Restricted accessResearch articleFirst published online 2025-04
Small Fishes,Big Issues: Species Delimitation in Hemigrammus Marginatus,Gill,1958 (Acestrorhamphidae: Pristellinae) from Brazilian Coastal Basins Based on Integrative Genetics
The small characins represent a systematic puzzle in the Neotropical ichthyofauna as a result of independent miniaturization processes, adaptive convergence and lack of diagnostic characters for several genera. In order to diminish the taxonomic uncertainties and the evolutionary pathways in Hemigrammus, we carried out an integrative genetic analysis in the putatively widespread Hemigrammus marginatus Ellis, 1958 by combining cytogenetic and molecular data based on the mitochondrial Cytochrome C Oxidase subunit I (COI). Specimens of H. marginatus from the type locality in Itapicuru River basin and other two populations from coastal rivers in northeastern Brazil were analyzed and compared with the available data from other regions in South America. Conspicuous macro and microkaryotypic differences were detected between the samples from northeastern and southern Brazil (Upper Paraná River basin). Likewise, the DNA barcoding and species delimitation analyses recovered distinct Molecular Operational Taxonomical Units within H. marginatus. Therefore, the population from the type locality should be referred to as H. marginatus stricto sensu, representing a restricted characin taxon from coastal drainages (including the São Francisco River basin) along northeastern Brazil, while other populations of this small characin fish need to be taxonomically revised and managed as unique lineages.
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References
1.
LimaFC, Urbano‐BonillaA, Prada‐PedrerosS. A new Hemigrammus from the upper Río Vaupés, Colombia (Characiformes: Characidae), with a discussion on the presence of an enlarged urogenital papilla in the family. J Fish Biol, 2020; 96(4):868–876; doi: 10.1111/jfb.14267
Toledo-PizaM, BaenaEG, DagostaFCP, et al.Checklist of the species of the Order Characiformes (Teleostei: Ostariophysi). Neotrop Ichthyol, 2024; 22:e230086; doi: 10.1590/1982-0224-2023-0086
4.
PrangG. An industry analysis of the freshwater ornamental fishery with particular reference to the supply of Brazilian freshwater ornamentals to the UK market. Uakari, 2008; 3(1):7–52; doi: 10.31420/uakari.v3i1.18
5.
Tribuzy-NetoIA, BeltrãoH, BenzakenZS, et al.Analysis of the ornamental fish exports from the Amazon State, Brazil. Bip, 2020; 46(4):e554; doi: 10.20950/1678-2305.2020.46.4.554
6.
MirandeJM. Phylogeny of the family Characidae (Teleostei: Characiformes): From characters to taxonomy. Neotrop Ichthyol, 2010; 8(3):385–568; doi: 10.1590/S1679-62252010000300001
7.
MattoxGM, BritzR, Toledo-PizaM, et al.Cyanogaster noctivaga, a remarkable new genus and species of miniature fish from the Rio Negro, Amazon basin (Ostariophysi: Characidae). Ichthyol Explor Freshw, 2013; 23(4):297–318.
8.
MirandeJM. Morphology, molecules and the phylogeny of Characidae (Teleostei, Characiformes). Cladistics, 2019; 35(3):282–300; doi: 10.1111/cla.12345
9.
MotaTF, FabrinT, DepraGC, et al.Molecular characterization of Moenkhausia (Pisces: Characiformes) populations with different lateral line developmental levels. An Acad Bras Cienc, 2018; 90(3):2815–2825; doi: 10.1590/0001-3765201820170493
10.
LimaFCT, MalabarbaLR, BuckupPA, et al.Characidae, genera incertae sedis. 2003. In: (ReisRE, KullanderSO, FerrarisCJJR eds.), Check List of the Freshwater Fishes of South and Central America. EDIPUCRS, Porto Alegre, Brazil, pp. 106–169.
11.
