Rapid sympatric ecological differentiation of crater lake cichlid fishes within historic times
© Elmer et al; licensee BioMed Central Ltd. 2010
Received: 15 December 2009
Accepted: 12 May 2010
Published: 12 May 2010
The Erratum to this article has been published in BMC Biology 2012 10:70
After a volcano erupts, a lake may form in the cooled crater and become an isolated aquatic ecosystem. This makes fishes in crater lakes informative for understanding sympatric evolution and ecological diversification in barren environments. From a geological and limnological perspective, such research offers insight about the process of crater lake ecosystem establishment and speciation. In the present study we use genetic and coalescence approaches to infer the colonization history of Midas cichlid fishes (Amphilophus cf. citrinellus) that inhabit a very young crater lake in Nicaragua-the ca. 1800 year-old Lake Apoyeque. This lake holds two sympatric, endemic morphs of Midas cichlid: one with large, hypertrophied lips (~20% of the total population) and another with thin lips. Here we test the associated ecological, morphological and genetic diversification of these two morphs and their potential to represent incipient speciation.
Gene coalescence analyses [11 microsatellite loci and mitochondrial DNA (mtDNA) sequences] suggest that crater lake Apoyeque was colonized in a single event from the large neighbouring great lake Managua only about 100 years ago. This founding in historic times is also reflected in the extremely low nuclear and mitochondrial genetic diversity in Apoyeque. We found that sympatric adult thin- and thick-lipped fishes occupy distinct ecological trophic niches. Diet, body shape, head width, pharyngeal jaw size and shape and stable isotope values all differ significantly between the two lip-morphs. The eco-morphological features pharyngeal jaw shape, body shape, stomach contents and stable isotopes (δ15N) all show a bimodal distribution of traits, which is compatible with the expectations of an initial stage of ecological speciation under disruptive selection. Genetic differentiation between the thin- and thick-lipped population is weak at mtDNA sequence (FST = 0.018) and absent at nuclear microsatellite loci (FST < 0.001).
This study provides empirical evidence of eco-morphological differentiation occurring very quickly after the colonization of a new and vacant habitat. Exceptionally low levels of neutral genetic diversity and inference from coalescence indicates that the Midas cichlid population in Apoyeque is much younger (ca. 100 years or generations old) than the crater itself (ca. 1 800 years old). This suggests either that the crater remained empty for many hundreds of years after its formation or that remnant volcanic activity prevented the establishment of a stable fish population during the early life of the crater lake. Based on our findings of eco-morphological variation in the Apoyeque Midas cichlids, and known patterns of adaptation in Midas cichlids in general, we suggest that this population may be in a very early stage of speciation (incipient species), promoted by disruptive selection and ecological diversification.
Since Darwin and Wallace, the study of island inhabitants has greatly influenced evolutionary and ecological research . In many respects, crater lakes are the aquatic equivalent to islands. Like islands, the small size, distinct boundaries, simplified biota, young age, geographical isolation and frequently well-known geological history of crater lakes makes them well-suited to study the diversification of sister taxa [1–3]. The physical isolation of crater lakes means that after the first seeding by a colonizing lineage evolution may proceed rapidly in situ. Indeed, crater lakes have provided the most compelling examples of sympatric ecological speciation of fishes, particularly cichlid fishes [4–7]. Intralacustrine speciation in depauperate habitats (for example, postglacial lakes and crater lakes) typically proceeds due to disruptive ecological selection , though the rapidity, strength and completeness of this process depends on a number of population and environmental factors [5, 9–11]. Well-known examples of this phenomenon include the benthic and planktivorous populations of sticklebacks [12, 13], introduced salmon populations  and whitefish [15, 16] in isolated lakes.
Cichlid fishes are pre-eminent non-model organisms for the study of ecological speciation because of their trophic polymorphism and rapid evolution [17, 18]. Mesoamerican fishes of the Midas cichlid species complex (the Amphilophus citrinellus species group) are particularly variable in trophic characteristics such as body shape and pharyngeal jaw morphology, as well as maintaining a striking colour polymorphism [19–21] (for a review of the crater lakes and species complex see ). One of the most renowned examples of ecological differentiation in the species complex is the elongate, open-water species Amphilophus zaliosus, which evolved from a high-bodied benthic ancestor by sympatric speciation within Nicaragua's oldest crater lake, Lake Apoyo [4, 20]. A similarly elongate but evolutionarily independent endemic species is also found in crater lake Xiloá [22–25]. An additional trophic polymorphism of Midas cichlids is fleshy, hypertrophied lips, which is believed to be involved in harvesting invertebrates from between cracks and recesses . This character is absent from most crater lake Midas cichlid populations [20, 22, 26] and is best known from Amphilophus labiatus, a species that occurs in the two largest and shallowest lakes in the region, the great lakes Managua and Nicaragua . Apoyeque is the only crater lake that harbours a sizeable population of Midas cichlids with hypertrophied lips; the origin and diversity of this population is the focus of our current study.
Despite the crater lake Apoyeque's young age and isolation, it is home to an abundant population of Midas cichlids (Amphilophus cf. citrinellus). There are two phenotypes living in the lake: one with normal citrinellus thin lips ('thin-lipped') and one with pronounced fleshy, or hypertrophied, lips ('thick-lipped'; Figure 1). The lake is otherwise depauperate in species, only housing some small live-bearing fishes (Family Poeciliidae) and, less abundantly, the predatory cichlid Parachromis managuense .
In the present study, we test whether the two phenotypes (morphs) of Midas cichlid in Apoyeque originated by sympatric differentiation promoted by ecological divergence. Given the intralacustrine diversification of Midas cichlids in other crater lakes, we hypothesize that this trait of thin- or thick- lips may be a novel or alternative axis of ecological differentiation exploited by crater lake cichlids. First, we infer the colonization history of crater lake Apoyeque using population genetic and coalescent approaches, in the context of neighbouring lakes Managua and Xiloá. We specifically assess evidence for a single or multiple colonization events and see whether the age of the population corresponds to the geological age of the lake. Second, we compare ecological (diet, trophic level and pharyngeal jaw shape) and morphological (body shape, head and body size) variation between thin- and thick-lipped fishes to see whether this character is associated with distinct niches, sex or maturity, or morphological difference. Third, we assess the role of disruptive natural selection in driving incipient speciation by comparing the distribution of phenotypes and genetic differentiation in Apoyeque's Midas cichlid population, with the expectation that ecological diversification should result in a bimodal distribution of ecologically relevant traits.
Colonization and population genetics of Apoyeque and neighbouring lakes
Genetic diversity and differentiation
Genetic diversity for Midas cichlid populations in three lakes: haplotype richness for mtDNA; observed heterozygosity, allelic richness, and private alleles for microsatellite markers (means ± standard deviations).
Crater Lake Apoyeque
Crater Lake Xiloá
Great Lake Managua
0.632 ± 0.176
0.727 ± 0.214
0.785 ± 0.151
5.97 ± 2.97
8.55 ± 3.71
11.24 ± 4.70
Private allelic richness
0.540 ± 0.532
1.14 ± 0.785
3.37 ± 1.83
The pronounced lack of microsatellite genetic variation within the Apoyeque Midas cichlid population, and its genetic distinctiveness from neighbouring lakes, can be visualized in the separation along axis 1 and the tight clustering of Apoyeque individuals in the Factorial Correspondence Analysis (Additional File 2). Midas cichlid fish populations in lakes Apoyeque, Xiloá and Managua are all significantly genetically differentiated from each other at mtDNA (FST = 0.072 - 0.262) and microsatellite loci (FST = 0.053 - 0.146; Additional File 3).
