Abstract

Some pelagic and usually large sized fishes are preferential targets for sport and commercial fishing. Despite their economic importance, cytogenetic data on their evolutionary processes and management are very deficient, especially due to logistical difficulties. Here, information for two of such charismatic species, the tarpon, Megalops atlanticus (Elopiformes: Megalopidae), and the sailfish, Istiophorus platypterus (Istiophoriformes: Istiophoridae), both with a wide Atlantic distribution, were provided. Cytogenetic data were obtained using conventional methods (Giemsa staining, Ag-NORs technique, and C-banding), base-specific fluorochrome staining and fluorescence in situ hybridization (FISH) with rDNA probes. Megalops atlanticus has 2n = 50 chromosomes, all acrocentric ones (NF = 50), while Istiophorus platypterus has 2n = 48 chromosomes, 2m + 2st + 44a (NF = 52). Megalops atlanticus populations from the South Atlantic and Caribbean share identical karyotypic patterns, likely associated with gene flow between them. In turn, I. platypterus presents karyotype similarities with phylogenetically close groups, such as Carangidae. The chromosomal characteristics of these species highlight their independent evolutionary paths. Additionally, the current data contribute to knowledge of new aspects of pelagic fish fauna and will support further comparative studies with congeneric species, clarifying evolutionary karyotype trends of these fish groups.

Keywords:
Animal cytogenetics; Chromosome evolution; rDNA; Species conservation

Resumo

Alguns peixes pelágicos de grande porte são alvos preferenciais para a pesca esportiva e comercial. Apesar de sua importância econômica, os dados citogenéticos sobre seus processos evolutivos e de manejo são muito deficientes, principalmente devido às dificuldades logísticas. Aqui são apresentadas informações cromossômicas de duas espécies carismáticas, o tarpão, Megalops atlanticus (Elopiformes: Megalopidae), e o agulhão-vela, Istiophorus platypterus (Istiophoriformes: Istiophoridae), ambos com ampla distribuição no oceano Atlântico. Os dados citogenéticos foram obtidos usando métodos convencionais (coloração em Giemsa, técnica de Ag-NORs e bandamento C), coloração com fluorocromos específicos e hibridização fluorescente in situ (FISH) com sondas DNAr. Megalops atlanticus possui 2n = 50 cromossomos, todos acrocêntricos (NF = 50), enquanto Istiophorus platypterus possui 2n = 48 cromossomos, 2m + 2st + 44a (NF = 52). Populações de M. atlanticus do Atlântico Sul e Caribe compartilham padrões cariotípicos idênticos, provavelmente associados ao fluxo gênico entre regiões. Por sua vez, I. platypterus apresenta semelhanças cariotípicas micro e macroestruturais com grupos filogeneticamente próximos, como Carangidae. As características cromossômicas destas espécies destacam seus caminhos evolutivos independentes. Adicionalmente, os dados apresentados contribuem com novos aspectos da fauna pelágica e apoiarão futuros estudos comparativos com espécies congenéricas, esclarecendo as tendências evolutivas do cariótipo destes grupos de peixes.

Palavras-chave:
Citogenética animal; Conservação de espécies; DNAr; Evolução cromossômica

INTRODUCTION

Pelagic ecosystems represent one of the largest environments on the planet and, in general, little is known about the evolutionary features of its ichthyofauna. Marine pelagic fishes can reach an extensive geographical distribution, a condition that has direct implications for their genetic and cytogenetic patterns (Galetti et al., 2000Galetti PM Jr, Aguilar CT, Molina WF. An overview of marine fish cytogenetics. Hydrobiologia. 2000; 420:55–62. https://doi.org/10.1007/978-94-017-2184-4_6
https://doi.org/10.1007/978-94-017-2184-... , 2006Galetti PM Jr, Molina WF, Affonso PRAM, Aguilar CT. Assessing genetic diversity of Brazilian reef fishes by chromosomal and DNA markers. Genetica. 2006; 126(1–2):161–77. https://doi.org/10.1007/s10709-005-1446-z
https://doi.org/10.1007/s10709-005-1446-... ; Soares et al., 2013Soares RX, Bertollo LAC, Costa GWWF, Molina WF. Karyotype stasis in four Atlantic Scombridae fishes: mapping of classic and dual-color FISH markers on chromosomes. Fish Sci. 2013; 79(2):177–83. https://doi.org/10.1007/s12562-013-0602-0
https://doi.org/10.1007/s12562-013-0602-... , 2017Soares RX, Cioffi MB, Bertollo LAC, Borges AT, Costa GWWF, Molina WF. Chromosomal evolution in large pelagic oceanic apex predators, the barracudas (Sphyraenidae, Percomorpha). Genet Mol Res. 2017; 16(2):gmr16029644. https://doi.org/10.4238/gmr16029644
https://doi.org/10.4238/gmr16029644... ). However, cytogenetic analyses in large marine fishes, especially the pelagic ones, are very scarce even in those of great economic value, mostly due to logistical restrictions involved (Soares et al., 2013Soares RX, Bertollo LAC, Costa GWWF, Molina WF. Karyotype stasis in four Atlantic Scombridae fishes: mapping of classic and dual-color FISH markers on chromosomes. Fish Sci. 2013; 79(2):177–83. https://doi.org/10.1007/s12562-013-0602-0
https://doi.org/10.1007/s12562-013-0602-... , 2014Soares RX, Bertollo LAC, Cioffi MB, Costa GWWF, Molina WF. Chromosomal distribution of two multigene families and the unusual occurrence of an X1X1X2X2/X1X2Y sex chromosome system in the dolphinfish (Coryphaenidae): An evolutionary perspective. Genet Mol Res. 2014; 13(2):2470–79. https://doi.org/10.4238/2014.April.3.19
https://doi.org/10.4238/2014.April.3.19... ).

