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A Phylogenetic Index for Cichlid Microsatellite Primers

A Phylogenetic Index for Cichlid Microsatellite Primers Hindawi Publishing Corporation International Journal of Zoology Volume 2010, Article ID 482732, 5 pages doi:10.1155/2010/482732 Research Article 1 2 1 Robert D. Kunkle, ChristianR.L.Reilly, andSuzyC.P.Renn Biology Department, Reed College, 3203 SE Woodstock Boulevard, Portland, OR 97202, USA Santa Catalina School, 1500 Mark Thomas Drive, Monterey, CA 93940, USA Correspondence should be addressed to Suzy C. P. Renn, renns@reed.edu Received 1 April 2010; Revised 28 September 2010; Accepted 12 October 2010 Academic Editor: Ajai Kumar Srivastav Copyright © 2010 Robert D. Kunkle et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microsatellites abound in most organisms and have proven useful for a range of genetic and genomic studies. Once primers have been created, they can be applied to populations or taxa that have diverged from the source taxon. We use PCR amplification, in a 96-well format, to determine the presence and absence of 46 microsatellite loci in 13 cichlid species. At least one primer set amplified a product in each species tested, and some products were present in nearly all species. These results are compared to the known phylogenetic relationships among cichlids. While we do not address intraspecies variation, our results present a phylogenetic index for the success of microsatellite PCR primer product amplification, thus providing information regarding a collection of primers that are applicable to wide range of species. Through the use of such a uniform primer panel, the potential impact for cross species would be increased. 1. Introduction makes MFRs likely to be selectively neutral, to the point that their sequence can be used as a molecular clock for Microsatellites, or short sequence repeats (SSRs), are short phylogeny studies [4]. Since MFR-derived primer pairs do (2–6 bp) DNA motifs that are repeated at least three, and not anneal to repeat regions, this disruption does not up to hundreds of, times consecutively [1]. SSRs abound interfere with these primers’ efficacy. Once MFR primers in most organisms, and fishes are no exception, with an have been created, therefore, depending on genome-wide estimated frequency of one locus per several kb of DNA [2]. mutation dynamics, they can likely be applied to populations The repetition of a microsatellite motif makes misalignment or taxa that have diverged from the source taxon. For of template and newly synthesized strands during DNA example, Rico et al. [5] were able to amplify a microsatellite replication very likely, resulting in a range of alleles differing region with the same MFR-derived primer set in two fish by whole numbers of repeats [3]. Such unstable mutation species that diverged 470 Mya. However, the pattern of dynamics hamper sequence-dependent function of a locus, MFR sequence conservation was sufficiently unpredictable to so with few exceptions, such as the human Huntington’s require locus-by-locus confirmation. If, at the outset of work locus, observed microsatellite regions are not transcribed. As on one taxonomic group, MFR primers are available from primarily neutral, polymorphic loci with a signature pattern previous work on a related group, some expense still must that facilitates isolation, microsatellites have proven useful be undertaken to determine which microsatellites are present for a range of genomic studies. and informative (i.e., variable) in the particular genomes and Despite broad utility, a researcher interested in applying populations of interest [6, 7]. The current study presents an microsatellite-based tools to linkage mapping or phyloge- index of putative MFR-specific primer sets tested in species netic analysis faces a significant investment in time and representing the major groups within the most speciose material to isolate repeat regions and create primers that family of fish (the Cichlidae). This information should anneal with the microsatellite flanking regions (MFRs) reduce the entrance cost to those interested in applying adjacent to the repeats. Their proximity to microsatellites microsatellite-based analyses to additional cichlid species. 