, Volume 789, Issue 1, pp 77–89 | Cite as

Mapping 45S and 5S ribosomal genes in chromosomes of Anostomidae fish species (Ostariophysi, Characiformes) from different Amazonian water types

  • Lucas Caetano de Barros
  • Pedro Manoel Galetti Junior
  • Eliana Feldberg


The fish from the family Anostomidae represent one of the most important groups of freshwater ichthyofauna from South America, with species of high economical value. The migratory characteristic of some species, through the several Amazonian environments, takes them into waters with different physico-chemical characteristics. Cytogenetic studies on the Anostomidae demonstrate that these fishes have a conserved diploid number and karyotype macrostructure. So, to verify if this conservation occurs also in the genomic level, the current study aimed at a chromosomal comparative physical mapping, using 45S and 5S rDNA, of seven species of anostomids: Leporinus fasciatus, L. agassizi, L. friderici, L. trifasciatus, Rhytiodus macrolepis, Laemolyta taeniata, and Schizodon fasciatus, collected in different Amazonian environments. The results obtained corroborate the conservation of the karyotype macrostructure. However, significant differences were found in the distribution of heterochromatin and on the pair bearing the nucleolus organizer region. The staining of 45S and 5S rDNA by FISH highlighted, for four of the seven species, more than one chromosome pair bearing the site 45S. The 5S rDNA, although present in only one chromosome pair, varied in its chromosome and karyotype position. Thus, although the Anostomidae family has a conserved chromosomic macrostructure the use of molecular techniques revealed the presence of chromosomic translocation during the evolution of these fishes.


Cytogenetics Chromosome Fish Aguas amazonicas 



The authors are grateful to Dr. Jansen A. Zuanon for identifying the specimens. This study was supported by the Brazilian agencies, Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq-LCB scholarship), Instituto Nacional de Pesquisas da Amazônia/Genética, Conservação e Biologia Evolutiva (INPA/GCBEv), Fundação de Amparo a Pesquisas do Estado do Amazonas (PRONEX FAPEAM/CNPq 003/2009), Center for Studies of Adaptation to Environmental Changes in the Amazon (INCT ADAPTA, FAPEAM/CNPq 573976/2008-2), and Edital MCT/CNPq/MEC/CAPES/FNDCT–Ação Transversal/FAPs No. 47/2010 (Rede BioPHAM).


  1. Aguilar, C. T. & P. M. Galetti Jr., 2008. Chromosome mapping of 5S rRNA genes differentiates Brazilian populations of Leporellus vittatus (Anostomidae, Characiformes). Genetics and Molecular Biology 31(1): 188–194.CrossRefGoogle Scholar
  2. Alves-Costa, F. A., A. P. Wasko, C. Oliveira, F. Foresti & C. Martins, 2006. Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini fishes. Genetica 127: 243–252.CrossRefPubMedGoogle Scholar
  3. Barros, C. L., U. Santos, M. B. Cioffi & J. Dergam, 2015. Evolutionary divergence among Oligosarcus spp. (Ostariophysi, Characidae) from the São Francisco and Doce River Basins: Oligosarcus solitarius Menezes, 1987 shows the highest rates of chromosomal evolution in the Veotropical region. Zebrafish 12: 102–110.CrossRefPubMedGoogle Scholar
  4. Bertollo, L. A. C., C. S. Takahashi & O. Moreira-Filho, 1978. Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erytrinidae). Revista Brasileira de Genética 1: 103–120.Google Scholar