A large phylogenetic spectrum of fish groups inhabit the pelagic ecosystems, including representatives of the orders Elopiformes and Istiophoriformes. Elopiformes presents itself as a sister group to all the others groups of the superorder Elopomorpha Chen et al., 2014Chen JN, López JA, Lavoué S, Miya M, Chen WJ. Phylogeny of the Elopomorpha (Teleostei): evidence from six nuclear and mitochondrial markers. Mol Phylogenet Evol. 2014; 70:152–61. https://doi.org/10.1016/j.ympev.2013.09.002
https://doi.org/10.1016/j.ympev.2013.09.... ) and comprises only two old and slightly diverse families, the Elopidae (with only the Elops genus, with 7 species) and Megalopidae (with only the Megalops genus, with 2 species), with an estimated origin of 215 Mya (Broughton et al., 2013Broughton RE, Betancur-R R, Li C, Arratia G, Ortí G. Multi-locus phylogenetic analysis reveals the pattern and tempo of bony fish evolution. PLoS Curr. 2013; (April 16): 1–37. https://dx.doi.org/10.5061/dryad.f1t15
https://dx.doi.org/10.5061/dryad.f1t15... ). Elopiformes (9 spp.) is hundreds of times less diverse than other Elopomorpha groups, such as Anguilliformes (995 spp.) Fricke et al., 2020Fricke R, Eschmeyer WN, Fong JD. Eschmeyer’s catalog of fishes: genera, species by family, subfamily [Internet]. San Francisco: California Academy of Science; 2020. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp
http://researcharchive.calacademy.org/re... ). Therefore, due to their phylogenetically position and evolutionary aspects, the cytogenetic patterns of Elopiformes are one important element that contributes to clarify the karyotype evolution in Teleostei as a whole.

Istiophoriformes includes the families Istiophoridae and Xiphiidae, also comprising important species in sport fishing, such as the sailfish I. platypterus (Shaw, 1792), globally distributed throughout the world’s tropical and subtropical marine water, and the swordfish Xiphias gladius Linnaeus, 1758 widely distributed in the Atlantic, Pacific and Indian Oceans (Fricke et al., 2020Fricke R, Eschmeyer WN, Fong JD. Eschmeyer’s catalog of fishes: genera, species by family, subfamily [Internet]. San Francisco: California Academy of Science; 2020. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp
http://researcharchive.calacademy.org/re... ). The origin of the Istiophoriformes probably occurred around ~71 Mya, in the Late Cretaceous (100.5–66 Mya), and the diversification of istiophorids and swordfishes originated around ~17.5 Mya, in the Early Miocene (23–16 Mya) (Santini et al., 2013Santini F, Sorenson L. First molecular timetree of billfishes (Istiophoriformes: Acanthomorpha) shows a Late Miocene radiation of marlins and allies. Ital J Zool. 2013; 80(4):481–89. https://doi.org/10.1080/11250003.2013.848945
https://doi.org/10.1080/11250003.2013.84... ).

Sailfishes are active predators distributed in pelagic ecosystems in tropical and temperate regions, morphologically characterized by a protruding upper jaw (Nakamura, 1985Nakamura I, editor. FAO species catalogue. Vol. 5. Billfishes of the world. An annotated and illustrated catalogue of marlins, sailfishes, spearfishes and swordfishes known to date. Rome: FAO Fisheries Synopsis; 1985. ), and considered to be among the fastest swimmers in the oceans (Svendsen et al., 2016Svendsen MB, Domenici P, Marras S, Krause J, Boswell KM, Rodriguez-Pinto I, Wilson AD, Kurvers RH, Viblanc PE, Finger JS, Steffensen JF. Maximum swimming speeds of sailfish and three other large marine predatory fish species based on muscle contraction time and stride length: a myth revisited. Biol Open. 2016; 5(10):1415–19. https://doi.org/10.1242/bio.019919
https://doi.org/10.1242/bio.019919... ). Despite their ecological and commercial importance, the global genetic population structure of sailfish is not well understood (Lu et al., 2015Lu C-P, Bremer JRA, McKenzie JL, Chiang W-C. Analysis of sailfish (Istiophorus platypterus) population structure in the North Pacific Ocean. Fish Res. 2015; 166(2015):33–38. https://doi.org/10.1016/j.fishres.2014.09.018
https://doi.org/10.1016/j.fishres.2014.0... ), and cytogenetic information on these fishes is still lacking.

