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Parallel evolution evidenced by molecular data in the banded-tetra (Astyanax fasciatus)
Biochemical Systematics and Ecology 70 (2017) 141e146
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Parallel evolution evidenced by molecular data in the bandedtetra (Astyanax fasciatus) Rubens Pazza*, Letícia Aparecida Cruvinel, Karine Frehner Kavalco rio de Gen gica e Evolutiva, Rodovia BR 354, Km 310 (1300m), Universidade Federal de Viçosa, Campus Rio Paranaíba, Laborato etica Ecolo 38810-000, PO Box 22, Rio Paranaíba, Minas Gerais, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2016 Received in revised form 24 October 2016 Accepted 29 October 2016
Astyanax is well known as a model for developmental biology studies, particularly with regard to Mexico's cave populations. More than 130 species of Astyanax are already known, most of which live in South America. The occurrence of cryptic species and species complexes elucidated by chromosomal and genetic studies demonstrates that the relationship between morphology and molecular evolution is quite complex within this group. In this work, we demonstrate that morphology does not follow the path of vicariant processes observed in Astyanax fasciatus populations, which separated about three million years ago, although molecular data suggests its separation in two species. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Phylogeography Tetra Molecular ecology Ecological genetics
1. Introduction Brazil is a huge country with some major hydrographic basins. The largest and best known of these is the Amazon, but the should also be highlighted for their wide area and endemism. Nevertheless, basins of the S~ ao Francisco and Upper Parana these basins are home to various common species, remnant of the geological processes of its genesis. ~o Francisco and Parana river basins and is located in a NorthThe Upper Paranaíba arc is the watershed between the Sa South arrangement in the Southern Brazil (Campos and Dardenne, 1997). This region seems to be responsible for the shared ichthyofauna between these two hydrographic basins (Hubert and Renno, 2006). The region, which in some places is elevated above 1100 m, began its geological elevation about 117 million years ago (Ma). This was then followed by draining during neotectonic events at the end of the Tertiary, around 3 Ma (Saadi et al., 1991). From this time of divergence, for the initial vicariant processes in the region, we tested if morphological evolution followed the genetic structure and phylogeography within one species of the genus Astyanax, Astyanax fasciatus, which was widely distributed among these watersheds, in order to determine whether this time of divergence was enough for differentiation as separate species. Although widely known, this species presents taxonomic problems since its description in the nineteenth century to more recent chromosomal characterizations. Its type location is referred to as “rivers from Brazil,” although Eigenmann suggests that the type location is the S~ ao Francisco river (Eigenmann, 1921). On the other hand, cytogenetic studies suggest that Astyanax fasciatus is more than one species but that it has a common denomination due to interesting chromosomal variation river basin with divergent cytotypes found in sympatry and alopatry (Pazza et al., 2006, 2008). in Upper Parana
* Corresponding author. E-mail address: [email protected] (R. Pazza). http://dx.doi.org/10.1016/j.bse.2016.10.024 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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R. Pazza et al. / Biochemical Systematics and Ecology 70 (2017) 141e146
Fig. 1. Map of the region of Upper Paranaíba showing collection sites. 1. Grande River; 2. Paranaíba River A; 3. Paraíso stream; 4. Paranaíba River B; 5. Mombaça River; 6. Das Araras weir; 7. Para River; 8. Santa Catarina River. Brazil map with major hydrographic basins in the detail.
2. Material and methods All the specimens and DNA samples used in this study were deposited in the DNA and Collection of Vertebrates and DNA of the Laboratory of Ecological and Evolutionary Genetics of the Federal University of Viçosa (Rio Paranaíba campus) (Suppl. 1) and were collected on the basis of field studies conducted during 2008e2010. Collecting permit SISBIO 15571-1 was issued to Prof. Rubens Pazza. The samples of Astyanax fasciatus analyzed in the current study originated from nine different locations, ~o Francisco rivers (Fig. 1). distributed throughout the hydrographic basins of the Rio Grande Paranaíba and Sa The following point-to-point measurements were taken from specimens using a digital caliper: standard length; head length; pre-dorsal distance; pre-pelvic distance; pre-pectoral distance; pre-anal distance; height of dorsal origin; height of tail peduncle; length of anal bases; length of dorsal bases; length of pelvic bases; length of pectoral bases; head height; snout length; eye diameter; interorbital diameter; and jaw length. The multivariate analysis was performed using the PAST software package (Hammer et al., 2001) after correction in order to avoid the effects of size deviations among different populations (size-free). Five representative individuals from each site sampled were used whenever possible. We examined mitochondrial segments of subunits 6 and 8 of the ATPase gene and Cytochrome Oxidase I (COI). For the polymerase chain reactions (PCRs) we used the pairs of primers ATP8.2-L8331 (50 -AAAGCRTTRGCCTTTTAAAGC-30 ) and CO3.2-H9236 (50 -GTTAGTGGTCAGGGCTTGGRTC-30 ) (Sivasundar et al., 2001); for ATPase and FishF1 (50 -TCAACCAACCACAAAGACATTGGCAC-30 ); and FishR1 (50 -TAGACTTCTGGGTGGCCAAAGAATCA-30 ) (Ward et al., 2005) for COI, which amplified a segment of approximately 900 and 650 bp, respectively. The amplification reactions were conducted in a thermal cycler with a total volume of 25 mL, containing 15 ng of DNA template, Tris-KCl (20 mM Tris-HCl pH 8.4 and 50 mM KCl), 1.5 mM MgCl2, 2.5 mM of each primer, 0.1 mM of each dNTP, and 2.5 U Taq-polymerase. The reaction conditions were as follows: initial denaturation of 94 C for 4 min, hydridization at 56 C for 30 s, and extension at 72 C for 2 min, followed by 40 cycles of 15 s at 94 C, 30 s at 56 C, 2 min at 72 C, and a final extension step for 10 min at 72 C (Prioli et al., 2002). After checking amplification via 1% agarose gel, the samples were sent to a purification and sequencing outsource service. The sequences obtained were visualized and edited using the program Chromas Lite v2.01, and further verified at GenBank (http://www.ncbi.nlm.nih.gov) using the Blastn program. Subsequently, the sequences were aligned using the algorithm ClustalW v1.6 (Thompson et al., 1994) by the software MEGA v5 (Tamura et al., 2011), applying the penalties for the
R. Pazza et al. / Biochemical Systematics and Ecology 70 (2017) 141e146
143
Fig. 2. Spatial distribution through principal components analysis (PCA). Morphometric data (size-free) from 65 samples of Astyanax fasciatus grouped by collection site. Grande River (dark blue); Paranaíba River A (light green); Paraíso stream (light blue); Paranaíba River B (yellow); Mombaça River (dark green); Do Boi river (light green); Das Araras weir (red); Para River (purple); Santa Catarina River (orange). Minimum span tree between populations (complete lines), and load and direction of variables (dots): 1. standard length; 2. head length; 3. pre-dorsal distance; 4. pre-pelvic distance; 5. pre-pectoral distance; 6. pre-anal distance; 7. height of dorsal origin; 8. height of tail peduncle; 9. length of anal bases; 10. length of dorsal bases; 11. length of pelvic bases; 12. length of pectoral bases; 13. head height; 14. snout length; 15. eye diameter; 16. interorbital diameter; and 17. jaw length. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
alignments pair-to-pair and multiplex, as well as for opening (20) and gap extension (6.66) (the alignment files will be deposited in TreeBase as the acception of the manuscript, and this sentence will be changed with the respective numbers). MEGA v5 software (Tamura et al., 2011) calculated the best model to use for applying the data, and from this we generated a phylogram of Maximum Likelihood Estimation (MLE). Statistical analyses of the sequences and haplotype analyses were performed using DnaSP v5 software (Librado and Rozas, 2009). The haplotype network was obtained using Network (FluxusTechnologies).
Fig. 3. Dendrogram of UPGMA based on the Euclidean distance. Dendrogram of the morphometric data of 65 samples of Astyanax fasciatus grouped by River collection site. Grande River (rg); Paranaíba A River (pnc); Paraíso stream (par); Paranaíba B River (pnp); Mombaça River (mom); Das Araras weir (aar); Para (sfpa); Santa Catarina River (sfsc). Bootstrap values refer to 1000 replications (values below 80 not shown).
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Fig. 4. Phylogram of maximum likelihood. Phylogram based on the analysis of 744 bp of the ATPase mitocondrial gene 6/8, using the algorithm HKY according to the evolutive model test. Bootstrap values refer to 100 replications. The nomenclature of the subjects is organized according to their registration codes. 1. Rio River Grande River (rg); 2. Paranaíba A River (pnc); 3. Paraíso stream (par); 4. Paranaíba B River (pnp); 5. Mombaça River (mom); 6. Das Araras weir (aar); 7. Para (sfpa); 8. Santa Catarina River (sfsc).
3. Results With the use of a size-free Principal Component Analysis (PCA), it was possible to obtain eigenvectors, with the first component being responsible for 95.5% of the variation. The morphometric analysis demonstrated clear structuring within some of the populations studied. PCA was efficient in separating each of the populations, although some of them are very ~o closely linked (Fig. 2). The populations of the Grande River, Paranaíba River (Carmo do Paranaíba), and Mombaça River (Sa Francisco River basin) proved to be very structured. This structuring was also clearly evidenced on the UPGMA dendrogram (Fig. 3) which, with elevated bootstrap values, demonstrated strong structuring among these populations. By using this ~o Francisco and analysis, it was possible to observe two major groups, both represented by populations belonging to the Sa River basins. The two populations descendant from the Paranaíba River were very distant, despite being from the same Parana river. Sequences of mitochondrial DNA ATPase gene were analyzed in 19 specimens, totaling 744 bp. Meanwhile, COI gene was analyzed in 28 specimens, totalizing near to 600 bp. Phylogeographic analyses were carried out only for the ATPase gene, COI showed low variation on one occasion, with 0.6% distance between the populations. The model that best explains the data obtained by ATPase sequence was HKY, according to the test performed in the Mega v5 software. The phylogram of ML (Fig. 4) ~o Francisco and the Parana river basins, despite the small shows clear separation between the haplotypes belonging to the Sa distance between them (p-distance ¼ 0.019). river The haplotype network showed the distribution of three main groups, one originating from the basin of the Parana and two originating from the S~ ao Francisco river (Fig. 5).