  5. Born, G. G. & L. A. C. Bertollo, 2000. An XX/XY sex chromosome system in a fish species, Hoplias malabaricus with a polymorphic NOR bearing X chromosome. Chromosome Research 8: 111–118.CrossRefPubMedGoogle Scholar
  6. Britski, H. A. & J. O. Birindelli, 2013. A new species of Leporinus agassiz (Characiformes: Anostomidae) from the rio Tocantins, Brazil. Neotropical Ichthyology 11(1): 25–32.CrossRefGoogle Scholar
  7. Brooks, L. D., 1988. The evolution of recombination rates. In Michod, R. E. & B. R. Levin (eds), The Evolution of Sex. Sianauer, Sunderland, MA: 87–105.Google Scholar
  8. Cabral-de-Mello, D. C., S. G. Oliveira, R. C. Moura & C. Martins, 2011. Chromosomal organization of the 18S and 5S rRNA and histone H3 genes in Scarabaeinae coleopterans: insights into the evolutionary dynamics of multigene families and heterochromatin. BMC Genetics 12: 88.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Caputo, V., M. Giovannotti, P. N. Cerioni, A. Splendiani, J. Tagliavini & E. Olmo, 2011. Chromosomal study of a lamprey (Lampetra zanandreai Vladykov, 1955) (Petromyzonida: Petromyzontiformes): conventional and FISH analysis. Chromosome Research 19: 481–491.CrossRefPubMedGoogle Scholar
  10. Carvalho, N. D. M., M. C. Gross, C. H. Schneider, M. L. Terencio, J. Zuanon & E. E. Feldberg, 2012. Cytogenetics of Synbranchiformes: a comparative analysis of two Synbranchus Bloch, 1795 species from the Amazon. Genetica 140: 149–158.CrossRefPubMedGoogle Scholar
  11. Centofante, L., L. A. C. Bertollo & O. Moreira-Filho, 2002. A ZZ/ZW sex chromosome system in a new species of the genus Parodon (Pisces, Parodontidae). Caryologia 55: 139–150.CrossRefGoogle Scholar
  12. Charlesworth, B., P. Snegowski & W. Stephan, 1994. The evolution dynamics of repetitive DNA in eukaryotes. Nature 371: 215–220.CrossRefPubMedGoogle Scholar
  13. Diniz, D., A. Laudicina & L. A. C. Bertollo, 2009. Chromosomal location of 18S and 5S rDNA sites in Triportheus fish species (Characiformes, Characidae). Genetics and Molecular Biology 32(1): 37–41.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Eschmeyer, W. N. & J. D. Fong, 2015. Species by family/subfamily. [Available on internet at]. Accessed on 2 July 2015.
  15. Feldberg, E., L. A. C. Bertollo, L. F. Almeida-Toledo, F. Foresti & O. Moreira-Filho, 1987. Biological aspects of Amazonian fishes. IX. Cytogenetic studies in two species of the genus Semaprochilodus (Pisces, Prochilodontidae). Genome 29: 1–4.CrossRefGoogle Scholar
  16. Ferreira, E. J. G., 1992. A ictiofauna do rio Trombetas na área de influência da futura usina hidrelétrica de Cachoeira Porteira, Pará. Tese de Doutorado, FUA/INPA, Manaus: 162 pp.Google Scholar
  17. Ferreira, I. A., C. Oliveira, P. C. Venere, P. M. Galetti & C. Martins, 2007. 5S rDNA variation and its phylogenetic inference in the genus Leporinus (Characiformes: Anostomidae). Genetica 129: 253–257.CrossRefPubMedGoogle Scholar
  18. Fujiwara, A., S. Abe, E. Yamaha, F. Yamazaki & M. C. Yoshida, 1998. Chromosomal localization and heterochromatin association of ribosomal RNA genes loci and silver stained nucleolar organizer regions in salmonid fishes. Chromosome Research 6: 463–471.CrossRefPubMedGoogle Scholar
  19. Galetti, P. M., A. C. G. Cesar & P. C. Venere, 1991a. Heterochromatin and NORs variability in Leporinus fish (Anostomidae, Characiformes). Caryologia 44: 287–292.CrossRefGoogle Scholar