Coalescence inference of origin and time of colonization
Apoyeque could only have reasonably been colonized from either of its neighbouring lakes, Managua or Xiloá: all other lakes are geographically much more distant (Figure 1). Inferred from gene flow analyses in Migrate[32, 33] (here a proxy for likely colonization), Lake Managua is the primary ancestral population of Apoyeque: migration from Managua to Apoyeque was estimated to be two to four times higher than the rate of migration from Xiloá to Apoyeque. Based on isolation-with-migration coalescence analyses (IMa), the divergence time between the populations of Apoyeque and its founding population from lake Managua (approximately equivalent to the time since colonization) was estimated to be about 100 years ago (89 years, 63 years and 132 years in three independent iterations; see Additional File 4 for likelihood values of all parameters). The effective population size of Apoyeque was estimated to be approximately 1000 times smaller than that of the ancestral population in great lake Managua (Additional File 4).
Apoyeque's extant Midas cichlid population probably originated from a single founding event. Historical migration (coalescence of genes from Managua to Apoyeque) was estimated to be negligible (~10 migrations/thousand generations) and there was a single probability peak for population divergence in the IMa analysis (Additional File 4).
Eco-morphological variation within Apoyeque
Frequency of thin- and thick-lipped individuals
We collected exemplars of thin-and thick-lipped mature adults and juvenile fishes in Apoyeque. Twenty percent of the adult individuals were thick-lipped (42 of 214 sexed adults) and 24% of the juveniles were thick-lipped (19 of 80 specimens classified by dissection as juvenile). Additionally, 15 thick-lipped and 86 thin-lipped specimens were not classified to age or sex (see Methods and Additional File 5). There is no difference in the number of thick-lipped fishes in any of the four groupings (female, male, juvenile and not noted; G2 = 5.15, df = 3, P = 0.161). Thus, from our total collection 19% of fishes were thick-lipped and 81% were thin-lipped.
Body shape and size
The body size (standard length) of adult thin- and thick-lipped Midas cichlid fishes did not differ (two factor ANOVA, morph effect, F1,202 = 0.652, P = 0.42; Additional File 6).
Specifically focusing on head morphology, head width at the eyes was different for thin- and thick-lipped fishes (ANCOVA on log-transformed head width with log-transformed standard length as a covariate, morph effect, F1,166 = 7.09, P = 0.008; sex effect, F1,166 = 0.144, P = 0.71; morph × sex interaction, F1,166 = 0.857, P = 0.36). The two morphs had similar head widths at the snout (posterior point of the mouth: ANCOVA, morph effect, F1,166 = 0.051, P = 0.82; sex effect, F1,166 = 0.557, P = 0.46; morph × sex interaction, F1,166 = 1.62, P = 0.21; the nostrils: ANCOVA, morph effect, F1,166 = 0.006, P = 0.94; sex effect, F1,166 = 0.377, P = 0.54; morph × sex interaction, F1,166 = 0.275, P = 0.60). In other words, the snout width of thin- and thick-lipped fish does not differ but the head is thinner and more tapered in the thick-lipped fishes.
Lip size did not differ between sexes (ANCOVA on log-transformed lip size with log-transformed standard length as a covariate, F1,203 = 0.158, P = 0.69). Thus, the extent of lip hypertrophy is not related to sex. However, adults (sexed males and females) had larger lips relative to their body size than juveniles (ANCOVA, F1,280 = 4.91, P = 0.028). Indeed, lip size clearly increased with body size (ANCOVA F1,280 = 179, P < 0.001). A plot of lip size by body length (including adult, juvenile and not noted specimens) demonstrates how the increase of lip size differs between the two morphs (Additional File 7; there is considerable overlap between thin- and thick-lipped fishes because we measured total lip area, not exclusively the protruding lip). The quadratic lines of best-fit differ markedly between thin- and thick-lipped fishes: the y-intercept is higher and the slope steeper in thick-lipped fishes. Thus lips get proportionally larger with maturity, age or size.
Muscle tissue of adult thin- and thick-lipped fishes had different carbon (∂13C) and nitrogen (∂15N) signatures (non-parametric MANOVA: F = 13.0, P < 0.001) (Figure 4c). Thin-lipped fishes were typically slightly 13C depleted (median ± IQR = -7.4 ± 0.85 ‰) compared to thick-lipped fishes (-7.1 ± 0.95 ‰) and had higher ∂15N values (10.2 ± 0.7 ‰ versus 9.4 ± 0.7 ‰), suggesting that, on average, thin-lipped fishes fed at a slightly higher trophic level.
Lower pharyngeal jaws
Disruptive selection and phenotypic differentiation
Statistical support for whether the distribution of phenotypes for eco-morphological traits is composed of one or two normal distributions.
AICc for unimodal
AICc for bimodal
P < 0.0001
Bimodal (strong support)
P < 0.0001
Bimodal (strong support)
P < 0.0002
Bimodal (strong support)
P < 0.0001
Bimodal (strong support)
Stable isotope (13C)
P < 0.0008
Unimodal = bimodal
Stable isotope (15N)
P < 0.0556
Bimodal (moderate support)
Genetic differentiation within Apoyeque
There was no genetic differentiation at microsatellite loci between the population of thin- and thick-lipped fishes in Apoyeque (FST < 0.001, P = 0.360). There was genetic differentiation in mtDNA haplotypes between the population of thin- and thick lipped fishes (FST = 0.018, P = 0.064), though it is not strongly statistically significant.
Rapid intralacustrine eco-morphological differentiation
Analyses of ecologically relevant traits indicate that thick- and thin-lipped fishes occupy two distinct niches in sympatry within the small and extremely young crater lake Apoyeque. The two morphs have a different diet, as reflected by Schoener's index (0.59) and a clustering into separate multi-dimensional diet space (Figure 4b). Thick-lipped fishes tend to have a diet richer in benthic crustacea and insect prey (Figure 4a). They are also enriched in δ13C and depleted in δ15N stable isotope values compared to thin-lipped fishes (Figure 4c), which may suggest a greater use of littoral prey . The pharyngeal jaws of thick-lipped fishes are significantly thinner in shape, i.e. characteristically more papilliform (Figure 5). Papilliform-jawed Midas cichlids in other crater lakes are more insectivorous [4, 37, 38], as they also tend to be in Apoyeque, reflecting the established correlation between diet and jaw in cichlids. In contrast, thin-lipped fishes tend to be more molariform, with thicker jaws and broader teeth (Figure 5). Molariform jaw morphology is associated with more efficient snail crushing and a benthic diet in Midas cichlids [38, 39] and cichlids generally , and also here in Apoyeque. We found that the shape of pharyngeal jaws was significantly affected by jaw size, which may support the hypothesis that pharyngeal jaws change shape over time depending on diet .
Lip hypertrophy is found in males and females at similar frequencies and the relative size of the lips does not differ between the sexes. Hence, lip hypertrophy is not a sex-linked character. Nor is lip hypertrophy a character that develops only after maturity: it was found at similar frequencies in adults and juveniles. However, lip size increases with body length, indicating that the hypertrophy may get more pronounced with age. The relationship of increasing lip size with body size is not the same for thin- and thick-lipped fishes. Instead, thick-lipped fishes show a much more dramatic increase of lip size with body size (Additional File 7). Therefore, it would be interesting to build upon our research by studying trophic variation at across life history stages, a possible role of plasticity or feedback between ecological niche and lip hypertrophy, and genetic influences on lip hypertrophy.
Although ecological differentiation between lip morphs is clear, overall body shape (Figure 3) and head widths differ between lip morphs only subtly, albeit significantly. Indeed, the body shape difference between other Midas cichlid species, such as syntopic thin-lipped A. citrinellus and thick-lipped A. labiatus in the great lakes Managua and Nicaragua, is much more pronounced [22, 41] than what we found between the orthologous thin- and thick-lipped morphs in Apoyeque. In this regard, it is important to note that the thin- and thick-lipped Midas cichlids in Apoyeque are morphologically, genetically and geographically distinct from the Midas cichlids in lakes Managua and Nicaragua (this study and ), indicating that they are not simply allotopic populations of A. citrinellus and A. labiatus.
