The neotropical cichlid genus Astronotus currently comprises two valid species: A. ocellatus Agassiz, 1831 and A. crassipinnis Heckel, 1840. The diagnosis is based on color pattern and meristics counts. However, body color pattern is highly variable between regions and the meristic counts show a considerable overlap between populations differing in color patterning. They do not represent true synapomorphies that diagnose species. Purportedly the only truly diagnostic character is the presence or absence of one or more ocelli at the base of the dorsal fin, diagnosing A. ocellatus and A. crassipinnis, respectively. Using the portion of the mitochondrial COI gene and EPIC nuclear markers, the validity of the dorsal ocelli as diagnostic character was tested in individuals sampled from ten localities in the Amazon basin. Analyses rejected the hypothesis that dorsal ocelli are diagnostic at the species level. However, they revealed the existence of five hypothetical, largely allopatrically distributed morphologically cryptic species. The phylogeographic structure is not necessarily surprising, since species of the genus Astronotus have sedentary and territorial habits with low dispersal potential. The distribution of these hypothetical species is coincident with patterns observed in other Amazonian aquatic fauna, suggesting the role of common historical processes in generating current biodiversity patterns. 1. Introduction The neotropical cichlid genus Astronotus currently comprises two valid species: A. ocellatus Agassiz, 1831 and A. crassipinnis Heckel, 1840 [1]. Kullander [1] reports a number of diagnostic characters, however, with the exception of the presence of ocelli at the base of the dorsal fin in A. ocellatus and their absence in A. crassipinnis, all other characters show considerable overlap in their statistical distributions. The two species are characterized by differences in the modal number of lateral line scales (35 to 40 in A. crassipinnis versus. 33 to 39 in A. ocellatus), and the number of rays and spines of the dorsal fin (modal XIII.20 in A. ocellatus versus modal XII.21-22 in A. crassipinnis). There are also reported differences in color hue and patterning where A. crassipinnis is darker than A. ocellatus, the first light vertical bar is above the anal fin base in A. ocellatus versus. more anteriorly in A. crassipinnis, and A. crassipinnis has two more or less well-separated dark vertical bars in the position of the first light bar in A. ocellatus. Although proposed as diagnostic characters, the position of the vertical bars and body
References
[1]
S. O. Kullander, Cichlid Fishes of the Amazon River Drainage of Peru, Museum of Natural History, Stockholm, Sweden, 1986.
[2]
H. L. Queiroz and M. Barcelos, Seminário Anual de Pesquisas do IDSM, Instituto do Desenvolvimento Sustentavel Mamirauá, Tefé, AM, Brazil, 2005.
[3]
G. M. D. Santos, E. J. G. Ferreira, and J. A. S. Zuanon, Peixes Comerciais de Manaus, IBAMA/Pró-Várzea, Manaus, AM, Brazil, 2006.
[4]
S. O. Kullander, “Family Cichlidae,” in Check List of the Freshwater Fishes of South and Central America, R. E. Reis, S. O. Kullander, and C. J. Ferraris, Eds., pp. 605–654, EDIPUCRS, Porto Alegre, Brazil, 2003.
[5]
P. D. N. Hebert, A. Cywinska, S. L. Ball, and J. R. DeWaard, “Biological identifications through DNA barcodes,” Proceedings of the Royal Society B, vol. 270, no. 1512, pp. 313–321, 2003.
[6]
M. Valdez-Moreno, N. V. Ivanova, M. Elías-Gutiérrez, S. Contreras-Balderas, and P. D. N. Hebert, “Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes,” Journal of Fish Biology, vol. 74, no. 2, pp. 377–402, 2009.
[7]
N. Hubert, R. Hanner, E. Holm et al., “Identifying Canadian freshwater fishes through DNA barcodes,” PLoS ONE, vol. 3, no. 6, Article ID e2490, 2008.
[8]
R. D. Ward, T. S. Zemlak, B. H. Innes, P. R. Last, and P. D. N. Hebert, “DNA barcoding Australia's fish species,” Philosophical Transactions of the Royal Society B, vol. 360, no. 1462, pp. 1847–1857, 2005.
[9]
J. April, R. L. Mayden, R. H. Hanner, and L. Bernatchez, “Genetic calibration of species diversity among North America's freshwater fishes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 26, pp. 10602–10607, 2011.
[10]
R. E. Reis, S. O. Kullander, and C. J. Ferraris, Eds., Check List of the Freshwater Fishes of South and Central America, EDIPUCRS, Porto Alegre, Brazil, 2003.
[11]
M. C. F. Santos, M. L. Ruffino, and I. P. Farias, “High levels of genetic variability and panmixia of the tambaqui Colossoma macropomum (Cuvier, 1816) in the main channel of the Amazon River,” Journal of Fish Biology, vol. 71, pp. 33–44, 2007.
[12]
T. Hrbek, I. P. Farias, M. Crossa, I. Sampaio, J. I. R. Porto, and A. Meyer, “Population genetic analysis of Arapaima gigas, one of the largest freshwater fishes of the Amazon basin: implications for its conservation,” Animal Conservation, vol. 8, no. 3, pp. 297–308, 2005.