MeloBF, OtaRP, BenineRC, et al.Phylogenomics of Characidae, a hyper-diverse Neotropical freshwater fish lineage, with a phylogenetic classification including four families (Teleostei: Characiformes). Zoological Journal of the Linnean Society, 2024; 202(1):zlae101.
12.
ArcilaD, OrtíG, VariR, et al.Genome-wide interrogation advances resolution of recalcitrant groups in the tree of life. Nat Ecol Evol, 2017; 1(2):20; doi: 10.1038/s41559-016-0020
13.
TeránGE, BenitezMF, MirandeJM. Opening the Trojan horse: Phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc, 2020; 190(4):1217–1234; doi: 10.1093/zoolinnean/zlaa019
14.
PanteE, SchoelinckC, PuillandreN. From integrative taxonomy to species description: One step beyond. Syst Biol, 2015; 64(1):152–160; doi: 10.1093/sysbio/syu083
15.
GavazzoniM, PavanelliCS, GraçaWJ, et al.Species delimitation in Psalidodon fasciatus (Cuvier, 1819) complex (Teleostei: Characidae) from three hydrographic basins. Bio J Linn Soc, 2023; 138(1):51–67; doi: 10.1093/biolinnean/blac139
16.
Mari-RibeiroIP, ScorsimB, RanucciL, et al.Integrating genetic and cytogenetic data: A diversity study of Astyanax and Psalidodon (Characidae) species from the paraná river basin. Zebrafish, 2024; 21(4):300–309; doi: 10.1089/zeb.2024.0134
17.
NirchioM, PaimFG, MilanaV, et al.Identification of a new mullet species complex based on an integrative molecular and cytogenetic investigation of Mugil hospes (Mugilidae: Mugiliformes). Front Genet, 2018; 9:17–19; doi: 10.3389/fgene.2018.00017
18.
AbrantesYG, RamosTPA, BentoDDM, et al.Molecular delimitation of the seasonal killifishes of the Hypsolebias antenori species group (Cyprinodontiformes, Rivulidae), with description of two new species from the Caatinga coastal basins, northeastern Brazil. Zootaxa, 2023; 5389(5):545–562; doi: 10.11646/zootaxa.5389.5.2
19.
SantosAGD, SoaresSC, NakayamaCM, et al.Chromosomal characterization of three species of Serrasalmini (Serrasalmidae: Characiformes). Genet Mol Biol, 2023; 46(4):e20230088; doi: 10.1590/1678-4685-GMB-2023-0088
20.
ReisRE. Check list of the freshwater fishes of South and Central America. Edipucrs, 2003.
21.
Vanegas-RíosJA, BritzkeR, MirandeJM. Geographic variation of Moenkhausia bonita (Characiformes: Characidae) in the rio de la Plata basin, with distributional comments on M. intermedia. Neotrop Ichthyol, 2019; 17(1):e170123; doi: 10.1590/1982-0224-20170123
22.
Portela-CastroALB, Júlio–JúniorHF. Karyotype relationships among species of the subfamily Tetragonopterinae (Pisces, Characidae): Cytotaxonomic and evolution aspects. Cytologia, 2002; 67:329–336; doi: 10.1508/cytologia.67.329
23.
FernandesMB, Bitencourt J deA, GaldinoJH, et al.“Split Happens”: A rare case of chromosomal mosaicism in characin from an impacted river basin. Braz Arch Biol Technol, 2024; 67(4240145); doi: 10.1590/1678-4324-2024240145
24.
ManigliaTC, PrioliAJ, BignottoTS, et al.Nucleotide diversity of Hemigrammus cf. marginatus (Characiformes, Characidae) in the upper Paraná River floodplain. Acta Sci Biol Sci, 2012; 34(3):303–309; doi: 10.4025/actascibiolsci.v34i3.6669
25.
OtaRP, LimaFC, PavanelliCS. A new species of Hemigrammus Gill, 1858 (Characiformes: Characidae) from the central and western Amazon and rio Paraná-Paraguai basins. Zootaxa, 2015; 3948(2):218–232; doi: 10.11646/zootaxa.3948.2.4
26.
