Metabolic divergence between sibling species of cichlids Pundamilia nyererei and Pundamilia pundamilia

This study compared Pundamilia nyererei and Pundamilia pundamilia males in routine metabolic rate (R_R) and in the metabolic costs males pay during territorial interactions (active metabolic rate, R_A). Pundamilia nyererei and P. pundamilia males housed in social isolation did not differ in R_R. In contrast to expectation, however, P. nyererei males used less oxygen than P. pundamilia males, for a given mass and level of agonistic activity. This increased metabolic efficiency may be an adaptation to limit the metabolic cost that P. nyererei males pay for their higher rate of aggressiveness compared to P. pundamilia males. Thus, the divergence between the species in agonistic behaviour is correlated with metabolic differentiation. Such concerted divergence in physiology and behaviour might be widespread in the dramatically diverse cichlid radiations in East African lakes and may be an important factor in the remarkably rapid speciation of these fishes. The results did not support the hypothesis that higher metabolic rates caused a physiological cost to P. nyererei males that would offset their dominance advantage.     

Isolation and characterization of tetranucleotide microsatellite markers in a mouth‐brooding haplochromine cichlid fish (Pseudocrenilabrus multicolor victoriae) from Uganda

Eight tetranucleotide microsatellite loci were isolated from the haplochromine cichlid fish, Pseudocrenilabrus multicolor victoriae, an important model species for studies in respiratory ecology, conservation, and evolution. We surveyed variation at these loci in 23 individuals from western Uganda, finding four to 19 alleles per locus and an average expected heterozygosity of 0.8575. These microsatellite loci will be used to examine gene flow and population structure in Ugandan P. m. victoriae.     

Unusually fine-scale genetic structuring found in rapidly speciating Malawi cichlid fishes

Mechanisms behind the explosive radiation of over 500 cichlid fish species from a single founding population in Lake Malawi during the last 700 000 years are poorly understood. Recent studies have suggested that the degree of population subdivision among the habitat patches within the lake may be responsible, but the evidence has been circumstantial: lack of a dispersal stage in haplochromine cichlids; genetic and colour variation among populations separated by large-scale geographical barriers; and fluctuating lake levels. One reason for the rapidity of speciation in these fishes may be that population subdivision is on a much finer scale than previously thought. Here we quantify the level of population subdivision and estimate migration at a scale of 700-1400 m, in order to investigate whether cichlid populations are sufficiently isolated from each other for allopatric divergence and perhaps speciation to take place. Using six microsatellite loci, we demonstrate the existence of highly significant genetic differentiation between subpopulations on adjacent headlands in each of four rock-dwelling haplochromine cichlid species. Our results suggest that these fish populations are divided into thousands of subunits among which genetic divergence is currently occurring, and that this may provide unprecedented opportunities for allopatric speciation. <br>     

Pliocene isolation of a north‐west Saharan cichlid fish

The evolutionary origin of the only north‐west Saharan haplochromine cichlid, Astatotilapia desfontainii, was explored using mitochondrial DNA sequences. Phylogenetic analyses revealed that this species belonged to the main East African–Nilotic haplochromine clade, but was a distinct lineage that diverged from modern haplochromines in the Pliocene. The results suggest that A. desfontainii is a relict haplochromine lineage that has endured major climate fluctuations in North Africa.     

Linkage Relationships and Haplotype Variation of the Major Histocompatibility Complex Class I <Emphasis Type="BoldItalic">A</Emphasis> Genes in the Cichlid Fish <Emphasis Type="BoldItalic">Oreochromis niloticus</Emphasis>

: The haplochromine cichlid species flocks of the East African Great Lakes are one of the best examples of adaptive radiation. Analysis of genetic variation among these species provides valuable information on species relationships and timing of speciation events. Although the haplochromine cichlids generally display little genetic variation, the major histocompatibility complex (Mhc) genes have been found to be highly variable. A study of the linkage relationships of the Mhc class I A genes in the cichlid fish Oreochromis niloticus was therefore undertaken. Class I loci were identified, and their segregation in seven mothers and their haploid embryos was determined. In total, 56 class I A sequences were found among the seven families. A strong concordance of segregation was observed in five haplotypes among the embryos, indicating a close linkage of all loci. The number of loci per haplotype varied from 11 to 17, while the total number of distinct loci found among all families was 22. These findings show that all class I A loci are linked in a single genetic cluster in O. niloticus.     

