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Although Trinidadian populations of the guppy, Poecilia reticulata, show considerable adaptive genetic differentiation, they have been assumed to show little or no reproductive isolation. We tested this assumption by crossing Caroni (Tacarigua River) and Oropuche (Oropuche R.) drainage populations from Trinidad's Northern Range, and by examining multiple aspects of reproductive compatibility in the F1, F2 and BC1 generations. In open-aquarium experiments, F1 males performed fewer numbers of mating behaviours relative to parental population controls. This is the first documentation of hybrid behavioural sterility within a species, and it suggests that such sterility may feasibly be involved in causing speciation. The crosses also uncovered hybrid breakdown for embryo viability, brood size and sperm counts. In contrast, no reductions in female fertility were detected, indicating that guppies obey Haldane's rule for sterility. Intrinsic isolation currently presents a much stronger obstacle to gene flow than behavioural isolation, and our results indicate that Trinidadian populations constitute a useful model for investigating incipient speciation.
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A burgeoning literature has accumulated over the past approximately 80 years that is dedicated to the cataloguing and description of barriers causing reproductive isolation, and it has provided many important insights into the mechanics of speciation (see Coyne & Orr, 2004 for review). For instance, we now know that multiple barriers typically isolate any given species pair (e.g. Ramsey et al., 2003), and that certain patterns characterize the evolution of these barriers. Notable patterns include Haldane's rule (Laurie, 1997) and the more rapid appearance of sterility over inviability problems in hybrids (e.g. Price & Bouvier, 2002). However, many fundamental issues remain unresolved.
One concerns the relative importance of intrinsic and prezygotic barriers in causing speciation. This issue is difficult to address because of the typically long timescale of speciation (e.g. see Coyne & Orr, 1989, 1997), which often obliges researchers to study taxa that have already speciated. Unfortunately, the resulting inferences may poorly reflect what actually happens during speciation (Presgraves, 2003; Reed & Markow, 2004). A particular problem with inter-specific studies is that they may suffer from an ascertainment bias that favours the detection of prezygotic over intrinsic barriers. This ascertainment bias becomes increasingly severe with time following speciation, as prezygotic isolation effects a greater proportional barrier to gene flow (Coyne & Orr, 2004).
Uncertainty over the relative importance of different barriers has been compounded by shortcomings in the treatment of intrinsic isolation. Crossing experiments have generally concentrated on a few groups (e.g. Lepidoptera and Drosophila), even though the tempo and mode of speciation may differ between groups. For example, it is possible that the genetic architecture of intrinsic isolation varies in parallel with the phylogenetic distribution of life-history traits (Rieseberg et al., 2000) or the importance of genomic imprinting (Reik & Walter, 1998; Vrana et al., 2000), meaning that the evolution of intrinsic isolation could show lineage-specific properties. In addition, crossing experiments have frequently failed to assay hybrid generations other than the F1 (Edmands, 2002; but see, e.g. Lu & Bernatchez, 1998), or components of intrinsic isolation other than physiological sterility and inviability. One component that is poorly understood is hybrid behavioural sterility, where hybrids fail to mate or reproduce because of a defect (neurological, pheromonal, etc.) that renders them incapable of effective courtship (Coyne & Orr, 2004). These problems exist despite the commonness of hybrid breakdown (Edmands, 2002) and an awareness that behavioural sterility can be important, at least between species. To our knowledge, behavioural sterility has not yet been assayed in intra-specific crosses.
Resolving such uncertainties is critical for a holistic understanding of speciation and requires further consolidation of the literature describing isolating traits. To this end, Trinidadian populations of Poecilia reticulata (the guppy; family Poeciliidae) may be of particular value as they show considerable adaptive genetic differentiation (Reznick et al., 1990), suggesting that they may serve as a model for the investigation of isolating barriers prior to speciation. Moreover, the large body of literature on Trinidadian guppies (reviewed in Houde, 1997 and Magurran, 2005) means that the evolution of reproductive barriers can be productively interpreted within specific ecological and genetic contexts.
Guppies are freshwater fish that are naturally distributed across the north-eastern corner of South America, including Trinidad and Tobago. They are notable for a number of reproductive traits, including internal fertilization, live-birth of young and the absence of any maternal provisioning of nutrients to eggs following fertilization (i.e. ovoviviparity) (Houde, 1997).
