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The polychromatic and trophically polymorphic Midas cichlid fish species complex (Amphilophus cf. citrinellus) is an excellent model system for studying the mechanisms of speciation and patterns of phenotypic diversification in allopatry and in sympatry. Here, we first review research to date on the species complex and the geological history of its habitat. We analyse body shape variation from all currently described species in the complex, sampled from six crater lakes (maximally 1.2–23.9 kyr old) and both great lakes in Nicaragua. We find that Midas cichlid populations in each lake have their own characteristic body shape. In lakes with multiple sympatric species of Midas cichlid, each species has a distinct body shape. Across the species complex, most body shape change relates to body depth, head, snout and mouth shape and caudal peduncle length. There is independent parallel evolution of an elongate limnetic species in at least two crater lakes. Mitochondrial genetic diversity is higher in crater lakes with multiple species. Midas cichlid species richness increases with the size and age of the crater lakes, though no such relationship exists for the other syntopic fishes. We suggest that crater lake Midas cichlids follow the predicted pattern of an adaptive radiation, with early divergence of each crater lake colonization, followed by intralacustrine diversification and speciation by ecological adaptation and sexual selection.
1. Introduction
A major challenge in speciation research is that environmental, biological, ecological and geographical conditions change over time and can obscure past evolutionary processes and events. Arguably, species complexes and incipient species are among the best ways to understand speciation, as species or populations in the complex might be at different stages of divergence and thereby offer a chronological perspective of change. Furthermore, since the divergence may be recent or ongoing, the current biotic and abiotic conditions are likely to be similar as those that are or were involved in the initial differentiation.
The species complex of Midas cichlid (Amphilophus cf. citrinellus (Günther)) fishes is an ideal model system for studying speciation in nature because of its biology and geographical setting (table 1). The complex is distributed across the great lakes and crater lakes of Nicaragua (figure 1) and, at lower densities, some of the larger nearby rivers (Barlow 1976; Smith & Bermingham 2005). The Midas cichlid species complex is highly variable within and across species, including a pronounced polychromatism and trophic polymorphism. Much of the early research on the species complex focused on behavioural aspects of mate choice and competition (e.g. Barlow 1973, 1986, 1998; Baylis 1976a,b; McKaye & Barlow 1976). That line of inquiry was followed by an interest in trophic ecology, especially feeding apparatus variation and phenotypic plasticity (Meyer 1989, 1990a,b). More recently and using newly available molecular tools, questions about phylogeography, population history and speciation have been addressed in this species complex (Wilson et al. 2000; Barluenga & Meyer 2004; Barluenga et al. 2006; Bunje et al. 2007; Elmer et al. 2009, 2010). Several species in this complex arose very recently and some through sympatric speciation (Wilson et al. 2000; Barluenga et al. 2006; Elmer et al. 2009). Each crater lake in Nicaragua is likely to harbour a different set of endemic species, which makes these lakes an excellent system to study the relative speed and strength by which geographical isolation and natural and sexual selection promote phenotypic diversification and speciation.
Here we summarize the state of research on this system, with a focus on the geography, ecology and genetics of speciation. Further, we present a distribution-wide analysis of body shape variation and genetic diversity in the Midas cichlid species complex that is based on both new morphometric data and mitochondrial DNA (mtDNA) sequence.
(a) Geographical setting
Western Nicaragua is underlain by one of the world's most volcanically active areas, known as the Central American Volcanic Arc. After a volcano erupts and subsequently cools the resultant cone-shaped crater fills with ground- and rainwater to become a crater lake (also known as a caldera lake or maar). Nicaraguan crater lakes are characteristically oligotrophic, with a high proportion of dissolved solids and no in- or out-flow (Barlow et al. 1976). After some time, these crater lakes are colonized by aquatic organisms, including cichlid fishes. At least eight Nicaraguan crater lakes have been successfully colonized by Midas cichlids (figure 1, table 1). It is not known for each case how or when crater lake colonizations happen; it is generally thought to be from natural phenomena like hurricanes or piscivorous birds, though some lakes may have been purposely colonized by humans (Villa 1976a). In general, crater lakes have an impoverished fauna and a lot of ecological niche space to be filled (Schluter 2000a). Because of their geographical isolation and known geological history, crater lakes are excellent natural systems for studying the evolution of species in both allopatry and sympatry.
Nicaragua is subject not only to volcanic but also tectonic activity, which has resulted in the two largest and oldest lakes in Central America: great lakes Managua and Nicaragua. These date to the Early Pleistocene (Kutterolf et al. 2007) and together cover approximately 9000 km2 (Cole 1976). Lake Managua, to the north, lies at 7 or 8 m higher elevation than Lake Nicaragua. Connectivity between these two lakes has varied depending on lake fluctuations (Swain 1966). River Tipitapa rarely but periodically connects Lake Managua to Nicaragua when water levels are high (Cole 1976). The great lakes are exceptionally shallow (mean depth around 8.6 m for Lake Managua and 12.4 m for Lake Nicaragua) and have gently sloping basins, allowing wind to stir the sediments (Cole 1976). This and a high prevalence of phytoplankton (Barlow 1976) make the water of the great lakes very turbid. Thus, this habitat is very different from the clear, deep waters of the crater lakes.
(b) Midas cichlid diversity
The A. citrinellus species complex is part of the most genus-rich clade of neotropical cichlids: the tribe Heroini (Říčan et al. 2008), which display a wide diversity of morphological, ecological and behavioural adaptations. Some of their morphologies are phylogenetically discordant because of homoplasy and convergent evolution (Říčan et al. 2008), as has been found for cichlids in general (Meyer 1993; Stiassny & Meyer 1999).
Based on evidence from geology, biogeography and population genetics, Nicaragua's great lakes are thought to contain the ancestral population of Midas cichlids from which all crater lake populations originate (Barlow 1976; Barluenga & Meyer 2004; Barluenga et al. 2006). Midas cichlids within a crater lake are genetically more similar to each other than to those studied in any other lake (Barluenga et al. 2006; Bunje et al. 2007; Elmer et al. 2009).
Currently, there are nine species described in the Midas species complex: Amphilophus amarillo Stauffer and McKaye (Lake Xiloá endemic), Amphilophus astorquii Stauffer, McCrary and Black (Lake Apoyo endemic), Amphilophus chancho Stauffer, McCrary and Black (Lake Apoyo endemic), A. citrinellus (Günther), Amphilophus flaveolus Stauffer, McCrary and Black (Lake Apoyo endemic), Amphilophus labiatus (Günther), Amphilophus sagittae Stauffer and McKaye (Lake Xiloá endemic), Amphilophus xiloaensis Stauffer and McKaye (Lake Xiloá endemic) and Amphilophus zaliosus Barlow (Lake Apoyo endemic) (table 1). Rapidly evolving genetic markers can discern many of these species (Barluenga & Meyer 2004; Barluenga et al. 2006; Elmer et al. 2009), though not all species have been suitably investigated. We expect that more species from the crater lakes will be described in the future and that the taxonomic validity of the original species A. 'citrinellus' will need to be revisited. Therefore, we refer to Midas cichlids found in multiple crater lakes as A. cf. citrinellus.
Midas cichlids are polymorphic for trophically relevant attributes such as body shape, pharyngeal jaw apparatus and hypertrophied lips. Limnetic species A. sagittae and A. zaliosus are elongate ecotypes with papilliform pharyngeal jaws, while benthic species such as A. astorquii, A. chancho and A. flaveolus in Apoyo and A. amarillo and A. xiloaensis in Xiloá are more high bodied and tend to have molariform pharyngeal jaws (Barlow & Munsey 1976; Vivas & McKaye 2001; Klingenberg et al. 2003; Parsons et al. 2003; Barluenga et al. 2006). Amphilophus labiatus has a slightly more elongate body, more pointed snout, lateral compression and, most obviously, dramatically hypertrophied lips compared with A. citrinellus (Barlow & Munsey 1976; Klingenberg et al. 2003). A similarly thick-lipped ecotype is found at moderate frequencies (approx. 20%) in Lake Apoyeque, where it occupies an ecological niche distinct from the more abundant thin-lipped morph (K. R. Elmer, T. K. Lehtonen & A. Meyer 2010, unpublished data), and much more rarely in lakes Masaya and Xiloá (Barlow 1976; McKaye et al. 2002).
Some species in the Midas cichlid species complex are polychromatic: the common colour morph is greyish with spotted, striped and barred patterns (referred to as the 'normal' or 'dark' morph) while the 'gold' morph is uniformly orange, yellow or even white (Barlow 1976). All fish begin life dark and some later lose their melanophores and become gold (or more rarely white, lacking both dark melanocytes and yellow xanthophores) permanently (Barlow 1976; Dickman et al. 1988). Though there is an environmental (e.g. diet) component to the brightness of the gold morph (Webber et al. 1973), amelanism is under genetic control (Henning et al. in press). Gold morphs are found in moderate to low frequencies (e.g. less than 20% in A. xiloaensis, less than 7% in A. sagittae) in crater lakes Xiloá, Masaya, Asososca León and Asososca Managua (Barlow 1976; Elmer et al. 2009; K. R. Elmer, T. K. Lehtonen & A. Meyer 2007, personal observation) and in great lakes Managua and Nicaragua (8–10%; Webber et al. 1973). Historically, the melanic morph has been called normal (or sometimes 'grey') because it is the most common colour morph, is drab like most freshwater fishes and is similar to generalized cichlids in the genus (Webber et al. 1973; Barlow 1976). Unfortunately, normal is a loaded semantic descriptor that is uninformative for readers not familiar with Midas cichlids. It also implies that the gold phenotype is 'abnormal' or mutant, which is not true because the system is a natural (probably stable) colour polymorphism. Consequently, we prefer the more neutral and inclusive term dark to describe the common melanic morph.
Strong assortative mating within the colour morphs has been shown in the field (McKaye 1980, 1986; Elmer et al. 2009) and laboratory (e.g. Barlow et al. 1977; Barlow 1986). In Lake Xiloá, this assortative mating may be resulting in incipient speciation, where gold and dark morphs are significantly genetically differentiated at neutral markers (Elmer et al. 2009). In contrast, gold and dark morphs in the great lakes show little or no genetic differentiation (Barluenga & Meyer 2004).
(c) Midas cichlid mating behaviour
In comparison to African cichlids, which are remarkably adapted to diverse and extremely tight ecological niches and show highly specialized sexual strategies (Salzburger & Meyer 2004; Salzburger 2009), Midas cichlids were thought to be rather unspecialized (Barlow 1976; Baylis 1976b). Though the sexes are generally isomorphic (i.e. show little or no difference in overall body shape), males tend to be larger, have longer dorsal and pectoral fins and bigger nuchal humps. Females typically choose to pair with males that are larger than themselves and aggressive (Barlow 1992), though the exact dynamics of mate choice and partner stability are complicated. Males and females pair at the beginning of the breeding season, acquire and defend a territory while raising and guarding their broods (Barlow 1986, 1992), though the extent of biparental care may vary across pairs and species (T. K. Lehtonen 2007, unpublished data). Mating is assortative by ecological species (Baylis 1976a; Vivas & McKaye 2001; Elmer et al. 2009), though this has not been researched in detail or for all species. Competition for breeding sites and predation on fry is intense (McKaye 1977; McKaye & McKaye 1977), which may exert strong selective pressure for suitable good mate communication and coordination within pairs (Baylis 1976b).
(d) Sympatric speciation
Because crater lakes are isolated environments, this biological system has become a model for sympatric speciation in nature (Baylis 1976a; Barluenga & Meyer 2004; Barluenga et al. 2006; Gavrilets et al. 2007; Luz-Burgoa et al. 2007; Elmer et al. 2009). In all cases where multiple Midas cichlid species have so far been recognized within a crater lake, they have different ecologies and morphologies, strongly suggestive of the effect of disruptive, and then divergent, natural selection to exploit different intralacustrine niches (Baylis 1976a; Stauffer & McKaye 2002; Barluenga et al. 2006; Stauffer et al. 2008) (sensu Schluter 2000b). Breeding of each species commonly occurs within a metre or two from sister taxa, which argues for strong pre-zygotic reproductive isolation mechanisms (Barlow & Munsey 1976; Stauffer et al. 2008; Elmer et al. 2009; T. K. Lehtonen 2005–2008, personal observation). Even when speciation is incomplete or undetermined, there can be intralacustrine ecological differentiation (e.g. Lake Apoyeque). Ecological differentiation can occur much faster than even rapidly evolving neutral molecular markers can track; thus the absence of genetic differentiation between ecotypes does not refute ecological speciation (Thibert-Plante & Hendry 2009).
Incipient sympatric speciation by sexual selection based on colour (either gold or dark) has been proposed in this group, first inferred from observational field studies (McKaye 1980; Meyer 1989, 1990a) and later by genetic inference (Wilson et al. 2000; Elmer et al. 2009). Theoretical verbal and mathematical models suggest such speciation is possible (Turner & Burrows 1995; Higashi et al. 1999; Takimoto et al. 2000; Kirkpatrick & Ravigné 2002), but the mode of gold inheritance (a dominant allele) (Barlow 1983; Henning et al. in press), effect of brood adoptions on sexual imprinting (Barlow 1992), frequency of mixed colour morph matings (Barlow 1992; Elmer et al. 2009) and possibly late gold colour change after sexual maturity is reached (Barlow 1998) might all be factors that could inhibit speciation based on colour.
