepod females were collected at fishing ports (Sète and Grau-du-Roi, France), from the gill chambers of their respective hosts (turbot and brill). In the laboratory, the collected eggs were incubated until hatching at 15°C in filtered sea water, following the method described by De Meeûs et al. (1990). Some of the 10 cm parasite-free turbots used for experimental infestations came from a fish farm. Other hosts were caught along the Languedoc coast (Mediterranean, France): 20-25 cm brills were caught at sea by craft fishermen and 5.6-7 cm brills and 3.5-17 cm turbots were caught inshore. After anaesthesia with 3-amino-benzoic-acid-ethyl-ester (Sigma A 5040), the fishes were observed under a binocular microscope. Only one 8 cm turbot was found infested with two chalimus 1 and was not used for the experiments. Infective stages and experimental infestations After hatching and development of the larvae, the infective stages (copepodids) were isolated, counted and placed in a 50 l tank containing fish to be infested. Daily observation of anaesthetized fishes commenced 3 days post-infestation. Attached parasites were then counted using a binocular microscope. Experiment I To test colonization rates, all the possible combinations of fish infestation (turbot or brill with parasite from turbot or brill) were monitored. Experiment 2 To test the power of discrimination, simultaneous infestations (1) of one turbot and one brill and (2) of two turbots and one brill were performed using brill parasites (L. europaensis).

Biodiversity and habitat selection in parasitic copepods

133

Experiment 3 A parasite-free turbot was introduced into a tank containing a brill exposed to L. thompsoni (turbot's parasite) infective stages 3 days previously. This measured the number of L. thompsoni copepodids refusing to settle on brill, but still able to infest a turbot after 3 days. Experiment 4 Turbots with a 3 day old L. europaensis infestation were transferred into a tank containing a parasite-free brill in order to test the capability of the infective stages to detach and colonize another host. Statistical treatment Whenever the data allowed for parametric tests, we used a Student t-test. In cases of nonnormally distributed data and/or of heterogeneity of variances, we used non-parametric tests: the Wilcoxon Mann-Whitney U-test (e.g. Sokal and Rolph, 1981) and Spearman's coefficient of rank correlation S (e.g. Scherrer, 1984). Multiple comparison tests increase the probability of rejecting the null hypothesis by chance. Thus, we also used the sequential Bonferroni technique (described by Rice, 1989). Results Experiment I Figure 1 shows the relationship between the host size and the fixation rates of parasites for each type of experiment. As far as the host range used in this study is concerned, fixation rates and host size are independent of one another (Fig. 1). Table 1 gives the number of trials, the mean, variance and standard error of fixation percentages observed for each type of infestation. Significance levels obtained for multiple comparisons are given in Table 2. For each kind of infestation, adult parasites could be obtained. Tables 1 and 2 reveal that L. thompsoni (the turbot parasite) is highly selective and that L. europaensis (brill parasite) is much more opportunistic. Lepeophtheirus thompsoni colonizes brill with much lower rates than its natural host, but L. europaensis seems to settle just as well on turbot as on its natural host. Also, L. europaensis infests turbot at lower rates than does L. thompsoni although the two parasite species infest their natural hosts (L. thompsoni on turbot and L. europaensis on brill) with similar fixation rates. It is however possible that some similarities came from the lack of power of the non-parametric tests we have used. Experiment 2 There is a strong preference of L. europaensis infective stages for their natural host (brill): 89% (s² = 65) of the fixed copepodids attached to brill (Table 3, experiment 1) (ts = 10.7, df = 4, p < 0.001, one-tailed test). This discrimination is also present when the number of alternative hosts (turbots) is twice as many as the number of brill (Table 4, experiment 2) (ts = 3.69, df = 2, p < 0.05, one-tailed test). The two experiments did not show any significant difference in the percentage of copepodids attached to the brill (Fs = 5.2, df = 2 and 4, p > 0.1; ts = 1.8, df = 6, p ≥ 0.1), although there was less attachment in experiment ii (73%, s² = 341). Experiment 3 Table 5 reveals that only 1% of L. thompsoni copepodids are able to settle on brill whereas 34% (variance s2 = 18) prefer to remain in the water and subsequently attach to a turbot introduced into the tank 3 days later. Host selection thus appears to come from a refusal to settle on brill.