2 International Journal of Zoology The cichlids of the Great Rift Lakes of Eastern Africa and Reverse primers, and .5 ng template DNA, using the ◦ ◦ are especially important as research model of evolu- following program: 30 cycles; 30 s at 95 C, 30sat56 C, tionary processes because their phylogenetic history has 1m at 72 C. Alternate reaction conditions were not tested been reconstructed to reveal multiple adaptive radiations; as it is our goal to identify, for use by other researchers, many are recent (<2 Mya) and some exceptionally recent those primer sets that are likely to be most amenable to (<12.500 ya). This has resulted in extensive diversification, multiplexing and high-throughput analysis. PCR products often exhibiting convergence of form, niche, and behav- were run on 4% agarose gel stained with ethidium bromide. ior [8]. The presence of many closely related species, Digital gel images were captured for analysis. Band presence, often sympatric and quite subtly diverged [9], enables relative brightness, approximate length, and the presence an appropriately subtle analysis of speciation genomics or absence of a doublet (two distinct bands suggesting and genetic basis for adaptive traits. For example, jaw heterozygous state or multiple loci) were assessed by eye. morphology-related genes have been studied in Malawi Failed reactions were repeated for confirmation of negative cichlids [10], and population structure has been addressed results. in the Tanganyika rock cichlid species [11]aswellasfor the Clustering of species according to the pattern of success- sympatrically speciating Midas cichlids from South America ful PCR products was performed using R software v2.0.1 [12]. [16]. The dissimilarity measures were obtained using the dist The most intensively studied cichlids are the widely function in the stats package based on Euclidean distance farmed, multigeneric tilapia species, in one of which, using product presence and absence information only. The Oreochromis niloticus, Lee and Kocher have developed consensus tree and bootstrap confidence values for each microsatellite isolation methods used to create several node were obtained with the consensus function in the hundred sets of MFR-specific primers [13], in addition to MAANOVA package [17]. The consensus tree dendrogram creating a linkage map of those loci [14]. Albertson et al. and confidence values were calculated as the proportion of also isolated microsatellites and created a linkage map for a 1000 trees that agreed with the original tree as obtained by hybrid of Labeotropheus fuelleborni and Metriaclima zebra, resampling with replacement, again using presence-absence two closely related species of Mbuna or rock cichlid from data only. Lake Malawi. In the creation of the Mbuna linkage map, 248 of the primer sets obtained from O. niloticus were also tested, 3. Results and 46 were found to produce product in the Mbuna hybrid [10]. These 46 loci represent all but three of the O. niloticus In total, 13 species were assayed including 9 from Africa and linkage groups from the available genetic map [14], with as 4 from South America. In general, the number and pattern many as four loci for linkage groups 3, 10, and 17. As O. of successful amplification products reflect phylogenetic niloticus and the Mbuna species share a common ancestor relationship (Figure 1). Among 9 African cichlid species, 2 with most of the Great Lakes cichlids approximately 18– are endemic to Lake Malawi and 7 to Lake Tanganyika. The 30 Mya (Figure 1(a)) [15], the 46 loci found in both species mean number of positive amplifications per species (out bore significant chances of being present in other African of 46) was 33.5 (s.d. ± 3.5) for Lake Malawi and 35.67 cichlids. This current study creates a phylogenetic index for (s.d. ± 4.03) for Lake Tanganyika. T. polylepis is excluded a wide range of cichlid species indicating the presence and from this average calculation due to its recent immigrant absence of PCR product using primers to these loci. Such an status and distant relationship to other Tanganyikan cichlids index is anticipated to aid cichlid researchers. The 46 primer [15, 18]. The 14 positive amplifications from T. polylepis sets were tested on a single genomic DNA samples extracted were the least of any African species, but still more than any from each of 13 cichlid species: Astatotilapia burtoni, Neo- of the South American cichlids. The two species from the lamprologus brichardi, Perissodus microlepis, Protomelas sim- Ectodini tribe showed the most similar pattern of microsatel- ilis, Metriaclima estherae, Tylochromis sp., Tropheus duboisi, lite amplification products, six primer sets amplified in Xenotilapia flavipinnis, Xenotilapia ochrogenys, Retroculus X. flavipinnis and not X. ochrogenys, but there were no xinguensis, Cichla temensis, Astronotus sp., and Satanoperca other differences in their amplification patterns. As