Abstract
The introduction of a few individuals to new, isolated habitats (founder effect) is expected to reduce the genetic variability of a population. At the beginning of the last century a few eastern mosquitofish (Gambusia holbrooki) individuals were introduced to Southern Europe from North America to control malaria‐carrying mosquitoes. We studied the effect of this severe bottleneck on genetic variability in four populations of eastern mosquitofish introduced to Spain and Italy in the early 1900s and compared them to a native population in North America. Using amplified polymorphic DNA–polymerase chain reaction (RADP–PCR) we found a strong reduction of genetic diversity, in terms of both number of polymorphic loci and heterozygosity, in all four European populations. Despite this great reduction in genetic diversity, eastern mosquitofish successfully invaded European internal waters causing eutrophication problems and threatening the extinction of local populations of native fish.
Introduction
Population bottlenecks can have significant effects on a population's genetic variability leading to short‐term evolutionary changes. Conservation genetic lessons have taught us about the potentially harmful effects of small population size because genetic drift and the effect of inbreeding are thought to contribute to the extinction of small populations (Frankham et al. Citation2002). The introduction and subsequent isolation of populations can provide a model to study such effects as the reduction of the effective population size (Wright Citation1938), loss of heterozygosity (Nei et al. Citation1975; Maruyama & Fuerst Citation1985b) and loss of alleles (Maruyama & Fuerst Citation1985a) as well the persistence and adaptability of the population in a new region. Invasions are rapid evolutionary events where populations are usually subjected to a bottleneck during the colonization or introduction event, followed by a rapid expansion (Sakai et al. Citation2001). Thus, newly arrived populations are likely to be less variable than the original population from whence they derived (Barrett & Kohn Citation1991), although this is not a rule. Indeed, newly introduced populations can be the result of individuals invading from different source populations, thus transforming the among‐population variation in the native range to within‐population variation in the newly established population (Baker Citation1992; Kolbe et al. Citation2004).
Invasive species may thrive in their introduced range due to the absence of competitors, predators, parasites, or diseases that help to regulate populations in the native range (Elton Citation1958; Orians Citation1986; Pimm Citation1991; Porter et al. Citation1997). This type of biotic release can increase colonization success and the subsequent rate of spread (Suarez et al. Citation1999). The loss of genetic variability associated with a small population size can also lead to changes in the behaviour, physiology and morphology of the invasive species in the introduced populations. These changes have been proved to be advantageous in the invasion success (Ross & Keller Citation1995; Ross et al. Citation1996; Suarez et al. Citation1999; Tsutsui et al. Citation2000).
The eastern mosquitofish, Gambusia holbrooki Girard, 1859, is a cyprinodontiformes freshwater fish naturally distributed on the Atlantic coast of the USA from New Jersey to Florida and the Gulf of Mexico (Rosen & Bailey Citation1963; Black & Howell Citation1979). Eastern mosquitofish were introduced to many countries to control mosquitoes at the beginning of the 20th century. In Southern Europe the eastern mosquitofish was first introduced to Spain. In 1922, Gambusia was introduced to Italy from Spanish populations (Black & Howell Citation1979). In 1924, mosquitofish from Italy were introduced to the Transcaucasic regions and from there to areas in the south and centre of the former USSR. Later, eastern mosquitofish were introduced to other malaric areas of the world (other European countries, East Asia, Australia and New Zealand) (Ronchetti Citation1968). Following the initial enthusiasm in the control of Anopheles larvae, Gambusia was introduced to all malaric areas in Italy (Ronchetti Citation1968). However, the intense predatory activity on insect larvae (not just Anopheles) and on zooplankton altered the biological equilibrium of the water systems, contributing to eutrophic effects (Veronesi et al. Citation1997). Gambusia aggressiveness and its great adaptability to new and human‐disturbed habitat have facilitated its diffusion (Smith et al. Citation1989), causing it to become a serious predator and competitor for many native species (Howe et al. Citation1997; Godinho & Ferreira Citation1998; Arthington & Marshall Citation1999), resulting, in some cases, to the local extinction of other fish (Deacon et al. Citation1964; Minckley & Deacon Citation1968; Gandolfi Citation1973). Nothing is known about the genetic variability of the actual European populations of eastern mosquitofish. In this viviparous species, females can give birth to 30–50 offspring and multiple paternity is widespread (Greene & Brown Citation1991; Zane et al. Citation1999). Thus, even if the initial number of adults introduced to Europe was small, the effective population size may have been larger due to multiple paternity if the introduction included pregnant females. Although Gambusia is presently distributed only in southern Europe, it has had enormous success in invading different habitats within its distribution range, with climates varying from truly Mediterranean with mild winters, similar to its original habitat, to temperate. Furthermore, thermal ponds and cave habitats have been also colonized (e.g. Camassa, Citation2001; Specziar Citation2004), suggesting that the reduction of genetic variability may have been less severe than expected (e.g. Congdon Citation1995).