  20. Galetti, P. M., C. A. Mestriner, P. C. Venere & F. Foresti, 1991b. Heterochromatin and karyotype reorganization in fish of the family Anostomidae (Characiformes). Cytogenetic Cell Genetics 56: 116–121.CrossRefGoogle Scholar
  21. Galetti, P. M., N. R. W. Lima & P. C. Venere, 1995a. A monophyletic ZW chromosome system in Leporinus (Anostomidae, Characiformes). Cytologia 60: 375–382.CrossRefGoogle Scholar
  22. Galetti, P. M., C. A. Mestriner, P. J. Monaco & E. M. Rasch, 1995b. Post-zygotic modification and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involving Leporinus friderici. Chromosome Research 3: 285–290.CrossRefPubMedGoogle Scholar
  23. Garavello, J. C. & H. A. Britski, 2003. Family Anostomidae (Headstanders). In Reis, R. E., S. O. Kullander & C. J. Ferraris Jr. (eds), Checklist of the Freshwater Fishes of South and Central America. EDIPUCRS, Porto Alegre: 71–86.Google Scholar
  24. Goulding, M., R. Barthem & E. J. Ferreira, 2003. The Negro and the Trombetas: black and clear waters from ancient lands. In: The Smithsonian Atlas of the Amazon. Smithsonian Institution Press, Washington, DC.Google Scholar
  25. Gregory, T. R., 2005. Genome size evolution in animals. In Gregory, T. R. (ed.), The Evolution of the Genome. Elsevier, San Diego, CA: 3–87.CrossRefGoogle Scholar
  26. Gross, M. C., C. H. Schneider, G. Valente, J. I. R. Porto, C. Martins & E. Feldberg, 2010. Comparative cytogenetic analysis of the genus Symphysodon (Discus Fishes, Cichlidae): chromosomal characteristics of retrotransposons and minor ribosomal DNA. Cytogenetics and Genome Research 1: 1–11.Google Scholar
  27. Guerra, M., 2004. FISH: Conceitos e Aplicações na Citogenética. Sociedade Brasileira de Genética, Ribeirão Preto.Google Scholar
  28. Haffer, J., 1982. General aspects of the refuge theory. In Prance, G. T. (ed.), Biological Diversification in the Tropics. Columbia University Press, New York: 6–26.Google Scholar
  29. Hatanaka, T., F. Henrique-Silva & P. M. Galetti, 2002. A polymorphic, telomeric-like sequence microsatellite in the Neotropical fish Prochilodus. Cytogenetics and Genome Research 98: 308–310.CrossRefPubMedGoogle Scholar
  30. Hillis, D. M. & M. T. Dixon, 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology 66: 411–453.CrossRefPubMedGoogle Scholar
  31. Howell, W. M. & D. A. Black, 1980. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: 1014–1015.CrossRefPubMedGoogle Scholar
  32. Ijdo, J. W., R. A. Wells, A. Baldini & S. T. Reeders, 1991. Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Research 19: 4780.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Junk, W. J. & K. Furch, 1993. A general review of tropical South American floodplains. Wetlands Ecology and Management 4: 231–238.Google Scholar
  34. Junk, W. J. & M. T. Z. Piedade, 2011. A classification of major naturally-occurring Amazonian lowland wetlands. Wetlands 31: 623–640.CrossRefGoogle Scholar
  35. Jurka, J., W. Bao & K. K. Kojima, 2011. Families of transposable elements, population structure and the origin of species. Biology Direct 6: 44.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Krichanã, S. R. L., 1999. Contribuição ao estudo citogenético da família Anostomidae (Pisces, Characiformes) na região Amazônica. Dissertação de mestrado, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos: 80 pp.Google Scholar