In the present study we provide a detailed karyotypic analysis of the tarpon, Megalops atlanticus Valenciennes, 1847 (Elopiformes: Megalopidae) and the sailfish, Istiophorus platypterus (Istiophoriformes: Istiophoridae), both representatives of marine species with a high economic importance, especially in the lucrative sportfishing market (Ault, Luo, 2013Ault JS, Luo J. A reliable game fish weight estimation model for Atlantic tarpon (Megalops atlanticus). Fishery Research. 2013; 139:110–17. https://doi.org/10.1016/j.fishres.2012.10.004
https://doi.org/10.1016/j.fishres.2012.1... ; Adams et al., 2019Adams A, Guindon K, Horodysky A, MacDonald T, McBride R, Shenker J, Ward R. Megalops atlanticus [Internet]. The IUCN Red List of Threatened Species; 2019. Available from: https://www.iucnredlist.org/search?query=megalops%20atlanticus&searchType=species
https://www.iucnredlist.org/search?query... ). These species occupy vast tropical and subtropical oceanic regions, where M. atlanticus inhabits coastal waters, including estuaries and lagoons, and I. platypterus is eminently oceanic (Nakamura, 1985Nakamura I, editor. FAO species catalogue. Vol. 5. Billfishes of the world. An annotated and illustrated catalogue of marlins, sailfishes, spearfishes and swordfishes known to date. Rome: FAO Fisheries Synopsis; 1985. ; Riede, 2004Riede K. Global register of migratory species - from global to regional scales. Final Report of the RandD-Projekt 808 05 081. Germany: Federal Agency for Nature Conservation; 2004.; Ault, 2010Ault JS. Silver King – a most perfect and ancient sportfish: the biology, ecology and management of Megalops atlanticus - and its precarious future. In: Mill AR, editor. A Passion for Tarpon. Mill Creek: Wild River Press; 2010. p.260–90. ). It was applied conventional and molecular cytogenetic procedures (Giemsa, Ag- NORs, C- and MM/DAPI banding, and mapping of the 18S and 5S rDNAs, in order to investigate the chromosomal patterns of the current species, provide a first basis to further interpopulation comparisons, and highlight the main cytogenetic divergences between Elopifomes and Istiophoriformes groups.

MATERIAL AND METHODS

Samples. Five juvenile individuals of Megalops atlanticus (Elopiformes: Megalopidae) and four individuals (undetermined sex) of Istiophorus platypterus (Istiophoridae) were collected from the Brazilian Northeast coast, in the Rio Grande do Norte State (M. atlanticus and I. platypterus – 06°20’S 35°15’W) (Fig. 1), through sport and commercial fishing vessels. Collections had the authorization of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), System of Authorization and Information about Biodiversity (SISBIO-Licenses N^o 19135–1, 131360–1 and 27027–2), a National System of Genetic Resource Management and Associated Traditional Knowledge (SISGEN). All cytogenetics procedures were performed at the Laboratory of Genetics of Marine Resources from the Federal University of Rio Grande do Norte.

FIGURE 1 |
Geographic distribution map of Megalops atlanticus (Megalopidae) and Istiophorus platypterus (Istiophoridae) across the Atlantic ocean. The shaded areas represents the occurrence and the yellow stars represent the collection sites of the species.

Chromosome preparation, C-banding, Ag-NOR and MM/DAPI staining. Chromosome preparations were performed from kidney tissues dissociated in 9.5 ml RPMI 1640 medium with 0.2 ml colchicine, for 30 min, followed by hypotonization with KCl 0.075, for 25 min at room temperature (Gold et al., 1990Gold JR, Lee C, Shipley NS, Powers PK. Improved methods for working with fish chromosomes with a review of metaphase chromosome banding. J Fish Biol. 1990; 37(4):563–75. https://doi.org/10.1111/j.1095-8649.1990.tb05889.x
https://doi.org/10.1111/j.1095-8649.1990... ). The cell suspension was dropped onto clean slides covered with a thin film of water at 60 ^oC. After drying, chromosomes were stained with Giemsa 10%, diluted in pH 6.8 phosphate buffer. Nucleolar organizing regions (NORs) and the constitutive heterochromatin were visualized by Silver nitrate staining (i.e., Ag-NORs) and C-banding, according to Howell, Black, (1980)Howell WM, Black DA. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer a 1-step method. Experientia. 1980; 36(8):1014–15. https://doi.org/10.1007/BF01953855
https://doi.org/10.1007/BF01953855... and Sumner, (1972)Sumner AT. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res. 1972; 75(1):304–06. https://doi.org/10.1016/0014-4827(72)90558-7
https://doi.org/10.1016/0014-4827(72)905... , respectively. Additionally, chromosomes were stained with Mithramycin (GC-specific) and DAPI (AT-specific) fluorochromes, according to Schweizer, (1976)Schweizer D. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma. 1976; 58(4):307–24. https://doi.org/10.1007/BF00292840
https://doi.org/10.1007/BF00292840... .