(e) Revealing morphological and genetic variation
The research field of geometric morphometrics allows hypothesis testing by describing and quantifying differences in shape between biological groups (Rohlf & Marcus 1993; Adams et al. 2004; Zelditch et al. 2004) because multivariate shape variation is statistically comparable. Founded on landmarks that describe overall body shape, and subsequent Procrustes superimposition that minimizes inter-individual variation, modern analyses of shape can discern much more subtle differences between groups than could traditional morphometrics (Rohlf & Marcus 1993; Parsons et al. 2003; Adams et al. 2004). The methods have been very successful in discerning biological groups of fishes, including cichlids, and relating the body shape differentiation to ecology or phylogeny (Ruber & Adams 2001; Klingenberg et al. 2003; Trapani 2003; Zelditch et al. 2004; Clabaut et al. 2007).
In the present study, we use geometric morphometrics to characterize body shape variation within and between eight lakes and all currently described species of the Midas cichlid species complex in Nicaragua. We further test whether abiotic environmental characteristics of each crater lake are correlated with genetic diversity and species richness in the Midas cichlid species complex. We take advantage of the crater lakes as natural replicate evolutionary experiments to infer some general patterns of the Midas cichlid adaptive radiation across time and space.
2. Material and methods
(a) Sample collection
Samples were collected in 2001, 2003, 2005 and 2007 from great lakes Nicaragua (one location: Las Isletas) and Managua (three locations: Miraflores, San Antonio and Tipitapa) and from six crater lakes (north to south): Asososca León, Apoyeque, Xiloá, Asososca Managua, Masaya and Apoyo (figure 1). Fish were collected using gill nets. Sample sizes for lakes Nicaragua and Managua were augmented by purchasing specimens from local fishermen.
In the field, fish were placed on their right side on a flat surface and photographed from directly above using a tripod and a Nikon Coolpix 995 or S4 digital camera. A scale ruler was included in each photograph. Tissue samples (fin clip and muscle) were collected from each specimen and stored in pure ethanol. Fish heads were taken as vouchers and stored in 70 per cent ethanol. Species were initially taxonomically classified on site. Later the specimen photographs also served in assigning each specimen to a species independently by more than one author. The currently available taxonomy was used.
A total of 1334 specimens from all nine currently recognized species of the Midas cichlid species complex were included in this study (table 2; electronic supplementary material, appendix table S1). This sampling represents the entire known crater lake and great lake distribution, except for Monte Galán and Tiscapa, for which our sample sizes were not sufficient for geometric morphometric analyses. For this study, we limited ourselves to the investigation of external phenotypic differences only between lakes and species. Intraspecific polymorphisms (e.g. in colour, jaw type or lip shape) have not been investigated here and will be the subject of future study. Males and females are combined because other research has indicated no significant body shape differences between the sexes (K. R. Elmer 2010, unpublished data). Previous morphometric studies on this species complex also combined sexes (e.g. Klingenberg et al. 2003; Parsons et al. 2003; Barluenga et al. 2006). Sampling locations within lakes have been pooled, because in none of the previous studies on crater lakes has any intralacustrine genetic structure been observed (Barluenga et al. 2006; Elmer et al. 2009). Juvenile fish (total body length approx. less than 10 cm) were excluded because fish of this size cannot be reliably assigned to species.
(b) Genetic diversity analyses
(i) DNA sequencing
Genomic DNA was extracted from tissues using a standard high-salt protocol (Bruford et al. 1998). The mtDNA control region was amplified with primers LProF (Meyer et al. 1994) and 12S5R (GGC GGA TAC TTG CAT GT) using standard PCR conditions. PCR products were cleaned using a FastAP Thermosensitive Alkaline Phophatase dephosphorylation protocol and cycle sequenced in the forward and reverse directions by BigDye Terminator Cycle Sequencing Ready Reaction using standard conditions and the same primers as in the PCR. After cleaning the single-stranded product with Zymo ZR-96 DNA Sequencing Clean-up Kit, samples were resuspended in water and electrophoresed in an ABI3130xl DNA sequencer (Applied Biosystems). Forward and reverse contigs were assembled in Sequencher v. 4.2.2. Additional sequences were obtained from GenBank.
(ii) Genetic diversity analyses
Sequences were aligned in MacClade v. 4 (Maddison & Maddison 2003) and trimmed to a common length that resulted in no terminal gaps (733 bp). The distribution of haplotypes was calculated in DnasP v. 5 (Librado & Rozas 2009) (20 sites with alignment gaps were not considered). Haplotype or allelic richness (rarefied to a sample size of 63 to avoid any bias induced by unequal sample sizes per crater lake) and gene diversity were calculated in Contrib v. 1.02 (Petit et al. 1998) for each crater lake.
(c) Geometric morphometrics
(i) Data acquisition
Fifteen landmarks that describe body shape were digitized in TpsDig 2.12 (Rohlf 2001) by a single investigator (H.K.) from the photograph of each specimen (figure 2). Two additional landmarks representing 10 cm on the size scale were used to determine the standard length of each fish, calculated in Past 1.89 (Hammer et al. 2001).
Specimens were grouped in such a way as to test three main questions:
—Morphological variation across all crater and great lakes. To investigate the overall morphometric variation in the entire Midas cichlid species complex across lakes, all specimens within each lake were pooled (n = 1334 across eight lakes; table 2) and comparisons drawn across lakes.
—Morphological variation within lakes with multiple species. To assess the variation among different Midas species within particular lakes, we created pooled and separate datasets for crater lakes Apoyo (four species) and Xiloá (three species) and for great lakes Managua (two species) and Nicaragua (two species) (table 2). Morphometric analyses were the same for all datasets.
—Diversity within crater lakes and correlations with lake characteristics. To test correlations between biological diversity in crater lakes and crater lake abiotic characteristics such as size and age of lakes, specimens in each crater lake were pooled for calculating partial disparity (PD) and mtDNA diversity.
(ii) Body shape analysis
Geometric morphometric methodologies largely follow Klingenberg et al. (2003). Analyses were performed in Morphoj 1.00k (Klingenberg 2008). The first step was a generalized least squares Procrustes superimposition (Dryden & Mardia 1998), in which the configuration of 15 landmarks for each specimen was scaled to unit centroid size, translated to a common position and rotated to minimize Procrustes distances between all landmark configurations (Dryden & Mardia 1998; Rohlf 1999; Zelditch et al. 2004). Therefore, the Procrustes distances become relative measures of shape differences between biological groups. The reliability of the Procrustes distance in distinguishing groups was assessed by permutation tests (1000 iterations) and p-values were corrected for non-independence (α/k − 1) (Rice 1989).
We applied a size correction to our data to account for any allometric effects associated with growth, i.e. any dependence of body shape on absolute body size (Loy et al. 1996; Reis et al. 1998; Klingenberg 2003). We divided the dataset into subgroups according to the comparison we were performing (e.g. by lake, or species within a lake). A multivariate regression of shape (dependent variable: Procrustes coordinates) on size (independent variable: centroid size) (Monteiro 1998) was performed for each subgroup using a permutation test against the null hypothesis of independence (10 000 iterations). The regression residuals were then used for all geometric morphometric analyses.
The thin plate spline technique (Dryden & Mardia 1998) was used to visualize shape changes in particular body regions. This method displays the average body shape for a set of individuals of interest compared with an 'average' (or 'consensus') body shape of all fish included in the analysis. The two body shapes (i.e. configurations of 15 landmarks) are superimposed onto a grid. The differences distort the grid in those body regions that differ the most between the average body shape of the groups being compared.
To assess the total amount of variation in body shape between groups, we used canonical variates analysis (CVA; when comparing multiple groups) and discriminant function analysis (DFA; when comparing pairs of groups). These are commonly used ordination analyses to capture the multi-dimensional variation that is inherent in body shapes (Mardia et al. 1979; Albrecht 1980; Klingenberg et al. 2003; Zelditch et al. 2004). These multivariate analyses reduce the amount of variation within groups and express the variation among groups in n dimensions, where n is the number of groups minus one. Multi-dimensional plots were used to describe the morphospace (i.e. the abstract space where each point represents a particular individual) and to distinguish between groups simultaneously. Each axis successively describes the greatest proportion of variation in body shape. Principal components analyses (PCAs) were also conducted. This ordination method is hypothesis-free in that it assumes no a priori groupings in assessing variation but can obscure true and biologically relevant variation between groups.
To quantify the contribution a group makes to the overall morphological variation across the crater lakes, we estimated PD for each group (i.e. lake) compared with the grand mean as: where Di is the Procrustes distance of the ith group from the grand mean and N is the total number of groups (Zelditch et al. 2004, p. 302).
3. Results
(a) Morphological variation across all lakes
Mean standard length (±standard deviation) of all specimens pooled was 13.1 ± 2.9 cm (n = 1334). Centroid size accounted for significant proportions of shape variation across lakes (minimum of 1.99% in Lake Apoyeque up to 8.47% in Lake Nicaragua, p < 0.01) and for all groups pooled (2.01%, p < 0.0001) (table 3). Therefore, body shape is correlated to centroid size and an allometry correction was applied for all analyses (as described in §2; table 3).
Each crater lake and great lake occupies a unique field of morphospace variation. Specimens from any given lake only slightly or moderately overlap with specimens from any other lake (figure 3). The distinctiveness of each lake is less pronounced but is still evident in a plot of the first two PCs, which makes no a priori grouping (electronic supplementary material, appendix figure S1). In the PCA and CVA, lakes Apoyeque, Masaya and Asososca León contain specimens with the most central body shapes in the analyses, i.e. that deviate least from the average Midas cichlid body shape. Specimens from great lakes Managua and Nicaragua form an overlapping cluster, indicating that the body shapes in those lakes are quite similar to each other but different from those in the crater lakes. Individuals in lakes Apoyo and Xiloá cluster together along CVs 1 and 2 and are set apart from the other lakes. Fish in Asososca Managua are the most distinct. Pronounced shape changes associated with CV1 are: lip/snout thickness (landmark (LM) 1 and 2), body height (LM 6) and width of the mid-body region (LM 5, 7–9), a shift of the anterior insertion of the anal fin (LM 10) and differences in the length of the caudal peduncle region (LM 11–15) (figure 4).
Procrustes distances are a metric to describe the difference in body shape between two groups. In a comparison of Procrustes distances between all lakes, Midas cichlid body shape always differs significantly (p < 0.001) (table 4). Average body shapes are the least different between crater lake Masaya and great lake Nicaragua (Procrustes distance 0.0385) and the most different between crater lake Asososca Managua and great lake Managua (Procrustes distance 0.1234).
(b) Morphological variation within lakes
(i) Great lakes Managua and Nicaragua
Amphilophus citrinellus and A. labiatus are considered to be two species that inhabit both great lakes (Astorqui 1971). However, the body shape of A. citrinellus is different in Lake Managua from its conspecifics in Lake Nicaragua (figure 5a, table 5). The body shape of A. labiatus is also different in each of the two great lakes (figure 5a, table 5).
Amphilophus citrinellus and A. labiatus in Lake Managua are completely morphologically distinguished by the DFA, i.e. there is no morphospace overlap between these two species. This pronounced differentiation is primarily due to a greater body depth of A. citrinellus compared with A. labiatus (LM 6). Shape differences in gill opercula–body intersection (LM 5), placement of the anal fin (LM 10) and caudal peduncle length (LM 14 and 15) are also important (figure 5b).
DFA also completely discriminates A. citrinellus from A. labiatus in Lake Nicaragua based on body shape. As in Lake Managua, A. labiatus has a more shallow body (LM 6), elongate head and voluminous lip/snout region (LM 1) relative to A. citrinellus (figure 5b).
(ii) Crater lake Apoyo: four species
There is a significant difference in body shape among all four species of Midas cichlids in Apoyo (figure 6, table 6). The fields of morphospace variation for A. astorquii and A. zaliosus are each tightly clustered, divergent from each other, and almost completely attributable to the body shape variation described in CV1. Amphilophus chancho and A. flaveolus are both high-bodied benthic species that show very little difference in morphospace between them and, in contrast to A. astorquii and A. zaliosus, are only separated along CV2. The shape change associated with CV1 across all specimens is primarily a broadening of the mid-body region (LM 5–9) and an elongation of the caudal peduncle (LM 10–15) (data not shown).