expected, sp. This sample covers most of the major African clades the more distantly related South American cichlid species and some South American clades. No cichlids from Indian showed significantly fewer successful microsatellite products. or Madagascan were examined. At least one primer set On average, 4.5 (s.d. ± 2.06) primers sets amplified in these amplified a product in each species tested, and some PCR species, and each species tested had a unique pattern of primer sets successfully amplified a product in nearly all test positive amplifications. species. Clustering analysis resulted in a dendrogram that separated the African Great Lakes cichlids from their sister genus, Tylochromis, and from the South Ameri- 2. Materials and Methods can species. There was insufficient statistical confidence Fin clips were collected in the field and placed immediately to distinguish relationships within the Great Lakes and in ethanol. Genomic DNA was extracted from each indi- accurately capture relationships among the South Amer- vidual using a standard proteinase K/Phenol protocol. PCR ican clades (Figure 1). These results agree, as far as res- was performed using the standard FastStart Taq protocol olution allows, with the mtDNA phylogeny studies [19, in 10 μL reactions, with 2.5 μMMgCl ,.25 mM Forward 20]. 2 International Journal of Zoology 3 0.99 0.98 0.99 0.97 0.59 0.89 Genbank accession Primer pair # GM201 1 BV005353 UNH104 1 G12257 GM166 2b BV005579 GM128 3 BV005322 GM150 3 BV005434 GM385 3 G68259 UNH971 3 G68259 UNH911 4 ∗∗ G68224 UNH169 5 BV005541 GM651 6 ∗ BV005541 UNH908 6 G68222 6 ∗∗ ∗ UNH948 G68246 GM431 7 BV005450 UNH896 G68214 UNH973 7 ∗ G68260 UNH2191 9a BV005710 UNH875 9a G68203 UNH843 9b G68182 GM294 10 BV005399 UNH937 10 G68241 UNH915 10 G68227 UNH932 10 ∗ G68238 ∗ ∗ ∗ ∗ GM401 11 BV005440 GM284 12 BV005395 UNH1009 12 G68285 UNH874 12 G68202 GM641 13a BV005536 UNH934 13b G68240 UNH989 14 ∗ ∗ G68269 UNH2150 15 BV005692 UNH2166 15 BV005702 UNH958 15 G68252 UNH138 16 G12290 GM323 17 BV005408 UNH362 17 ∗∗ G54844 UNH951 17 G68248 UNH974 17 G68261 UNH1003 18 G68280 ∗∗ UNH906 18 G68220 GM320 19 BV005406 GM634 19 BV005531 UNH933 19 G68239 GM138 21 BV005578 GM538 22 BV005485 ∗ ∗ UNH919 22 G68230 UNH216 23 G12367 Figure 1: Successful PCR amplification with microsatellite primers in 13 cichlid species using primers designed to Tilapia. Color indicates relative intensity of the PCR band black: brightest, dark grey: visible, light grey: faint, white: absent. Definitive doublets are indicated with an asterisk. The dendrogram represents the consensus cluster confidence values indicated based upon 1000 trees, with resampling, using presence or absence of product and Euclidean distance measures. O.niloticus Linkage group A. burtoni P. microlepis N. brichardi T. duboisi P. similis M. estherae X. flavipinnis X. ochrogenys Tylochromis sp. Astronotus sp. R. xinguinsis C. temensis Satanoperca sp. 4 International Journal of Zoology 4. Discussion They thank H. Machado for comments on the paper. This work has been supported by a Grant to S.C.P.R. from the M. The data presented here demonstrate that the previously J. Murdock Charitable Trust (no.:2006253:JVZ:2/22/2007). isolated MFR primer sets should be useful for population studies in many cichlid taxa, particularly throughout the East References African radiation. While the range of species used in this study cannot definitively predict which primer sets will yield [1] D.Tautz,M.Trick,and G. A. Dover, “Cryptic simplicity in informative (i.e., variable) genetic information for every DNA is a major source of genetic variation,” Nature, vol. 322, no. 6080, pp. 652–656, 1986. cichlid species, it does provide a measure of the expected [2] D.A.Chistiakov,B.Hellemans,and F. A. M. Volckaert, success rate for a given phylogenetic position. Furthermore, “Microsatellites and their genomic distribution, evolution, the availability of this primer set in a 96-well format will function and applications: a review with special reference to facilitate rapid screening for any species of interest. fish genetics,” Aquaculture, vol. 255, no. 1–4, pp. 1–29, 2006. The band brightness aspect of the data may estimate [3] H. Ellegren, “Microsatellites: simple sequences with complex sequence divergence in these MFR’s. It is possible that highly evolution,” Nature Reviews Genetics, vol. 5, no. 6, pp. 435–445, diverged loci will not amplify as efficiently, and further divergence would prohibit amplification all together; this [4] R.Zardoya,D.M.Vollmer,C.Craddock, J. T. Streelman, S. should be anticipated when a distantly related (e.g., South Karl, and A. Meyer, “Evolutionary conservation of microsatel- American) species is studied. As Ellegren [3] made clear, lite flanking regions and their use in resolving the phylogeny mutation rates vary between loci, individuals, and taxa, due of cichlid fishes (Pisces: Perciformes),” Proceedings of the Royal Society B, vol. 263, no. 1376, pp. 1589–1598, 1996. to disabled mismatch repair and proofreading, chromatin [5] C. Rico, I. Rico, and G. Hewitt, “470 million years of conser- structure variation, or other mechanisms. Therefore, we vation of microsatellite loci among fish species,” Proceedings of cannot infer sequence similarity by a measure of band the Royal Society B, vol. 263, no. 1370, pp. 549–557, 1996. brightness, and this information provides only a rough [6] L. Zane, L. Bargelloni, and T. Patarnello, “Strategies for guideline. Similarly, an allele of a given length may have microsatellite isolation: a review,” Molecular Ecology, vol. 11, arisen from either a lengthening or from a shortening no. 1, pp. 1–16, 2002. mutation, meaning that its exact relationship to other [7] T. C. Glenn and N. A. Schable, “Isolating microsatellite DNA alleles is unclear. In addition, as with absolute mutation loci,” Methods in Enzymology Part B, vol. 395, no. 6879, pp. rates, the relative frequencies of shortening and lengthening 202–222, 2005. vary within genomes and taxa. Therefore, estimating a [8] G. W. Barlow, Cichlid Fishes: Nature’s Grand Experiment in given allele’s ancestry requires considerable groundwork Evolution, Perseus Books, Cambridge, Mass, USA, 2000. [9] T. D. Kocher, “Adaptive evolution and explosive speciation: the to describe the variation at that locus for any species of cichlid fish model,” Nature Reviews Genetics,vol. 5, no.4,pp. interest. For research over a fairly short scale of divergence, 288–298, 2004. where novel alleles are at a minimum, this groundwork [10] R. C. Albertson, J. T. Streelman, and T. D. Kocher, “Directional will require amplification from several individuals’ genomic selection has shaped the oral jaws of Lake Malawi cichlid DNA to estimate whether enough polymorphism exists to fishes,” Proceedings of the National Academy of Sciences of the allow for distinction between lineages. Here, (Figure 1)wedo United States of America, vol. 100, no. 9, pp. 5252–5257, 2003. report all observed variation in relative brightness (denoted [11] N. Duftner, S. Koblmul ¨ ler, and C. Sturmbauer, “Evolutionary by shading) of the imaged PCR products as well as the relationships of the Limnochromini, a tribe of benthic deep- presence or absence of a doublet (denoted by the asterisk) in water cichlid fish endemic to Lake Tanganyika, East Africa,” order to provide all possible information regarding potential Journal of Molecular Evolution, vol. 60, no. 3, pp. 277–289, polymorphism of each locus. However, it must be noted that 2005. [12] P. M. E. Bunje, M. Barluenga, and A. Meyer, “Sampling genetic only a single individual was assayed in the current study and diversity in the sympatrically and allopatrically speciating resolution was ∼20 bp or greater. Therefore, further work Midas cichlid species complex over a 16 year time series,” BMC is required to describe polymorphic loci in any particular Evolutionary Biology, vol. 7, article 25, 2007. species of interest. We have not conducted such intraspecific [13] W.-J. Lee and T. D. Kocher, “Microsatellite DNA markers analysis in this study because demonstration of variation and for genetic mapping in Oreochromis niloticus,” Journal of Fish utility of the loci as genetic markers must be established for Biology, vol. 49, no. 1, pp. 169–171, 1996. the exact species and population of interest and could not be [14] B.-Y. Lee, W.-J. Lee, J. T. Streelman et al., “A second-generation inferred across species boundaries. genetic linkage map of tilapia (Oreochromis spp.),” Genetics, By contacting the corresponding author, the full set of vol. 170, no. 1, pp. 237–244, 2005. 