  37. Krinski, D. & C. S. Miyazawa, 2013. Análises cariotípicas em Leporellus vittatus e Leporinus striatus (Teleostei, Characiformes, Anostomidae) da Bacia do Alto Paraguai, Mato Grosso. Brasil. Estudo Biology 35(85): 113–120.CrossRefGoogle Scholar
  38. Levan, A., K. Fredga & A. A. Sandberg, 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201–220.CrossRefGoogle Scholar
  39. Long, E. O. & I. B. Dawid, 1980. Repeated genes in eukaryotes. Annu Rev Biochem 49: 727–764.CrossRefPubMedGoogle Scholar
  40. Mantovani, M., D. S. L. Abel, O. Moreira-Filho, 2005. Conserved 5S and variable 45S rDNA chromosomal localization revealed by FISH in Astyanax scabripinnis (Pisces, Characidae). Genetica 123: 211–216.CrossRefPubMedGoogle Scholar
  41. Margarido, V. P. & P. M. Galetti, 2000. Amplification of a GC-rich heterochromatin in the freshwater fish Leporinus desmotes (Characiformes, Anostomidae). Genetics Molecular Biology 23: 569–573.CrossRefGoogle Scholar
  42. Marreta, M. E., F. L. C. Faldoni & P. P. Parise-Maltempi, 2012. Cytogenetic mapping of the W chromosome in the genus Leporinus (Teleostei, Anostomidae) using a highly repetitive DNA sequence. Journal of Fish Biology 80: 630–637.CrossRefPubMedGoogle Scholar
  43. Martins, C., 2007. Chromosomes and repetitive DNAs: a contribution to the knowledge of fish genome. In Pisano, E., C. Ozouf-Costaz, F. Foresti & B. G. Kapoor (eds), Fish Cytogenetics. Science Publisher, Inc, Enfield: 421–453.Google Scholar
  44. Martins, C. & P. M. Galetti, 1998. Chromosome diversity in Neotropical fishes: NOR studies. Italian Journal of Zoology 65: 53–56.CrossRefGoogle Scholar
  45. Martins, C. & P. M. Galetti, 1999. Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes). Chromosome Research 7: 363–367.CrossRefPubMedGoogle Scholar
  46. Martins, C. & P. M. Galetti, 2000. Conservative distribution of 5S rDNA loci in Schizodon (Pisces, Anostomidae) chromosomes. Chromosome Research 8: 353–355.CrossRefGoogle Scholar
  47. Martins, C. & P. M. Galetti, 2001a. Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers. Genome 44: 903–910.CrossRefPubMedGoogle Scholar
  48. Martins, C. & P. M. Galetti, 2001b. Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes? Genetica 111: 439–446.CrossRefPubMedGoogle Scholar
  49. Martins, C. & A. P. Wasko, 2004. Organization and evolution of 5S ribosomal DNA in the fish genome. In Williams, C. L. (ed.), Focus on Genome Research. Nova Science Publishers, New York: 289–318.Google Scholar
  50. Martins, C., A. P. Wasko, C. Oliveira & J. M. Wright, 2000. Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the tilapiine cichlid fish, Oreochromis niloticus. Hereditas 133: 39–46.CrossRefPubMedGoogle Scholar
  51. Martins, C., D. C. Cabral-de-Mello, G. T. Valente, J. Mazzuchelli, S. G. Oliveira & D. Pinhal, 2011. Animal Genomes Under the Focus of Cytogenetics, 1st edn. Nova Science Publisher, Hauppauge: 160 pp.Google Scholar
  52. Meyne, J., R. L. Ratliff & R. K. Moyzis, 1989. Conservation of the human telomere sequence TTAGGGn among vertebrates. Proceedings of the National Academy of Sciences of the United States of America 86: 7049–7053.CrossRefPubMedPubMedCentralGoogle Scholar
  53. Molina, W. F. & P. M. Galetti, 2007. Early replication banding in Leporinus species (Osteichthyes, Characiformes) bearing differentiated sex chromosomes (ZW). Genetica 130: 153–160.CrossRefPubMedGoogle Scholar