Each species in Apoyo clearly has a different average body shape (figure 6b). Amphilophus astorquii is a relatively compact fish with a robust mid-body (LM 3–10), a short caudal peduncle (LM 11–15) and eyes shifted rostrally (LM 3 and 4). Amphilophus chancho has a shorter lower jaw (LM 5), a larger head and a deep body (LM 6–10) and a short caudal peduncle (LM 13–15). Amphilophus flaveolus is characterized by a more bulky and steep forehead region (LM 6) and is the most similar to the consensus shape. Amphilophus zaliosus is the most morphologically distinct species: its body is very narrow (LM 6–10), lower jaw very long (LM 5) and caudal peduncle narrow and elongate (LM 11–15).
(iii) Crater lake Xiloá: three species
There are significant body shape differences between all three Xiloá Midas cichlid species (figure 7, table 7). Amphilophus sagittae has the most distinct morphology, evidenced by high interspecific Procrustes distances and differentiation along CV1. Amphilophus amarillo and A. xiloaensis are primarily differentiated along CV2. CV1 encodes a shape change in head size, mid-body depth (LM 6–10) and caudal peduncle length (LM 11–15) (data not shown). Amphilophus amarillo is the most similar to the average Xiloá Midas cichlid shape, except for being slightly more high bodied (LM 6) and having the anal fin shifted ventro-rostrally (figure 7b). Amphilophus sagittae has a relatively small head and eye (LM 1–5), is elongated through the mid-body and caudal peduncle (LM 5, 6, 9–10, 13–15) and differs most from consensus. Amphilophus xiloaensis is slightly more deep bodied (LM 6–9) with a shorter caudal peduncle (LM 13–15) and a longer lower jaw (LM 2 and 5).
(iv) Parallelism across crater lakes Apoyo and Xiloá
Midas cichlids in lakes Apoyo and Xiloá share similar morphospaces (figure 3). Species in different lakes but with similar ecologies showed equivalent shifts in body shape (e.g. lateral compression, elongated candal peduncle) (figures 6b,7b). This is especially clear in the limnetic species A. sagittae and A. zaliosus. A PCA to compare the morphospace variation between all endemic species of Apoyo and Xiloá demonstrates that these elongate limnetics have their morphospace explained best by the same directions on the PC, with the older species A. zaliosus showing the more extreme variation (figure 8). This suggests that similar ecological forces through divergent selection caused the parallel evolution of a limnetic species in both crater lakes Apoyo and Xiloá.
(c) Biological diversity within crater lakes
One hundred and forty-four new mtDNA sequences (GenBank accession numbers GU016624–GU016703, GU016998–GU017061) were combined with previously available sequences (electronic supplementary material, appendix table S2) for a total dataset of 929 sequences from all crater lakes. These collapsed to 73 different haplotypes for the entire Midas cichlid species complex. Lake Xiloá had the highest rarefied allelic richness (16.48) and Apoyeque the lowest (1.90) (figure 9; electronic supplementary material, appendix table S3). This pattern was generally similar to that observed for gene diversity, which is a measure that is not corrected for sample size differences (electronic supplementary material, appendix table S3).
We were interested in lake and faunal features that may suggest why some crater lakes house multiple species while most other crater lakes apparently contain only a single species of Midas cichlid. Because only six crater lakes could be included in this study, this small sample size renders rigorous statistical analyses difficult. Nonetheless, some relevant patterns emerge.
Crater lakes with one species (A. cf. citrinellus) have significantly lower allelic richness than lakes with multiple Midas cichlid species (mean ± s.d.: 5.02 ± 3.24 versus 15.14 ± 1.90; t-test assuming equal variances: t = 3.94, d.f. = 4, two-tailed p = 0.017) (calculated in JMP v. 7; SAS 2008) (figure 9). PD is higher in lakes with one species (5.81 × 10−4 ± 2.73 × 10−4) than it is in lakes with multiple species (3.11 × 10−4 ± 2.17 × 10−4), though the relationship is not significant (t-test assuming equal variances: t = −1.197, d.f. = 4, two-tailed p = 0.297) (figure 9). Allelic richness tends to decrease weakly with increasing PD (F1,4 = 1.374, p = 0.31) (figure 9).
Midas cichlid species richness increases significantly with crater lake age (F1,4 = 10.59, p = 0.031) and size (F1,4 = 5.586, p = 0.077) (figure 10). This effect is leveraged by Apoyo, which is the most species-rich, oldest and largest of the crater lakes. Xiloá has apparently relatively high species richness for a mid-aged lake age of small size. The same relationship between species richness and crater lake size and age does not hold when comparing syntopic fish species other than Midas cichlids, including (crater lake age: F1,4 = 0.035, p = 0.861; crater lake size: F1,4 = 0.045, p = 0.842) or excluding (crater lake age: F1,4 = 0.014, p = 0.911; crater lake size: F1,4 = 0.003, p = 0.960) alien species (table 1). Any undescribed species, in the Midas complex or otherwise, would affect the inferences drawn here, as we will discuss below.
4. Discussion
(a) Each lake has a unique Midas cichlid morphology
Morphometric analysis of the entire Midas cichlid species complex across six crater lakes and both great lakes revealed that Midas cichlids from each lake have their own characteristic body shapes (figure 3). Each lake is significantly different from all other lakes, whether crater or great lake (table 4). This includes lakes that supposedly have the same species composition, e.g. A. cf. citrinellus in crater lakes or A. labiatus and A. citrinellus in great lakes.
Populations in crater lakes Asososca Managua and Apoyo are the most divergent in body shape (figure 3, table 4). Specimen morphospaces from some crater lakes overlap, particularly from lakes Asososca León and Apoyeque, and Xiloá and Apoyo, and from the great lakes. This suggests that similar body shapes independently evolved in species of Midas cichlids that inhabit those pairs of lakes. The morphospaces of specimens from all other lakes, however, are almost completely non-overlapping.
The greatest apportioning of body shape change across the complex occurs in the head and mouth shape, head size, body depth and length of the caudal peduncle (figure 4). Interestingly, these are also body shape characteristics that have been found in other cichlid studies to relate to trophic niche and ecological selection (e.g. Rüber & Adams 2001; Hulsey & García de León 2005; Barluenga et al. 2006; Clabaut et al. 2007; Konow et al. 2008). Thus, though we have not addressed trophic differences here, our findings in light of previous research on eco-morphology in the species complex (Barlow & Munsey 1976; Meyer 1989, 1990a,b; Vivas & McKaye 2001; Barluenga et al. 2006) suggest a strong role for ecological selection and adaptation in the diversification of Midas cichlid body shapes.
The body shapes of Midas cichlids from the great lakes are distinct from those inhabiting the crater lakes. Such a pronounced body shape variation between great lakes and crater lakes is not unexpected because the clear, deep waters of crater lakes are quite different from the shallow and turbid environment in the great lakes of Nicaragua. Nonetheless, some crater lakes are very young, especially Asososca Managua (1.2 kyr ago) and Apoyeque (1.8 kyr ago) (table 1). Therefore, the change in body shape that comes with colonization of a new crater lake must occur quite quickly. Such morphological changes are expected as a first macrohabitat shift, or colonization of a new environment, during the formation of an adaptive radiation (Schluter 2000a; Gavrilets & Losos 2009). Drift or random sampling of the ancestral population may also be important (Mayr 1954). The role of local adaptation and ecological character displacement very likely interact strongly in crater lakes with multiple ecological species, e.g. minimally Apoyo, Xiloá and nominally also Apoyeque (K. R. Elmer, T. K. Lehtonen & A. Meyer 2010, unpublished data). An analysis of body shape with ecological niche and inferred adaptation (sensu Schluter 1993) across lakes would be important in order to clarify whether similar or different selection regimes act in each of these crater lakes.
(b) Variation in crater lakes with one described species
The crater lakes with A. cf. citrinellus—Apoyeque, Asososca León, Asososca Managua and Masaya—may hold several species that are as of yet undiscovered or formally described. For example, morphs with hypertrophied lips occur to different extents in lakes Apoyeque and Masaya (Waid et al. 1999). In Asososca Managua, which has the highest PD, we collected a relatively elongate A. cf. citrinellus at low frequencies (K. R. Elmer, H. Kusche & A. Meyer 2009, unpublished data), and future research may identify it as an incipient limnetic ecotype or species. Asososca Managua has a large water volume, the smallest littoral zone of all crater lakes (Waid et al. 1999) and clear water: thus, it is feasible that rapid speciation to exploit the limnetic niche might occur (sensu Barluenga et al. 2006).
Lake Masaya is maximally 6000 years old. Given the examples of other crater lakes of the region with similar age, Lake Masaya might be expected to harbour multiple species, though they have not yet been described. However, the lake has experienced frequent volcanic activity (Kutterolf et al. 2007), even as recently as 1800 years ago. It is unknown to what extent these eruptions affect a crater lake's fauna, but they could have repeatedly eliminated or decimated the entire fish population. Additionally, historical documents indicate that there were no large fish, such as Midas cichlids, in Masaya 450 years ago (Villa 1976a), which raises the interesting possibility that the Midas population in Lake Masaya is only a few centuries old.
(c) Patterns of diversity in Nicaraguan great lakes
In both Nicaraguan great lakes there is significant body shape differentiation between the species A. citrinellus and A. labiatus (figure 5, table 5). Though the taxonomy has been far from clear (Astorqui 1971; Villa 1976b), these species are recognized as having distinct morphologies (Astorqui 1971; Klingenberg et al. 2003), though little or no difference in meristic characters has been documented (Barlow & Munsey 1976). The body shape of A. labiatus is rather elongated, and it has a more pointed snout relative to that of A. citrinellus (figure 5b). Additionally, A. labiatus has large, hypertrophied lips, which gave it its name.
Intriguingly, both A. citrinellus and A. labiatus have body shapes that are significantly different from their conspecifics in the neighbouring great lake (figure 5a, table 5). Differences among populations of A. citrinellus and A. labiatus between the two great lakes may indicate phenotypic divergence by drift promoted by long periods of geographical isolation. Amphilophus citrinellus in lakes Managua and Nicaragua are significantly genetically differentiated at nuclear microsatellites and mtDNA (Barluenga & Meyer 2004), suggesting a strong role for geographical isolation. Alternatively, the significant intraspecific, interpopulation body shape differences may represent local adaptation to different lake conditions. Abiotic environmental factors such as turbidity, depth, slope and temperature are very similar for both great lakes (Cole 1976), but the lakes differ dramatically in water chemistry (Cole 1976). Lake Managua is much more concentrated in dissolved solids, probably because it has no major out-flow (Cole 1976). Our results for A. labiatus in Managua and A. citrinellus in Nicaragua should be treated with some caution, as the former has a relatively small sample size (table 2) and the latter relies on samples drawn only from the west–northwestern area of the lake. Additional studies to discern the ecological causes of the intraspecific variation between the populations in lakes Managua and Nicaragua are underway, as these populations represent the source of biological variation that colonized the isolated crater lakes.
(d) Midas cichlid diversity and the question of species
An overall assessment of the Midas cichlid species complex was due, given that in recent years six new species have been described (Stauffer & McKaye 2002; Stauffer et al. 2008). Prior to those species descriptions, Klingenberg et al. (2003) analysed body shape differences among the species that were at that time properly taxonomically described: A. citrinellus, A. labiatus and A. zaliosus. The authors found that some species had body shapes more similar to the syntopic populations of their sister taxa than to allotopic populations of their apparent conspecifics. Our current findings indicate that the earlier lack of interspecific resolution in the study of Klingenberg et al. (2003) was due to an A. citrinellus sample that included specimens from different lakes. Thus, this 'lumping' of species would have exaggerated perceived 'intraspecific' variation of A. citrinellus. Future study will probably identify that we too have lumped species that might await taxonomic description. Certainly, more ecological and behavioural research is needed, but it would seem clear already that some of the crater lakes contain as yet undescribed species.
Whether populations of Midas cichlids in each lake should be considered different species—given the significant morphological variation and distinctiveness we have shown here—depends on one's species concept (see Mallet 1995; Sites & Marshall 2003; Wiens 2004). Allopatric populations of Midas cichlids in different crater lakes cannot come into physical contact or hybridize in nature, so they are isolated genetic units on independent evolutionary trajectories. However, reproductive isolation is not complete and Midas cichlids from different species and lakes will breed in captivity if no homospecifics are offered (Baylis 1976b) and there is no evidence for F1 hybrid inviability (to date only tested between A. 'citrinellus' × A. zaliosus, Baylis (1976a)). Thus, we suggest that populations in each crater lake are diverging because of geographical isolation, drift and, ultimately, local adaptation (Templeton 1980). Conversely, diversification within lakes probably occurs under sympatric conditions, depending on specific lake environments and disruptive ecological selection (discussed below). Thus, paradoxically, the question of crater lake Midas cichlid species is less clear in allopatry than in sympatry.
(e) Crater lake characteristics and species richness