46 primes used in this study is freely available in a 96-well [15] M. J. Genner, O. Seehausen, D. H. Lunt et al., “Age of cichlids: format and diluted to a working concentration for use in new dates for ancient lake fish radiations,” Molecular Biology and Evolution, vol. 24, no. 5, pp. 1269–1282, 2007. PCR with any species of interest. [16] R Development Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Com- Acknowledgments puting, Vienna, Austria, 2006, http://www.R-project.org. [17] H. Wu, M. K. Kerr, X. Cui, and G. A. Chruchill, “MAANOVA: a The authors are grateful for the donation of tissue samples by software package for the analysis of spotted cDNA microarray D. Joyce (University of Hull), Malawi species, and S. Willis experiments,” in The Analysis of Gene Expression Data: Meth- (University of Nebraska, Lincoln), South American species. ods and Software, Springer, New York, NY, USA, 2003. International Journal of Zoology 5 [18] M. Koch,S.Koblmu ¨ller, K. M. Sefc,N.Duftner,C.Katongo, and C. Sturmbauer, “Evolutionary history of the endemic Lake Tanganyika cichlid fish Tylochromis polylepis:arecent intruder to a mature adaptive radiation,” Journal of Zoological Systematics and Evolutionary Research, vol. 45, no. 1, pp. 64– 71, 2007. [19] I. P. Farias, G. Ort´ ı, I. Sampaio, H. Schneider, and A. Meyer, “Mitochondrial DNA phylogeny of the family cichlidae: monophyly and fast molecular evolution of the neotropical assemblage,” Journal of Molecular Evolution, vol. 48, no. 6, pp. 703–711, 1999. [20] W. Salzburger, T. Mack, E. Verheyen, and A. Meyer, “Out of Tanganyika: genesis, explosive speciation, key-innovations and phylogeography of the haplochromine cichlid fishes,” BMC Evolutionary Biology, vol. 5, no. 1, pp. 1–15, 2005. 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A Phylogenetic Index for Cichlid Microsatellite Primers

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Copyright © 2010 Robert D. Kunkle et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Publishing Corporation International Journal of Zoology Volume 2010, Article ID 482732, 5 pages doi:10.1155/2010/482732 Research Article 1 2 1 Robert D. Kunkle, ChristianR.L.Reilly, andSuzyC.P.Renn Biology Department, Reed College, 3203 SE Woodstock Boulevard, Portland, OR 97202, USA Santa Catalina School, 1500 Mark Thomas Drive, Monterey, CA 93940, USA Correspondence should be addressed to Suzy C. P. Renn, renns@reed.edu Received 1 April 2010; Revised 28 September 2010; Accepted 12 October 2010 Academic Editor: Ajai Kumar Srivastav Copyright © 2010 Robert D. Kunkle et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microsatellites abound in most organisms and have proven useful for a range of genetic and genomic studies. Once primers have been created, they can be applied to populations or taxa that have diverged from the source taxon. We use PCR amplification, in a 96-well format, to determine the presence and absence of 46 microsatellite loci in 13 cichlid species. At least one primer set amplified a product in each species tested, and some products were present in nearly all species. These results are compared to the known phylogenetic relationships among cichlids. While we do not address intraspecies variation, our results present a phylogenetic index for the success of microsatellite PCR primer product amplification, thus providing information regarding a collection of primers that are applicable to wide range of species. Through the use of such a uniform primer panel, the potential impact for cross species would be increased. 1. Introduction makes MFRs likely to be selectively neutral, to the point that their sequence can be used as a molecular clock for Microsatellites, or short sequence repeats (SSRs), are short phylogeny studies [4]. Since MFR-derived primer pairs do (2–6 bp) DNA motifs that are repeated at least three, and not anneal to repeat regions, this disruption does not up to hundreds of, times consecutively [1]. SSRs abound interfere with these primers’ efficacy. Once MFR primers in most organisms, and fishes are no exception, with an have been created, therefore, depending on genome-wide estimated frequency of one locus per several kb of DNA [2]. mutation dynamics, they can likely be applied to populations The repetition of a microsatellite motif makes misalignment or taxa that have diverged from the source taxon. For of template and newly synthesized strands during DNA example, Rico et al. [5] were able to amplify a microsatellite replication very likely, resulting in a range of alleles differing region with the same MFR-derived primer set in two fish by whole numbers of repeats [3]. Such unstable mutation species that diverged 470 Mya. However, the pattern of dynamics hamper sequence-dependent function of a locus, MFR sequence conservation was sufficiently unpredictable to so with few exceptions, such as the human Huntington’s require locus-by-locus confirmation. If, at the outset of work locus, observed microsatellite regions are not transcribed. As on one taxonomic group, MFR primers are available from primarily neutral, polymorphic loci with a signature pattern previous work on a related group, some expense still must that facilitates isolation, microsatellites