Disruptive selection in sympatry is expected to result in a bimodal distribution of quantitative eco-morphological traits. This is because intraspecific competition and negative frequency dependent selection result in higher fitness of extreme over intermediate phenotypes (reviewed in ). Such a pattern has been demonstrated under ecological speciation in nature but is notoriously difficult to quantitate (for example [43–46]). In Apoyeque Midas cichlids, lip size and a variety of eco-morphological traits that are reflective of trophic niche-body shape, pharyngeal jaw shape, stomach contents and δ15N-are all found at frequencies predicted under frequency-dependent fitness and disruptive selection. Specifically, these traits better fit a bimodal rather than unimodal distribution (Table 2, Additional File 8). Secondary contact could also result in a bimodal distribution of phenotype, but cichlid ecology, a single colonization event and no genetic evidence for secondary admixture (discussed below), suggests this is not the case in Apoyeque. Extensive empirical and theoretical research suggests that disruptive selection can drive multimodal eco-morphological differentiation and, with even partial non-random mating, result in incipient adaptive speciation [42, 47, 48].
Despite the bimodality of phenotype, we find no equivalent differentiation in nuclear genetic markers between thin- and thick-lipped Midas cichlids in Apoyeque (microsatellite FST < 0.001, P = 0.360). This absence of differentiation may be due to very low population genetic polymorphism, a young population with younger divergence, or weak or absent assortative mating; field observations will be necessary. However, we do find population differentiation in the mtDNA haplotypes (FST = 0.018, P = 0.064). While marginally statistically non-significant, this represents a substantial sympatric genetic differentiation within Apoyeque based on eco-morphology. To put it in context, this is one-third of the differentiation found among allopatric Midas cichlid populations of lakes Managua vs. Xiloá (FST = 0.053, P < 0.001, Additional File 3). More striking is that the mtDNA divergence between morphs within Apoyeque exceeds that of the different thin- and thick-lipped species, A. citrinellus and A. labiatus, in the great lake Nicaragua (FST = 0.004, P = non-significant  also based on mtDNA control region). It is presently unclear what processes are at work that would result in panmixia at nuclear loci and differentiation at mtDNA in this young, sympatric, genetically depauperate Midas cichlid population and this should be the focus of future research.
Our finding of eco-morphological differentiation with low genetic differentiation could suggest that lip morph is plasticity, polymorphism or incipient speciation. We discuss these alternatives below.
Midas cichlid species and ecological traits
Hypertrophied lips are an overt species-specific character related to ecology in cichlids . It has evolved independently in different environments and species complexes of various New World (for example ) and Old World cichlids ( and references therein; [17, 52, 53]). It is considered to be a diagnostic trait: among the scores of described cichlid species (1611 Cichlidae in FishBase.org, 5 November 2009), we know of none for which the character of hypertrophied lips is a described intraspecific polymorphism (though there is debate about Crenicichla iguassuensis, see [54, 55]).
For example, the Nicaraguan great lake Midas cichlid species A. citrinellus and A. labiatus are discerned primarily by lip morphology, although they also differ in head shape, snout angle, feeding behaviour, anterior teeth, fin position and length [20, 56], pharyngeal jaws [24, 41] and body shape [22, 41]-much like the two morphs of Midas cichlid in Apoyeque. In contrast to these differences, some meristic values overlap between A. citrinellus and A. labiatus and the extent of lip hypertrophy varies within populations . Further, neutral genetic differentiation between those two species is low or absent . This is a conundrum because young eco-morphological variants (for example, benthic or limnetic species) in various crater lakes can be discerned genetically (for example [4, 25, 57]). Thus, early researchers of Nicaragua's cichlids vacillated on the issue of polymorphism versus speciation but concluded that the weigh of evidence argued for two Midas cichlid species in the great lakes, A. citrinellus and A. labiatus [20, 56] and all currently published research proceeds under the assumption that they are different species. In the laboratory, A. labiatus mate assortatively and resultant offspring have thick lips (KRE and AM unpublished data), suggesting that this trait is involved in sexual isolation by assortative mating and has a strong genetic component. Mate choice experiments between A. citrinellus and A. labiatus have, to our knowledge, never been published. Certainly, further research is needed on 'lippiness' and this is a research topic that we are currently undertaking from ecological, behavioural and genetic perspectives.
Therefore, although at present we cannot completely rule out that being thin- or thick-lipped is an intrapopulation polymorphism or plasticity in Apoyeque, we consider it quite unlikely. Instead, we suggest that the ecological differentiation between thick- and thin-lipped eco-morphs in Apoyeque represents a case of ecologically distinct, sympatric incipient sister species. Moreover, lips may be one of the very few examples for a 'magic trait' -a trait that combines disruptive natural selection and assortative mating and that also (sensu ) might lead to speciation in sympatry. Low or absent genetic differentiation between morphs does not contradict incipient ecological speciation because it is an overly conservative estimator  and neither microsatellites nor mtDNA sequences robustly differentiate other species of Midas cichlid that differ in lip character . It may be that the same ecological, genetic or behavioural factors responsible for low meristic and genetic divergence between A. citrinellus and A. labiatus in Nicaragua's great lakes are also at play in Apoyeque. Further research on the association of eco-morphological divergence, such as lip hypertrophy, with reproductive isolation is needed in all Midas cichlids.
In other Nicaraguan crater lakes, intralacustrine divergence of Midas cichlids has occurred with a different set of ecological traits-primarily body elongation . In some cases this progressed to full species status with phenotypic, meristic and molecular divergence, such as in lakes Apoyo [4, 57] and Xiloá [25, 61]. Though thick-lipped Midas cichlids are rather common in Apoyeque (~20% of the population), they are found only sporadically in crater lake Masaya (personal observation, KRE, AM and ) and extremely rarely in crater lake Xiloá (personal observation, KRE, TKL and AM and [20, 26]), and are believed to be completely absent in many crater lakes (for example, Apoyo, Asososca Managua and Asososca León). This suggests that hypertrophied lips is not the ancestral state in this species complex and instead may only arise under particular ecological conditions and genetic backgrounds.
Crater lake colonization
The dearth of genetic variability sampled in Apoyeque Midas cichlids is consistent with coalescence analyses that indicated a very recent founding of Apoyeque (approximately 100 years or generations ago) by a single colonization event. The coalescent methods we employed here have been used to infer the age of African cichlid species because the recent speciation in many cichlid species hinders lineage-based phylogenetic methods [62, 63]. Even if this method underestimates the colonization time, all geological and genetic evidence indicate that the Apoyeque population is extremely young (that is, maximally 1800 years old) and the most genetically depauperate of all the crater lake Midas cichlid populations .
How a high walled crater lake could be colonized by fishes is a tantalizing question for history. Some possibilities include being transported by birds (for example, ospreys, which fly moderate distances with fish in their talons), momentary subterranean aquatic connections caused by volcanic and tectonic movements or being flung in by hurricanes or funnel winds with water transfer between neighbouring lakes; spectacular though these phenomena seem, they have been reported scientifically for many years (reviewed in [64, 65]). It is also possible that the population of fish in Apoyeque was seeded by humans, as is proposed for other lakes , but this seems less plausible because the lake is quite difficult to access and is not located next to any human settlement. The presence of a large population of small poeciliid fish in Apoyeque, which would not likely be purposely introduced for food or sport, argues that natural colonizations may be more likely than human introductions. Regardless of the historical mode of arrival, our data suggest that the extant population of Midas cichlid fishes colonized Apoyeque only once and very recently.
Is crater lake colonization rare? If crater lake Apoyeque is about 1800 years old, but the Midas cichlid population within is only about 100 years old, did the lake stand devoid of fish all the time in between? One possibility is that cycles of extinction and re-colonization may be relatively frequent in the early life of a crater lake. Occurrences such as remnant volcanic activity, fumerole activity  or periodic gas releases and rising of toxic waters [68, 69] may exterminate a crater lake's fish fauna. For example, locals of the area report that Apoyeque occasionally 'boils' and 'all' of the fish die (personal communication to KRE). Such a fish kill was observed in January 2008 in Lake Masaya, though clearly not all fish died (personal observation, TKL). In that case, Apoyeque may have been colonized previously, followed by an extinction event, and the extant population only arrived within the past 100 years. Additional comparative phylogeographic investigations across lakes and species will help in addressing these questions, but ultimately a crater lake's colonization history may remain speculative.