Crater lakes harbouring multiple described endemic Midas cichlid species tend to have populations with higher genetic diversity and lower PD than populations in lakes of only A. cf. citrinellus (figure 9). Species-rich lakes may have higher genetic diversity because of intralacustrine population genetic structuring. Perhaps for similar reasons, temperate lakes with benthic and limnetic stickleback ecotypes are also more haplotype-rich than lakes with a generalist ecotype (Taylor & McPhail 1999). Alternatively, allelic richness may simply increase linearly with time when crater lakes are isolated and/or with lake size (size is correlated with genetic diversity in stickleback lacustrine populations; Caldera & Bolnick 2008). Reduced PD suggests a reduction in interindividual body shape variation, perhaps with increasing specialization.
Midas cichlid species-rich crater lakes are older and larger than crater lakes with one species (figure 10). Waid et al. (1999) also found Nicaraguan crater lake size and age to be important in the number of fish species therein. There is no relationship between crater lake age or size and the number of other fish species therein. This contrast argues for a rapid speciation rate in Midas cichlids compared with the other syntopic crater lake cichlids such as Neetroplus nematopus or Parachromis managuensis.
The population in Lake Xiloá has an exceptionally high haplotype diversity relative to the lake's young age. Xiloá has at least once in the past been merged with Lake Managua (Villa 1976c). An historical influx of A. citrinellus and A. labiatus into the Xiloá Midas cichlid population could have increased diversity within the crater lake by contributing new alleles or promoting character displacement into benthic and limnetic ecotypes (Grant 1972; Schluter 2000b), a pattern that is common in freshwater fishes (Robinson & Wilson 1994). Finally, Lake Xiloá has a heterogeneous bottom profile, with steep slopes to the north and shallow, sandy substrate to the south, which effectively increases the number of potential habitats (Lim et al. 1976) and may promote intralacustrine diversification and speciation. The combination of ecological and genetic factors promoting speciation in isolated populations may together further increase species richness (Emerson & Kolm 2005).
Some Nicaraguan lakes are better studied than others. Arguably, we know of multiple species in Apoyo and Xiloá because they are the cleanest, most accessible and therefore best studied of Nicaragua's crater lakes. Finding and describing new species in other crater lakes could change these perceived relationships.
(f) Intralacustrine diversity in crater lakes Apoyo and Xiloá
In both crater lakes that house multiple described species of Midas cichlid, we find that all species can be statistically significantly distinguished in body shape from their sympatric sister taxa. Body shape differentiation among species in Lake Apoyo is higher than it is in Lake Xiloá (tables 6 and 7).
In Lake Apoyo, Nicaragua's largest and oldest crater lake, there is pronounced body shape variation among the four endemic cichlid species (figure 6, table 6). Amphilophus zaliosus, the species with the illustrative common name 'arrow cichlid', is the most morphologically distinct. It is characterized by a narrow, elongate shape, reflecting adaptation to the limnetic niche (Barlow & Munsey 1976; Barluenga et al. 2006). Amphilophus astorquii, a benthic, colony-breeding species, has a short mid-body and is also quite distinct from the other Apoyo species; it is particularly separated from A. zaliosus along CV1. Amphilophus chancho, also a benthic species, has a bulky mid-body and a short lower jaw, which might reflect adaptation to prey types (Meyer 1989, 1990b) different from the other benthic species. The most basal shape in Lake Apoyo can be found in A. flaveolus. Larger sample sizes of A. chancho and A. flaveolus would give us greater power to describe their differences, which are subtle but morphometrically and meristically consistent and significant (present study; Stauffer et al. 2008). Species in Apoyo are endemic, reproduce assortatively in close geographical proximity (Baylis 1976a; T. K. Lehtonen 2008, unpublished data) and are ecologically distinct sister taxa (Barluenga et al. 2006; Stauffer et al. 2008). Thus, the system has been argued as a strong case for sympatric speciation by disruptive ecological selection and associated divergence in assortative mating (Baylis 1976a; Barluenga et al. 2006; Gavrilets et al. 2007; Stauffer et al. 2008). However, to date, this has only been researched in A. zaliosus (Barlow & Munsey 1976; Baylis 1976a; Barluenga et al. 2006).
Lake Xiloá is the only other crater lake that currently has several described endemic Midas cichlid species. All species are significantly different from each other in body shape (table 7, figure 7). As in Apoyo, Xiloá's limnetic species, A. sagittae, is the most distinct in its elongate body shape and small head (figure 7b). It was also named for its 'arrow' body shape (Stauffer & McKaye 2002), like A. zaliosus, and feeds on a predatory diet (Vivas & McKaye 2001). Amphilophus amarillo closely approximates the consensus shape and is only distinguished from A. xiloaensis along CV2. Amphilophus amarillo and A. xiloaensis are both high bodied putatively benthic species that feed on a diet richer in snails (Vivas & McKaye 2001).
There are ecological differences between all three species in the pharyngeal jaw apparatus, choice of breeding substrates (Vivas & McKaye 2001) and nest site depth (Elmer et al. 2009). Xiloá Midas cichlids are a distinct genetic cluster compared with neighbouring crater and great lakes (Bunje et al. 2007; K. R. Elmer, A. Kautt & A. Meyer 2010, unpublished data). Given the sister taxon relationships, ecological distinctiveness, assortative mating, novel environment and complete endemism, there is a strong argument for sympatric speciation of all the extant species in crater lake Xiloá.
(g) Adaptive radiation and parallel evolution
In Apoyo and Xiloá, we identify a pattern of morphological and genetic variation that agrees with expectations of intralacustrine divergence into available ecological niches, resulting in eco-morphological adaptation and subsequent sympatric speciation. Parallel evolution of a limnetic species in multiple crater lakes has been hypothesized (McKaye et al. 2002; Stauffer & McKaye 2002; Barluenga et al. 2006; Stauffer et al. 2008) but not previously demonstrated.
Here, we show the independent parallel body shape evolution of an elongate limnetic ecotype in crater lakes Apoyo and Xiloá (figures 6b,7b). Amphilophus sagittae is limnetic (Vivas & McKaye 2001) and ecologically equivalent to A. zaliosus (Stauffer & McKaye 2002), which is also limnetic (Barlow & Munsey 1976; Barluenga et al. 2006). The original species description of A. zaliosus noted that 'fish similar in appearance' were living in Xiloá (Barlow & Munsey 1976), but until now no formal morphological comparison of similarity and parallelism had been pursued. As first suggested generally by Barlow & Munsey (1976), the morphospace of species is generally equivalent in lakes Apoyo and Xiloá (figure 3). Morphological variation is described in the same direction of shape change into benthic and limnetic forms (figure 8). Body shapes of A. zaliosus and A. sagittae are generally more similar to each other than they are to other species in their native lake. Both species have slender, elongate bodies and longer caudal peduncles compared with their syntopic, less morphologically differentiated species (figures 6,7).
In Lake Apoyo, A. zaliosus originated sympatrically from an 'A. citrinellus' ancestor within the last 10 000 years (Barluenga et al. 2006; Elmer et al. 2010). Amphilophus sagittae is almost certainly younger, since Lake Xiloá itself is only 6100 years old. The less dramatic morphological differentiation between A. sagittae and its syntopic Midas cichlids compared with the distinctiveness of A. zaliosus relative to its syntopics might be due to the comparatively recent speciation. Rapid evolution of a limnetic morph, even under crater lake sympatric conditions, is in agreement with theory and model predictions (Gavrilets et al. 2007).
Ecomorphological parallelism across independent lakes is a prediction of ecological speciation and strong evidence for adaptation to local environment, rather than variation owing to drift (Schluter 1996). Such divergence can occur quickly (Hendry et al. 2007), especially if adaptation or speciation is based on standing genetic variation, i.e. alleles that are present in the ancestral population, rather than novel mutations (reviewed in Barrett & Schluter 2007). The limnetic ecological niche associated with the deep open water found in crater lakes is a novel habitat for Midas cichlids compared with the shallow ancestral habitat of the great lakes. Thus, disruptive selection on eco-morphology and concomitant assortative mating is thought to drive speciation in the crater lakes (Barlow & Munsey 1976; Baylis 1976a; Barluenga et al. 2006). In the temperate zone, competition and predation are thought to be the main drivers of diversification rather than lake physical characteristics (Robinson & Wilson 1994; Vamosi 2003), though some physical and chemical attributes may be important as well (Vamosi 2003; Landry et al. 2007; Gow et al. 2008).
Ecological speciation by disruptive selection is thought to be difficult to accomplish (Felsenstein 1981; Kirkpatrick & Ravigné 2002; Bürger et al. 2006; Nosil et al. 2009). It requires that multiple factors occur in concert: strong trade-offs in efficiencies to maximize fitness (Levins 1962); spatial and/or ecological conditions that maintain polymorphism and frequency-dependent selection (e.g. Udovic 1980; Otto et al. 2008); the evolution of reproductive isolation between ecologically dissimilar types (Maynard Smith 1966; Rice & Salt 1990; Kirkpatrick & Ravigné 2002); and a mechanism for ecologically relevant mate choice (Higashi et al. 1999; Takimoto et al. 2000). Conversely, under the forces of natural selection, parallel evolution may be rather likely (Orr 2005) and disruptive selection rather common (Martin & Pfennig 2009). Correspondingly, a number of taxa do show parallel evolution of eco-morphological traits (reviewed in Schluter 2000a) and cichlid fishes in particular include convincing cases of parallel evolution (Meyer 1993; Galis & Metz 1998; Stiassny & Meyer 1999; Turner 2008; Salzburger 2009). The only known limnetic neotropical cichlid species are A. zaliosus (Barlow & Munsey 1976) and A. sagittae (Stauffer & McKaye 2002), so ecological speciation into this niche may be rare. However, our findings suggest that intralacustrine diversification can occur very quickly when environmental conditions are suitable.
5. Conclusion
Understanding the environmental and biological conditions that result in an acceleration or stasis of speciation is important if we are to test rigorously the current theories of ecological speciation and adaptive radiation (Schluter 2000a; Seehausen 2006; Nosil et al. 2009). Our findings suggest that Midas cichlids in Nicaraguan crater lakes follow an expected pattern of adaptive radiation: divergence in macrohabitat (lake colonization), then microhabitat (benthics and limnetics within lakes) and associated divergence in adaptive traits and assortative mating (Gavrilets & Losos 2009). Albeit the Midas cichlid species complex does not match the species richness of, for example, the African cichlids (Kocher 2004), we consider the Midas cichlid species complex across crater lakes to be an incipient adaptive radiation (sensu Schluter 2000a). This groundwork should allow for predictive hypothesis testing in future research. It also supports the notion that the crater lakes in Nicaragua represent repeated 'natural experiments' (Wilson et al. 2000; Barluenga et al. 2006).
Because all Nicaraguan crater lakes are unique with respect to colonization history and environmental factors, it may be that different histories influence the rate of speciation in various lakes (e.g. age and isolation in Apoyo, flux in Xiloá). We are restricted in replicates of these natural experiments to the number of crater lakes that exist. Nonetheless, many natural model systems with sympatric ecotypes of freshwater fishes in young, isolated and simplified lakes have a similar number of replicate environments, e.g. five lakes with sympatric benthic and limnetic stickleback ecotypes in British Columbia (Gow et al. 2008), six St John River lakes with dwarf and normal lake whitefish ecotypes (Landry et al. 2007), three Cameroonian crater lakes (Schliewen et al. 1994; Schliewen & Klee 2004). These systems have offered unparalleled insights into adaptive radiation and speciation in fishes.
Myriad aspects of the Nicaraguan Midas cichlid species complex call for further ecological and evolutionary investigation. For example, quantifying ecological and genetic diversification that occurs shortly after colonizing a new lake; testing the extent of reproductive isolation within ecotypes across lakes (i.e. parallel speciation sensu Schluter & Nagel 1995); assessing the adaptive roles of morphological and colour polymorphisms. Because of parallel evolution across lakes and the emerging genome resources (whole genome sequence: International Cichlid Genome Consortium 2006), the Midas cichlids also offer an exciting avenue for inferring genomic and transcriptomic bases to phenotypic variation (Elmer et al. 2010) and the role of standing genetic variation versus novel mutation in parallel evolution and natural selection (Schluter & Conte 2009; Via 2009). The success of other freshwater fishes with divergent sympatric ecotypes offers a roadmap for further experimental and conceptual challenges (e.g. Peichel et al. 2001; Kingsley et al. 2004; Rogers & Bernatchez 2005; Jeukens et al. 2009; Schluter 2009) that can be tackled with the diverse Midas cichlid species complex.
We thank M. Barluenga and W. Salzburger for some specimen photographs. Many thanks to W. Challenger and A. Cardini for statistical advice and A. Freundt and S. Kutterolf for help sourcing geological information. This research was supported by an Alexander von Humboldt and Natural Sciences and Engineering Research Council fellowship to K.R.E., Alexander von Humboldt and Academy of Finland fellowships to T.K.L. and grants of the DFG and the University of Konstanz to A.M. A.M. thanks the Institute for Advanced Study in Berlin for a fellowship that aided in the preparation of this manuscript and H. Ellegren for the invitation to the Wren-Foundation Kristineberg workshop on speciation and for permitting us to contribute to this special issue.
↵† Present address: School of Biological Sciences, Monash University, Victoria 3800, Australia.
One contribution of 11 to a Theme Issue 'Genomics of speciation'.
- © 2010 The Royal Society