have proven useful be undertaken to determine which microsatellites are present for a range of genomic studies. and informative (i.e., variable) in the particular genomes and Despite broad utility, a researcher interested in applying populations of interest [6, 7]. The current study presents an microsatellite-based tools to linkage mapping or phyloge- index of putative MFR-specific primer sets tested in species netic analysis faces a significant investment in time and representing the major groups within the most speciose material to isolate repeat regions and create primers that family of fish (the Cichlidae). This information should anneal with the microsatellite flanking regions (MFRs) reduce the entrance cost to those interested in applying adjacent to the repeats. Their proximity to microsatellites microsatellite-based analyses to additional cichlid species. 2 International Journal of Zoology The cichlids of the Great Rift Lakes of Eastern Africa and Reverse primers, and .5 ng template DNA, using the ◦ ◦ are especially important as research model of evolu- following program: 30 cycles; 30 s at 95 C, 30sat56 C, tionary processes because their phylogenetic history has 1m at 72 C. Alternate reaction conditions were not tested been reconstructed to reveal multiple adaptive radiations; as it is our goal to identify, for use by other researchers, many are recent (<2 Mya) and some exceptionally recent those primer sets that are likely to be most amenable to (<12.500 ya). This has resulted in extensive diversification, multiplexing and high-throughput analysis. PCR products often exhibiting convergence of form, niche, and behav- were run on 4% agarose gel stained with ethidium bromide. ior [8]. The presence of many closely related species, Digital gel images were captured for analysis. Band presence, often sympatric and quite subtly diverged [9], enables relative brightness, approximate length, and the presence an appropriately subtle analysis of speciation genomics or absence of a doublet (two distinct bands suggesting and genetic basis for adaptive traits. For example, jaw heterozygous state or multiple loci) were assessed by eye. morphology-related genes have been studied in Malawi Failed reactions were repeated for confirmation of negative cichlids [10], and population structure has been addressed results. in the Tanganyika rock cichlid species [11]aswellasfor the Clustering of species according to the pattern of success- sympatrically speciating Midas cichlids from South America ful PCR products was performed using R software v2.0.1 [12]. [16]. The dissimilarity measures were obtained using the dist The most intensively studied cichlids are the widely function in the stats package based on Euclidean distance farmed, multigeneric tilapia species, in one of which, using product presence and absence information only. The Oreochromis niloticus, Lee and Kocher have developed consensus tree and bootstrap confidence values for each microsatellite isolation methods used to create several node were obtained with the consensus function in the hundred sets of MFR-specific primers [13], in addition to MAANOVA package [17]. The consensus tree dendrogram creating a linkage map of those loci [14]. Albertson et al. and confidence values were calculated as the proportion of also isolated microsatellites and created a linkage map for a 1000 trees that agreed with the original tree as obtained by hybrid of Labeotropheus fuelleborni and Metriaclima zebra, resampling with replacement, again using presence-absence two closely related species of Mbuna or rock cichlid from data only. Lake Malawi. In the creation of the Mbuna linkage map, 248 of the primer sets obtained from O. niloticus were also tested, 3. Results and 46 were found to produce product in the Mbuna hybrid [10]. These 46 loci represent all but three of the O. niloticus In total, 13 species were assayed including 9 from Africa and linkage groups from the available genetic map [14], with as 4 from South America. In general, the number and pattern many as four loci for linkage groups 3, 10, and 17. As O. of successful amplification products reflect phylogenetic niloticus and the Mbuna species share a common ancestor relationship (Figure 1). Among 9 African cichlid species, 2 with most of the Great Lakes cichlids approximately 18– are endemic to Lake Malawi and 7 to Lake Tanganyika. The 30 Mya (Figure 1(a)) [15], the 46 loci found in both species mean number of positive amplifications per species (out bore significant chances of being present in other African of 46) was 33.5 (s.d. ± 3.5) for Lake