Development and characterization of 14 microsatellite loci in the Neotropical fish Geophagus brasiliensis (Perciformes,Cichlidae)

Fourteen polymorphic microsatellite loci were isolated and characterized for the Neotropical cichlid Geophagus brasiliensis and tested on 30 individuals belonging to a single population. Among the 14 loci described, four showed potential presence of null alleles, inferred from the excess of homozygous genotypes, and three of these loci showed significant deviations from Hardy–Weinberg equilibrium. Fifty‐nine different alleles were detected (ranging from two to eight alleles per locus), with estimates of observed and expected heterozygosity ranging from 0·167 to 0·700 and from 0·269 to 0·825. Cross‐amplification of primers was successful in five other cichlid species.     

Preference polymorphism for coloration but no speciation in a population of Lake Victoria cichlids

Female mating preference based on male nuptial coloration hasbeen suggested to be an important source of diversifying selectionin the radiation of Lake Victoria cichlid fish. Initial variationin female preference is a prerequisite for diversifying selection;however, it is rarely studied in natural populations. In clearwater areas of Lake Victoria, the sibling species Pundamiliapundamilia with blue males and Pundamilia nyererei with redmales coexist, intermediate phenotypes are rare, and most femaleshave species-assortative mating preferences. Here, we studya population of Pundamilia that inhabits turbid water wheremale coloration is variable from reddish to blue with most malesintermediate. We investigated male phenotype distribution andfemale mating preferences. Male phenotype was unimodally distributedwith a mode on intermediate color in 1 year and more blue-shiftedin 2 other years. In mate choice experiments with females ofthe turbid water population and males from a clearer water population,we found females with a significant and consistent preferencefor P. pundamilia (blue) males, females with such preferencesfor P. nyererei (red) males, and many females without a preference.Hence, female mating preferences in this population could causedisruptive selection on male coloration that is probably constrainedby the low signal transduction of the turbid water environment.We suggest that if environmental signal transduction was improvedand the preference/color polymorphism was stabilized by negativefrequency-dependent selection, divergent sexual selection mightseparate the 2 morphs into reproductively isolated species resemblingthe clear water species P. pundamilia and P. nyererei.     

Development of microsatellite markers for the critically endangered Limonium dufourii (Girard) Kuntze (Plumbaginaceae)

Limonium dufourii is an endemic plant from the eastern Mediterranean coast of Spain with a triploid chromosome number and apomictic reproduction. We have isolated and characterized 13 polymorphic microsatellite loci from an enriched library in order to investigate its population genetic structure. Simple sequence repeat (SSR) loci were screened in 120 individuals from the six extant populations of this species. They show an average of 5.76 alleles per locus, ranging from 2 to 18, with seven loci exhibiting heterozygosities larger than 0.60. Three loci present one single allele in each individual, whereas one locus presents three alleles in every individual analysed.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of starry flounder (Platichthys stellatus) and cross-species amplification

Starry flounder (Platichthys stellatus) is a rare fish species in China. Here, we reported 12 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of starry flounder (P. stellatus). The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from two to six, from 0.2500 to 1.0000 and from 0.4512 to 0.7667, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in P. stellatus. Guidong Miao and Changwei Shao have contributed equally.     

Developmental plasticity and variability in the formation of egg-spots,a pigmentation ornament in the cichlid Astatotilapia calliptera

Vertebrate pigmentation patterns are highly diverse, yet we have a limited understanding of how evolutionary changes to genetic, cellular, and developmental mechanisms generate variation. To address this, we examine the formation of a sexually-selected male ornament exhibiting inter- and intraspecific variation, the egg-spot pattern, consisting of circular yellow-orange markings on the male anal fins of haplochromine cichlid fishes. We focus on Astatotilapia calliptera, the ancestor-type species of the Malawi cichlid adaptive radiation of over 850 species. We identify a key role for iridophores in initializing egg-spot aggregations composed of iridophore-xanthophore associations. Despite adult sexual dimorphism, aggregations initially form in both males and females, with development only diverging between the sexes at later stages. Unexpectedly, we found that the timing of egg-spot initialization is plastic. The earlier individuals are socially isolated, the earlier the aggregations form, with iridophores being the cell type that responds to changes to the social environment. Furthermore, we observe apparent competitive interactions between adjacent egg-spot aggregations, which strongly suggests that egg-spot patterning results mostly from cell-autonomous cellular interactions. Together, these results demonstrate that A. calliptera egg-spot development is an exciting model for investigating pigment pattern formation at the cellular level in a system with developmental plasticity, sexual dimorphism, and intraspecific variation. As A. calliptera represents the ancestral bauplan for egg-spots, these findings provide a baseline for informed comparisons across the incredibly diverse Malawi cichlid radiation.     