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This design is easy to assemble and because it's made out of paper it can be temporarily hung on the wall without much effort.
Are you creating any 3d paper projects for spring?
Quick project tip : When creating projects for a party or special event, your Silhouette paired with a patterned paper stack will be a time saver! The Silhouette to do all of the cutting, and a paper pad that comes with pre coordinated colors and patterns to take out all of the guesswork. Genius!
Happy making!

subset: commoncrawl


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Thursday, October 22, 2009
Tutu Giveaway!
Allison's Wonderland is in need of some fabulous photos of cute kids in her tutus for use on her esty store site! If you have a professional photography session coming up (or you are a pro yourself) and would like a tutu, please comment below! Allison will choose two little ladies to receive a tutu. In exchange, you send us 2-3 digital images (or more!) from your photo shoot to use on www.myallisonswonderland.etsy.com. Please note that I will need permission from your photographer, and their information to give them credit. Photos that focus on the tutu in addition to the adorable child are preferred. Tutus will be custom made to your waist, length and color preference (from our in stock colors). In your comment, please let me know the age of your child, and when your photo session is. Winners will be chosen on Saturday, October 31st!
6 comments:
Hi Allison! I'm planning to have a 2 month photo session for my daughter and DH and I at the end of November. I'd love to include pics of her in a tutu!!!ReplyDelete
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Does a 18 month old count or do you want younger babies? If so count me in!ReplyDelete
We take monthly "professional" pictures at our house but can do them anytime.
Older kids and babies count too! I need all ages!ReplyDelete
I happen to have a pretty cute kid and a wonderful friend & neighbor who is a professional photographer so you should for sure pick us! :)ReplyDelete
But...if you are looking for an itty bitty baby I have another very good friend who has a 3 1/2 year old little girl and a newbie baby girl too. The newbie was a preemie and is just now getting to 8 pounds..at 5 months. The momma is a budding photographer and I'm sure she would love to take some photos for you.
But then again...you could always just pick me. ;)
Would love to have Julia model one of your tutus! My husband is a photographer, so he could do as many sessions as needed to get just the right picture for your store! Julia is almost 5 months old.ReplyDelete