  54. Moraes-Neto, A., M. Silva, D. A. Motos, M. R. Vicari, M. C. Almeida, M. J. Collares-Pereira & R. F. Artoni, 2011. Karyotype variability in neotropical catfishes of the family Pimelodidae (Teleostei, Siluriforme). Neotropical Ichthyology 9: 97–105.CrossRefGoogle Scholar
  55. Muller, H. J., 1964. The relation of recombination to mutational advance. Mutation Research 1: 2–9.CrossRefGoogle Scholar
  56. Nagamachi, C. Y., J. C. Pieczarka, P. C. M. O’Brien, J. Á. Pinto, S. M. Malcher, A. L. Perreira, J. D. Rissino, A. C. Mendes-Oliveira, R. V. Rossi & M. A. Ferguson-Smith, 2013. Fish with whole chromosome and telomeric probes demonstrates huge karyotypic reorganization with ITS between two species of Oryzomyini langguthi karyotype. Chromosome Research 21: 107–119.CrossRefPubMedGoogle Scholar
  57. Ohno, S., 1967. Sex Chromosomes and Sex-Linked Genes. Springer, Berlin.CrossRefGoogle Scholar
  58. Oliver, K. R. & W. K. Greene, 2011. Mobile DNA and the TE-Thrust Hypothesis: supporting evidence from the primates. Mobile DNA 2: 8.CrossRefPubMedPubMedCentralGoogle Scholar
  59. Parise-Maltempi, P. P., C. Martins, C. Oliveira & F. Foresti, 2007. Identification of a new repetitive element in the sex chromosomes of Leporinus elongatus (Teleostei: Characiformes: Anostomidae): new insights into the sex chromosomes of Leporinus. Cytogenetic and Genome Research 116: 218–223.CrossRefPubMedGoogle Scholar
  60. Parise-Maltempi, P. P., L. S. Edson, R. Willem, D. Frances, C. M. O’Brien, T. Vladimir Patricia & A. F. S. Malcolm, 2013. Comparative analysis of sex chromosomes in Leporinus species (Teleostei, Characiformes) using chromosome painting. BMC Genetics 14: 60.CrossRefPubMedPubMedCentralGoogle Scholar
  61. Pendás, A. M., P. Móran, L. P. Freije & E. Garcia-Vásquez, 1994. Chromosomal location and nucleotide sequence of two tandem repeats of the Atlantic salmon 5S rDNA. Cytogenetic Cell Genetic 67: 31–36.CrossRefGoogle Scholar
  62. Pereira, M. A., C. Oliveira, F. Foresti & E. L. Maistro, 2002. Cytogenetic and nuclear DNA content analysis in Anostomidae fishes from the Sapucaí River, Minas Gerais State, Brazil. Cytologia 68: 289–296.CrossRefGoogle Scholar
  63. Pinkel, D., T. Straume & J. W. Gray, 1986. Cytogenetic analysis using quantitative, high- sensitivity, fluorescence hybridization. Proceedings of the Natural Academy of Science of United States of America 83: 2934–2938.CrossRefGoogle Scholar
  64. Poltronieri, J., V. Marquioni, L. A. C. Bertollo, E. Kejnovsky, W. F. Molina, T. Liehr & M. B. Cioffi, 2013. Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes. Cytogenetic and Genome Research 142: 40–45.CrossRefPubMedGoogle Scholar
  65. Renno, J. F., P. Berreb, T. Boujard & R. Guyomard, 1990. Intraspecific genetic differentiation of Leporinus friderici. (Anostomidae, Pisces) in French Guiana and Brazil: a genetic approach to the refuge theory. Journal of Fish Biology 36: 85–95.CrossRefGoogle Scholar
  66. Sambrook, J. & D. W. Russell, 2001. Molecular Cloning: A Laboratory Manual, Vol. I. Cold Spring Harbor Press, Cold Spring Harbor, NY.Google Scholar
  67. Santos, G. M., 1991. Pesca e ecologia dos peixes de Rondônia. Tese de Doutorado, INPA/FUA, Manaus, AM: 213 pp.Google Scholar
  68. Santos, G. M. & M. Jégu, 1989. Inventário taxonômico e redescrição das espécies de anostomídeos (Characiformes, Anostomidae) do baixo rio Tocantins, PA, Brasil. Acta Amazonica 19: 159–213.CrossRefGoogle Scholar