Repetitive DNA mapping with fluorescence in situ hybridization (FISH). FISH (fluorescence in situ hybridization) was performed according to Pinkel et al., (1986)Pinkel D, Straume T, Gray JW. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. P Natl Acad Sci USA. 1986; 83(9):2934–38. https://doi.org/10.1073/pnas.83.9.2934
https://doi.org/10.1073/pnas.83.9.2934... . The 5S rDNA (~200 bp) and 18S rDNA (1400 bp) probes were obtained by polymerase chain reaction (PCR), from the nuclear DNA of Rachycentron canadum (Carangiformes), using the primers A 5′-TAC GCC CGA TCT CGT CCG ATC-3 ′, B 5′-CAG GCT GGT ATG GCC GTA AGC-3 ′ (Pendás et al., 1994Pendás AM, Morán P, Freije JP, Garcia-Vásquez E. Chromosomal location and nucleotide sequence of two tandem repeats of the Atlantic salmon 5S rDNA. Cytogenet Cell Genet. 1994; 67(1):31–36. https://doi.org/10.1159/000133792
https://doi.org/10.1159/000133792... ) and NS1 5′-GTA GTC ATA TGC TTG TCT C-3 ′ / NS8 5 ′ -TCC GCA GGT TCA CCT ACG GA-3 ′ (White et al., 1990White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T, editors. PCR Protocols: A guide to methods and applications. New York: Academic Press Inc; 1990. p.315–22.). The probes were labeled by nick translation with biotin-14-dATP and digoxigenin-11-dUTP (Roche, Mannheim, Germany) and detected with streptavidin-FITC (Vector Laboratories), and anti-digoxigenin-rhodamine (Roche, Mannheim, Germany), respectively.

Microscopy and image processing. At least 30 metaphases of each individual were analyzed and the best results were photographed in an Olympus ™ BX51 epifluorescence microscope coupled to the digital image capture system Olympus DP73 (Olympus Corporation, Ishikawa, Japan), using the cellSens software (Version 1.9 Digital, Tokyo, Kanto, Japan). The fundamental number was based on the number of chromosome arms and the chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a), according to the arms ratio (Levan et al., 1964Levan A, Fredga K, Sandberg A. Nomenclature for centromeric position on chromosomes. Hereditas. 1964; 52(2):201–20. https://doi.org/10.1111/j.1601-5223.1964.tb01953.x
https://doi.org/10.1111/j.1601-5223.1964... ).

Abbreviations. 18S – 18S ribosomal RNA; 2n – Diploid number; 5S – 5S ribosomal RNA; a – Acrocentric chromosome(s); Ag-NORs – Nucleolar Organizing Regions evidenced through silver nitrate impregnation; AT – Adenine/Thymine; DAPI – 4′,6-diamidino-2-phenylindole; FISH – Fluorescence in situ hybridization; FITC – Fluorescein isothiocyanate; GC – Guanine/Cytosine; ICMBio – Chico Mendes Institute for Biodiversity Conservation; KCl – Potassium chloride; m – Metacentric chromosome(s); MM – Mithramycin; Mya – Millions of years ago; NF – Fundamental number; NORs – Nucleolar organizing regions; PCR – Polymerase chain reaction; rDNA – Ribosomal DNA; SISBIO – System of Authorization and Information about Biodiversity; SISGEN – National System of Genetic Resource Management and Associated Traditional Knowledge; st – Subtelocentric chromosome(s); µm – micrometer.

RESULTS

Megalops atlanticus has 2n = 50 chromosomes, all acrocentric (NF = 50), while I. platypterus has 2n = 48, and the karyotype composed of 2m + 2st + 44a chromosomes (NF = 52) (Fig. 2). No heteromorphic chromosomes were evidenced among the individuals of species.

In both species, heterochromatic blocks occur mainly in the centromeric regions (e.g., M. atlanticus – pairs 8, 10, 12; I. platypterus – pairs 10, 11, 14), but also in the terminal regions of some pairs (e.g., M. atlanticus – pairs 5, 7, 17; I. platypterus – pairs 5, 8, 11) (Fig. 2). The Ag-NORs sites are found in a single chromosome pair, although specific to each species. Thus, in M. atlanticus they are interstitially located in the long arms of the smallest 25^th pair, while in I. platypterus they are terminally located in the short arms of the 2^nd pair (Fig. 2, highlighted). These sites are in agreement with the location of the 18S rDNA hybridization signals, being also MM+/DAPI- stained, which characterizes them as GC-rich regions (Fig. 2, highlighted).

FIGURE 2 |
Karyotypes of Megalops atlanticus (Megalopidae) and Istiophorus platypterus (Istiophoridae) after Giemsa staining, C-banding and FISH procedures. The small left boxes highlight the Ag-NORs and MM+/DAPI- sites, and the right ones the 18S (red) and 5S (green) rDNA sites. Scale bar = 5 µm.

The 5S rDNA sites are located in the short arms of the pair 7, in M. atlanticus and in the terminal region of the long arms of the pair 9, in I. platypterus (Fig. 2), both acrocentric chromosomes. The (TTAGGG)_n probe hybridized exclusively on the terminal regions of the chromosomes of M. atlanticus. In some metaphases of this species, recurrent radial chromosome arrangements were observed (Fig. 2, larger box).

DISCUSSION

Cytogenetic data for large pelagic fishes are sporadic and usually restricted to the description of the diploid chromosome number (Doucette, Fitzsimons, 1988Doucette AJ Jr, Fitzsimons JM. Karyology of elopiform and clupeiform fishes. Copeia. 1988; 1988(1):124–30. https://doi.org/10.2307/1445931
https://doi.org/10.2307/1445931... ; Khuda-Bukhsh et al., 1995Khuda-Bukhsh AR, Rahman A, Chanda T, Nayak K, Khuda-Bukhsh. Diploid numbers and chromosome formulae of some 29 species of Indian teleosts (Pisces). Chromosome Inf Serv. 1995; 58:38–39.; Arai, 2011Arai R. Fish Karyotypes: a check list. Tokyo: Springer; 2011.). This lack of karyotype data for several groups impairs comparative analyzes on their chromosomal relationships and evolutionary trends. In this sense, this study provides classical and molecular cytogenetic data for two representative species, M. atlanticus and I. platypterus.