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Here is a department that's at least working hard to confront their situation.
Tuesday, February 05, 2008
"New Humanities"
Check out this video about the transformation of English at Rutgers, done by the department chair. The video itself is slick and about hustling money, I guess, but the idea of a "Center for New Humanities" has got real appeal. Around 5:45, note that he says the humanities "somewhat lost its way" and, more importantly, argues that the "real function of the humanities" is to be creative "to improve the quality of the world we live in".
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A good neighbourhood restaurant is a wonderful thing. That place you can walk to, even in the grimmest weather, enjoy genuinely familiar service, see those people from down the road. To some it might sound awful; a break from the anonymous cover that London affords. Not I.
It's something I've thought a lot about recently - a colleague was recently telling me about neighbourhoods that benefit from good systems or good levels of empathy. Good systems (transport) mean people come and go easily, and so lack empathy. Empathetic neighbourhoods typically have poor systems, but people rub together well - using local facilities. I like to think this is true of much of Hackney - our relative disconnecteness means we spend more time on our doorstep, and benefit from the warmth of familiar contact.
Shane's on Chatsworth in Lower Clapton is a fine example of a neighbourhood restaurant, and not just because I can walk there in two minutes. It's at the centre of the neighbourhood, sandwiched between a fruit and veg shop and an old electrical store, with big windows looking onto the street - it's part of it. It's small, bright and personal, no more than 30 covers, so it always feels intimate and cosy. Shane, the proprietor and chef, pops up to make sure everyone is happy and enjoying the food.
And they are. The food is excellent. Modern British some might say, focusing on well sourced game and seafood from not too far away, some foraged leafs. It isn't pious about it, which is refreshing. We were there on a Saturday in November, and enjoyed the riches of the latest haul of game from Shane's supplier.
Starters were simple and understated: we particularly enjoyed the venison scotch eggs with a semi-soft quail's egg inside. The venison was really simply and effectively seasoned and had really high meat content. It came with lightly pickled autumn veg.
We also enjoyed the mixed game terrine - proper chunks of pheasant, pigeon and venison, brushed with an almost sweet jelly and served with pickled pears. All flavours amazing together - meat and fruit, an ancient combination.
The wild fallow deer came highly recommended and my fellow diners certainly enjoyed it. It was so tender, but still bright pink inside. Served with a celariac puree and red and gold beetroot, and the flavours were perfectly autumnal.
I opted for wood pigeon with a herby parsnip puree and kale with chestnuts. It was unbelievable. The pigeon was perfectly cooked - seared and charred on the outside, pink and juicy inside, a small amount of luscious jus. The parnsip mash was intense, rich and bursting with woody sweet flavours. Kale and chestnuts is a favourite combination - just couldn't get enough.
I had a sample of a friend's hake and shellfish stew - delicious, subtly and served with warm crusty bread.
The meal was incredibly filling, despite portions being sensible in size. But we had to try desserts - clementine posset, beetroot and chocolate cake with candied fennel, and a bread and butter pudding. All were delicious but the clementine posset was particularly good - so zesty and refreshing after all that rich food. Clementine is underused as an ingredient, and it's just perfect at this time of year.
With two glasses of wine each, starters, mains, desserts and a tip it came to £34 a head. It's definitely a fair price for the quality, though some local people were surprised at the prices when they saw the first menu up in the window earlier in the year. Shane has responded and now there's a range of menus and offers to make it more affordable - there's now a two/three course with wine option for £18/£21, afternoon tapas and wine offers that are very fair priced. I loved it, and am so pleased to have it in the neighbourhood. I'll keep going back to check out the menu as the seasons change.


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Aggressive cost cutting helped Vonage eke out a narrow profit in its second-quarter results. Still, I believe the earnings news is a last hurrah rather than a sign of things to come.
How can Vonage stay afloat in competitive sea roiled by triple-play cable MSOs and telecommunications carriers on one side and voracious wireless operators on the other? The latter, in particular, are mercilessly eating into Vonage's subscriber base.
Vonage was a pioneer in bringing VoIP services to the masses. Barring a magical reinvention, it doesn't have a bright future.


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Sunday, March 28, 2010
A First!
I guess there's a first time for everything.
Have your best Palm Sunday NOW! ;-)
17 comments:
- Jason Kanz said...
Wow. Have the read you? Have the read Osteen? That's like comparing chocolate bars and broccoli.
- March 28, 2010 at 8:31 AM
- Jared said...
Wait -- which one am I?
Please say chocolate. :-)
- March 28, 2010 at 8:44 AM
- Jeff Sahol said...
Don't let it go to your head, Jared.
- March 28, 2010 at 9:47 AM
- Mike Leake said...
Oh come on, you have to link to that. That is hilarious...or sad...maybe both.
- March 28, 2010 at 11:56 AM
- Jared said...
Jeff, LOL
- March 28, 2010 at 11:57 AM
Well Jared - if you're sitting in coach on a plane anytime soon, and you get told by the stewardess that you've been inexplicably bumped to first class.......just do us a huge favor and tell us that "something cool" happened, rather than that "God's special blessing is on your life"............
......btw - I'd like to see the context of this also..... because one of the main reasons that you're awesome is that you're the opposite of those guys.
- March 28, 2010 at 1:40 PM
- Jared said...
nhe, I hate to introduce the morass to you, but if you're a glutton for punishment, the thread is here:
- March 28, 2010 at 1:43 PM
Hahaha! That's incredible! I just love it. I want to read it all over again.
Jared, didn't the same thing happen to you over at Evangel?
- March 28, 2010 at 3:34 PM
- Ken Stoll said...
all you need is a cheesy smile, a perm, some new suits, a big bushy gotee and some sweat on your brow... oh, and you need to do an about turn on your theology.
- March 28, 2010 at 4:21 PM
- Mike Leake said...
wow, thanks for the link. I think I'm going to stop reading your blog. Not because I'm offended but I'm convinced that you are too young to blog.
Keep up the good work my friend!
- March 28, 2010 at 5:14 PM
- Byron Harvey said...
Dude, what's your point? That's exactly what I think of when I think of you: Jakes, Osteen, Wilson, and Hinn, the Four Horsemen of...something.
- March 28, 2010 at 8:02 PM
Jared - I liked the lady on there who gave us a link and told us that we all need to listen to a 55 minute sermon from her pastor....then we would all understand what you meant.
Yikes!.....fortunately, my short attention span is better suited to your controversial 3 sentence blog posts......even if you were a heretic, at least you'd be giving it to me in small doses......my being a chill-out loving slacker has its advantages.
- March 28, 2010 at 8:08 PM
- Doug said...
At least no one tried the "athlete's foot" defense...
Part of me is just really sad/angry about this, but I'm trying to see what I need to learn here. It shows how deeply we need to keep giving the Gospel to each other. We (and I really do mean "me" in there too, though at times I can be arrogant and forget that) really would prefer a Savior whose yoke is a little heavier, and whose burden came not so light. It'd make us feel better about the fact that we can't pay Him back for loving us.
Keep preaching, Jared. And please don't ever wear a silk suit. This is not negotiable. ;-)
- March 29, 2010 at 1:44 AM
Doug - I've found that I Cor 3:10-15 gives those who prefer a heavier yoke some comfort.
They do of course have to realize that (technically) they don't get to keep the gold for themselves - however it gives them something to strive for (all of us actually, since Eph 2:8-9 also includes vs. 10).
- March 29, 2010 at 12:29 PM
- Philip said...
Jared, just read the thread...
Wow. I don't know if this makes you feel better but...
but I've been told I remind people of Joel Osteen by how I look! Yipes.
And I know that Paul said, "Don't let anyone look down on you because you are young" but how is one supposed to do that? I wish he would have said. I have yet to be able to keep anyone from looking down on me because I'm young. (I also look young, which makes a difference. People are way more prejudiced then they realize.)
That "young" comment was out of line.
- March 29, 2010 at 4:31 PM
- Jared said...
Phil, yeah. That one was the most condescending, I thought, and made worse b/c it was passive aggressive. Saying I'm too young to know better is just a "nice" way to say I'm too stupid. But I'll get older/smarter, like him.
I didn't really respond (although I asked how young was too young) b/c by that point I had decided defending/clarifying would be a matter of pride and I wanted to just embrace confidence in knowing who I am in Christ and satisfaction that what I said was said clear enough for anyone who had the ears to hear.
Also: You're older than me! Geezer.
- March 29, 2010 at 4:35 PM
- Marty said...
Truly odd. Just keep preaching the word brother. And laugh. You have to laugh at things like this.
- March 29, 2010 at 9:20 PM


text: Greetings, An update to all Orion Subscribers *****OPENING CEREMONY***** Sunday January 12, 10:15-10:30 am, Humanities Bldg. 2713 The Orion Center is pleased to invite you to the Opening Ceremony of its Second International Symposium. We are honored to have as distinguished inaugural speakers: Professor M. Milson, Dean of the Faculty of Humanities Professor Y. Zakovitch, Head of the Institute of Jewish Studies. The first paper of the Symposium will commence immediately following the Opening Ceremony at 10:30 am and will be given by the Director of the Orion Center, Professor Michael E. Stone. *****PROGRAM ADDITIONS AND CHANGES****** *Sunday January 12* 10:15-10:30 am Opening Ceremony, Inaugural Speakers: Professor M. Milson, Dean of the Faculty of Humanities Professor Y. Zakovitch, Head of Institute of Jewish Studies 10:30 am M.E. Stone, "The Axis of History at Qumran" 2:30-3:30 pm Orion Web Site Visit and Web Bibliography Demonstration, Orion Center Scholars' Room Humanities Building. Room 6115 (Tel. 58881230) *Monday January 13* 12:30-2 pm Orion Web Site Visit (details as above) 3:30-4 pm Martha Himmelfarb is unable to attend and sends her regrets. M.E. Stone and E. Chazon will present the Orion Center and elicit the participants' suggestions for future programming