  69. Santos, G. M. & M. Jégu, 1996. Inventário Taxonômico dos Anostomídeos (Pisces, Anostomidae) da Bacia do Rio Uatumã – AM, Brasil, com descrição de duas Espécies Novas. Acta Amazonica 26: 151–184.CrossRefGoogle Scholar
  70. Santos, G. M. & J. Zuanon, 2008. Leporinus amazonicus, a new anostomidae species from the Amazon lowlands, Brazil (Osteichthyes: Characiformes). Zootaxa 1815: 35–42.Google Scholar
  71. Schemberger, M. O., E. Bellafronte, V. Nogaroto, M. C. Almeida, G. S. Schuhli, R. F. Artoni, O. Moreira-Filho & M. R. Vicari, 2011. Differentiation of repetitive DNA sites and 98 sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes). Genetica 139: 1499–1508.CrossRefPubMedGoogle Scholar
  72. Schlade-Bartusiak, K., T. Costa, A. M. Summers, M. J. Nowaczyk & D. W. Cox, 2005. Fish-Mapping of telomeric 14q32 deletions: search for the cause of seizures. American Journal of Medical Genetics 183A: 218–224.CrossRefGoogle Scholar
  73. Silva, E. L., S. B. Rafael & P. P. Parise-Maltempi, 2012. Chromosome mapping of repetitive sequences in Anostomidae species: implications for genomic and sex chromosome evolution. Molecular Cytogenetics 5: 45.CrossRefPubMedPubMedCentralGoogle Scholar
  74. Sioli, H., 1950. Das Wasser im Amazonasgebiet. Forsch. Fortschr. In Lowe-McConnel, R. H. (ed.), Estudos ecológicos de comunidades de peixes tropicais. EDUSP, São Paulo: 345–373.Google Scholar
  75. Sliwinska-Jewsiewicka, A., M. Kucinski, L. Kirtiklis, S. Dobosz, K. Ocalewicz & M. Jankun, 2013. Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814). Genetica 143: 425–432.CrossRefGoogle Scholar
  76. Splendore de Borba R., E. Lourenço da Silva & P. P. Parise-Maltempi, 2013. Chromosome mapping of retrotransposable elements Rex1 and Rex3 in Leporinus Spix, 1829 species (Characiformes: Anostomidae) and its relationships among heterochromatic segments and W sex chromosome. Mobile Genetic Elements 3: e27460.Google Scholar
  77. Steinemann, S. & M. Steinemann, 2005. Retroelements: tools for sex chromosome evolution. Cytogenetic and Genome Research 110: 134–143.CrossRefPubMedGoogle Scholar
  78. Sumner, A. T., 1972. A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research 75: 304–306.CrossRefPubMedGoogle Scholar
  79. Terencio, M. L., C. H. Schneider, M. C. Gross, M. R. Vicari & E. Feldberg, 2012. Stable karyotypes: a general rule for the fish of the family Prochilodontidae? Hydrobiologia 686: 147–156.CrossRefGoogle Scholar
  80. Vicari, M. R., M. C. Almeida, L. A. C. Bertollo, O. Moreira-Filho & R. F. Artoni, 2006. Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA in the fish Prochilodus lineatus (Characiformes, Prochilodontidae). Genetics and Molecular Biology 4: 621–625.CrossRefGoogle Scholar
  81. Vicente, V. E., C. M. Jesus & O. Moreira-Filho, 2001. Chromosomal localization of 5S and 18S rRNA genes in three Parodon species (Pisces, Parodontidae). Caryologia 54: 365–369.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Lucas Caetano de Barros
    • 1
  • Pedro Manoel Galetti Junior
    • 2
  • Eliana Feldberg
    • 1
  1. 1.Programa de Pós-Graduação em Genética, Conservação e Biologia EvolutivaInstituto Nacional de Pesquisas da AmazôniaManausBrazil
  2. 2.Departamento de Genética e EvoluçãoUniversidade Federal de São CarlosSão CarlosBrazil

Personalised recommendations