4. Discussion river basins, as shown by The data demonstrates clear structuring among the populations from S~ ao Francisco and Parana ATPase gene sequencing. On the other hand, it was difficult to separate the samples that originated from these two
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Fig. 5. Haplotype network. The distance between each node is proportional to the substitutions. Populations sampled: 1. Rio Grande River (light blue); 2. River Paranaíba A River (dark blue); 3. Paraíso stream (red); 4. Paranaíba B River (light green); 5. Mombaça River (brown); 6. Das Araras weir (purple); 7. Para (pink); 8. Santa Catarina River (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
hydrographic basins purely on the basis of morphometric characters. Despite the absence of morphological divergences, ~o Francisco and Parana populations are different species. molecular data suggests that Sa ATPase mitochondrial sequencing has been used in studies of genetic diversity and phylogenetic analysis of neotropical fish. Sivasundar et al. (2001) used the ATPase mitochondrial sequences six and eight when studying populations of Prochilodus (Characiformes) from different hydrographic basins, obtaining distinctive haplotypic standards for each of them. Perdices et al. (2002) used mitochondrial sequences, among which were ATPase six and eight, to study phylogenetic and distribution of species of the genus Rhamdia (Siluriformes) in Mesoamerica. These studies outlined the process of vicariance of these Central American fish from those of South America (Perdices et al., 2002). On the other hand, COI sequences are chosen for the barcoding endeavor, especially for fish species (Hebert and Gregory, 2005). However, it seems that this sequence is not so useful to distinguish among Astyanax species (Carvalho et al., 2011). We found a lower distance between 2n ¼ 46 and 2n ¼ 48 cytotypes with COI sequence than with ATPase sequence. Furthermore, Ornelas-García et al. (2008), and Pereira et al. (2011) suggest that low genetic diversity by using DNA barcoding (COI sequences) in Astyanax could be the result ~o Francisco and Parana populations is of recent evolutionary radiation process. Even so, the divergence rate between Sa consistent for different species, separated after the vicariant event of the Upper Paranaíba arc uplift. and Sa ~o Francisco rivers are separated by the uplift of the Upper Paranaíba arc, The hydrographic basins of the Parana which began around 117e119 Ma (Campos and Dardenne, 1997). Draining of this region occurred during the events of the neotectonic at the end of the Tertiary period, around 3 Ma (Saadi et al., 1991). Such events were decisive with regard to the geomorphology of the region, and could have influenced the distribution and structuring of the fish population. Indeed, this region may be responsible for the distribution of the species between the two hydrographic basins (Hubert and Renno, 2006). Bermingham et al. (1997) reported a substitution rate of 1.3% per million years through ATPase 6 sequencing in neotropical fish. If we apply this rate of substitution to the current data, we obtain a divergence time of at least 1.5 million years among the rivers. This estimation agrees with the dating of geological events of the Quapopulations of the S~ ao Francisco and Parana ternary that occurred within the same basins (Campos and Dardenne, 1997). The polytomy observed in data from the phylogram (Fig. 4), which represents populations originating from the basins of ~o Francisco and Paran the Sa a rivers, can be explained by the fast dispersion and diversification following the population separation process. This is mirrored, in a way, in both hydrographic basins, which are quite similar to that observed by Perdices et al. (2002) with Rhamdia laticauda and Rhamdia guatemalensis (Siluriformes). Finding morph groups close or even intermixed by the analysis of grouping of UPGMA (Fig. 3) can indicate that the differentiation between the populations is either very small or that the environments in which they were found was very similar and was, therefore, able to produce morphological homoplasy. Eigenmann (1921) comments on the problems associated with the identification of Astyanax fasciatus as noted by Cuvier in 1819, which affects both its museum conservation and identification at the collection site, which is why its distribution is attributed to “rivers from Brazil.” According to the author, this Characidae species is widely distributed throughout the neotropical region, giving rise to countless distinctive forms due to its isolation within different rivers. Some of those forms are sufficiently distinctive to the extent of being considered a different species, such as A. mexicanus, A. aeneus, and A. parahybae (Eigenmann, 1921). and Sa ~o Francisco rivers was This overlap of morphology between individuals originating from the basins of the Parana also observed by Moreira-Filho and Bertollo (1991) when they analyzed different populations of Astyanax scabripinnis. As with the observations within the current paper, although some population types could be distinguished by morphometric analyses, others had morphological characteristics common to both the populations in the basins, despite showing distinctive cytogenetic characteristics (Moreira-Filho and Bertollo, 1991). According to Eigenmann (1921), A. scabripinnis and A. fasciatus, together with A. taeniatus, form a species triangle whose morphological distinction is complex when compared with