Founder events, novel habitats and speciation
Nicaraguan crater lake environments, being small, deep, clear, mineral-rich isolated waters, are very different from the large, shallow, turbid waters of the ancestral great lakes. This represents a major shift in ecological niche for Midas cichlids and may allow for the rapid evolution of new phenotypes [70, 71]. To date, we have largely relied on models to suggest how long phenotypic divergence and speciation may take after colonisation of a new environment. Those estimates vary greatly: some model suggest 10 000 generations may be sufficient for the emergence of ecologically differentiated species under favourable conditions [5, 72], or possibly much faster [60, 73]. Based on field collected data, especially for fish, other authors [14–16, 74–76] argue that ecological speciation or morphological diversification can occur on timescales of only tens to hundreds of generations. Here, we have demonstrated that ecological divergence can begin in sympatry on historic time scales of a minimum of 100 years.
The results of our study suggest: (i) a case of ecological and morphological differentiation of sympatric Midas cichlids to exploit divergent niches, with associated weak population divergence at mtDNA; and (ii) that this eco-morphological divergence occurred very quickly after colonization of a crater lake habitat. Midas cichlids in Apoyeque are geographically and genetically isolated from neighbouring populations, and morphologically distinctive  and, thus, evolutionarily independent from other populations. Whether thin- and thick-lipped Midas cichlids in crater lake Apoyeque are a single polymorphic species or two incipient species cannot yet be determined with certainty but, based on cichlid biology in general and patterns of genetic and eco-morphological variation in Apoyeque in particular, we suggest that thin- and thick-lipped fishes may be sympatric incipient species.
Specimens were collected in November and December 2007 using gill nets in lakes Apoyeque (12.15°N, 86.20°W) and Xiloá (12.13°N, 86.19°W) (Figure 1). Specimens from Managua were collected previously by AM and M Barluenga. Fish were digitally photographed in a standardized position. Maturity and sex were determined by dissection for a subset of specimens (see Additional File 5). Tissue and voucher specimens (whole fish or head) were collected for every specimen and stored in ethanol. Stomachs that held contents were collected and stored in ethanol. Apoyeque fishes were classified as having normal lips ('thin-lipped') or hypertrophied lips (if the upper lip protruded forward beyond the snout profile; 'thick-lipped') based on standardized photos (Figure 1). Although there is variation in the extent of hypertrophy of the 'thick-lipped' lips (within Apoyeque and across the Midas species complex [20, 66]), the great majority of individuals were readily assignable.
A subset of adult specimens from Apoyeque was used to assess variation in pharyngeal jaw shape, body size, head width, and body shape (thick-lipped ♀ n = 23, thick-lipped ♂ n = 13, thin-lipped ♀ = 66, thin-lipped ♂ n = 69; Additional File 5). A more extensive group of specimens was used for lip size covariate analysis (pooled for the two morphs: ♀ n = 106, ♂ n = 100, juveniles n = 77) and the plot of lip size by body size (thin-lipped n = 309, thick-lipped n = 71; Additional File 5, Additional File 6, Additional File 7). The final microsatellite data set comprised: Apoyeque thin-lipped n = 317, Apoyeque thick-lipped n = 76, Xiloá n = 50, Managua n = 185 (Additional File 5). The mtDNA data set comprised: Apoyeque thin-lipped n = 232, Apoyeque thick-lipped n = 58, Xiloá n = 29, Managua n = 70 new or resequenced (Additional File 5). Juvenile and 'not noted' fishes were included in genetic analyses and the lip size plot but were not analysed for eco-morphological variation in order to avoid confounding effects of developmental stage on niche and body shape.
Body size and shape variation
A single observer measured head width from voucher specimens using digital callipers at: (1) posterior point of mouth; (2) nostrils; and (3) posterior point of eye. Standard body length and the volume of the lower and upper lip (lip size) were measured in ImageJ v 1.41o . In order to test the influence of sex or maturity on lip size, the effect of body size was standardized by including standard length as a covariate in the lip size analyses. Initially, we included the full model using log-transformed body size and lip size values. After the interaction term was found to be non-significant, it was removed and we proceeded testing for the main effects.
Thirteen landmarks to describe body shape were digitized from standardized photographs in TPSDig version 2.12  (Figure 2) and analysed with classifier variables (sex, lip morph) in MorphoJ version 1.00j  using least-squares Procrustes superimposition  and geometric morphometric methods. Body shape analyses have been described elsewhere . There was no significant influence of body size on body shape in any group (thick-lipped ♀ P = 0.76, thick-lipped ♂ P = 1.0, thin-lipped ♀ P = 0.62, thin-lipped ♂ P = 0.19; 10 000 permutations) so no allometric correction was applied. Body shapes of males and females do not differ (Procrustes distance = 0.008, P = 0.12) so sexes were subsequently combined.
Ecological niche assessment
Lower pharyngeal jaws were extracted and cleaned in a solution of 10% trypsin for 17 h before being imaged using a desktop scanner. Jaws are an approximately symmetrical structure  so only one side was used to infer shape. Ten landmarks (approximately following ) were digitally assigned using TPSDig (Figure 5a). Jaw shape was analyzed in MorphoJ by the same approaches used for body shape. Male and female jaws do not differ in shape (Procrustes = 0.006, P = 0.79: allometry corrected) so sexes were subsequently combined.
Gut contents were categorized under a stereoscope (observer blind to fish's morph) for groupings benthic crustacea, plants and seeds, fish and fish parts, snail shells, insect pieces and larvae, brown algae, white algae [determined by polymerase chain reaction (PCR) to be Oomycota]. Volume of each category was determined by comparing it to a microlitre standard after 30 s centrifugation . In order to test whether the two morphs differed in diet, we applied a generalized linear mixed model. Volume was used as a response variable with food category and morph as fixed effects. Identity code of each individual was entered as a random effect. The model was fitted using maximum likelihood and a likelihood ratio (G 2 ) test was used to calculate the P-value for the interaction term between food category and morph. In order to improve the normality of the residual errors, the food volume values were log + 1 transformed prior to the analysis. In addition, we calculated Schoener's index of diet difference  and used monotonic multidimensional scaling (MDS) with Guttman loss function to scale pair-wise proportional similarities in gut content into a two-dimensional plot. Digested, unrecognizable material in the guts was excluded from all diet analyses.
Carbon (∂13C) and nitrogen (∂15N) stable isotope values were determined following . Values of ∂13C were normalized for lipid content . Non-parametric MANOVA in Past was used to compare groups statistically.
Normality of each eco-morphological trait was assessed by plotting and observing a frequency histogram of values and normal probability plots (using raw data, PC1 or MDS1; Additional File 8) and the statistical test of goodness-of-fit to a normal distribution was calculated by Shapiro-Wilk W in JMP version 7 . For all non-normally distributed traits, we tested whether the plots are best fit by a single normal distribution or a bimodal distribution using a two-component normal mixture model in discmixtureprogs version 0.5 mix2 http://www.bioss.ac.uk/~markb/mixtures/. This method uses numerical integration to calculate mean, variance and proportion for each distribution by integration over a posterior density . Bayesian mixture models were run for 500 iterations (bins) and convergence assessed by stabilized trace plots and a posterior distribution of theda and sigma values that started at, and returned to, zero or near-zero (not shown). The fit of a single-component model and a two-component model to our data was calculated with AICs (second-order, or corrected for sample size). The difference between models (ΔAICc) was calculated from the AICc for a single unimodal distribution minus the AICc for a two-component distribution (reviewed in [88, 89]). We interpreted ΔAICc values following established guidelines: 0-5 are equivalent support for either model, 5-8 is moderate support for one model over the other, >8 is strong support for one model over the other. A negative ΔAICc value supports a single normal distribution and positive ΔAICc value supports a two-component distribution over a single-component [44, 89].