In this study we compared the genetic variability of four populations in Italy and Spain where eastern mosquitofish were successfully introduced and one native population in Florida. To obtain a large number of loci to analyse, RAPD–PCR was used to assess the genetic variability.
Materials and methods
Extraction of total genomic DNA, using the method described in Kocher et al. (Citation1989), was performed for 79 individuals of Gambusia holbrooki from one American (Styx river, Alachua, Florida), two Spanish (Esla river, Leon and Tormes river, Salamanca) and two Northern Italian (Fimon, Vicenza and Idrovia, Padova) populations.
To identify our Gambusia as G. holbrooki from the similar species Gambusia affinis Baird & Girard, 1853, we sequenced a short fragment of 300 bp of the mitochondrial cytochrome b for a few individuals of each population using the universal primers Cytb1 and Cytb2 (Kocher et al. Citation1989). The cytochrome b of G. holbrooki presents several characteristic mutations that distinguish it from G. affinis (Lydeard et al. Citation1995).
RAPD–PCR were performed using the set OPE (Operon Technologies). Of the 20 primers tested, five (OPE01, 06, 09, 11 and 12) were chosen to screen the populations on the basis of the clarity of the band patterns and the repeatability of the amplifications. RAPD–PCR were performed in a volume of 25 µl containing 50 mM KCl, 10 mM TRIS–HCl, 2 mM MgCl2, 0.025% Triton X‐100, 200 µM dNTP, 5 µM primer, 1 unit Taq polymerase (Promega) and 20 ng DNA. DNA amplifications were performed in a Perkin Elmer Cetus 380 model. The amplification mixture was subjected to a denaturation step for 5 min at 94°C followed by 40 cycles of 10 s at 94°C, 1 min at 38°C and 1 min at 72°C. A final step of 3 min at 72°C was performed after the 40 cycles. Half of the PCR product was run in a 1.2% agarose gel stained with ethidium bromide to a final concentration of 0.5 µg ml−1. Gels were run for 5 h at 5 V cm−1 in TBE 1X. Each band was considered as a locus and each individual was represented by a sequence of 0 and 1 indicating the presence (1) or absence (0) of each band for the five different primers. Reproducibility of the banding pattern for each primer was tested with new amplifications using newly extracted DNA from a subsample of each population.
Allele frequency, effective allele number (n e) (Hartl & Clark Citation1989) and percentage of polymorphic loci (p) for each population were calculated using POPGENE v. 1.32 (Yeh & Boyle Citation1997). Gene diversity (H) for each population and the overall genetic differentiation among populations (F ST) were also calculated with Hickory ver. 1.03 (Holsinger et al. Citation2002) using the free model option which does not require the assumption that populations are in Hardy–Weinberg equilibrium. Nucleotide diversity (π) within and between populations was estimated from the RAPD bands using RAPDDIP (kindly provided by Andrew G. Clark) (Clark & Lanigan Citation1993). A neighbour joining tree with bootstrap supports showing the relationships between populations was obtained from Nei's genetic distance (Nei Citation1972) using DISPAN (Ota Citation1993). Population structure was analysed by AMOVA using GenAlEx ver. 6 (Peakall & Smouse Citation2005). The statistical significance of each Φ ST value was assessed under the null hypothesis of genetic homogeneity, performing 1000 permutations of the original data set.