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R. Pazza et al. / Biochemical Systematics and Ecology 70 (2017) 141e146
specimens from different locations. It is not surprising that the two groups that are more widely distributed (A. scabripinnis and A. fasciatus) have been designated as a complex species with peculiar chromosomal characteristics (Moreira-Filho and Bertollo, 1991; Artoni et al., 2006; Pazza et al., 2006). On the other hand, when analyzed separately, the Astyanax scabripinnis populations studied by Moreira-Filho and Bertollo (1991) appear to be well structured. The same thing occurs in four populations of A. scabripinnis taken from the basin of the Upper Paran a River with regard to diploid number and distinctive karyotyping formula (Mizoguchi and Martins-Santos, 1998), as well as in two alopatric populations of A. fasciatus with distinct chromosome numbers (Pazza et al., 2008). In view of the results obtained, it is possible to hypothesize that the fast divergence among populations of Astyanax fasciatus from both hydrographic basins after the vicariant event of Upper Paranaiba arc uplift was accompanied by morphologic variation, reinforced by isolation in small inbreeding populations facing similar selective pressures. This explains the genetic structuring found in the basins of the S~ ao Francisco and Paran a rivers, with regard to parallel evolution in relation to the morphological characteristics. Ornelas-García et al. (2008) and Strecker et al. (2012) observed a similar phenomenon in populations of superficial and cave Astyanax mexicanus resulting from independent invasions toward the north of Mexico and our findings are showing that such phenomena is not restricted to great morphological effects. Acknowledgements Pesquisa do Estado de Minas Gerais (Foundation of Support to This research was supported by Fundaç~ ao de Amparo a Research from Minas Gerais State e APQ00756-09) (FAPEMIG). The voucher numbers and GeneBank access of all analyzed individuals are in the Supplementary material. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2016.10.024. References Artoni, R.F., Shibatta, O.A., Gross, M.C., Schneider, C.H., Almeida, M.C., et al., 2006. Astyanax aff. fasciatus Cuvier, 1819 (Teleostei, Characidae): evidences of a , Brazil). Neotrop. Ichthyol. 4, 197e202. species complex in the upper rio Tibagi basin (Parana Bermingham, E., McCafferty, S.S., Martin, A.P., 1997. Fish biogeography and molecular clocks: perspectives from the Panamian Isthmus. In: Kocher, T., Stepien, C.A. (Eds.), Molecular Systematics of Fishes. Academic Press, San Diego, CA, pp. 113e128. ~o tecto ^ nica da bacia Sanfranciscana. Rev. Bras. Geocienc. 27, 283e294. Campos, J.E.G., Dardenne, M.A., 1997. Origem e evoluça Carvalho, D.C., Oliveira, D.A.A., Pompeu, P.S., Leal, C.G., Oliveira, C., Hannern, R., 2011. Deep barcode divergence in Brazilian freshwater fishes: the case of the ~o Francisco River basin. Mitochondrial DNA 22, 80e86. Sa Eigenmann, C.H., 1921. The American Characidae. Part 3. Camb. - Mem. Mus. Comp. Zool. 43, 209e310. Hammer, O., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics Software Package for education and Data Analysis. Palaeontol. Electron. 4, 4e9. Hebert, P.D.N., Gregory, T.R., 2005. The promise of DNA barcoding for taxonomy. Mol. Syst. Biol. 54, 852e859. Hubert, N., Renno, J.-F., 2006. Historical biogeography of South American freshwater fishes. J. Biogeogr. 33, 1414e1436. Librado, P., Rozas, J., 2009. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451e1452. Mizoguchi, S.M.H.M., Martins-Santos, I.C., 1998. Cytogenetic and morphometric differences in populations of Astyanax “scabripinnis” (Pisces, Characidae) from Maring a region, PR, Brazil. Genet. Mol. Biol. 21, 55e61. Moreira-Filho, O., Bertollo, L.A.C., 1991. Astyanax scabripinnis (Pisces, Characidae): a species complex. Genet. Mol. Biol. 14, 331e357. Ornelas-García, C.P., Domínguez-Domínguez, O., Doadrio, I., 2008. Evolutionary history of the fish genus Astyanax Baird & Girard (1854) (Actinopterygii, Characidae) in Mesoamerica reveals multiple morphological homoplasies. BMC Evol. Biol. 8, 340. Pazza, R., Kavalco, K.F., Bertollo, L.A.C., 2006. Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 1-Karyotypic analysis, Ag-NORs and mapping of the 18S and 5S ribosomal genes in sympatric karyotypes and their possible hybrid forms. Cytogenet. Genome Res. 112, 313e319. Pazza, R., Kavalco, S.A.F., Penteado, P.R., Kavalco, K.F., Almeida-Toledo, L.F., 2008. The species complex Astyanax fasciatus Cuvier, 1819 (Teleostei, Characiformes): a multidisciplinary approach. J. Fish. Biol. 72, 2002e2010. Perdices, A., Bermingham, E., Montilla, A., Doadrio, I., 2002. Evolutionary history of the genus Rhamdia (Teleostei: pimelodidae) in Central America. Mol. Phylogenet Evol. 25, 172e189. Pereira, L.H.G., Pazian, M.F., Hanner, R., Foresti, F., Oliveira, C., 2011. DNA barcoding reveals hidden diversity in the neotropical freshwater fish Piabina argentea (Characiformes: Characidae) from the Upper Paran a basin of Brazil. Mitochond. DNA 22, 87e96. Prioli, S.M.A.P., Prioli, A.J., Júlio Jr., H.F., Pavanelli, C.S., de Oliveira, A.V., et al., 2002. Identification of Astyanax altiparanae (Teleostei, Characidae) in the Iguaçu river, Brazil, based on mitochondrial DNA and RAPD markers. Genet. Mol. Biol. 25, 421e430. ~es sobre a neotecto ^nica e morfoge ^nese de minas gerais. SBG. In: Simpo sio Nacional de Estudos Saadi, A., Hasui, Y., Magalh~ aes, F.S., 1991. Informaço ^nicos, 3. Boletim Resumos Expandidos, Rio Claro, pp. 105e106. Tecto Sivasundar, A., Bermingham, E., Ortí, G., 2001. Population structure and biogeography of migratory freshwater fishes (Prochilodus: characiformes) in major South American Rivers. Mol. Ecol. 10, 407e417. Strecker, U., Hausdorf, B., Wilkins, H., 2012. Parallel speciation in Astyanax cave fish (Teleostei) in Northern Mexico. Mol. Phylogenet Evol. 62, 62e70. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., et al., 2011. MEGA5: molecular evolutionary genetics Analysis using maximum Likelihood, evolutionary Distance, and maximum parsimony Methods. Mol. Biol. Evol. 28, 2731e2739. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673e4680. Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., Hebert, P.D.N., 2005. DNA barcoding Australia's fish species. Philos. T. Roy. Soc. B. 360, 1847e1857.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Parallel evolution evidenced by molecular data in the bandedtetra (Astyanax fasciatus) Rubens Pazza*, Letícia Aparecida Cruvinel, Karine Frehner Kavalco rio de Gen gica e Evolutiva, Rodovia BR 354, Km 310 (1300m), Universidade Federal de Viçosa, Campus Rio Paranaíba, Laborato etica Ecolo 38810-000, PO Box 22, Rio Paranaíba, Minas Gerais, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2016 Received in revised form 24 October 2016 Accepted 29 October 2016
Astyanax is well known as a model for developmental biology studies, particularly with regard to Mexico's cave populations. More than 130 species of Astyanax are already known, most of which live in South America. The occurrence of cryptic species and species complexes elucidated by chromosomal and genetic studies demonstrates that the relationship between morphology and molecular evolution is quite complex within this group. In this work, we demonstrate that morphology does not follow the path of vicariant processes observed in Astyanax fasciatus populations, which separated about three million years ago, although molecular data suggests its separation in two species. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Phylogeography Tetra Molecular ecology Ecological genetics
1. Introduction Brazil is a huge country with some major hydrographic basins. The largest and best known of these is the Amazon, but the should also be highlighted for their wide area and endemism. Nevertheless, basins of the S~ ao Francisco and Upper Parana these basins are home to various common species, remnant of the geological processes of its genesis. ~o Francisco and Parana river basins and is located in a NorthThe Upper Paranaíba arc is the watershed between the Sa South arrangement in the Southern Brazil (Campos and Dardenne, 1997). This region seems to be responsible for the shared ichthyofauna between these two hydrographic basins (Hubert and Renno, 2006). The region, which in some places is elevated above 1100 m, began its geological elevation about 117 million years ago (Ma). This was then followed by draining during neotectonic events at the end of the Tertiary, around 3 Ma (Saadi et al., 1991). From this time of divergence, for the initial vicariant processes in the region, we tested if morphological evolution followed the genetic structure and phylogeography within one species of the genus Astyanax, Astyanax fasciatus, which was widely distributed among these watersheds, in order to determine whether this time of divergence was enough for differentiation as separate species. Although widely known, this species presents taxonomic problems since its description in the nineteenth century to more recent chromosomal characterizations. Its type location is referred to as “rivers from Brazil,” although Eigenmann suggests that the type location is the S~ ao Francisco river (Eigenmann, 1921). On the other hand, cytogenetic studies suggest that Astyanax fasciatus is more than one species but that it has a common denomination due to interesting chromosomal variation river basin with divergent cytotypes found in sympatry and alopatry (Pazza et al., 2006, 2008). in Upper Parana
* Corresponding author. E-mail address: [email protected] (R. Pazza). http://dx.doi.org/10.1016/j.bse.2016.10.024 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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Fig. 1. Map of the region of Upper Paranaíba showing collection sites. 1. Grande River; 2. Paranaíba River A; 3. Paraíso stream; 4. Paranaíba River B; 5. Mombaça River; 6. Das Araras weir; 7. Para River; 8. Santa Catarina River. Brazil map with major hydrographic basins in the detail.