134

De Meeûs et al.

Figure 1. Relationships between host size and fixation percentage: (a) infestation of turbot by turbot copepods, (b) infestation of brill by brill copepods, (c) infestation of brill by turbot copepods, (d) infestation of turbot by brill copepods. S: Spearman coefficient (probabilities are given for one-tailed tests).

Table 1. Fixation percentages obtained during single fish experiments CT Turbot n Mean s2 smean Brill n Mean s2 smean

15 41.3 534 6 9 1.8 1.1 0.35

CB 8 20.5 99.7 3.5 5 36.5 338.9 8.2

CT, L. thompsoni, copepods from turbot; CB, L. europaensis copepods from brill. n, number of experiments; s2, variance; smean , standard error of the mean.


135

Table 2.Results of Mann-Whitney U-tests for multiple comparisons between single fish experiments

CT/B CB/B CBRF

CT/'F

CT/B

CB/B

p = 0.0001 (S) p 1 = 0.0083 p = 0.93

-

-

p = 0.0025 (S) p 4 = 0.017 p = 0.0005 (S) p 2 = 0.01

-

p = 0.0019 (S) p 3 = 0.0125

p = 0.1073

CT/T indicates infestation of turbot (T) with turbot copepod (CT), and CB/B indicates infestation of brill( B) with brill copepod (CB). CT/B indicates infestation of brill (B) with turbot copepod (CT). (S): significant after the sequential Bonferroni procedure. For the sequential Bonferroni technique, the successive 'table-wide' significant levels (p i = α/k ) are given for the significance level α = 0.05, k being the number of remaining tests.

Table 3. Discriminating power of copepod infective stages: simultaneous infestations of one brill and one turbot by L. europaensis (natural parasite of the brill) Experiment number 1 Infective copepodids 130 Fish species Turbot Brill Fixed copepodids 6 108 Percentage fixed on brill 95

2

3

4

5

107 Turbot Brill 6 32 84




Table 4. Discrimination power of copepodids infective stages: simultaneous infestations of two turbots and one brill by L. europaensis (natural parasite of the brill) Experiment number

Fish species Infective copepodids Fixed copepodids Percentage fixed on brill

1

2

3

Turbot Turbot Brill 100 12 13 27 52

Turbot Turbot Brill 103 5 10 54 78

Turbot Turbot Brill 100 5 2 49 87.5

Table 5. Nature of the choice process: proportion of L. thompsoni copepodids (parasite of turbot) that refuse to attach to brill and are later recovered from a turbot introduced subsequently Experiment number

Infective copepodids Fixed copepodids on brill Fixed copepodids on turbot Percentage not found on brill and attached to turbot

1

2

3

4

5

98 2 38 39

101 1 30 30

101 0 30 30

100 1 34 34

102 1 38 38

The turbot was introduced into the tank containing the brill 3 days after infestation with L. thompsoni (turbot's parasite).

136

De Meeûs et al.

Experiment 4 Host discrimination by brill parasites was weaker after attachment (Table 6). Only a small proportion (19%) of L. europaensis previously attached to the turbot moved to brill (host switching) after we introduced brill to the tank (a significantly lower fixation rate than in experiment 2i, p = 0.05, one-tailed Wilcoxon Mann-Whitney).