Like some other marine pelagic fishes (Accioly et al., 2012Accioly IV, Bertollo LA, Costa GW, Jacobina UP, Molina WF. Chromosomal population structuring in carangids (Perciformes) between the north-eastern and south-eastern coasts of Brazil. Afr J Mar Sci. 2012; 34(3):383–89. https://doi.org/10.2989/1814232X.2012.689671
https://doi.org/10.2989/1814232X.2012.68... ; Soares et al., 2013Soares RX, Bertollo LAC, Costa GWWF, Molina WF. Karyotype stasis in four Atlantic Scombridae fishes: mapping of classic and dual-color FISH markers on chromosomes. Fish Sci. 2013; 79(2):177–83. https://doi.org/10.1007/s12562-013-0602-0
https://doi.org/10.1007/s12562-013-0602-... , 2014Soares RX, Bertollo LAC, Cioffi MB, Costa GWWF, Molina WF. Chromosomal distribution of two multigene families and the unusual occurrence of an X1X1X2X2/X1X2Y sex chromosome system in the dolphinfish (Coryphaenidae): An evolutionary perspective. Genet Mol Res. 2014; 13(2):2470–79. https://doi.org/10.4238/2014.April.3.19
https://doi.org/10.4238/2014.April.3.19... ), istiophorids with species with large distributions provide a valuable model on karyotype evolution in such ecosystem. However, as commonly found, considerable gaps occur with regard to their cytogenetic characteristics. All cytogenetic information for the Istiophoriformes Order comes down exclusively to the data presented here for I. platypterus. Despite this, it is feasible to compare the chromosome patterns of this species with phylogenetically close groups, such as the barracudas (Sphyraenidae), remoras (Echeneidae), archer fishes (Toxotidae), snooks (Centropomidae), jacks (Carangiformes), flatfishes (Pleuronectiformes), all included in a common clade, the Carangimorphariae one (Betancur-R. et al., 2013Betancur-R R, Broughton RE, Wiley EO, Carpenter K, López JA, Chenhong L, Holcroft NI, Arcila D, Sanciangco M, Cureton JC, Zhang F, Buser T, Campbell MA, Ballesteros JA, Roa-Varon A, Willis S, Borden AC, Rowley T, Reneau PC, Hough D, Lu G, Grande T, Arratia G, Orti G. The tree of life and a new classification of bony fishes. PLoS Curr. 2013; 1–54. https://doi.org/10.1371/currents.tol.53ba26640df0ccaee75bb165c8c26288
https://doi.org/10.1371/currents.tol.53b... ). It is noteworthy that a large amount of the Carangimorphariae species has 2n = 48 chromosomes (Arai, 2011Arai R. Fish Karyotypes: a check list. Tokyo: Springer; 2011.), but a remarkable diversity in their structural patterns can also be found. In fact, some groups of this clade have exclusively 2n = 48 acrocentric chromosomes, such as Centropomidae (Borges et al., 2019Borges AT, Cioffi MB, Bertollo LAC, Soares RX, Costa GWWF, Molina WF. Paracentric inversions differentiate the conservative karyotypes in two Centropomus species (Teleostei: Centropomidae). Cytogenet Genome Res. 2019; 157(4):239–48. https://doi.org/10.1159/000499748
https://doi.org/10.1159/000499748... ) and Toxotidae (Supiwong et al., 2017Supiwong W, Jiwyam W, Sreeputhorn K, Maneechot N, Bertollo LAC, Cioffi MB, Getlekha N, Tanomtong A. First report on classical and molecular cytogenetics of archerfish, Toxotes chatareus (Perciformes: Toxotidae). The Nucleus. 2017; 60(3):349–59. https://doi.org/10.1007/s13237-017-0216-5
https://doi.org/10.1007/s13237-017-0216-... ), while other ones like Sphyraenidae (Soares et al., 2017Soares RX, Cioffi MB, Bertollo LAC, Borges AT, Costa GWWF, Molina WF. Chromosomal evolution in large pelagic oceanic apex predators, the barracudas (Sphyraenidae, Percomorpha). Genet Mol Res. 2017; 16(2):gmr16029644. https://doi.org/10.4238/gmr16029644
https://doi.org/10.4238/gmr16029644... ), Carangidae (Accioly et al., 2012Accioly IV, Bertollo LA, Costa GW, Jacobina UP, Molina WF. Chromosomal population structuring in carangids (Perciformes) between the north-eastern and south-eastern coasts of Brazil. Afr J Mar Sci. 2012; 34(3):383–89. https://doi.org/10.2989/1814232X.2012.689671
https://doi.org/10.2989/1814232X.2012.68... ), Echeneidae (Rishi, 1973Rishi KK. A preliminary report on the karyotypes of eighteen marine fishes. Res Bull Panjab Univ. 1973; 24(3/4):161–62.; Vasiliev, 1980Vasiliev VP. Chromosome numbers of fishes. J Ichthyol. 1980; 20:387–422. ; Arkhipchuk, 1999Arkhipchuk VV. Chromosome database. Database of Dr. Victor Arkhipchuk; 1999.; Accioly, 2007Accioly IV. Levantamento cariotípico em espécies de peixes marinhos costeiros de fundo arenoso (Osteichthypes, Perciformes). [Master’s Thesis]. Natal: Universidade Federal do Rio Grande do Norte; 2007. Available from: https://repositorio.ufrn.br/jspui/handle/123456789/16774
https://repositorio.ufrn.br/jspui/handle... ) and especially Pleuronectiformes (Azevedo et al., 2005Azevedo MFC, Oliveira C, Pardo BG, Martinez P, Foresti F. Chromosome banding and 18S rDNA in situ hybridization analysis of seven species of the family Achiridae (Teleostei: Pleuronectiformes). Genetica. 2005; 125(2–3):125–32. https://doi.org/10.1007/s10709-005-4921-7
https://doi.org/10.1007/s10709-005-4921-... , 2007Azevedo MFC, Oliveira C, Pardo BG, Martinez P, Foresti F. Cytogenetic characterization of six species of flatfishes with comments to karyotype differentiation patterns in Pleuronectiformes (Teleostei). J Fish Biol. 2007; 70:1–15.), exhibit diversification in the karyotype number and structure.