2. Material and methods All the specimens and DNA samples used in this study were deposited in the DNA and Collection of Vertebrates and DNA of the Laboratory of Ecological and Evolutionary Genetics of the Federal University of Viçosa (Rio Paranaíba campus) (Suppl. 1) and were collected on the basis of field studies conducted during 2008e2010. Collecting permit SISBIO 15571-1 was issued to Prof. Rubens Pazza. The samples of Astyanax fasciatus analyzed in the current study originated from nine different locations, ~o Francisco rivers (Fig. 1). distributed throughout the hydrographic basins of the Rio Grande Paranaíba and Sa The following point-to-point measurements were taken from specimens using a digital caliper: standard length; head length; pre-dorsal distance; pre-pelvic distance; pre-pectoral distance; pre-anal distance; height of dorsal origin; height of tail peduncle; length of anal bases; length of dorsal bases; length of pelvic bases; length of pectoral bases; head height; snout length; eye diameter; interorbital diameter; and jaw length. The multivariate analysis was performed using the PAST software package (Hammer et al., 2001) after correction in order to avoid the effects of size deviations among different populations (size-free). Five representative individuals from each site sampled were used whenever possible. We examined mitochondrial segments of subunits 6 and 8 of the ATPase gene and Cytochrome Oxidase I (COI). For the polymerase chain reactions (PCRs) we used the pairs of primers ATP8.2-L8331 (50 -AAAGCRTTRGCCTTTTAAAGC-30 ) and CO3.2-H9236 (50 -GTTAGTGGTCAGGGCTTGGRTC-30 ) (Sivasundar et al., 2001); for ATPase and FishF1 (50 -TCAACCAACCACAAAGACATTGGCAC-30 ); and FishR1 (50 -TAGACTTCTGGGTGGCCAAAGAATCA-30 ) (Ward et al., 2005) for COI, which amplified a segment of approximately 900 and 650 bp, respectively. The amplification reactions were conducted in a thermal cycler with a total volume of 25 mL, containing 15 ng of DNA template, Tris-KCl (20 mM Tris-HCl pH 8.4 and 50 mM KCl), 1.5 mM MgCl2, 2.5 mM of each primer, 0.1 mM of each dNTP, and 2.5 U Taq-polymerase. The reaction conditions were as follows: initial denaturation of 94 C for 4 min, hydridization at 56 C for 30 s, and extension at 72 C for 2 min, followed by 40 cycles of 15 s at 94 C, 30 s at 56 C, 2 min at 72 C, and a final extension step for 10 min at 72 C (Prioli et al., 2002). After checking amplification via 1% agarose gel, the samples were sent to a purification and sequencing outsource service. The sequences obtained were visualized and edited using the program Chromas Lite v2.01, and further verified at GenBank (http://www.ncbi.nlm.nih.gov) using the Blastn program. Subsequently, the sequences were aligned using the algorithm ClustalW v1.6 (Thompson et al., 1994) by the software MEGA v5 (Tamura et al., 2011), applying the penalties for the
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Fig. 2. Spatial distribution through principal components analysis (PCA). Morphometric data (size-free) from 65 samples of Astyanax fasciatus grouped by collection site. Grande River (dark blue); Paranaíba River A (light green); Paraíso stream (light blue); Paranaíba River B (yellow); Mombaça River (dark green); Do Boi river (light green); Das Araras weir (red); Para River (purple); Santa Catarina River (orange). Minimum span tree between populations (complete lines), and load and direction of variables (dots): 1. standard length; 2. head length; 3. pre-dorsal distance; 4. pre-pelvic distance; 5. pre-pectoral distance; 6. pre-anal distance; 7. height of dorsal origin; 8. height of tail peduncle; 9. length of anal bases; 10. length of dorsal bases; 11. length of pelvic bases; 12. length of pectoral bases; 13. head height; 14. snout length; 15. eye diameter; 16. interorbital diameter; and 17. jaw length. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
alignments pair-to-pair and multiplex, as well as for opening (20) and gap extension (6.66) (the alignment files will be deposited in TreeBase as the acception of the manuscript, and this sentence will be changed with the respective numbers). MEGA v5 software (Tamura et al., 2011) calculated the best model to use for applying the data, and from this we generated a phylogram of Maximum Likelihood Estimation (MLE). Statistical analyses of the sequences and haplotype analyses were performed using DnaSP v5 software (Librado and Rozas, 2009). The haplotype network was obtained using Network (FluxusTechnologies).