Mitochondrial DNA sequencing and analysis
Samples were sequenced for mtDNA using standard methods described elsewhere . Diversities were calculated in Contrib version 1.02  rarefied to the smallest sample size (Xiloá, n = 29). Sequences were collapsed into haplotypes ignoring missing data in DnaSP version 4 . FST estimates were calculated in Arlequin version 3.11  using a Tamura  + gamma (unequal base frequencies, unequal substitution rates, alpha = 0.74) corrected distance matrix to approximate the best model of evolution that was inferred to be HKY  + I + G from Modeltest version 3.7  in PAUP* version 4 . Statistical power was determined by a 10 000 step Markov Chain method. Sequences from Apoyeque were searched in on-line nucleotide BLAST http://blast.ncbi.nlm.nih.gov/ for identical sequences (Identity = 100%) in the nr/nt database.
Using all newly determined sequences and all Midas cichlid complete control region sequences from nearby lakes Xiloá, Nicaragua, Managua, and Tisma Pond available in Genbank (n = 459; as of 19 Jan. 2009), a median-joining haplotype network  was calculated in Network version 188.8.131.52 http://www.fluxus-engineering.com. Transversions tend to be rarer in mitochondrial DNA and were, therefore, weighted three-times transitions. The MJ network was maximum parsimony post-processed to display the shortest tree compatible with the predictions of coalescence . In order to simplify visualization, singleton haplotypes are not drawn in the network, except from Apoyeque.
Microsatellite genotyping and analyses
Eleven microsatellite loci were amplified using standard PCR conditions: Abur151, Abur82 , M1M(=Acit1), M2(=Acit2), M7(=Acit3), M12(=Acit4) , Unh002 , Unh012, Unh013 , Burtkit F 474/R672  and TmoM7 . Fluorescently labelled fragments were analysed on an ABI3130XL (Applied Biosystems, CA, USA) and sized according to Rox 500 internal standard in GeneMapper version 4.0 (Applied Biosystems).
Genotyping quality for each population was assured by testing in Micro-Checker with 5 000 randomizations and a 95% confidence interval. Any influence of null alleles was always low (Brookfield 1  < 0.06), suspected in only two or fewer loci and with no consistent deviation across loci or populations, so all loci were retained. Expected heterozygosity, inbreeding coefficient FIS and inter-population FST were calculated in Microsatellite Analyzer. Rarefied allelic richness and private allelic richness (standardized to the lowest number of alleles)  were calculated in HP-Rare version 1.0 . Hardy Weinberg exact tests and linkage disequilibrium were calculated in Genepop '007 [109, 110]. Multiple tests were Bonferroni corrected. Genetic variation was analysed in a two-dimensional individual-based factorial correspondence analysis in Genetix.
The population (Managua or Xiloá) that founded Apoyeque was estimated from the microsatellite dataset using Migrate version 2.4.3 [32, 33]. We applied the Bayesian inference  and restricted Apoyeque gene flow to immigration. Apoyeque was reduced to n = 200 to decrease disparity between population sample sizes, as such disparity can cause problems for parameter convergence. We applied a static heating scheme of four chains with the temperatures 10 000.00, 3.00, 1.50 and 1.00. Mutation rates varied between loci and were estimated from the data. Three independent runs were conducted, each consisting of 4 000 000 MCMC steps and a burn-in period of 100 000 steps. Albeit absolute posterior parameter estimates deviated slightly due to the stochastic nature of the coalescence analyses, all runs resulted in similar estimates. This suggests adequate convergence and a good match between the data and the models.
Divergence time between the founding population, Managua, and Apoyeque was calculated in IMa[113, 114] using all mtDNA and microsatellite data combined. We used a geographic heating scheme with 60 concurrent chains and applied values of 0.9 and 0.95 for the heating parameters g1 and g2 to achieve adequate swapping among chains (> 0.4 even for low numbered chains). Each run began with a different random number seed and ran for 1 000 000 steps following a burn-in period of 250 000 steps. Mutation rate is estimated at 5 × 10-4 per locus per year . Migration rates and population sizes were restricted based on priors that generated generally well-behaved posterior distributions in optimizing runs (q1 1, q2 10, qA 200, m1 100, m2 5). We initially restricted divergence time to 2 000 year (t 1) to match Apoyeque's geological age but this resulted in an unstable peak at the maximal divergence, probably due to ancestral polymorphism. A peak at about 100 years was resilient to varying maximal divergence values. Three runs with maximal divergence 10 000 years (t 5) were iterated with different starting seeds to confirm consistency of the results and they all converged on similar values with the highest posterior density between 60 and 150 years.
Akaike Information Criterion
isolation-with-migration coalescence analysis
polymerase chain reaction.
Research was conducted with collection permits and approval of MARENA, Nicaragua. We thank: H Recknagel, C Rudolf-Chang and M von Ludner for assistance in the laboratory; N Probst and M Muschick for advice about fish stomachs; M Barluenga and W Salzburger for samples from Lake Managua; P Beerli for assistance with Migrate; PA Svensson for help with diet data analyses; C Bogs for algae identification by PCR; A Freundt and S Kutterolf for helpful geological information; M Brewer for an update to the mix2 software; Konstanz Ecology and Evolution journal club; and five anonymous reviewers for comments that improved the manuscript. CH thanks the Max Planck Gesellschaft (MPG) and D Tautz for support and H Buhtz for isotope analyses. This research was funded by: the Alexander von Humboldt Foundation (KRE, TKL); NSERC (KRE); Academy of Finland (TKL); Finnish Cultural Foundation (TKL); MPG (CH); University of Konstanz (AM); and the Deutsche Forschungsgemeinschaft (AM).
- Losos JB, Ricklefs RE: Adaptation and diversification on islands. Nature. 2009, 457: 830-836. 10.1038/nature07893.PubMedGoogle Scholar
- Grant PR, Grant BR: Adaptive radiation of Darwin's finches: Recent data help explain how this famous group of Galapagos birds evolved, although gaps in our understanding remain. Am Sci. 2002, 90: 130-141. 10.1511/2002.2.130.Google Scholar
- Savolainen V, Anstett MC, Lexer C, Hutton I, Clarkson JJ, Norup VV, Powell MP, Springate D, Salamin N, Baker WJ: Sympatric speciation in palms on an oceanic island. Nature. 2006, 441: 210-213. 10.1038/nature04566.PubMedGoogle Scholar
- Barluenga M, Stölting KN, Salzburger W, Muschick M, Meyer A: Sympatric speciation in Nicaraguan crater lake cichlid fish. Nature. 2006, 439: 719-723. 10.1038/nature04325.PubMedGoogle Scholar
- Gavrilets S, Vose A, Barluenga M, Salzburger W, Meyer A: Case studies and mathematical models of ecological speciation. 1. Cichlids in a crater lake. Mol Ecol. 2007, 16: 2893-2909. 10.1111/j.1365-294X.2007.03305.x.PubMedGoogle Scholar