Results
Before proceeding with the population analysis, a subsample of individuals from each population was sequenced for a fragment of mitochondrial cytochrome b to identify our samples as G. holbrooki. Sequences of G. affinis and G. holbrooki from Lydeard et al. (Citation1995) were used as controls. All our individuals showed the cytochrome b sequence characteristic of G. holbrooki.
RAPD amplifications with the five primers resulted in a total of 49 bands or loci with a size ranging from 300 bp to 1500 bp. The number of loci per primer was 9 for OPE01, 10 for OPE06, 11 for OPE09, 9 for OPE11 and 10 for OPE12, respectively. Of the 49 loci, seven were monomorphic across all populations (table ). Thirty‐nine loci were polymorphic in the North American population while 21 and 8 loci were polymorphic in the Italian and Spanish populations, respectively. Of the 39 polymorphic loci in the American population, 12 were fixed as presence of band and seven as absence of band in all European populations. Three loci (OPE11‐1, 11‐4 and 11‐5) were fixed (1) in the American and Spanish populations but were polymorphic in the Italian populations (see table ). The average number of alleles per locus and the percentage of polymorphic loci were lower in all European introduced populations than in the American native population, as indicated in table . The percentage of polymorphic loci in the European population ranged from 10.2 to 38.8%, while the American population was 79.6% (table ). This reduction of polymorphism in European populations was also reflected in the gene and nucleotide diversity. Gene diversity and nucleotide diversity were lower in the European populations than in the North America population (table ). Gene and nucleotide diversity were higher in the Italian populations than in the Spanish populations (table ). The overall F ST among populations was 0.372 (95% Credibility Interval (CI), 0.295–0.457). This high genetic differentiation among populations was not exclusively due to the North American population; F ST among European populations still indicated strong genetic structure (F ST = 0.236; 95% CI, 0.145–0.348). Pairwise population genetic distances and nucleotide diversity are shown in table . Population relationships are represented by the NJ tree of figure . The North American population was the most divergent from all other populations while the Spanish populations were the two most similar populations. AMOVA clearly indicates a strong genetic differentiation between the two continents: 24% of the total variation was attributed to variation among the two continents (P = 0.001) and 11% of the variance was attributed to population variation within groups (P < 0.001). When the populations from the European continent only were considered, there was no differentiation among the Spanish and Italian population groups (variance among group = 0, P = 0.629), while 25% of the total variation was attributed to population variation within groups (P = 0.001).
Table I. Allele frequencies per locus and population. Only the frequency of one allele (presence) is reported. Legend: Fl, Florida; It, Italy; Sp, Spain.
Table II. The number of individuals analysed (n), the effective number of alleles per locus (n e), the percentage of polymorphic loci (p), the gene diversity (H) with the 95% credibility interval between brackets and the nucleotide diversity (π) are indicated for each population. Legend: Fl, Florida; It, Italy; Sp, Spain.
Table III. Nei's genetic distance (Nei Citation1972) (above the diagonal) and nucleotide diversity (below the diagonal) between populations obtained with RAPDDIPP (Clark & Lanigan Citation1993). Standard errors are indicated between brackets. Legend: Fl, Florida; It, Italy; Sp, Spain.
Figure 1 Population relationships ofGambusia holbrooki obtained with a neighbour joining tree and the Nei (Citation1972) genetic distance for the RAPD data set. Numbers at the nodes indicate bootstrap support.