OtaRR, DepráGDC, GraçaWJD, et al.Peixes da planície de inundação do alto rio Paraná e áreas adjacentes: Revised, annotated and updated. Neotrop Ichthyol, 2018; 16(2):e170094; doi: 10.1590/1982-0224-20170094
27.
LeeMR, ElderFFB. Yeast stimulation of bone marrow mitosis for cytogenetic investigations. Cytogenet Cell Genet, 1980; 26(1):36–40; doi: 10.1159/000131419
28.
BlessingJJ, MarshallJC, BalcombeSR. Humane killing of fishes for scientific research: A comparison of two methods. J Fish Biol, 2010; 76(10):2571–2577; doi: 10.1111/j.1095-8649.2010.02633.x
29.
NettoM, PaulsE, de Mello AffonsoPRA. A standard protocol for obtaining fish chromosomes under post-mortem conditions. Micron, 2007; 38(3):214–217; doi: 10.1016/j.micron.2006.07.019
30.
LevanA, FredgaK, SandbergAA. Nomenclature for centromeric position on chromosomes. 1964.
SumnerAT. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res, 1972; 75(1):304–306; doi: 10.1016/0014-4827(72)90558-7
33.
HowellWMT, BlackDA. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: A 1-step method. Experientia, 1980; 36(8):1014–1015; doi: 10.1007/BF01953855
34.
SchmidM. Chromosome banding in amphibia. IV. Differentiation of GC-and AT-rich chromosome regions in Anura. Chromosoma, 1980; 77(1):83–103; doi: 10.1007/BF00292043
35.
HatanakaT, GalettiPM-.Jr, Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes, Prochilodontidae). Genetica, 2004; 122(3):239–244; doi: 10.1007/s10709-004-2039-y
36.
MartinsC, GalettiPM-.Jr, Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes). Chromosome Res, 1999; 7(5):363–367.
37.
FolmerRH, FolkersPJ, KaanA, et al.Secondary structure of the single‐stranded DNA binding protein encoded by filamentous phage Pf3 as determined by NMR. Eur J Biochem, 1994; 224(2):663–676; doi: 10.1111/j.1432-1033.1994.00663.x
38.
PaithankarKR, PrasadK. Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res, 1991; 19(6):1346.
39.
ThompsonJD, GibsonT, HigginsDG. Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics, 2002;Chapter 2:Unit 2.3; doi: 10.1002/0471250953.bi0203s00
KimuraM. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol, 1980; 16(2):111–120; doi: 10.1007/BF01731581
42.
FelsensteinJ. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 1985; 39(4):783–791; doi: 10.1111/j.1558-5646.1985.tb00420.x
43.
HebertPDN, StoeckleMY, ZemlakTS, et al.Identification of birds through DNA barcodes. PLoS Biol, 2004; 2(10):e312; doi: 10.1371/journal.pbio.0020312
44.
TamuraK, StecherG, PetersonD, et al.MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol, 2013; 30(12):2725–2729; doi: 10.1093/molbev/mst197
45.
TrifinopoulosJ, NguyenLT, Von HaeselerA, et al.W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res, 2016; 44(W1):W232–W235; doi: 10.1093/nar/gkw256
46.
HoangDT, ChernomorO, VonH, et al.UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol, 2018; 35(2):518–522; doi: 10.1093/molbev/msx281
TanabeAS. Kakusan: A computer program to automate the selection of a nucleotide substitution model and the configuration of a mixed model on multilocus data. Mol Ecol Notes, 2007; 7(6):962–964; doi: 10.1111/j.1471-8286.2007.01807.x
49.
RambautA, SuchardMA, XieD, et al.MCMC trace analysis tool. Version v1. 6.0. 2013.
50.
RambautA. FigTree. v1. 4.2: Tree drawing tool in computer program and documentation distributed by the author. 2014.
51.
PuillandreN, BrouilletS, AchazG. ASAP: Assemble species by automatic partitioning. Mol Ecol Resour, 2021; 21(2):609–620; doi: 10.1111/1755-0998.13281
52.