- Schliewen U, Rassmann K, Markmann M, Markert J, Kocher TD, Tautz D: Genetic and ecological divergence of a monophyletic cichlid species pair under fully sympatric conditions. Mol Ecol. 2001, 10: 1471-1488. 10.1046/j.1365-294X.2001.01276.x.PubMedGoogle Scholar
- Schliewen UK, Tautz D, Paabo S: Sympatric speciation suggested by monophyly of crater lake cichlids. Nature. 1994, 368: 629-632. 10.1038/368629a0.PubMedGoogle Scholar
- Schluter D: The Ecology of Adaptive Radiations. 2000, New York: Oxford University PressGoogle Scholar
- Nosil P, Harmon LJ, Seehausen O: Ecological explanations for (incomplete) speciation. Trends Ecol Evol. 2009, 24: 145-156. 10.1016/j.tree.2008.10.011.PubMedGoogle Scholar
- Waxman D, Gavrilets S: 20 questions on adaptive dynamics. J Evol Biol. 2005, 18: 1139-1154. 10.1111/j.1420-9101.2005.00948.x.PubMedGoogle Scholar
- Orr MR, Smith D: Ecology and speciation. Trends Ecol Evol. 1998, 13: 502-506. 10.1016/S0169-5347(98)01511-0.PubMedGoogle Scholar
- Schluter D: Ecological speciation in postglacial fishes. Phil Trans R Soc B. 1996, 351: 807-814. 10.1098/rstb.1996.0075.Google Scholar
- McKinnon JS, Rundle H: Speciation in nature: the threespine stickleback model system. Trends Ecol Evol. 2002, 17: 480-488. 10.1016/S0169-5347(02)02579-X.Google Scholar
- Hendry AP, Wenburg JK, Bentzen P, Volk EC, Quinn TP: Rapid evolution of reproductive isolation in the wild: Evidence from introduced salmon. Science. 2000, 290: 516-518. 10.1126/science.290.5491.516.PubMedGoogle Scholar
- Lu G, Bernatchez L: Correlated trophic specialization and genetic divergence in sympatric lake whitefish ecotypes (Coregonus clupeaformis): support for the ecological speciation hypothesis. Evolution. 1999, 59: 1491-1505. 10.2307/2640895.Google Scholar
- Østbye K, Amundsen PA, Bernatchez L, Klemetsen A, Knudsen R, Kristoffersen R, TF N, Hindar K: Parallel evolution of ecomorphological traits in the European whitefish Coregonus lavaretus (L.) species complex during postglacial times. Mol Ecol. 2006, 15: 3983-4001. 10.1111/j.1365-294X.2006.03062.x.PubMedGoogle Scholar
- Meyer A: Phylogenetic relationships and evolutionary processes in East African cichlid fishes. Trends Ecol Evol. 1993, 8: 279-285. 10.1016/0169-5347(93)90255-N.PubMedGoogle Scholar
- Salzburger W: The interaction of sexually and naturally selected traits in the adaptive radiations of cichlid fishes. Mol Ecol. 2009, 18: 169-185. 10.1111/j.1365-294X.2008.03981.x.PubMedGoogle Scholar
- Barlow GW: The Midas cichlid in Nicaragua. Investigations of the Ichthyology of Nicaraguan Lakes. Edited by: Thorson TB. 1976, Lincoln: University of Nebraska Press, 333-358.Google Scholar
- Barlow GW, Munsey JW: The red devil-Midas-arrow cichlid species complex in Nicaragua. Investigations of the Ichthyology of Nicaraguan Lakes. Edited by: Thorson TB. 1976, Lincoln: University of Nebraska Press, 359-369.Google Scholar
- Meyer A: Morphometrics and allometry in the trophically polymorphic cichlid fish, Cichlasoma citrinellum: alternative adaptations and ontogenetic changes in shape. J Zool. 1990, 221: 237-260. 10.1111/j.1469-7998.1990.tb03994.x.Google Scholar
- Elmer KR, Kusche H, Lehtonen TK, Meyer A: Local variation and parallel evolution: morphological and genetic diversity across a species complex of Neotropical crater lake cichlid fishes. Phil Trans R Soc B. 2010, 365: 1769-1782. 10.1098/rstb.2009.0271.Google Scholar
- Bunje PME, Barluenga M, Meyer A: Sampling genetic diversity in the sympatrically and allopatrically speciating Midas cichlid species complex over a 16 year time series. BMC Evol Biol. 2007, 7: 25-10.1186/1471-2148-7-25.PubMed CentralPubMedGoogle Scholar
- Barluenga M, Meyer A: The Midas cichlid species complex: incipient sympatric speciation in Nicaraguan cichlid fishes?. Mol Ecol. 2004, 13: 2061-2076. 10.1111/j.1365-294X.2004.02211.x.PubMedGoogle Scholar
- Elmer KR, Lehtonen TK, Meyer A: Color assortative mating contributes to sympatric divergence of neotropical crater lake cichlid fish. Evolution. 2009, 63: 2750-2757. 10.1111/j.1558-5646.2009.00736.x.PubMedGoogle Scholar
- McKaye KR, Stauffer JR, Berghe van den EP, Vivas R, Perez LJL, McCrary JK, Waid R, Konings A, Lee WJ, Kocher TD: Behavioral, morphological and genetic evidence of divergence of the Midas cichlid species complex in two Nicaraguan crater lakes. Cuadernos Investigación UCA. 2002, 12: 19-47.Google Scholar
- Kutterolf K, Freundt A, Pérez W, Wehrmann H, Schmincke HU: Late Pleistocene to Holocene temporal succession and magnitudes of highly-explosive volcanic eruptions in west-central Nicaragua. J Volcanol Geotherm Res. 2007, 163: 55-82. 10.1016/j.jvolgeores.2007.02.006.Google Scholar
- Volcan Apoyeque. [http://www.ineter.gob.ni/geofisica/vol/apoyeque/index.html]
- Laguna Apoyeque. [http://www.ineter.gob.ni/Direcciones/Recursos%20Hidricos/HIDROGRAFIA%20WEB/Lagunas/Apoyeque.htm]
- Waid RM, Raesly RL, McKaye KR, McCrary JK: Zoogeografía íctica de lagunas cratéricas de Nicaragua. Encuentro. 1999, 51: 65-80.Google Scholar
- Barlow GW, Baylis JR, Roberts D: Chemical analyses of some crater lakes in relation to adjacent Lake Nicaragua. Investigations of the Ichthyology of Nicaraguan Lakes. Edited by: Thorson TB. 1976, Lincoln: University of Nebraska Press, 17-20.Google Scholar
- Beerli P, Felsenstein J: Maximum-likelihood estimation of migration rates and effective population numbers in two populations using a coalescent approach. Genetics. 1999, 152: 763-773.PubMed CentralPubMedGoogle Scholar
- Beerli P, Felsenstein J: Maximum likelihood estimation of a migration matrix and effective population sizes in n subpopulations by using a coalescent approach. Proc Natl Acad Sci USA. 2001, 98: 4563-4568. 10.1073/pnas.081068098.PubMed CentralPubMedGoogle Scholar
- Wallace RK: An assessment of diet-overlap indexes. Trans Am Fish Soc. 1981, 110: 72-76. 10.1577/1548-8659(1981)110<72:AAODI>2.0.CO;2.Google Scholar
- McCrary JK, Madsen H, Gonzáles L, Luna I, López LJ: Comparison of gastropod mollusc (Apogastropoda: Hydrobiidae) habitats in two crater lakes in Nicaragua. Rev Biol Trop. 2008, 56: 113-120.PubMedGoogle Scholar
- Post DM: Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology. 2002, 83: 703-718. 10.1890/0012-9658(2002)083[0703:USITET]2.0.CO;2.Google Scholar
- Vivas R, McKaye KR: Habitat selection, feeding ecology, and fry survivorship in the Amphilophus citrinellus species complex in Lake Xiloá. J Aquacult Aq Sci. 2001, IX: 32-48.Google Scholar
- Meyer A: Costs and benefits of morphological specialization: feeding performance in the trophically polymorphic neotropical cichlid fish, Cichlasoma citrinellum. Oecologia. 1989, 80: 431-436. 10.1007/BF00379047.Google Scholar
- Meyer A: Ecological and evolutionary consequences of the trophic polymorphism in Cichlasoma citrinellum (Pisces: Cichlidae). Biol J Linn Soc. 1990, 39: 279-299. 10.1111/j.1095-8312.1990.tb00517.x.Google Scholar