In a broader phylogenetic context, the karyotype of I. platypterus (2m + 2st + 44a; NF = 52) and some features of the repetitive DNA organization in the chromosomes show similarities with species of the Sphyraenidae (Soares et al., 2017Soares RX, Cioffi MB, Bertollo LAC, Borges AT, Costa GWWF, Molina WF. Chromosomal evolution in large pelagic oceanic apex predators, the barracudas (Sphyraenidae, Percomorpha). Genet Mol Res. 2017; 16(2):gmr16029644. https://doi.org/10.4238/gmr16029644
https://doi.org/10.4238/gmr16029644... ), and Carangidae (Accioly et al., 2012Accioly IV, Bertollo LA, Costa GW, Jacobina UP, Molina WF. Chromosomal population structuring in carangids (Perciformes) between the north-eastern and south-eastern coasts of Brazil. Afr J Mar Sci. 2012; 34(3):383–89. https://doi.org/10.2989/1814232X.2012.689671
https://doi.org/10.2989/1814232X.2012.68... ) families, thus supporting a phylogenetic proximity among them. This is true for the independent distribution of the 18S rDNA/Ag-NOR and 5S rDNA sites on chromosomes, a common condition found in different tribes of Carangidae, and also frequent in teleosts (Gornung, 2013Gornung E. Twenty years of physical mapping of major ribosomal RNA genes across the teleosts: A review of research. Cytogenet Genome Res. 2013; 141(2–3):90–102. https://doi.org/10.1159/000354832
https://doi.org/10.1159/000354832... ). Besides, the terminal location of the 18S rDNA sequences in one of the largest chromosomes of the karyotype is a shared characteristic with several other Carangidae groups (Accioly et al., 2012Accioly IV, Bertollo LA, Costa GW, Jacobina UP, Molina WF. Chromosomal population structuring in carangids (Perciformes) between the north-eastern and south-eastern coasts of Brazil. Afr J Mar Sci. 2012; 34(3):383–89. https://doi.org/10.2989/1814232X.2012.689671
https://doi.org/10.2989/1814232X.2012.68... ; Jacobina et al., 2013Jacobina UP, Vicari MR, Martinez PA, Cioffi MB, Bertollo LAC, Molina WF. Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation. Helgol Mar Res. 2013; 67(3):499–506. https://doi.org/10.1007/s10152-012-0338-8
https://doi.org/10.1007/s10152-012-0338-... ), thus suggesting they hold extensive homeologous linkage groups as a plesiomorphic condition.

Megalops atlanticus, with habitats preferably coastal, and I. platypterus, which occurs in oceanic regions (Nakamura, 1985Nakamura I, editor. FAO species catalogue. Vol. 5. Billfishes of the world. An annotated and illustrated catalogue of marlins, sailfishes, spearfishes and swordfishes known to date. Rome: FAO Fisheries Synopsis; 1985. ), represent model species with high migratory capacity in the marine environment. These species make up groups of low diversity, formed by one genus and two species (Fricke et al., 2020Fricke R, Eschmeyer WN, Fong JD. Eschmeyer’s catalog of fishes: genera, species by family, subfamily [Internet]. San Francisco: California Academy of Science; 2020. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp
http://researcharchive.calacademy.org/re... ) exemplifying the small potential for diversification (Gaither et al., 2016Gaither MR, Bowen BW, Rocha LA, Briggs JC. Fishes that rule the world: circumtropical distributions revisited. Fish Fish. 2016; 17(3):664–79. https://doi.org/10.1111/faf.12136
https://doi.org/10.1111/faf.12136... ), and consequently processes of slow karyotype evolution of large migratory species (RXS, pers. obs.) in the marine environment.