[13]
I. P. Farias, J. P. Torrico, C. García-Dávila, M. D. C. F. Santos, T. Hrbek, and J. F. Renno, “Are rapids a barrier for floodplain fishes of the Amazon basin? A demographic study of the keystone floodplain species Colossoma macropomum (Teleostei: Characiformes),” Molecular Phylogenetics and Evolution, vol. 56, no. 3, pp. 1129–1135, 2010.
[14]
J. S. Batista and J. A. Alves-Gomes, “Phylogeography of Brachyplatystoma rousseauxii (Siluriformes-Pimelodidae) in the Amazon Basin offers preliminary evidence for the first case of “homing” for an Amazonian migratory catfish,” Genetics and Molecular Research, vol. 5, no. 4, pp. 723–740, 2006.
[15]
D. Toffoli, T. Hrbek, M. L. G. de Araújo, M. P. de Almeida, P. Charvet-Almeida, and I. P. Farias, “A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes),” Genetics and Molecular Biology, vol. 31, no. 1, pp. 324–336, 2008.
[16]
I. P. Farias and T. Hrbek, “Patterns of diversification in the discus fishes (Symphysodon spp. Cichlidae) of the Amazon basin,” Molecular Phylogenetics and Evolution, vol. 49, no. 1, pp. 32–43, 2008.
[17]
M. V. Amado, T. Hrbek, and I. P. Farias, “A molecular perspective on systematics, taxonomy and classification Amazonian discus fishes of the genus Symphysodon,” International Journal of Evolutionary Biology, vol. 2011, Article ID 360654, 16 pages, 2011.
[18]
S. C. Willis, M. S. Nunes, C. G. Monta？a, I. P. Farias, and N. R. Lovejoy, “Systematics, biogeography, and evolution of the Neotropical peacock basses Cichla (Perciformes: Cichlidae),” Molecular Phylogenetics and Evolution, vol. 44, no. 1, pp. 291–307, 2007.
[19]
R. A. Chaves, Universidade Federal do Pará (UFPA), EmpresaBrasileira de Pesquisa Agropecuária—Amaz？nia Oriental (EMPBRAPA-Oriental), and Universidade Federal Rural da Amaz？nia (UFRA), M.Sc., 2007.
[20]
C. Li and J. J. M. Riethoven, “Exon-primed intron-crossing (EPIC) markers for non-model teleost fishes,” BMC Evolutionary Biology, vol. 10, no. 1, article 90, 2010.
[21]
K. R. Paithankar and K. S. N. Prasad, “Precipitation of DNA by polyethylene glycol and ethanol,” Nucleic Acids Research, vol. 19, no. 6, p. 1346, 1991.
[22]
T. Hall, “BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT,” Nucleic Acids Symposium Series, vol. 41, no. 1, pp. 95–98, 1999.
[23]
J. D. Thompson, D. G. Higgins, and T. J. Gibson, “CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research, vol. 22, no. 22, pp. 4673–4680, 1994.
[24]
T. H. Jukes and C. R. Cantor, “Evolution of protein molecules,” in Mammalian Protein Metabolism, H. N. Munro, Ed., pp. 21–123, Academic Press, New York, NY, USA, 1969.
[25]
O. Gascuel, “BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data,” Molecular Biology and Evolution, vol. 14, no. 7, pp. 685–695, 1997.
[26]
N. Puillandre, A. Lambert, S. Brouillet, and G. Achaz, “ABGD, Automatic Barcode Gap Discovery for primary species delimitation,” Molecular Ecology, vol. 21, no. 8, pp. 1864–1877, 2012.
[27]
S. Kumar, M. Nei, J. Dudley, and K. Tamura, “MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences,” Briefings in Bioinformatics, vol. 9, no. 4, pp. 299–306, 2008.
[28]
M. Kimura, “A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences,” Journal of Molecular Evolution, vol. 16, no. 2, pp. 111–120, 1980.
[29]
P. D. N. Hebert, E. H. Penton, J. M. Burns, D. H. Janzen, and W. Hallwachs, “Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 41, pp. 14812–14817, 2004.
[30]
R. A. Collins, L. M. Boykin, R. H. Cruickshank, and K. F. Armstrong, “Barcoding's next top model: an evaluation of nucleotide substitution models for specimen identification,” Methods in Ecology and Evolution, vol. 3, no. 3, pp. 457–465, 2012.
[31]
J. I. Davis and K. C. Nixon, “Populations, genetic variation, and the delimitation of phylogenetic species,” Systematic Biology, vol. 41, no. 4, pp. 421–435, 1992.
[32]
J. Rach, R. DeSalle, I. N. Sarkar, B. Schierwater, and H. Hadrys, “Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata,” Proceedings of the Royal Society B, vol. 275, no. 1632, pp. 237–247, 2008.
[33]
G. Jobb, A. Von Haeseler, and K. Strimmer, “TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics,” BMC Evolutionary Biology, vol. 4, article 18, 2004.