- Greenwood PH: Environmental effects on the pharyngeal mill of a cichild fish, Astatoreochromis alluaudi and their taxonomic implications. Proc Linn Soc Lond. 1965, 176: 1-10.Google Scholar
- Klingenberg CP, Barluenga M, Meyer A: Body shape variation in cichlid fishes of the Amphilophus citrinellus species complex. Biol J Linn Soc. 2003, 80: 397-408. 10.1046/j.1095-8312.2003.00246.x.Google Scholar
- Rueffler C, Van Dooren TJM, Leimar O, Abrams PA: Disruptive selection and then what?. Trends Ecol Evol. 2006, 21: 238-245. 10.1016/j.tree.2006.03.003.PubMedGoogle Scholar
- Martin RA, Pfennig DW: Disruptive selection in natural populations: the roles of ecological specialization and resource competition. Am Nat. 2009, 174: 268-281. 10.1086/600090.PubMedGoogle Scholar
- Hendry AP, Grant PR, Grant BR, Ford HA, Brewer MJ, Podos J: Possible human impacts on adaptive radiation: beak size bimodality in Darwin's finches. Proc R Soc Lond B. 2006, 273: 1887-1894. 10.1098/rspb.2006.3534.Google Scholar
- Schluter D, McPhail JD: Ecological character displacement and speciation in sticklebacks. Am Nat. 1992, 140: 85-10.1086/285404.PubMedGoogle Scholar
- Bolnick DI: Can intraspecific competition drive disruptive selection? An experimental test in natural populations of sticklebacks. Evolution. 2004, 58: 608-618.PubMedGoogle Scholar
- Udovic D: Frequency-dependent selection, disruptive selection, and the evolution of reproductive isolation. Am Nat. 1980, 116: 621-641. 10.1086/283654.Google Scholar
- Pennings PS, Kopp M, Meszéna G, Dieckmann U, Hermisson J: An analytically tractable model for competitive speciation. Am Nat. 2008, 171: E44-E71. 10.1086/523952.PubMedGoogle Scholar
- Keenleyside MHA: Cichlid Fishes: Behaviour, Ecology and Evolution. 1991, London: Chapman and HallGoogle Scholar
- de Lucena CAS, Kullander SO: The Crenicichla (Teleostei: Cichlidae) species of the Uruguai River drainage in Brazil. Ichthyol Explor Fres. 1992, 3: 97-192.Google Scholar
- Arnegard ME, Snoeks J: New three-spotted cichlid species with hypertrophied lips (Teleostei: Cichlidae) from the deep waters of Lake Malawi/Nyasa, Africa. Copeia. 2001, 2001: 705-717. 10.1643/0045-8511(2001)001[0705:NTSCSW]2.0.CO;2.Google Scholar
- Kocher TD, Conroy JA, McKaye KR, Stauffer JR: Similar morphologies of cichlid fish in Lakes Tanganyika and Malawi are due to convergence. Mol Phylogen Evol. 1993, 2: 158-165. 10.1006/mpev.1993.1016.Google Scholar
- Genner MJ, Seehausen O, Lunt DH, Joyce DA, Shaw PW, Carvalho GR, Turner GF: Age of cichlids: new dates for ancient lake fish radiations. Mol Biol Evol. 2007, 24: 1269-1282. 10.1093/molbev/msm050.PubMedGoogle Scholar
- Renesto E, Zawadzki CH, Revaldaves E: Biochemical taxonomy of Crenicichla (Pisces: Perciformes: Cichlidae) of the Iguaçu River, Brazil. Braz Arch Biol Techn. 2001, 44: 15-22.Google Scholar
- Kullander SO, Norén M, Frioriksson GB, de Lucena CAS: Phylogenetic relationships of species of Crenicichla(Teleostei: Cichlidae) in southern South America based on the mitochondrial cytochrome b gene. J Zool Syst Evol Res. [doi: 10.1111/j.1439-0469.2009.00557.x]
- Villa J: Systematic status of the cichlid fishes Cichlasoma dorsatum, C. granadense and C. nigritum Meek. Investigations of the Ichthyology of Nicaraguan Lakes. Edited by: Thorson TB. 1976, Lincoln: University of Nebraska Press, 101-113.Google Scholar
- Elmer KR, Fan S, Gunter HM, Jones JC, Boekhoff S, Kuraku S, Meyer A: Rapid evolution and selection inferred from the transcriptomes of sympatric crater lake cichlid fishes. Mol Ecol. 2010, 19 (Suppl 1): 197-211. 10.1111/j.1365-294X.2009.04488.x.PubMedGoogle Scholar
- Gavrilets S: Fitness Landscapes and the Origin of Species. 2004, Princeton: Princeton University PressGoogle Scholar
- Maynard Smith J: Sympatric speciation. Am Nat. 1966, 100: 637-650. 10.1086/282457.Google Scholar
- Thibert-Plante X, Hendry AP: Five questions on ecological speciation addressed with individual-based simulations. J Evol Biol. 2009, 88: 109-123. 10.1111/j.1420-9101.2008.01627.x.Google Scholar
- Stauffer JR, McKaye KR: Descriptions of three new species of cichlid fishes (Teleostei: Cichlidae) from Lake Xiloá, Nicaragua. Cuadernos Investigación UCA. 2002, 12: 1-18.Google Scholar
- Hey J, Won YJ, Sivasundar A, Nielsen R, Markert J: Using nuclear haplotypes with microsatellites to study gene flow between recently separated Cichlid species. Mol Ecol. 2004, 13: 909-919. 10.1046/j.1365-294X.2003.02031.x.PubMedGoogle Scholar
- Won YJ, Sivasundar A, Wang Y, Hey J: On the origin of Lake Malawi cichlid species: A population genetic analysis of divergence. Proc Natl Acad Sci USA. 2005, 102: 6581-6586. 10.1073/pnas.0502127102.PubMed CentralPubMedGoogle Scholar
- Thomson DP: Introduction to Meterology. 1849, London: William Blackwood and SonsGoogle Scholar
- Hora SL: Catching fishes with the hand in India. Science. 1950, 11: 263-264. 10.1126/science.111.2880.263-b.Google Scholar
- Villa J: Ichthyology of the Lakes of Nicaragua: historical perspective. Investigations of the Ichthyology of Nicaraguan Lakes. Edited by: Thorson TB. 1976, Lincoln: University of Nebraska Press, 101-113.Google Scholar
- Brinton DG: On an ancient human footprint from Nicaragua. Proc Am Phil Soc. 1887, 24: 437-444.Google Scholar
- Caliro S, Chiodini G, Izzo G, Minopli C, Signorini A, Avino R, Granieri D: Geochemical and biochemical evidence of lake overturn and fish kill at Lake Averno, Italy. J Volcanol Geotherm Res. 2008, 178: 305-316. 10.1016/j.jvolgeores.2008.06.023.Google Scholar
- Tassi F, Vaselli O, Fernandez E, Duarte E, Martinez M, Delgado Huertas A, Bergamaschi F: Morphological and geochemical features of crater lakes in Costa Rica: an overview. J Limnol. 2009, 68: 193-205.Google Scholar
- Mayr E: Change of genetic environment and evolution. Evolution as a Process. Edited by: Huxley J, Hardy AC, Ford EB. 1954, New York: Collier Books, 157-180.Google Scholar
- Barrett RDH, Schluter D: Adaptation from standing genetic variation. Trends Ecol Evol. 2007, 23: 38-44. 10.1016/j.tree.2007.09.008.PubMedGoogle Scholar
- Gavrilets S, Vose A: Dynamic patterns of adaptive radiation. Proc Natl Acad Sci USA. 2005, 102: 18040-10.1073/pnas.0506330102.PubMed CentralPubMedGoogle Scholar
- Doebeli M: A quantitative genetic competition model for sympatric speciation. J Evol Biol. 1996, 9: 893-909. 10.1046/j.1420-9101.1996.9060893.x.Google Scholar
- Pearse DE, Hayes SA, Bond MH, Hanson CV, Anderson EC, Macfarlane RB, Garza JC: Over the falls? Rapid evolution of ecotypic differentiation in steelhead/rainbow trout (Oncorhynchus mykiss). J Hered. 2009, 100: 515-525. 10.1093/jhered/esp040.PubMedGoogle Scholar
- Hart JJ, Gross MR, Heino M, Hilborn R, Kope RG, Law R, Reynolds JD: Evolutionary consequences of fishing and their implications for salmon. Evol Appl. 2008, 1: 388-408. 10.1111/j.1752-4571.2008.00020.x.Google Scholar
- Hendry AP, Nosil P, Rieseberg LH: The speed of ecological speciation. Funct Ecol. 2007, 21: 455-464. 10.1111/j.1365-2435.2007.01240.x.PubMed CentralPubMedGoogle Scholar