ZhangJ, KapliP, PavlidisP, et al.A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 2013; 29(22):2869–2876; doi: 10.1093/bioinformatics/btt499
53.
FujisawaT, BarracloughTG. Delimiting species using single-locus data and the generalized mixed yule coalescent approach: A revised method and evaluation on simulated data sets. Syst Biol, 2013; 62(5):707–724; doi: 10.1093/sysbio/syt033
54.
DrummondAJ, SuchardMA, XieD, et al.Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol, 2012; 29(8):1969–1973; doi: 10.1093/molbev/mss075
55.
ThomazAT, ArcilaD, OrtíG, et al.Molecular phylogeny of the subfamily stevardiinae gill, 1858 (Characiformes: Characidae): Classification and the evolution of reproductive traits. BMC Evol Biol, 2015; 15:146–125; doi: 10.1186/s12862-015-0403-4
56.
BarretoSB, SilvaAT, Batalha‐FilhoH, et al.Integrative approach reveals a new species of Nematocharax (Teleostei: Characidae). J Fish Biol, 2018; 93(6):1151–1162; doi: 10.1111/jfb.13834
57.
PazzaR, DergamJA, KavalcoKF. Trends in karyotype evolution in Astyanax (Teleostei, Characiformes, Characidae): Insights from molecular data. Front Genet, 2018; 9:131; doi: 10.3389/fgene.2018.00131
58.
SouzaTBD, FerreiraDC, SilvaHPD, et al.DNA barcoding of Pyrrhulina australis (Characiformes: Lebiasinidae) reveals unexpected cryptic diversity in the group. Neotrop Ichthyol, 2023; 21(4):e230037; doi: 10.1590/1982-0224-2023-0037
59.
Moreira-FilhoO, BertolloLAC. Astyanax scabripinnis (Pisces, Characidae): A species complex. Rev Bras Genet, 1991; 4:331–357.
60.
MedradoAS, RibeiroMS, AffonsoP, et al.Cytogenetic divergence in two sympatric fish species of the genus Astyanax Baird and Girard, 1854 (Characiformes, Characidae) from northeastern Brazil. Genet Mol Biol, 2012; 35(4):797–801; doi: 10.1590/S1415-47572012005000075
61.
CursinoMS, SalvianoMB, AbrilVV, et al.The role of chromosome variation in the speciation of the red brocket deer complex: The study of reproductive isolation in females. BMC Evol Biol, 2014; 14(1):40–13; doi: 10.1186/1471-2148-14-40
62.
SalvianoMB, CursinoMS, ZanettiEDS, et al.Intraspecific chromosome polymorphisms can lead to reproductive isolation and speciation: An example in red brocket deer (Mazama americana). Biol Reprod, 2017; 96(6):1279–1287; doi: 10.1093/biolre/iox041
63.
NascimentoVD, CoelhoKA, NogarotoV, et al.Do multiple karyomorphs and population genetics of freshwater darter characines (Apareiodon affinis) indicate chromosomal speciation? Zool Anz, 2018; 272:93–103; doi: 10.1016/j.jcz.2017.12.006
64.
Almeida-ToledoLFD, BigoniAP, BernardinoG, et al.Karyotype and NOR conservatism with heterochromatin reorganization in Neotropical Bryconids. Caryologia, 1996; 49(1):35–43; doi: 10.1080/00087114.1996.10797348
65.
FernandesCA, Martins-SantosIC. Cytogenetic characterization of two populations of Astyanax scabripinnis (Pisces, Characiformes) of the Ivaí Basin PR Brazil. Cytologia, 2003; 68(3):289–293; doi: 10.1508/cytologia.68.289
66.
PeresWAM, BertolloLAC, Moreira FilhoO. Physical mapping of the 18S and 5S ribosomal genes in nine Characidae species (Teleostei, Characiformes). Genet Mol Biol, 2008; 31(1 suppl):222–226; doi: 10.1590/S1415-47572008000200009
67.