Despite the great dependence on coastal environments, the tolerance to wide variations in salinity and oxygen (Adams et al., 2019Adams A, Guindon K, Horodysky A, MacDonald T, McBride R, Shenker J, Ward R. Megalops atlanticus [Internet]. The IUCN Red List of Threatened Species; 2019. Available from: https://www.iucnredlist.org/search?query=megalops%20atlanticus&searchType=species
https://www.iucnredlist.org/search?query... ), migratory habits (Ault et al., 2007Ault JS. Biology and management of the world tarpon and bonefish fisheries. Boca Ratón: CRC Press, 2007.) and the dispersive potential of larvae (McMillen-Jackson et al., 2005McMillen-Jackson AL, Bert TM, Cruz-Lopez H, Seyoum S, Orsoy T, Crabtree RE. Molecular genetic variation in tarpon (Megalops atlanticus Valenciennes) in the northern Atlantic Ocean. Mar Biol. 2005; 146(2):253–61. https://doi.org/10.1007/s00227-004-1432-5
https://doi.org/10.1007/s00227-004-1432-... ), provide favorable conditions for the genetic homogeneity of M. atlanticus (McMillen-Jackson et al., 2005McMillen-Jackson AL, Bert TM, Cruz-Lopez H, Seyoum S, Orsoy T, Crabtree RE. Molecular genetic variation in tarpon (Megalops atlanticus Valenciennes) in the northern Atlantic Ocean. Mar Biol. 2005; 146(2):253–61. https://doi.org/10.1007/s00227-004-1432-5
https://doi.org/10.1007/s00227-004-1432-... ). It seems that the set of these factors contributes to the karyotype sharing exhibited among populations of the Caribbean (Doucette, Fitzsimons, 1988Doucette AJ Jr, Fitzsimons JM. Karyology of elopiform and clupeiform fishes. Copeia. 1988; 1988(1):124–30. https://doi.org/10.2307/1445931
https://doi.org/10.2307/1445931... ), with those now presented for the Western Atlantic.

Megalops atlanticus shows microstructural cytogenetic traits also considered as plesiomorphic for several teleosts, such as reduced heterochromatic content, single Ag-NOR/18S rDNA sites (Galetti et al., 2000Galetti PM Jr, Aguilar CT, Molina WF. An overview of marine fish cytogenetics. Hydrobiologia. 2000; 420:55–62. https://doi.org/10.1007/978-94-017-2184-4_6
https://doi.org/10.1007/978-94-017-2184-... ), in non-syntenic arrangement with 5S rDNA sequences (Gornung, 2013Gornung E. Twenty years of physical mapping of major ribosomal RNA genes across the teleosts: A review of research. Cytogenet Genome Res. 2013; 141(2–3):90–102. https://doi.org/10.1159/000354832
https://doi.org/10.1159/000354832... ). On the other hand, its 2n value (2n = 50) differs from those found for the congeneric species, Megalops cyprinoides (Broussonet, 1782), distributed in the Indian and Pacific oceans (Carpenter, Niem, 2001Carpenter KE, Niem VH. FAO species identification guide for fishery purposes. The living marine resources of the Western Central Pacific. V. 5. Bony fishes part 3 (Menidae to Pomacentridae). Rome: FAO; 2001. ; Nelson et al., 2016Nelson JS, Grande TC, Wilson MVH. Fishes of the world. Fifth edition. New Jersey: John Wiley and Sons; 2016.). In fact, karyotypes with 2n = 46 (Rishi, Haobam, 1984Rishi KK, Haobam MS. Somatic chromosomes in a marine fish, Megalops cyprinoides (Broussonet) (Megalopidae: Elopiformes). Chromosome Inf Serv. 1984; 36:22–24.) and 2n = 52 chromosomes (Khuda-Bukhsh et al., 1995Khuda-Bukhsh AR, Rahman A, Chanda T, Nayak K, Khuda-Bukhsh. Diploid numbers and chromosome formulae of some 29 species of Indian teleosts (Pisces). Chromosome Inf Serv. 1995; 58:38–39.), were reported for M. cyprinoides from two different Indian locations, thus suggesting a more diversified evolutionary condition for this species.

Biogeographically, M. atlanticus and M. cyprinoides represent two lineages historically isolated by the closing of the Isthmus of Panama – 15–3.1 Mya (Coates, Obando, 1996Coates AG, Obando JA. The geologic evolution of the Central American isthmus. In: Jackson JBC, Budd AF, Coates AG, editors. Evolution and environments in tropical America. Chicago: University of Chicago Press; 1996. p.21–56.; Montes et al., 2015Montes C, Cardona A, Jaramillo C, Pardo A, Silva JC, Valencia V, Ayala C, Pérez-Angel LC, Rodriguez-Parra LA, Ramirez V, Niño H. Middle Miocene closure of the Central American seaway. Science. 2015; 348(6231):226–29. https://doi.org/10.1126/science.aaa2815
https://doi.org/10.1126/science.aaa2815... ), separating the Atlantic from the Pacific oceans, and by the Benguela current – 2 Mya. (Shannon, 1985Shannon LV. The Benguela ecosystem Part 1: Evolution of the Benguela, physical features and processes. Oceanogr Mar Biol. 1985; 23:105–82.; Marlow et al., 2000Marlow JR, Lange CB, Wefer G, Rosell-Mele A. Upwelling intensification as part of the Pliocene-Pleistocene climate transition. Science. 2000; 290(5500):2288–91. https://doi.org/10.1126/science.290.5500.2288
https://doi.org/10.1126/science.290.5500... ), segregating the Atlantic and Indian marine fauna (Henriques et al., 2016Henriques R, Potts WM, Sauer WH, Santos CV, Kruger J, Thomas JA, Shaw PW. Molecular genetics, life‐history and morphological variation in a coastal warm‐temperate sciaenid fish: evidence for an upwelling‐driven speciation event. J Biogeogr. 2016; 43(9):1820–31. https://doi.org/10.1111/jbi.12829
https://doi.org/10.1111/jbi.12829... ). However, the opening of the Panama Canal, approximately 100 years ago, provided a new migration route for M. atlanticus, from the Caribbean Sea to the Pacific Ocean, and its wide geographical expansion in the Pacific Ocean extending for ~ 2600 km, from Guatemala to the Colombia / Ecuador border (Castellanos-Galindo et al., 2019Castellanos-Galindo G, Robertson D, Pacheco-Chaves B, Angulo A, Chong-Montenegro C. Atlantic Tarpon in the Tropical Eastern Pacific 80 years after it first crossed the Panama Canal. Rev Fish Biol Fish. 2019; 29(2):401–16. https://dx.doi.org/ 10.1007/s11160-019-09565-z
https://dx.doi.org/... ). Given to its migratory potential, the biological invasion of M. atlanticus in the Pacific Ocean causes concern for biological conservation. Although no information on sympatry has already been reported, the physical contact could theoretically allow for a genetic introgression between the two Megalops species. However, although possible, cytogenetic data demonstrate the occurrence of a heterodiploid condition between them, thus potentiating possible post-zygotic barriers (Yakimowski, Rieseberg, 2014Yakimowski S, Rieseberg LH. The role of homoploid hybridization in evolution: A century of studies synthesizing genetics and ecology. Am J Bot. 2014; 101(8):1247–58. https://doi.org/10.3732/ajb.1400201
https://doi.org/10.3732/ajb.1400201... ), due to anomalous segregation of their chromosome sets.