Phylogenetic studies using the mitochondrial control region have shown that populations from Trinidad's Oropuche Drainage form a monophyletic clade, which has diverged substantially from populations from Trinidad's Caroni Drainage (mean pairwise sequence divergence: 0.042) and from mainland South America (mean divergence: 0.034) (Alexander & Breden, 2004). So, although the Caroni and Oropuche drainages, located in Trindad's Northern Range (Fig. 1), are separated by as little as 70 m during the wet season (Magurran, 2005; p. 118), their populations are among the most genetically divergent currently known from throughout the guppy's natural distribution. This incongruity is difficult to explain because the region's geological history is highly complex (Donovan & Jackson, 1994). Carvalho et al. (1991; see also Fajen & Breden, 1992) have suggested that independent colonization of the Caroni and Oropuche drainages by different lineages from the Venezuelan mainland may be responsible. Unfortunately, no independent biogeographical or fossil evidence are available to aid calibration of the guppy molecular clock, and the divergence of the Caroni and Oropuche populations cannot be reliably dated. Complementing the phylogeographic data, allozyme studies have also found that, within Trinidad, most genetic diversity exists between the Caroni and Oropuche drainages (Carvalho et al., 1991).
Research on the Northern Range populations has dealt with the adaptive geographic differentiation seen in many morphological, behavioural and life-history traits. For example, male colour patterns (Endler, 1980), brood size (Reznick et al., 1990) and schooling propensity (Seghers, 1974) often show consistent and large differences between the upstream and downstream compartments of individual rivers, or between equivalent compartments of different rivers (e.g. Endler, 1980). Such differentiation often has a large hereditary basis (e.g. Reznick et al., 1990; Magurran & Seghers, 1994), and has been attributed, in part, to differences in predation regime and resource availability (reviewed in Endler, 1995; Arendt & Reznick, 2005; Magurran, 2005). However, the considerable phenotypic differences between populations are not paralleled by large changes in female mating preferences. Instead, behavioural isolation is uniformly slight and often statistically undetectable, even between populations from different drainages (Endler & Houde, 1995; Magurran et al., 1996). This discrepancy has led some authors to consider that ‘the divergence of guppies has not proceeded beyond a preliminary stage in speciation (Houde, 1997, p. 152)’, and to speculate whether certain factors may actually be impeding speciation (e.g. Magurran, 1998, 2001; see also Discussion).
Yet, in the absence of methodical studies of alternative isolating barriers, any conclusions about the nature of reproductive divergence in guppies are provisional. Little is known, for example, about intrinsic isolation. Several preliminary surveys have suggested that F1 hybrids between Caroni and Oropuche drainage populations are viable, but these studies assayed only obvious, easily scored traits in the F1 generation. These studies remain unpublished and are alluded to in Endler (1995), Endler & Houde (1995) and Houde (1997).
We crossed the Caroni and Oropuche drainage populations to systematically test for reductions in hybrid fertility and viability. We paid particular attention to behavioural sterility to establish whether it can evolve prior to speciation. We considered post-F1 generations in case hybrid breakdown existed, and to enable preliminary biometrical analyses that might suggest the existence of large-X effects.
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Figure 2. Male mating behaviours in the parental and reciprocal F1 lines. O = Oropuche, T = Tacarigua. Shared numbers indicate significant differences (P < 0.05) following Games–Howell post hoc multiple comparisons that were sequential Bonferroni-corrected (Rice, 1989). Sample sizes (n) for each line are: T = 28, O = 29, T × O = 31, O × T = 27.
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Female mortality contributed to roughly one half of the instances of cross failure (data not shown). Approximately 70–80% of attempted crosses yielded progeny, and there were no marked differences in cross success between lines (Table 1). However, brood sizes differed significantly between lines (ancova: F9,214 = 6.76, P < 0.001) (Fig. 3). Post-hoc multiple comparisons showed that these differences were restricted to comparisons between parental or F1 and post-F1 lines. For example, the Tacarigua control (P1) differed significantly from F2, BC2 and BC4 (P < 0.05). Similarly, F1R differed from all post-F1 hybrid lines apart from F2R (P < 0.05). Planned comparisons were also conducted between each of the (T × O) backcrosses into Tacarigua (BC1 and BC3) and the Tacarigua control (P1), but each comparison was significant (P < 0.05), meaning that any maternal effects on brood size could not be detected.