Discussion Lepeophtheirus thompsoni is a highly selective parasite which almost always settles on turbot. It rarely settles on brill, although brill is ecologically and genetically closely related to turbot (Norman, 1934). Moreover, brill is a suitable host for the development of L. thompsoni, as shown by our experiments. Lepeophtheirus europaensis is opportunistic. It can infest brill (its natural host) and turbot with equal success when only one host species is available. Nevertheless, L. europaensis strongly prefers to colonize brill when given the choice between the two host species. Its preference for brill decreases after attachment on turbot; few move when given the chance. For such parasites, the infective stage is quickly followed by a chalimus stage, anchored to the host integument with a frontal filament (Lewis, 1963). Thus, host choice must occur before attachment, the parasite either refusing or accepting to settle on a host when close to it. Because sexual reproduction occurs within the host, such habitat selection could be responsible for the maintenance of diversity within this parasite community (Rice, 1987; De Meeûs et al., 1993). Theoretical models show that habitat selection of the Doyle (1975) type, following a Markovian chain of trial and error, can maintain an adaptive polymorphism even when the maximum number of trials is limited (De Meeûs et al., 1993). It has been experimentally demonstrated that habitat selection can maintain a deleterious allele much longer in a heterogeneous environment than in a homogeneous one (Jones and Probert, 1980), but natural examples of this phenomenon are few (Futuyma and Moreno, 1988). The biological system considered in this paper involves the sympatric co-existence of two different genomes, totally isolated within their habitat (Zeddam et al., 1988) (their respective sympatric hosts), though perfectly capable of developing and meeting on the same host and cross-breeding in a fertile manner (De Meeûs et al., 1990). Our results suggest that the maintenance of biological diversity in this host-parasite system is the result of habitat (i.e. host) preference of the Doyle (1975) model type. Because reproduction

Table 6. Discrimination power of copepod infective stages once attached to the host: transfer of L. europaensis copepodids intially attached to a turbot Experiment number

Infective copepodids Fixed copepodids on turbot Copepodids distribution after transfer of the turbot in the brill's tank Percentage of host switching

1

2

101 16 Turbot 10

200 96 Turbot 87

Brill 5 31

Brill 7 7

In each experiment the turbot, infested 3 days beforehand, is transferred to the tank containing a parasite-free brill.


137

occurs within hosts, this results in the maintenance of species diversity aswell (Rice, 1987; De Meeûs et al., 1993). Without this habitat selection, much of the existing polymorphism would probably be lost and only one species would remain on all the available hosts. According to the theory, the pattern observed requires soft selection (density dependence within each habitat) (De Meeûs et al., 1993). Without density dependence and because of the strong host preference observed, only one host species would be exploited by the most efficient specialist (De Meefis et al., 1993). In the Mediterranean, L. europaensis exploits two hosts, brill and flounder (flounder inhabits lagoons and is not totally sympatric with turbot and brill). In the Atlantic, flounder is exploited by two other copepod species Lepeophtheirus pectoralis and Acanthochondria depressa (Boxshall, 1974), both absent from the Mediterranean, while turbot and brill are parasitized by the same species as in the Mediterranean (Zeddam et al., 1988). The three host species, like most flatfish, are of northern origin and could have invaded the Mediterranean only during the last glaciations (Quignard, 1972). Perhaps, during this migration, flounder may have lost its original ecto-parasites (L. pectoralis and A. depressa). Then the more opportunistic L. europaensis could have colonized flounder. In any case, the absence of L. europaensis on flounder in the Atlantic strongly suggests that competition plays an important role in determining the host range for these parasitic copepods. Such behaviour seems in accordance with the effect of density-dependent habitat selection in competing species, particularly in the case of interactions between a 'tolerant' and an 'intolerant' species (asymmetrical competition) (see Rosenzweig (1991) for a review). Habitat preference of a tolerant species will disappear when that species is alone at high densities. But it is restored when a competing species (the intolerant) is added. This may be occurring in the system studied here. In the presence of L. thompsoni, L. europaensis (the tolerant species) limits itself to brill. However, L. thompsoni (the intolerant competitor) always restricts itself to turbot. In addition, L. europaensis shows its tolerance by using flounder in the Mediterranean where A. depressa and L. pectoralis are absent. However, the pattern of host preferences of all those copepods with regard to all the available hosts has not been experimentally studied in the Atlantic. Also, we do not yet know what prevents L. thompsoni from being more tolerant. In conclusion, our empirical results support the theory that a Doyle (1975)-type habitat selection, acting through a soft-selection process that remains to be clarified, maintains both genetic and species diversities in the biological system studied. To our knowledge, demon-strations of such phenomena are not widespread.