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today consisted of canning tomatoes with my mom.
my mom rocks. she is a canning superstar-- i want to can like her when i grow up.
i have 28 pretty quarts of diced tomatoes cooling on the counter and 10+ bags in the
freezer. not bad for an afternoon of work and a morning of prep (plus clean up from
yesterdays picnic). my hands smell like tomatoes. we had mexican take out tonight. i am full.
watching "the ghosts of girlfriends past"
yesterday was one of the best days.
picnicking in the park with friends + veggie hot dogs+ croquet = too much fun
i am blessed.
i am thankful.
i am tired.
tomorrow morning i am canning chunky applesauce for my husband...yum


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Stage 3 - Who is This Super-Hero?
Time to start work on our "gun". Given our theme, we obviously need a phallic image. We were going to go with a simple pixelated erection, but google image search came up with this winner:
"My parents laughed when I told them I wanted to major in Drama,
but look at me now."
(You can be as cool as this guy for just $49.99.)
We think the model in the picture above has had enough humiliation for one lifetime. We'd like to replace his face with that of an utterly debased public figure, with not a shred of dignity and no redeeming features. We also need someone who's light on his feet. Who better than ex-Whitehouse Chief-of-Staff and soon to be king of the Cook County kleptocracy, Rahm Emanuel?
Ballet Rahmbert.
One complication is his finger: lost in a tragic goosing accident when Nancy hiccuped.
(We'll ignore it : pretend he's wearing a "fingldo" prosthetic.)
(We'll ignore it : pretend he's wearing a "fingldo" prosthetic.)
Behold! Rahm, the Hebrew God of Dicks.
We know Rahm's light on his feet. Is he light on his balls?
Looks good. Now we just need to add a black background and mask trailing frames...
The Balled Avenger rides again.
Tomorrow we'll get him "shooting". (Unless the Illinois Supreme Court enjoins us not to.)
P.S. Google is pure genius. Just previewing this brought up ads for "email marketing". Don't mention H3RB4L V14GR4!


text:
Here is a video I took on my phone when she walked in. I sound like a 4-toothed hag from the depths of Mississippi. Brittany sounds like a cackling witch! This video makes me teary-eyed though- Megan was SO surprised!
We had the BEST weekend. I seriously love these girls SO much. We have all been best friends since the 6th GRADE!!! Can you believe that?? We were inseparable in high school, all went to different colleges, and now live between 2 states- all but 2 several hours apart. We talk or text weekly and pray for each other daily. It's rare that we are all together, and in fact it has been over 9 months- last Christmas that we were all together. Sweet Megan has had a tough time adjusting to a new city where she knew NO ONE. She has the cutest house and coolest job and I'm so glad I finally got to visit her!
What is amazing to me about our friendships- is that we literally can pick up right where we left off. We also don't have to be entertained. We always have plenty to talk about and spent many hours on Meg's back porch talking about life (and reminiscing about the occasional high school shenanigan we participated in.) We did some shopping on Saturday, then ventured out for a field trip to the site of the Branch-Davidians compound in Waco. It was super creepy, but the history and story was very interesting. There isn't much to do in Waco so we had to get creative!
One of the porch convos- me explaining that I have man hands. My ring in the middle, Megan and Kori's rings wont fit past my knuckles. :)
Jessica, me, Britt, Meg, Kori, and Tiffany
I love these girls so much! We are having our annual Christmas party in Fayetteville this year and I can hardly wait to see these girls again!


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Admiralty Courtand sentenced to hang on the pirates' gallows at Wapping Old Stairs.
MORE to BROWSE - Pages that might be of Interest
8 February 2011
Alice Tankerfelde -
Admiralty Courtand sentenced to hang on the pirates' gallows at Wapping Old Stairs.
6 comments:
Interesting that Alice was tried by the Admiralty because the crime was committed on water!ReplyDelete
The most notorious pirate to be hanged in this way at Wapping was Captain William Kidd in 1701.
Poor guy was "pushed off" (i.e. hanged) twice as the first time the rope broke.
A condemned man (or woman) was measured for "chains" (a sort of metal cage to contain the corpse) before they were hanged - an act said to be more frightening than the hanging itself.
In Kidd's case, after the tide had washed the body 3 times his corpse was tarred and left on the gibbet t rot - as a warning to others.
Fascinating, Helen.ReplyDelete
I may be wrong but I thought that if an execution failed at least once, the criminal was pardoned and freed?
This might depend upon the era/crime/case?
as far as hanging pirates was concerned its twice Hus - third time lucky!ReplyDelete
Some did not die though - there's an account of one poor man hanged for piracy who woke up laid out on the disection table at the RN college!
WOW! The way this was written was laid out so well, it kept me intrigued and made me almost feel like I could visualize being there. Kudos to Ms. Watson for luring the reader in... it certainly kept my attention and almost makes me want to get the book. I'm definitely going to bookmark this site. Great Job!ReplyDelete
- AnonymousFebruary 09, 2011 2:35 am
This is fascinating, Mari! I am so intrigued by the murderesses of the past. One that you might be interested in is Florie Maybrick (who was accused and convicted of murdering her husband, though she probably didn't).ReplyDelete
I enjoy your writing style and hope that you post some more of this type. I also was curious--did your investigation give any indication as to why those two particular men were their targets? Were they business foes of Wolfe?
Keep up the good work!
I really enjoyed this article! It's a slice of history not often covered. Kudos to the author and the blog!ReplyDelete
Thank you for leaving a comment - it should appear immediately, but Blogger sometimes chucks its teddies out of the cot and has a tantrum (especially if you are a Wordpress person) If you are having problems, contact me on author AT helenhollick DOT net and I will post it for you. Sometimes a post will appear as 'anonymous' instead of your name or avatar - I draw attention to this being a Blogger Blooper and not of MY doing... However ...SPAMMERS or distasteful rudeness will be stamped on, squashed, composted and very possibly cursed - if you spam my blog, next time something nasty happens to you just remember that I DID warn you...


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Jack Hazlett has been working at home as much as he can since his wife died, but every now and then he has to show up at the office.
Victoria "Vicky" Lowell arrived on Hazlett's doorstep with the intention of buying the men's bicycle he had advertised for her young son. She didn't anticipate him leaving her with his children all day without a how-do-you-do.
Vicky is excited about her new job. She's currently in an abusive relationship and relishes the opportunity the money will make for her and little Tommy. But when Hank turns his abuse on Tommy, Vicky has had enough. She turns to Jack and he allows the two to move in with him and his three little girls.
Although Vicky and Jack have no desire for a relationship, the working nanny and employer scene seem to suit them just fine, they still begin to have feelings for one another. The sense of family grows, but when Tommy's dad arrives wanting partial custody, Jack knows he needs to do something. He offers Vicky the haven of a marriage in name only. The girls will have a mother, Tommy will have a father, they will both continue with their lives. But Jack and Vicky cannot hide their true feelings for one another indefinitely, and eventually, hopefully, Jack will learn to smile and Vicky will learn to look him in the eyes.
Where Dreams Are Born is a captivating, heart-wrenching story of love, loss and hope. The characters are realistic, the plot tight and compelling. A winning story of coming to terms that loving someone requires a leap of faith sometimes and that dreams can come true, even from the least expected places. A stellar read!


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|Big Red and Baxter both keep me company in Living Room during Winter
Some days I really miss touring on my Bike. Sure I bike around town every day, even in Winter, but it's not the same. I miss the simple life of just riding my bike to the next destination, not caring what's happening in the world. Riding through some of the most beautiful country in the world.
I am already thinking of ideas for next year...though I probably will stay a bit closer next Summer.
While I miss my Bike Touring during the Winter, I miss my home and birds while Bike Touring in the Summer. It sure is nice being home with my birds right now, as I always feel so bad leaving them for a time while I travel as I love them dearly.
|Did someone say Popcorn Time?
|Popcorn Time: The Aftermath. Popcorn carnage everywhere!


text:
|go ahead... be a heretic
Regex substitution modifier /rby nonsequitur (Monk)
|on Dec 15, 2012 at 00:25 UTC ( #1008916=perlquestion: print w/replies, xml )
|Need Help??
nonsequitur has asked for the
wisdom of the Perl Monks concerning the following question:
I have been trying to use the regex substitution modifier "r" to work with no success. I finally tried running the examples from perlrequick and cannot get them to work either. This example is from the section titled "The non-destructive modifier s///r".
When I run it I get this error.I get the same error using strict and declaring the variables.
Can anyone tell me what's wrong?
Back to Seekers of Perl Wisdom