TonelloS, BlancoDR, CerqueiraFJ, et al.High rDNA polymorphisms in Astyanax lacustris (Characiformes: Characidae): New insights about the cryptic diversity in A. bimaculatus species complex with emphasis on the Paraná River basin. Neotrop Ichthyol, 2022; 20(2):e210147; doi: 10.1590/1982-0224-2021-0147
68.
CarottiE, CarducciF, CanapaA, et al.Transposable elements and teleost migratory behaviour. Int J Mol Sci, 2021; 22(2):602; doi: 10.3390/ijms22020602
69.
PiscorD, PaizLM, BaumgärtnerL, et al.Chromosomal mapping of repetitive sequences in Hyphessobrycon eques (Characiformes, Characidae): A special case of the spreading of 5S rDNA clusters in a genome. Genetica, 2020; 148(1):25–32; doi: 10.1007/s10709-020-00086-3
70.
Almeida-ToledoLFD, Ozouf-CostazC, ForestiF, et al.Conservation of the 5S-bearing chromosome pair and co-localization with major rDNA clusters in five species of Astyanax (Pisces, Characidae). Cytogenet Genome Res, 2002; 97(3–4):229–233; doi: 10.1159/000066609
71.
GavazzoniM, PavanelliCS, GraçaWJ, et al.Detection of natural hybridization and delimitation of two closely related operational taxonomic units of the Astyanax fasciatus (Teleostei: Characidae) complex through integrative approaches. Bio J Linn Soc, 2020; 129(3):687–700; doi: 10.1093/biolinnean/blz199
72.
CasciottaJ, AlmirónA, SánchezS, et al.The genus Gymnotus (Gymnotiformes: Gymnotidae) in Argentina. How bad taxonomy results in poor regulations and no conservation. J Appl Ichthyol, 2013; 29(1):208–212; doi: 10.1111/jai.12028
73.
NúñezR, GenaroJA, MillerJY, et al.DNA barcodes of the greater antillean butterflies (Lepidoptera: Papilionoidea) suggest a richer, more isolated fauna and higher endemism. Biol J Linn Soc, 2023; 140(3):447–458; doi: 10.1093/biolinnean/blad015
74.
RatnasinghamS, HebertPD. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS One, 2013; 8(7):e66213; doi: 10.1371/journal.pone.0066213
75.
OliveiraRCD, TencattLFC, DepráGDC, et al.A new species of Aequidens (Cichliformes: Cichlidae) from the Rio Paraguai basin, Brazil. Neotrop Ichthyol, 2024; 22(2):e230106; doi: 10.1590/1982-0224-2023-0106
76.
PlanquetteP, KeithP, Le BailPY. Atlas des poissons d’eau douce de Guyane (tome 1). Collection Du Patrimoine Naturel, 1996; 22:429p.
77.
WardRD, HannerR, HebertPDN. The campaign to DNA Barcode all fishes, FISHBOL. J Fish Biol, 2009; 74(2):329–356; doi: 10.1111/j.1095-8649.2008.02080.x
78.
CassemiroFA, AlbertJS, AntonelliA, et al.Landscape dynamics and diversification of the megadiverse South American freshwater fish fauna. Proc Natl Acad Sci U S A, 2023; 120(2):e2211974120; doi: 10.1073/pnas.2211974120
79.
AnjosMDS, QueirozLJ, PenidoIDS, et al.A taxonomically complex catfish group from an underrepresented geographic area: Systematics and species limits in Hypostomus Laceperde, 1803 (Siluriformes, Loricariidae) from Eastern South America. J Zool Syst Evol Res, 2021; 59(8):1994–2009; doi: 10.1111/jzs.12572
80.
LimaRC, de LimaSR, RochaMS, et al.Identification of fish specimens of the Tocantins River, Brazil, using DNA barcoding. J Fish Biol, 2024; 104(6):1924–1939; doi: 10.1111/jfb.15721
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