References Boxshall, G.A. (1974) Infections with parasitic copepods in North Sea marine fishes. J. Mar. Biol. Ass. UK 54, 355-72. De Meeûs, T., Renaud, F. and Gabrion, C. (1990) A model for studying isolation mechanisms in parasite populations: the genus Lepeophtheirus (Copepoda, Caligidae). J. Exp. Zool. 254, 207-14. De Meeûs, T., Michalakis, Y., Renaud, F. and Olivieri I. (1993) Polymorphism in heterogeneous environments, evolution of habitat selection and sympatric speciation. Soft and hard selection models. Evol. Ecol. 7, 175-98. Dempster, E. R. (1955) Maintenance of genetic heterogeneity. Cold Spring Harbor Symp. Quant. Biol. Sci. 20, 25-32. Doyle, R.W. (1975) Settlement of planktonic larvae: a theory of habitat selection in varying environments. Am. Nat. 109, 113-26. Futuyma, D.J. and Moreno, G. (1988) The evolution of ecological specialisation. Ann. Rev. Ecol. Syst. 19, 20733.

138

De Meeûs et al.

Garcia-Dorado, A. (1986) The effect of niche preference on polymorphism protection in a heterogeneous environment. Evolution 40, 936-45. Hedrick, P.W. (1990) Genotypic habitat selection: a new model and its application. Heredity 65, 145-9. Johannessen, A. (1978) Early stages of Lepeophtheirus salmonis (Copepoda, Caligidae). Sarsia 63, 169-76. Jones, J.S. and Probert, R.F. (1980) Habitat selection maintains a deleterious allele in a heterogeneous environment. Nature 287, 632-3. Kabata, Z. (1972) Developmental stages of Caligus clemensi (Copepoda; Caligidae). J. Fish Res. Bd. Canada 29, 1571-93. Levene, H. (1953) Genetic equilibrium when more than one ecological niche is available. Am. Nat. 87, 331-3. Lewis, A.G. (1963) Life history of the caligid copepod Lepeophtheirus dissimulatus Wilson, 1905 (Crustacea, Caligoida). Pacific Sci. 17, 195-242. Maynard Smith, J. (1962) Disruptive selection, polymorphism and sympatric speciation. Nature 195, 60-2. Maynard Smith, J. (1966) Sympatric speciation. Am. Nat. 100, 637-49. Maynard Smith, J. and Hoekstra, R.F. (1980) Polymorphism in a varied environment: how robust are the models? Genet. Res. Camb. 35, 45-57. Norman, J.R. (1934) A Systematic Monograph of flatfishes (Heterosomata) Vol. 1: Psenodidae, Bothidae, Pleuronectidae. British Museum (ed.), London, UK. Quignard, J.P. (1972). La Méditerranée, creuset ichthyologique. Bull. Zool. 45, 23-36. Rausher, M.D. (1984) The evolution of habitat preference in subdivided populations. Evolution 38, 596-608. Rice, W.R. (1987) Speciation via habitat specialisation: the evolution of reproductive isolation as a correlated character. Evol. Ecol. 1, 301-14. Rice, W.R. (1989) Analyzing tables of statistical tests. Evolution 43, 223-5. Rosenzweig, M.L. (1991) Habitat selection and population interactions: the search for mechanisms. Am. Nat. 137, S5-S28. Scherrer, B. (1984) Biostatistique. Gaêtan Morin. Chicoutimi, Canada. Sokal, R.R. and Rohlf, F.J. (1981) Biometry. Freeman and Co, New York. Zeddam, J.L., Berrebi, P., Renaud, F., Raibaut, A. and Gabrion, C. (1988) Characterisation of two species of Lepeophtheirus (Copepoda, Caligidae) from flatfishes. Description of Lepeophtheirus europaensis sp. Nov. Parasitology 96, 129-44.