In relation to virgin female fecundity, ancovas detected no significant differences between lines in the weight or number of mature eggs (weight: F8,215 = 0.704, P ≫ 0.05; number: F8,259 = 0.831, P ≫ 0.05) (Table 2). Similarly, male fertility did not differ between groups when testes weight was assayed (ancova: F8,242 = 0.58, P > 0.05) (Table 2). To analyse the sperm count data, reciprocal crosses were pooled within hybrid lines to increase replicate sizes. An ancova on the data generated a significant statistic (F3,96 = 3.59, P < 0.05), and Bonferroni post hoc multiple comparisons showed that the F2 line had significantly reduced sperm counts relative to both parental lines and to the F1 population (P < 0.05) (Fig. 4).
Table 2. Descriptive statistics for male and female fertility.
|Line (♀ × ♂)||Female fecundity||Male fertility|
|No. mature eggs||Dry weight of eggs (mg)||Testes weight (mg)|
|Mean (n)||SE||Mean (n)||SE||Mean (n)||SE|
|T||3.20 (20)||0.38||5.16 (16)||0.65||0.59 (31)||0.04|
|O||5.74 (34)||0.52||6.92 (29)||1.26||0.58 (43)||0.02|
|T × O||4.77 (34)||0.55||5.29 (32)||0.64||0.55 (24)||0.02|
|O × T||4.39 (23)||0.71||4.88 (22)||0.59||0.61 (23)||0.04|
|(O × T) × (O × T)||4.91 (46)||0.43||6.47 (35)||1.04||0.53 (28)||0.03|
|(T × O) × (T × O)||4.96 (24)||0.66||4.72 (20)||1.30||0.61 (30)||0.02|
|(T × O) × T||4.59 (27)||0.52||6.80 (20)||1.22||0.55 (21)||0.03|
|(O × T) × T||5.14 (29)||0.57||5.39 (19)||0.68||0.48 (19)||0.04|
|T × (T × O)||4.8 (24)||0.58||5.79 (24)||1.26||0.58 (25)||0.03|
Figure 4. Sperm counts for pooled parental and hybrid lines. T = Tacarigua, O = Oropuche. Sample sizes (n) for each class are: T = 36, O = 21, F1 = 23, F2 = 18.
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Juvenile survival did not differ between lines, as judged by the likelihood ratio test implemented in betabino (Jiggins et al., 2001) (L9,197 = 11.929, P ≫ 0.05) (Table 3). But embryo viability did show significant differences (Kruskal-Wallis H test: = 30.10, P < 0.001) (Table 3). Games–Howell post hoc tests showed that the pooled backcross class differed from each of the P1, P2, and pooled F1 and F2 classes (P < 0.01). Maternal effects on embryo viability were again tested for using planned comparisons that involved both the (T × O) and (O × T) backcrosses into Tacarigua – all contrasts were nonsignificant (P > 0.05), making any such effects undetectable.
Table 3. Percentiles for juvenile survival and embryonic inviability.
|Line (♀ × ♂)||Juvenile survival (proportion of offspring from a brood surviving to maturity)||Embryo inviability (no. dead embryos per mother)|
|n||Median||Interquartile range||n||Median||75th + 90th percentiles|
|T||24||1.00||0.88–1.00||20||0||0 + 1.0|
|O||21||1.00||0.91–1.00||20||0||0 + 1.0|
|T × O||24||1.00||0.93–1.00||40||0||0 + 1.8|
|O × T||27||0.96||0.84–1.00|| || || |
|(O × T) × (O × T)||20||1.00||0.85–1.00||40||0||0 + 0|
|(T × O) × (T × O)||20||1.00||1.00–1.00|| || || |
|(T × O) × T||21||0.86||0.65–1.00||121||0||3 + 7.9|
|(O × T) × T||18||0.94||0.65–1.00|| || || |
|T × (T × O)||22||1.00||0.75–1.00|| || || |
Finally, neither adult brood sex ratios [generalized linear models (GLM) with brood size as WLS weight: F8,195 = 1.69, P > 0.05] nor male or female body condition (males: ancova: F8,242 = 0.74, P ≫ 0.05; females: ancova: F8,215 = 1.39, P > 0.05) differed between lines.