- Rasband WS: ImageJ. 1997, Bethesda: US National Institutes of Health, [http://rsb.info.nih.gov/ij/]Google Scholar
- Rohlf FJ: TPSDig2: a program for landmark development and analysis. 2007, New York: Department of Ecology and Evolution, State University of New York, Stony Brook, [http://life.bio.sunysb.edu/morph/]Google Scholar
- Klingenberg CP: MorphoJ. 2008, Manchester: Faculty of Life Sciences, Univ. of Manchester, [http://www.flywings.org.uk/MorphoJ_page.htm]Google Scholar
- Dryden IL, Mardia KV: 1998, Statistical Shape Analysis Chichester: Wiley
- Klingenberg CP, Barluenga M, Meyer A: Shape analysis of symmetric structures: quantifying variation among individuals and asymmetry. Evolution. 2002, 56: 1909-1920.PubMedGoogle Scholar
- Hyslop EJ: Stomach contents analysis-a review of methods and their application. J Fish Biol. 1980, 17: 411-429. 10.1111/j.1095-8649.1980.tb02775.x.Google Scholar
- Schoener TW: Nonsynchronous spatial overlap of lizards in patchy habitats. Ecology. 1970, 51: 408-418. 10.2307/1935376.Google Scholar
- Harrod C, Grey J: Isotopic variation complicates analysis of trophic relations within the fish community of Plußsee: a small, deep, stratifying lake. Arch Hydrobiol. 2006, 167: 281-299. 10.1127/0003-9136/2006/0167-0281.Google Scholar
- Kiljunen M, Grey J, Sinisalo T, Harrod C, Immonen H, Jones RI: A revised model for lipid-normalising d13C values from aquatic organisms, with implications for isotope mixing models. J Appl Ecol. 2006, 43: 1213-1222. 10.1111/j.1365-2664.2006.01224.x.Google Scholar
- Hammer Ø, Harper DAT, Ryan PD: PAST: Paleontological statistics software package for education and data analysis. Palaeontol Electron. 2001, 41: 9-Google Scholar
- SAS: JMP Statistical Discovery version 7. 2007, NC, USA: SAS Institute IncGoogle Scholar
- Brewer MJ: Discretisation for inference on normal mixture models. Stat Comput. 2003, 13: 209-219. 10.1023/A:1024214615828.Google Scholar
- Burnham KP, Anderson DR: Model Selection and Multimodel Inference: a Practical Information-Theoretic Approach. 2002, New York: Springer-Verlag, IncGoogle Scholar
- Petit RJ, El Mousadik A, Pons O: Identifying populations for conservation on the basis of genetic markers. Conserv Biol. 1998, 12: 844-855. 10.1046/j.1523-1739.1998.96489.x.Google Scholar
- Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics. 2003, 19: 2496-2497. 10.1093/bioinformatics/btg359.PubMedGoogle Scholar
- Excoffier L, Laval LG, Schneider S: Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evol Bioinform. 2005, 1: 47-50.Google Scholar
- Tamura K: Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C content biases. Mol Biol Evol. 1992, 9: 678-687.PubMedGoogle Scholar
- Hasewaga M, Kishino H, Yano T: Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol. 1985, 22: 160-174. 10.1007/BF02101694.Google Scholar
- Posada D, Crandall K: MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998, 14: 817-818. 10.1093/bioinformatics/14.9.817.PubMedGoogle Scholar
- Swofford DL: PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4. 2003, Sunderland: Sinauer AssociatesGoogle Scholar
- Bandelt HJ, Forester P, Röhl A: Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999, 16: 37-48.PubMedGoogle Scholar
- Posada D, Crandall KA: Intraspecific gene genealogies: trees grafting into networks. Trends Ecol Evol. 2001, 16: 37-45. 10.1016/S0169-5347(00)02026-7.PubMedGoogle Scholar
- Sanetra M, Henning F, Fukamachi S, Meyer A: A microsatellite-based genetic linkage map of the cichlid fish, Astatotilapia burtoni (Teleostei): A comparison of genomic architectures among rapidly speciating cichlids. Genetics. 2009, 182: 387-397. 10.1534/genetics.108.089367.PubMed CentralPubMedGoogle Scholar
- Noack K, Wilson AB, Meyer A: Broad taxonomic applicability of microsatellites developed for the highly polymorphic neotropical cichlid, Amphilophus citrinellum. Anim Genet. 2000, 31: 151-152. 10.1046/j.1365-2052.2000.00592.x.PubMedGoogle Scholar
- Kellogg KA, Markert JA, Stauffer JR, Kocher TD: Microsatellite variation demonstrates multiple paternity in lekking cichlid fishes from Lake Malawi, Africa. Proc R Soc Lond B. 1995, 260: 79-84. 10.1098/rspb.1995.0062.Google Scholar
- Salzburger W, Braasch I, Meyer A: Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes. BMC Biol. 2007, 5: 51-10.1186/1741-7007-5-51.PubMed CentralPubMedGoogle Scholar
- Zardoya R, Vollmer DM, Craddock C, Streelman JT, Karl S, Meyer A: Evolutionary conservation of microsatellite flanking regions and their use in resolving the phylogeny of cichlid fishes (Pisces: Perciformes). Proc R Soc Lond B. 1996, 263: 1589-1598. 10.1098/rspb.1996.0233.Google Scholar
- van Oosterhout C, Hutchison WF, Wills DPM, Shipley P: Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes. 2004, 4: 535-538. 10.1111/j.1471-8286.2004.00684.x.Google Scholar
- Brookfield JFY: A simple new method for estimating null allele frequency from heterozygote deficiency. Mol Ecol. 1996, 5: 453-455.PubMedGoogle Scholar
- Dieringer D, Schlötter C: Microsatellite Analyser (MSA): a platform independent analysis tool for large microsatellite data sets. Mol Ecol Notes. 2003, 3: 167-169. 10.1046/j.1471-8286.2003.00351.x.Google Scholar
- Kalinowski ST: Counting alleles with rarefaction: Private alleles and hierarchical sampling designs. Conserv Genet. 2004, 5: 539-543. 10.1023/B:COGE.0000041021.91777.1a.Google Scholar
- Kalinowski ST: HP-Rare 1.0: a computer program for performing rarefaction on measures of allelic richness. Mol Ecol Notes. 2005, 5: 187-189. 10.1111/j.1471-8286.2004.00845.x.Google Scholar
- Raymond M, Rousset F: An exact test for population differentiation. Evolution. 1995, 49: 1280-1283. 10.2307/2410454.Google Scholar
- Rousset F: Genepop'007: a complete re-implementation of the genepop software for Windows and Linux. Mol Ecol Resour. 2008, 8: 103-106. 10.1111/j.1471-8286.2007.01931.x.PubMedGoogle Scholar
- Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F: GENETIX 4.05, Logiciel Sous Windows TM Pour Al Génétique De Populations. 1996, Montpellier: Laboratoire Génome, Populations, InteractionsGoogle Scholar
- Beerli P: Comparison of bayesian and maximum-likelihood inference of population genetic parameters. Bioinformatics. 2006, 22: 341-345. 10.1093/bioinformatics/bti803.PubMedGoogle Scholar
- Hey J, Nielsen R: Multilocus methods for estimating population sizes, migration rates and divergence time, with applications to the divergence of Drosophila pseudoobscura and D. persimilis. Genetics. 2004, 167: 747-760. 10.1534/genetics.103.024182.PubMed CentralPubMedGoogle Scholar
- Hey J, Nielsen R: Integration within the Felsenstein equation for improved Markov Chain Monte Carlo methods in population genetics. Proc Natl Acad Sci USA. 2007, 104: 2785-2790. 10.1073/pnas.0611164104.PubMed CentralPubMedGoogle Scholar
- Wirth T, Bernatchez L: Decline of North Atlantic eels: a fatal synergy?. Proc R Soc Lond B. 2003, 270: 681-688. 10.1098/rspb.2002.2301.Google Scholar
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