Fig. 3. Dendrogram of UPGMA based on the Euclidean distance. Dendrogram of the morphometric data of 65 samples of Astyanax fasciatus grouped by River collection site. Grande River (rg); Paranaíba A River (pnc); Paraíso stream (par); Paranaíba B River (pnp); Mombaça River (mom); Das Araras weir (aar); Para (sfpa); Santa Catarina River (sfsc). Bootstrap values refer to 1000 replications (values below 80 not shown).
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Fig. 4. Phylogram of maximum likelihood. Phylogram based on the analysis of 744 bp of the ATPase mitocondrial gene 6/8, using the algorithm HKY according to the evolutive model test. Bootstrap values refer to 100 replications. The nomenclature of the subjects is organized according to their registration codes. 1. Rio River Grande River (rg); 2. Paranaíba A River (pnc); 3. Paraíso stream (par); 4. Paranaíba B River (pnp); 5. Mombaça River (mom); 6. Das Araras weir (aar); 7. Para (sfpa); 8. Santa Catarina River (sfsc).
3. Results With the use of a size-free Principal Component Analysis (PCA), it was possible to obtain eigenvectors, with the first component being responsible for 95.5% of the variation. The morphometric analysis demonstrated clear structuring within some of the populations studied. PCA was efficient in separating each of the populations, although some of them are very ~o closely linked (Fig. 2). The populations of the Grande River, Paranaíba River (Carmo do Paranaíba), and Mombaça River (Sa Francisco River basin) proved to be very structured. This structuring was also clearly evidenced on the UPGMA dendrogram (Fig. 3) which, with elevated bootstrap values, demonstrated strong structuring among these populations. By using this ~o Francisco and analysis, it was possible to observe two major groups, both represented by populations belonging to the Sa River basins. The two populations descendant from the Paranaíba River were very distant, despite being from the same Parana river. Sequences of mitochondrial DNA ATPase gene were analyzed in 19 specimens, totaling 744 bp. Meanwhile, COI gene was analyzed in 28 specimens, totalizing near to 600 bp. Phylogeographic analyses were carried out only for the ATPase gene, COI showed low variation on one occasion, with 0.6% distance between the populations. The model that best explains the data obtained by ATPase sequence was HKY, according to the test performed in the Mega v5 software. The phylogram of ML (Fig. 4) ~o Francisco and the Parana river basins, despite the small shows clear separation between the haplotypes belonging to the Sa distance between them (p-distance ¼ 0.019). river The haplotype network showed the distribution of three main groups, one originating from the basin of the Parana and two originating from the S~ ao Francisco river (Fig. 5).
4. Discussion river basins, as shown by The data demonstrates clear structuring among the populations from S~ ao Francisco and Parana ATPase gene sequencing. On the other hand, it was difficult to separate the samples that originated from these two
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Fig. 5. Haplotype network. The distance between each node is proportional to the substitutions. Populations sampled: 1. Rio Grande River (light blue); 2. River Paranaíba A River (dark blue); 3. Paraíso stream (red); 4. Paranaíba B River (light green); 5. Mombaça River (brown); 6. Das Araras weir (purple); 7. Para (pink); 8. Santa Catarina River (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
hydrographic basins purely on the basis of morphometric characters. Despite the absence of morphological divergences, ~o Francisco and Parana populations are different species. molecular data suggests that Sa ATPase mitochondrial sequencing has been used in studies of genetic diversity and phylogenetic analysis of neotropical fish. Sivasundar et al. (2001) used the ATPase mitochondrial sequences six and eight when studying populations of Prochilodus (Characiformes) from different hydrographic basins, obtaining distinctive haplotypic standards for each of them. Perdices et al. (2002) used mitochondrial sequences, among which were ATPase six and eight, to study phylogenetic and distribution of species of the genus Rhamdia (Siluriformes) in Mesoamerica. These studies outlined the process of vicariance of these Central American fish from those of South America (Perdices et al., 2002). On the other hand, COI sequences are chosen for the barcoding endeavor, especially for fish species (Hebert and Gregory, 2005). However, it seems that this sequence is not so useful to distinguish among Astyanax species (Carvalho et al., 2011). We found a lower distance between 2n ¼ 46 and 2n ¼ 48 cytotypes with COI sequence than with ATPase sequence. Furthermore, Ornelas-García et al. (2008), and Pereira et al. (2011) suggest that low genetic diversity by using DNA barcoding (COI sequences) in Astyanax could be the result ~o Francisco and Parana populations is of recent evolutionary radiation process. Even so, the divergence rate between Sa consistent for different species, separated after the vicariant event of the Upper Paranaíba arc uplift. and Sa ~o Francisco rivers are separated by the uplift of the Upper Paranaíba arc, The hydrographic basins of the Parana which began around 117e119 Ma (Campos and Dardenne, 1997). Draining of this region occurred during the events of the neotectonic at the end of the Tertiary period, around 3 Ma (Saadi et al., 1991). Such events were decisive