Chromosomal diversification also occurs between Megalops (Megalopidae) and Elops (Elopidae) species, two sister clades of Elopiformes (Tab. 1), in which Elops saurus Linnaeus, 1766 shows 2n = 48; 6m/st + 42st/a; NF = 54 (Doucette, Fitzsimons, 1982Doucette AJ Jr, Fitzsimons JM. Karyology of the ladyfish Elops saurus. Jpn J Ichthyol. 1982; 29(2):223–26. https://doi.org/10.11369/jji1950.29.223
https://doi.org/10.11369/jji1950.29.223... ), while E. smithi McBride, Rocha, Ruiz-Carus & Bowen, 2010, has 2n = 50; 6m + 4st + 40a; NF = 60 (Sousa et al., 2019Sousa RPC, Sodré D, Costa RM, Vallinoto M, Oliveira EHC, Silva-Oliveira GC, Sampaio I, Guimarães-Costa A. Range distribution and contributions to taxonomy of Elops smithi (Elopiformes: Elopidae). An Acad Bras Cienc. 2019; 91(4):e20181240. https://doi.org/10.1590/0001-3765201920181240
https://doi.org/10.1590/0001-37652019201... ). Such differentiations in number and structure suggest that both fusion and fission events have played a role in the karyotype evolution of these Elopiformes families, although apparently associated with other complementary chromosome rearrangements paracentric inversions, translocations, duplications and deletions (Sousa et al., 2019Sousa RPC, Sodré D, Costa RM, Vallinoto M, Oliveira EHC, Silva-Oliveira GC, Sampaio I, Guimarães-Costa A. Range distribution and contributions to taxonomy of Elops smithi (Elopiformes: Elopidae). An Acad Bras Cienc. 2019; 91(4):e20181240. https://doi.org/10.1590/0001-3765201920181240
https://doi.org/10.1590/0001-37652019201... ). However, the reduced amount of cytogenetic information, coupled with conspicuous karyotypic differences, does not allow for accurate inferences on the evolutionary trends inside this order.

A significant portion of large pelagic marine fish is seriously threatened (Croll, Tershy, 2008Croll DA, Tershy BR. Pelagic predators. In: Jørgensen SE, Fath BD, editors. Encyclopedia of ecology. Amsterdam: Elsevier BV; 2008. p.2670–72.) and still lacks on their genetic aspects (Manel et al., 2020Manel S, Guerin P, Mouillot D, Blanchet S, Velez L, Albouy C, Pellissier L. Global determinants of freshwater and marine fish genetic diversity. Nat Commun. 2020; 11(692): 1–09. https://doi.org/10.1038/s41467-020-14409-7
https://doi.org/10.1038/s41467-020-14409... ), including their cytogenetic patterns (Soares et al., 2013Soares RX, Bertollo LAC, Costa GWWF, Molina WF. Karyotype stasis in four Atlantic Scombridae fishes: mapping of classic and dual-color FISH markers on chromosomes. Fish Sci. 2013; 79(2):177–83. https://doi.org/10.1007/s12562-013-0602-0
https://doi.org/10.1007/s12562-013-0602-... ). In this sense, the present results offer inedit and complimentary cytogenetic data about two important pelagic species, in order to elucidate their karyotype organization. The chromosomal aspects reflect independent evolutionary paths and instigate the extension of the data to other congeneric species and populations, thus providing valuable tools to clarify the evolutionary relationships still largely unknown to Elopiformes.

ACKNOWLEDGEMENTS

The authors are particularly grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq # 442664/2015-0), for the financial support and to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the doctoral fellowship granted to R.X. Soares. We also thank Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for the collection licenses (# 19135-1, # 131360-1 and # 27027-2) and José Garcia Júnior for help with taxonomic identifications of specimens.

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