Morphometric differentiation among haplochromine cichlid fish species of a satellite lake of Lake Victoria

Lake Victoria holds a young but species‐rich assemblage of cichlid fishes, which form a monophyletic assemblage with additional species from surrounding water bodies, termed the Lake Victoria superflock. Lake Victoria is surrounded by smaller lakes that are somewhat disconnected from the main lake. Lake Kanyaboli is such a small lake, having markedly reduced species diversity, in part comprised of Lake Victoria species and endemics. Here, we studied the modern haplochromine component of the cichlid fauna, represented by Lipochromis maxillaris, Astatotilapia nubila, Xystichromis phytophagus and Astatotilapia sp. ‘Bigeye’, as well as a number of unidentified modern haplochromine specimens. We used landmark‐based geometric morphometrics to study the degree of morphological divergence among those young entities. Twenty landmarks and 14 interlandmark distances were used for shape analysis. Multivariate analysis revealed significant differences between all four species, but principal component analysis and canonical variate analysis did not clearly discriminate between A. nubila and X. phytophagus, demonstrating great overall morphological similarity despite clear dietary differences. Besides coloration there was sexual dimorphism in body proportions, so that only male individuals were analysed further. In all four species, the observed similarities and differences in body shape conform to the type of ecological specialization of the fish. Most unidentified specimens overlapped the range of A. nubila and X. phytophagus, while the assignment test based on the canonical variate analysis suggested 70% of the three overlapping entities as separate units. To test their reproductive distinctness and to demonstrate potential hybridization, nuclear genetic data are needed.     

Signal and preference divergence among populations of the non‐endemic basal Lake Malawi cichlid fish Astatotilapia calliptera (Perciformes: Cichlidae)

In Lake Malawi and Lake Victoria, cichlid fishes have diversified into hundreds of species, many reproductively isolated by mate choice. Territorial males tend to be more aggressive to similar‐coloured males, facilitating coexistence of divergent colour morphs or species. Behavioural mate choice and aggression biases of species and allopatric populations of specialized rocky shore cichlids are influenced by divergent signals such as male colour. Believed to be basal to the Lake Malawi haplochromine radiation, and inhabiting shallow weedy areas of the lake and neighbouring water bodies, Astatotilapia calliptera also shows allopatric variation in colour. Here, it is demonstrated that such signal divergence is associated with tendencies of females to mate with males of their own population and also for males to preferentially attack males of their own population, indicating that preference divergence related to signal divergence in allopatry may have operated throughout the adaptive radiation of the Malawian cichlids. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 180–188.     

Microsatellite markers for <Emphasis Type="Italic">Pseudopanax</Emphasis> trees and their cross-species amplification in the Araliaceae

In order to study hybridisation, taxonomic boundaries and phylogeography in the genus Pseudopanax (Araliaceae), we developed seven novel microsatellite loci from enriched genomic libraries of P. lessonii and P. crassifolius. These loci were characterised in 16 individuals from a single population of P. crassifolius, and displayed 2–11 alleles per locus. Observed heterozygosities ranged from 0.063 to 0.688. Most loci were polymorphic in the closely related Pseudopanax species, and several loci amplified widely across the Araliaceae.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Ten microsatellite loci for the reef‐building coral Acropora millepora (Cnidaria,Scleractinia) from the Great Barrier Reef,Australia

Here we present nine novel, polymorphic microsatellite loci developed for the scleractinian coral Acropora millepora (Acroporidae) from the Great Barrier Reef. In addition, we have assessed the specificity and polymorphism of five microsatellite loci previously developed for a Caribbean congener and one locus developed for an Indo‐Pacific congener. Only one of the latter six loci produced reliable results, yielding a total of 10 polymorphic microsatellite loci for A. millepora. Variability was tested on 20–23 individuals from a single population, plus another ～10 individuals from each of three different populations, resulting in five to 20 alleles per locus.     