Discussion
Founder effect is usually expected when a species is introduced to a new habitat; however, in cases where samples from multiple sources have been introduced (Baker Citation1992) or where multiple introduction occurred (Kolbe et al. Citation2004) the genetic diversity of the introduced population may be similar to or higher than that of a single native population. This is not the case for G. holbrooki. The genetic analysis of RAPD markers clearly showed a large reduction of the genetic diversity in European populations of eastern mosquitofish following their introduction to Europe at the beginning of the 20th century. The Alachua population showed a higher amount of polymorphic loci and higher levels of heterozygosity than the European populations. The Alachua population was also clearly differentiated from all European populations as indicated by the AMOVA analysis. The Alachua population also showed a higher amount of polymorphic loci and higher levels of heterozygosity than the other populations in Florida and South Georgia assessed with allozymes (Hernandez‐Martich et al. Citation1995; Hernandez‐Martich & Smith Citation1997), indicating that RAPD markers are more suitable for assessing genetic variability than allozyme markers in this species. However, we have to note that heterozygosity is not considered a good indicator of previous founder effects or bottlenecks due to the effects of drift on the allele frequencies (Leberg Citation1992).
Despite the large reduction of genetic diversity found in the European populations, a high population genetic structure could still be seen. However, all genetic variation was observed among populations and none between the two European countries. Spanish Gambusia holbrooki were introduced to central Italy and this initial Italian population served as the source for the establishment of the two northern Italian populations under study (Ronchetti Citation1968; Gandolfi Citation1973). Following this serial introduction, we should expect lower polymorphism in the Italian populations than in the Spanish ones. Moreover, due to the longer and colder winter in northern Italy than in central Italy and Spain, these populations of eastern mosquitofish may have been subjected to more frequent bottlenecks (Ronchetti Citation1968). Surprisingly, the level of polymorphic loci, heterozygosity and the estimated nucleotide diversity was higher in the northern Italian populations than in the Spanish populations. This apparently paradoxical result may be due to the demographic history of the populations sampled in this study. For instance, in the years 1930–40 mosquitofish were extensively used for the control of mosquitoes in Italy and the northern populations were reinforced with new introductions from other Italian populations (Veronesi et al. Citation1997), possibly increasing genetic diversity. The two Spanish populations considered in this study, in turn, could have gone through serious bottlenecks losing a larger part of their genetic diversity compared to the Italian populations. This scenario could also explain why all the genetic variation was observed among populations and none between countries. Alternatively, there may have been unreported successive introductions into Italy of eastern mosquitofish directly from its original range resulting in a larger initial genetic pool for the Italian populations. Only a larger population survey would allow us to distinguish between these three alternative (although not mutually exclusive) explanations.
Although eastern mosquitofish in Europe show a reduced genetic variability, this has not limited the colonization success and the diffusion of this fish in the European internal waters, contrary to what has been reported in studies on the effect of the reduction of genetic variability on the growth and health of experimental populations (Leberg Citation1990; Stockwell & Leberg Citation2002). The establishment and adaptation of Gambusia to these new environments is not surprising, however, given the great adaptability of this species, as attested by the enormous success of the worldwide introductions (García‐Berthou et al. Citation2005). The reasons for this success are not clear. The native populations of eastern mosquitofish are exposed to a variety of predators (e.g. Horth Citation2004) and the absence of these predators in Europe may have favoured mosquitofish diffusion, although ecologically vicariant predators are present in Europe and probably the predation pressure is not very different from that in the original habitats. On the other hand, the species is highly aggressive and ecologically competitive, as proved by the tremendous effects on the local fauna following introduction. For instance, some 35 fish species worldwide have declined in abundance or range as a result of interactions with Gambusia (Lloyd Citation1990). Parasite resistance could also be an important factor. About 23 different parasites have been reported in native Gambusia populations (Swanson & Cech Citation1996). Gambusia holbrooki seems resistant to the parasites that instead infect other related fish, such as the Mediterranean killifish Aphanius fasciatus Valenciennes, 1821 (Gandolfi Citation1973), and the relative absence of specific parasites in Europe may be one reason for its success here despite the reduced genetic variability associated with introduction. Whatever the reason for the success of G. holbrooki as an invading species, our results demonstrate that invasion success is not necessarily associated with the maintenance of the original genetic variability.
Acknowledgements
We warmly thank Gary K. Meffe, Benito Fraile and José R. Alonso for providing population samples from Florida and Spain. Claudia Signori helped with the genetic analyses and Guglielmo Marin generously offered hospitality in his lab. Maxine Iversen and two anonymous reviewers provided comments on a previous version of the manuscript.
Related Research Data
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