[34]
M. Hasegawa, H. Kishino, and T. Yano, “Dating of the human-ape splitting by a molecular clock of mitochondrial DNA,” Journal of Molecular Evolution, vol. 22, no. 2, pp. 160–174, 1985.
[35]
H. Akaike, “A new look at the statistical model identification,” IEEE Transactions on Automatic Control, vol. 19, no. 6, pp. 716–723, 1974.
[36]
H. Shimodaira, “An approximately unbiased test of phylogenetic tree selection,” Systematic Biology, vol. 51, no. 3, pp. 492–508, 2002.
[37]
C. D. L. Orme, R. P. Freckleton, G. H. Thomas, T. Petzoldt, S. A. Fritz, and N. J. B. Isaac, “CAPER: comparative analyses of phylogenetics and evolution in R,” Methods in Ecology and Evolution, vol. 3, no. 1, pp. 145–151, 2012.
[38]
L. Excoffier and H. E. L. Lischer, “Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows,” Molecular Ecology Resources, vol. 10, no. 3, pp. 564–567, 2010.
[39]
E. Paradis, “Pegas: an R package for population genetics with an integrated-modular approach,” Bioinformatics, vol. 26, no. 3, pp. 419–420, 2010.
[40]
M. A. Smith, B. L. Fisher, and P. D. N. Hebert, “DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar,” Philosophical Transactions of the Royal Society B, vol. 360, no. 1462, pp. 1825–1834, 2005.
[41]
P. D. N. Hebert, M. Y. Stoeckle, T. S. Zemlak, and C. M. Francis, “Identification of birds through DNA barcodes,” PLoS Biology, vol. 2, no. 10, article e310, 2004.
[42]
A. Gómez, P. J. Wright, D. H. Lunt, J. M. Cancino, G. R. Carvalho, and R. N. Hughes, “Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon,” Proceedings. Biological sciences / The Royal Society, vol. 274, no. 1607, pp. 199–207, 2007.
[43]
R. Meier, G. Zhang, and F. Ali, “The use of mean instead of smallest interspecific distances exaggerates the size of the “barcoding gap” and leads to misidentification,” Systematic Biology, vol. 57, no. 5, pp. 809–813, 2008.
[44]
C. P. Meyer and G. Paulay, “DNA barcoding: error rates based on comprehensive sampling,” PLoS biology, vol. 3, no. 12, article e422, 2005.
[45]
M. Elias, R. I. Hill, K. R. Willmott et al., “Limited performance of DNA barcoding in a diverse community of tropical butterflies,” Proceedings of the Royal Society B, vol. 274, no. 1627, pp. 2881–2889, 2007.
[46]
M. Wiemers and K. Fiedler, “Does the DNA barcoding gap exist? A case study in blue butterflies (Lepidoptera: Lycaenidae),” Frontiers in Zoology, vol. 4, article 8, 2007.
[47]
J. S. Ready, E. J. G. Ferreira, and S. O. Kullander, “Discus fishes: mitochondrial DNA evidence for a phylogeographic barrier in the Amazonian genus Symphysodon (Teleostei: Cichlidae),” Journal of Fish Biology, vol. 69, pp. 200–211, 2006.
[48]
G. Pilleri and M. Gihr, “Observations on the Bolivian, Inia boliviensis, (D'Orbigny, 1834) and the Amazonian bufeo, Inia geoffrensis (Blainville, 1817), with a description of a new subspecies (Inia geoffrensis humboldtiana),” Experientia, vol. 24, no. 9, pp. 932–934, 1968.
[49]
E. Banguera-Hinestroza, H. Cárdenas, M. Ruiz-García et al., “Molecular identification of evolutionarily significant units in the Amazon river dolphin Inia sp. (Cetacea: Iniidae),” Journal of Heredity, vol. 93, no. 5, pp. 312–322, 2002.
[50]
S. O. Kullander and E. J. G. Ferreira, “A review of the South American cichlid genus Cichla, with descriptions of nine new species (Teleostei: Cichlidae),” Ichthyological Exploration of Freshwaters, vol. 17, no. 4, pp. 289–398, 2006.
[51]
D. E. Pearse, A. D. Arndt, N. Valenzuela, B. A. Miller, V. Cantarelli, and J. W. Sites Jr., “Estimating population structure under nonequilibrium conditions in a conservation context: continent-wide population genetics of the giant Amazon river turtle, Podocnemis expansa (Chelonia; Podocnemididae),” Molecular Ecology, vol. 15, no. 4, pp. 985–1006, 2006.
[52]
B. de Thoisy, T. Hrbek, I. P. Farias, W. R. Vasconcelos, and A. Lavergne, “Genetic structure, population dynamics, and conservation of Black caiman (Melanosuchus niger),” Biological Conservation, vol. 133, no. 4, pp. 474–482, 2006.
[53]
N. V. Meliciano, Instituto Nacional de Pesquisas da Amaz？nia (INPA) and Universidade Federal do Amazonas (UFAM), M.Sc., 2008.