text:
Sunday, 8 January 2012
Happy New Year
Ok, I'm a bit late. A bit busy. Had a good start to the year. In Germany at the moment working at
Studio Soi
on The Amazing World of Gumball. I'm here a couple more weeks. Just happy to be away for a bit. Anyway this is a quick post, with drawings done with the brushes app on I phone....
And Again....
Posted by
libra bear
22 comments:
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text:
After a very long time without a computer or an in-home internet connection, I have returned! Ah, my little blog. Reunited and it feels so good! Though I have no plans whatsoever for blog posts at this juncture, I sure am glad to have my personal forum at my disposal once again. We'll see what the future holds, there's always plenty to talk about. As it is, I just wanted to put a finger to the pulse for a second, but keep your eyes peeled for more activity from ole PJE in the near future. Also, bad as it is, I've all but wiped my past posts because I'm really not sure what direction I want to take the blog and it was quite a mish-mash of topics before. For now I'll leave a few oldies but goodies up and just figure out the rest from there. Cheers!
Category Archives: Uncategorized
Cloth Diapering on a Budget
Some people do it to save the planet, some people do it because it's cute, and some people do it to save money. Cloth diapering, the fluffiest and most stylish way to do all three!
Personally, I cloth diaper primarily for the budget benefits. (Don't misunderstand me – I think cloth is cute and want ALL the diapers!) We aren't what you'd call rich, and pack after pack of disposables gets to be pretty expensive. We currently only have one kiddo in diapers, but we're about to have two in diapers at once, and it wasn't so long ago that we went through Round One of having two in diapers at once. Sorry, but I'm not willing to pay that much for something that catches excrement once and then needs to be thrown away. My budget is just too tight for that!
Now, if you've already looked into cloth you know that the up-front cost is pretty steep. So, we'll address that first. How can you save money using cloth if you can't pay for the cloth to begin with?
*Note: This post is going to assume familiarity with cloth diaper types and terms. If there's anything you don't understand, just ask! Or Google it. Google is fun.
1. Cheapest of the Cheap
One thing many folks do is head to their friendly neighborhood Walmart and pick up a pack or two of flour sack towels for something like $4.88. Two packs will get you 10 flour sack towels, which can be used in the same way as flat diapers and will diaper a baby of pretty much any age for at least one day. Of course, you'll need a cover of some sort, and my suggestion is to go ahead and put your dollars into covers. There are some relatively inexpensive brands, and on the whole you don't need that many covers, especially if you're using PUL covers without mesh or cloth inners – they can be wiped clean between diaper changes (instead of needing to be washed after being used once) as long as no poop or pee gets on the elastic casings or the outside of the cover.
If you want to do less creative folding and quicker diapering of baby you can look into grabbing a dozen prefolds. Green Mountain Diapers carries great prefolds, FYI, and sells them by the dozen! Just keep in mind that these are going to cost roughly three times as much as flour sack towels (though a $30 dozen of diapers is still not bad!). Prefolds are sized but this doesn't necessarily mean you need to buy a new dozen every time baby outgrows the last set. Even though my kiddo has outgrown them, I keep a dozen GMD mediums around and use them trifolded inside of his diapers. I use mostly pocket diapers, so I use the prefolds as doublers, but the idea is the same. You can trifold the prefold and lay it in a cover and it will do the job nicely, even after baby has outgrown that size. I find this works best with size medium prefolds, they seem to fit perfectly into most covers.
2. Movin' on up!
Personally, I see nothing wrong with investing (when possible) in a smattering of good pocket diapers, AI2s, and AIOs. You don't necessarily need to buy them new, as fun as it is to get a brand new diaper. There's nothing wrong with growing your stash with used diapers as long as you strip, or at least initially sanitize them before putting them on your baby. Buying used is an excellent way to add variety to your stash, in terms of both convenience and effectiveness (we all know that every baby is unique and every diaper is constructed differently and absorbs differently, and you need to find what works). And, I'm not going to lie, as thrifty as it is to diaper using only flats and/or prefolds, it's not as much fun. So finding a way to combine budget with convenience, fit, and fun is a reasonable goal. This is where buy/sell/trade groups and Craigslist come in. You can find all kinds of pre-loved diapers exponentially cheaper than retail, and if you happen to be crafty you can find diapers with issues like shot elastic or worn out hook & loop for really cheap and then restore them to good usable quality!
Don't feel bad about wanting the occasional new diaper, though. Getting something new is really gratifying, we can be honest here. That's where co-ops come in. I've personally never used a co-op but I know people who participate all the time! Many diaper companies will place a discount on diapers sold in bulk, and co-ops are designed to make the purchase of a bulk lot possible by having many people contribute to the cost. Then everyone gets their cut of the order, and it ends up being cheaper per diaper. The downside is that co-op orders can take a long time to arrive, but patience is a virtue, right?
Another thing to do is to simply shop clearance on cloth diaper websites, take advantage of deals and discount codes, compare prices between websites (including shipping!) and space out purchases so that big lump sums aren't hitting your wallet.
3. Share the Love
When the savings become really obvious is when you have more than one child using the same stash of diapers. In my case, many of my diapers will have been worn by three children! Three children, and no need to buy a whole new stash for each! That's not to say I've never had elastic break, or lost a diaper. Heck, I've had someone – who is still a mystery – somehow vanish an entire pail liner full of dirties, presumably because they didn't agree with me cloth diapering (In the beginning I had no support at all in the endeavor, and lots of opposition!). So I have had to rebuild my stash after a few hits, but never from the bottom up! And even with needing to replace diapers, the savings inherent in not buying boxes of disposable diapers for THREE children are still phenomenal! Most people will not lose an entire pail liner's worth of diapers at once, so it's safe to say that most folks won't need to rebuild like I did! Even better!
4. Touchy Subject
I'm a firm believer that cloth diapers are just like any other oft-worn cloth in that they launder the same way. So another way I save money is by not buying into "cloth diaper safe" detergents, which cost a lot for only a little and have weak cleaning power at best. That's no bargain as far as I'm concerned. I'm also not the least bit concerned with warranties, which calculate out to less than $2/month for one year anyway in most cases, and usually carry an insane number of stipulations in order for the company to honor them at all. People get pretty amped about this, but the science and the experience speak for themselves – cloth diapers do just fine – in fact, they do AMAZINGLY – in regular amounts of regular detergent. So I use the same detergent on my diapers as I do on my family's clothing. In fact, I often put clothing in with my diapers after the diapers have been through their pre-rinse. Not buying separate detergents and combining loads is a no-brainer when it comes to saving money! Not to mention it makes laundry a lot simpler. As an added bonus, washing diapers as I do (regular amount of regular detergent, and only doing a pre-rinse and a regular wash – no extra rinses) means I'm saving water as well! Rinse, rinse, rinse is a thing of the past.
Sure, you can find plenty of people who will argue that cloth diapers aren't really saving money at all (or the environment, or that they're not even cute), but I've got the experience. I can tell you right now that for me, cloth has been an incredible help to us financially! And it can for you too.
Cleaning House (Virtually!)
I started this blog when I deleted my old Facebook profile as a way of continuing to update loved ones about my usual goings-on and to provide some access to pictures of and anecdotes about the kids. The blog has mostly been a mish-mash of random posts since then and I really wanted to sharpen my focus. I care most about parenting, so – much as it may be considered a no-no – I moved a whole bunch of my old posts to the trash. I didn't have the heart to outright delete them, but they may yet go the way of the buffalo. I've got to learn to let go sometime, ha!
So after a long hiatus and no small amount of internal debate I have returned with a vision for this blog and will henceforth post accordingly. This may mean my posts are more sporadic, sometimes many posts close together, sometimes fewer posts with farther in between, but I have not left this blog for dead! See you all soon!


text:
Thanks to Jennifer Bremer for writing this article & everyone at the American Maine Anjou Association for thinking of us. With everyone's support our beef business continues to grow & the success of our Ranch is due to all of you who support us!!
I have updated our website recently & will continue to add more pictures.
THANKS for all your support & encouragement!!!
Friday, October 15, 2010
Wow! We were featured in the latest issue of the AMAA Voice
Posted by GreenRanchingMom at 11:40 AM
1 comment:
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text:
Sav Zigzag 8.2
Sav Zigzag is a mobile object-relational DBMS that represents a Java library (API interface) working on PC, PDA and mobile phones.
This API interface forms new powerful Java/Zigzag language, more efficient than PL/SQL. Zigzag algebraic expressions enable to process tabular and XML data. Unlike XSLT, Zigzag is not data filter; it reads many of XML files in one database and then generates and prints new XML files. The some peculiarities of database: many-valued data, multi-level classification, class inheritance, dynamic structure, and automatic data integrity control. The Sav Zigzag, working under one JVM, provides synchronous connection many of users with many of local databases.


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{delight by design, amsale, janne petters, martha stewart weddings}
good morning and welcome to Weddings Fresh color inspiration week! i am so excited to explore chic color palettes that will add elegance, sophistication and maybe a little drama to your wedding day. today, we combine the innocence of blush and soft rose with the magical warmth of gold. an ideal palette for romance.


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Rotarians in Benin organised a massive media blitz in the run up to and on World Polio Day today, organising a feast of activities – from radio interviews on OCEAN FM on Polio and the implications of Rotarians in this fight to the End by the NPPC Chair Ashok Mirchandini -to Past President, Prof Hypolete Agboton (a member of the National Eradication Experts Committee), doing an animated hour-long phone-in show on CAPP FM. Chair Ashok also did a 52 minute show on LC2 TV on all aspects of polio eradication. This was followed by a 15 minute interview on National TV ORTB, with Zenabs THIS CLOSE AD serving as a backdrop.
Newspaper THIS CLOSE ads and interviews with Chair Ashok in dailies completed the well-organised media blitz spearheaded by Rotary Public Image coordinator for Zone 20A – Boris Crestia.
Ashok Mirchandani, who is also the Zone Challenge Coordinator, got Rotarians to double their TRF recognition points by contributing to the Polio Fund during the week leading from 18-24 October. "This will add to the Polio Gates Challenge. If online contributions were not mandatory, the contributions would have been much greater, but we hope they will come up with a double the points scheme for Africa to enable us to Kick Polio Out of Africa".
The next NID rounds in Benin have been pushed forward three weeks to 19 -21 November. The West African state of Benin this week, has been hit by the worst floods in living memory. Two-thirds of the country is affected by the flooding, with around 700,000 people having lost their homes. This could impact negatively on polio eradication efforts.
The KPOA team send their condolences to the people of Benin in their tragic loss and are with them in spirit at this sad and difficult time.


text:
America's Incredible Pizza Company coupon
Coupon From
America's Incredible Pizza Company
2850 S. Campbell Ave.
Springfield, MO 65807
Map it
Phone Number
Get a 99 cent All-You-Can-Eat Buffet with a $17 Game Card Purchase
Not valid with any other offer or discount. Can not be used towards a private birthday party, fundraising event or private group event. Valid at our Memphis, Springfield, St. Louis, San Antonio, Oklahoma City and Tulsa locations only.
Dec. 31st, 2018
Print This Offer


text:
image credit
A chance fossil discovery in Winifred, Montana, USA a decade ago has led to the identification of an audacious new species of horned dinosaur. The dinosaur is one among a growing number of newly discovered ceratopsids (four-legged dinosaurs generally characterized by horns on the face and elaborate head frills).
Its scientific name is Spiclypeus shipporum. About half of the skull, as well as parts of the dinosaur's legs, hips and backbone had been preserved in the silty hillside that once formed part of an ancient floodplain.


text:
That's the way it is for people who enjoy what they do. Whether firefighters are paid or volunteer, the vast majority love their work with a passion that transcends the job itself. Firefighting isn't just what they do; it's who they are.
I watch young firefighters (anymore, they're all young to me!) at the inception of their (hopefully) long careers, and I wonder if they, too, will have the sensation that their careers have flown by when they retire. Yes, there will be ups and downs, and those occasional times when the clock seems to run in reverse. But overall, will the years pass before they even know time is racing by?
The only way to know is to keep flipping the calendar pages and see what happens.
Have a happy, productive, and prosperous 2015.
|(Photo by R. Widmar © 2014)