Malawi and 35.67 cichlids. This current study creates a phylogenetic index for (s.d. ± 4.03) for Lake Tanganyika. T. polylepis is excluded a wide range of cichlid species indicating the presence and from this average calculation due to its recent immigrant absence of PCR product using primers to these loci. Such an status and distant relationship to other Tanganyikan cichlids index is anticipated to aid cichlid researchers. The 46 primer [15, 18]. The 14 positive amplifications from T. polylepis sets were tested on a single genomic DNA samples extracted were the least of any African species, but still more than any from each of 13 cichlid species: Astatotilapia burtoni, Neo- of the South American cichlids. The two species from the lamprologus brichardi, Perissodus microlepis, Protomelas sim- Ectodini tribe showed the most similar pattern of microsatel- ilis, Metriaclima estherae, Tylochromis sp., Tropheus duboisi, lite amplification products, six primer sets amplified in Xenotilapia flavipinnis, Xenotilapia ochrogenys, Retroculus X. flavipinnis and not X. ochrogenys, but there were no xinguensis, Cichla temensis, Astronotus sp., and Satanoperca other differences in their amplification patterns. As expected, sp. This sample covers most of the major African clades the more distantly related South American cichlid species and some South American clades. No cichlids from Indian showed significantly fewer successful microsatellite products. or Madagascan were examined. At least one primer set On average, 4.5 (s.d. ± 2.06) primers sets amplified in these amplified a product in each species tested, and some PCR species, and each species tested had a unique pattern of primer sets successfully amplified a product in nearly all test positive amplifications. species. Clustering analysis resulted in a dendrogram that separated the African Great Lakes cichlids from their sister genus, Tylochromis, and from the South Ameri- 2. Materials and Methods can species. There was insufficient statistical confidence Fin clips were collected in the field and placed immediately to distinguish relationships within the Great Lakes and in ethanol. Genomic DNA was extracted from each indi- accurately capture relationships among the South Amer- vidual using a standard proteinase K/Phenol protocol. PCR ican clades (Figure 1). These results agree, as far as res- was performed using the standard FastStart Taq protocol olution allows, with the mtDNA phylogeny studies [19, in 10 μL reactions, with 2.5 μMMgCl ,.25 mM Forward 20]. 2 International Journal of Zoology 3 0.99 0.98 0.99 0.97 0.59 0.89 Genbank accession Primer pair # GM201 1 BV005353 UNH104 1 G12257 GM166 2b BV005579 GM128 3 BV005322 GM150 3 BV005434 GM385 3 G68259 UNH971 3 G68259 UNH911 4 ∗∗ G68224 UNH169 5 BV005541 GM651 6 ∗ BV005541 UNH908 6 G68222 6 ∗∗ ∗ UNH948 G68246 GM431 7 BV005450 UNH896 G68214 UNH973 7 ∗ G68260 UNH2191 9a BV005710 UNH875 9a G68203 UNH843 9b G68182 GM294 10 BV005399 UNH937 10 G68241 UNH915 10 G68227 UNH932 10 ∗ G68238 ∗ ∗ ∗ ∗ GM401 11 BV005440 GM284 12 BV005395 UNH1009 12 G68285 UNH874 12 G68202 GM641 13a BV005536 UNH934 13b G68240 UNH989 14 ∗ ∗ G68269 UNH2150 15 BV005692 UNH2166 15 BV005702 UNH958 15 G68252 UNH138 16 G12290 GM323 17 BV005408 UNH362 17 ∗∗ G54844 UNH951 17 G68248 UNH974 17 G68261 UNH1003 18 G68280 ∗∗ UNH906 18 G68220 GM320 19 BV005406 GM634 19 BV005531 UNH933 19 G68239 GM138 21 BV005578 GM538 22 BV005485 ∗ ∗ UNH919 22 G68230 UNH216 23 G12367 Figure 1: Successful PCR amplification with microsatellite primers in 13 cichlid species using primers designed to Tilapia. Color indicates relative intensity of the PCR band black: brightest, dark grey: visible, light grey: faint, white: absent. Definitive doublets are indicated with an asterisk. The dendrogram represents the consensus cluster confidence values indicated based upon 1000 trees, with resampling, using presence or absence of product and Euclidean distance measures. O.niloticus Linkage group A. burtoni P. microlepis N. brichardi T. duboisi P. similis M. estherae X. flavipinnis X. ochrogenys Tylochromis sp. Astronotus sp. R. xinguinsis C. temensis Satanoperca sp. 4 International Journal of Zoology 4. Discussion They thank H. Machado for comments on the paper. This work has been supported by a Grant to S.C.P.R. from the M. The data presented here demonstrate that the previously J. Murdock Charitable Trust (no.:2006253:JVZ:2/22/2007). isolated MFR primer sets should be useful for population studies in many cichlid taxa, particularly throughout the East References African radiation. 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