Introgressive hybridization between color morphs in a population of cichlid fishes twelve years after human-induced secondary admixis

In the extremely species-rich haplochromine cichlid fishes of the East African Great Lakes, prezygotic isolation between closely related species is often maintained by color-assortative mating. In 1998, local fisherman working for the ornamental fish trade released different color morphs of the cichlid genus Tropheus into a small harbor basin in the southern part of Lake Tanganyika. This artificial amalgamation of color morphs provides a unique possibility to study mating patterns in cichlids in a natural environment over time. In a precursor study, we analyzed genotypes and phenotypes of almost 500 individuals sampled between 1999 and 2001 and uncovered a marked degree of color-assortative mating, which depended on the level of color pattern dissimilarity between morphs. Twelve years after introduction of nonindigenous morphs, we again sampled Tropheus individuals from the harbor basin and an adjacent, originally pure population and analyzed phenotypes (coloration) and genotypes (mitochondrial control region and 9 microsatellite loci) to assess the current status of the admixed population. Principal component analyses of color score data and population assignment tests demonstrate an increasing level of introgressive hybridization between morphs but also some ongoing color-assortative mating within morphs. The observed mating pattern might have been influenced by fluctuating environmental conditions such as periodic algal blooms or increased sedimentation causing turbid conditions in an otherwise clear lake.     

A BAC library of the East African haplochromine cichlid fish Astatotilapia burtoni

A BAC library was constructed from Astatotilapia burtoni, a haplochromine cichlid that is found in Lake Tanganyika, East Africa, and its surrounding rivers. The library was generated from genomic DNA of blood cells and comprises 96,768 individual clones. Its median insert size is 150 kb and the coverage is expected to represent about 14 genome equivalents. The coverage evaluation was based on genome size estimates that were obtained by flow cytometry. In addition, hybridization screens with five probes largely corroborate the above coverage estimate, although the number of clones ranged from 5 to 22 authenticated clones per single copy probe. The BAC library described here is expected to be useful to the scientific community interested in cichlid genomics as an important resource to gain new insights into the rapid evolution of the great species diversity of haplochromine cichlid fishes.     

Developmental effects of visual environment on species‐assortative mating preferences in Lake Victoria cichlid fish

Local adaptation can be a potent force in speciation, with environmental heterogeneity leading to niche specialization and population divergence. However, local adaption often requires nonrandom mating to generate reproductive isolation. Population divergence in sensory properties can be particularly consequential in speciation, affecting both ecological adaptation and sexual communication. Pundamilia pundamila and Pundamilia nyererei are two closely related African cichlid species that differ in male coloration, blue vs. red. They co‐occur at rocky islands in southern Lake Victoria, but inhabit different depth ranges with different light environments. The species differ in colour vision properties, and females exert species‐specific preferences for blue vs. red males. Here, we investigated the mechanistic link between colour vision and preference, which could provide a rapid route to reproductive isolation. We tested the behavioural components of this link by experimentally manipulating colour perception – we raised both species and their hybrids under light conditions mimicking shallow and deep habitats – and tested female preference for blue and red males under both conditions. We found that rearing light significantly affected female preference: shallow‐reared females responded more strongly to P. pundamilia males and deep‐reared females favoured P. nyererei males – implying that visual development causally affects mate choice. These results are consistent with sensory drive predictions, suggesting that the visual environment is key to behavioural isolation of these species. However, the observed plasticity could also make the species barrier vulnerable to environmental change: species‐assortative preferences were weaker in females that were reared in the other species’ light condition.     

A set of cross‐species amplifying microsatellite markers developed from DNA sequence databanks in Picea (Pinaceae)

Seven codominant genetic markers (of which six are located in gene untranslated transcribed regions) were developed from Picea DNA sequences available in databanks. Primers were designed to specifically amplify by polymerase chain reaction tri‐, di‐ or mononucleotide repeated motifs. They provided single locus length polymorphism on a representative sample of 93 Picea abies individuals. The usefulness of each locus in mapping, population genetics or gene flow studies was assessed. A weak geographical genetic differentiation was revealed. The loci were also amplified in P. glauca, P. engelmannii and P. omorika demonstrating potential transferability across Picea species.     

Isolation and characterization of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.