with regard to the geomorphology of the region, and could have influenced the distribution and structuring of the fish population. Indeed, this region may be responsible for the distribution of the species between the two hydrographic basins (Hubert and Renno, 2006). Bermingham et al. (1997) reported a substitution rate of 1.3% per million years through ATPase 6 sequencing in neotropical fish. If we apply this rate of substitution to the current data, we obtain a divergence time of at least 1.5 million years among the rivers. This estimation agrees with the dating of geological events of the Quapopulations of the S~ ao Francisco and Parana ternary that occurred within the same basins (Campos and Dardenne, 1997). The polytomy observed in data from the phylogram (Fig. 4), which represents populations originating from the basins of ~o Francisco and Paran the Sa a rivers, can be explained by the fast dispersion and diversification following the population separation process. This is mirrored, in a way, in both hydrographic basins, which are quite similar to that observed by Perdices et al. (2002) with Rhamdia laticauda and Rhamdia guatemalensis (Siluriformes). Finding morph groups close or even intermixed by the analysis of grouping of UPGMA (Fig. 3) can indicate that the differentiation between the populations is either very small or that the environments in which they were found was very similar and was, therefore, able to produce morphological homoplasy. Eigenmann (1921) comments on the problems associated with the identification of Astyanax fasciatus as noted by Cuvier in 1819, which affects both its museum conservation and identification at the collection site, which is why its distribution is attributed to “rivers from Brazil.” According to the author, this Characidae species is widely distributed throughout the neotropical region, giving rise to countless distinctive forms due to its isolation within different rivers. Some of those forms are sufficiently distinctive to the extent of being considered a different species, such as A. mexicanus, A. aeneus, and A. parahybae (Eigenmann, 1921). and Sa ~o Francisco rivers was This overlap of morphology between individuals originating from the basins of the Parana also observed by Moreira-Filho and Bertollo (1991) when they analyzed different populations of Astyanax scabripinnis. As with the observations within the current paper, although some population types could be distinguished by morphometric analyses, others had morphological characteristics common to both the populations in the basins, despite showing distinctive cytogenetic characteristics (Moreira-Filho and Bertollo, 1991). According to Eigenmann (1921), A. scabripinnis and A. fasciatus, together with A. taeniatus, form a species triangle whose morphological distinction is complex when compared with
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specimens from different locations. It is not surprising that the two groups that are more widely distributed (A. scabripinnis and A. fasciatus) have been designated as a complex species with peculiar chromosomal characteristics (Moreira-Filho and Bertollo, 1991; Artoni et al., 2006; Pazza et al., 2006). On the other hand, when analyzed separately, the Astyanax scabripinnis populations studied by Moreira-Filho and Bertollo (1991) appear to be well structured. The same thing occurs in four populations of A. scabripinnis taken from the basin of the Upper Paran a River with regard to diploid number and distinctive karyotyping formula (Mizoguchi and Martins-Santos, 1998), as well as in two alopatric populations of A. fasciatus with distinct chromosome numbers (Pazza et al., 2008). In view of the results obtained, it is possible to hypothesize that the fast divergence among populations of Astyanax fasciatus from both hydrographic basins after the vicariant event of Upper Paranaiba arc uplift was accompanied by morphologic variation, reinforced by isolation in small inbreeding populations facing similar selective pressures. This explains the genetic structuring found in the basins of the S~ ao Francisco and Paran a rivers, with regard to parallel evolution in relation to the morphological characteristics. Ornelas-García et al. (2008) and Strecker et al. (2012) observed a similar phenomenon in populations of superficial and cave Astyanax mexicanus resulting from independent invasions toward the north of Mexico and our findings are showing that such phenomena is not restricted to great morphological effects. Acknowledgements Pesquisa do Estado de Minas Gerais (Foundation of Support to This research was supported by Fundaç~ ao de Amparo a Research from Minas Gerais State e APQ00756-09) (FAPEMIG). The voucher numbers and GeneBank access of all analyzed individuals are in the Supplementary material. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2016.10.024. References Artoni, R.F., Shibatta, O.A., Gross, M.C., Schneider, C.H., Almeida, M.C., et al., 2006. Astyanax aff. fasciatus Cuvier, 1819 (Teleostei, Characidae): evidences of a , Brazil). Neotrop. Ichthyol. 4, 197e202. species complex in the upper rio Tibagi basin (Parana Bermingham, E., McCafferty, S.S., Martin, A.P., 1997. Fish biogeography and molecular clocks: perspectives from the Panamian Isthmus. In: Kocher, T., Stepien, C.A. (Eds.), Molecular Systematics of Fishes. Academic Press, San Diego, CA, pp. 113e128. ~o tecto ^ nica da bacia Sanfranciscana. Rev. 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