Research Article |
Corresponding author: Valentina Milana ( valentina.milana@uniroma1.it ) Academic editor: Ekaterina Gornung
© 2017 Mauro Nirchio, Claudio Oliveira, Zoila R. Siccha-Ramirez, Viviani F. de Sene, Luciana Sola, Valentina Milana, Anna Rita Rossi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nirchio M, Oliveira C, Siccha-Ramirez ZR, de Sene VF, Sola L, Milana V, Rossi AR (2017) The Mugil curema species complex (Pisces, Mugilidae): a new karyotype for the Pacific white mullet mitochondrial lineage. Comparative Cytogenetics 11(2): 225-237. https://doi.org/10.3897/CompCytogen.v11i2.11579
|
Recent molecular phylogenetic analyses have shown that the Mugil curema Valenciennes, 1836 species complex includes M. incilis Hancock, 1830, M. thoburni (Jordan & Starks, 1896) and at least four “M. curema” mitochondrial lineages, considered as cryptic species. The cytogenetic data on some representatives of the species complex have shown a high cytogenetic diversity. This research reports the results of cytogenetic and molecular analyses of white mullet collected in Ecuador. The analyzed specimens were molecularly assigned to the Mugil sp. O, the putative cryptic species present in the Pacific Ocean and showed a 2n = 46 karyotype, which is composed of 2 metacentric and 44 subtelocentric/acrocentric chromosomes. This karyotype is different from the one described for M. incilis (2n = 48) and from those of the two western Atlantic lineages Mugil curema (2n = 28), and Mugil margaritae (2n = 24). Data suggest the need for a morphological analysis to assign a species name to this Pacific lineage.
Fish, Mugilidae , cytochrome oxidase subunit I, cytotaxonomy, molecular systematics
The family Mugilidae currently comprises 20 genera and 74 species (
In the last decade, molecular phylogenetic and phylogeographic analyses have revealed that the morphological features commonly used to identify species seem to be insufficient, both to describe the great diversity of species within Mugilidae and to infer the phylogenetic relationships among the species (
Cytotaxonomy has been proven to be a powerful tool in revealing different lineages/species within Mugilidae. For example, the presence of different cytogenetic features (
This paper reports the cytogenetic analysis of samples of the white mullet collected in Ecuador (Pacific Ocean); according to
Seventeen juvenile specimens (undetermined sex), morphologically classified as white mullet (Mugil curema) according to
Total genomic DNA was extracted from muscle according to Aljanabi and Martínez (1997).
GenBank accession number (A.N.), sampling areas and references of the Mugil sp. COI sequences used in phylogenetic analyses.
Individual (Voucher number) | A.N. | Sampling area | Reference |
---|---|---|---|
0102 (UTMACH0102) | KU504271 | Ecuador | Present paper |
0103 (UTMACH0103) | KU504271 | Ecuador | Present paper |
104 | KU504271 | Ecuador | Present paper |
105 | KU504271 | Ecuador | Present paper |
119 | KU504272 | Ecuador | Present paper |
120 | KU504271 | Ecuador | Present paper |
121 | KU504271 | Ecuador | Present paper |
122 | KU504271 | Ecuador | Present paper |
123 | KU504271 | Ecuador | Present paper |
124 | KU504271 | Ecuador | Present paper |
76104 (LBP 76104) | KU504271 | Ecuador | Present paper |
76105 (LBP 76105) | KU504271 | Ecuador | Present paper |
76107 (LBP 76107) | KU504271 | Ecuador | Present paper |
76129 (LBP 76129) | KU504271 | Ecuador | Present paper |
76130 (LBP 76129) | KU504271 | Ecuador | Present paper |
76131(LBP 76131) | KU504271 | Ecuador | Present paper |
76132 (LBP 76132) | KU504271 | Ecuador | Present paper |
415 | JQ060604 | El Salvador |
|
426 | JQ060600 | El Salvador |
|
429 | JQ060601 | El Salvador |
|
430 | JQ060602 | El Salvador |
|
432 | JQ060603 | El Salvador |
|
293 | JQ060573 | Western Panama |
|
294 | JQ060574 | Western Panama |
|
413 | JQ060592 | Perù |
|
420 | JQ060595 | Ecuador |
|
423 | JQ060597 | Western Mexico |
|
425 | JQ060599 | Western Mexico |
|
406 | JQ060588 | Western Mexico |
|
422 | JQ060596 | Western Mexico |
|
396 | JQ060580 | Togo |
|
397 | JQ060581 | Togo |
|
390 | JQ060575 | Senegal |
|
391 | JQ060576 | Senegal |
|
392 | JQ060577 | Senegal |
|
393 | JQ060578 | Benin |
|
394 | JQ060579 | Benin |
|
399 | JQ060582 | Venezuela |
|
400 | JQ060583 | Venezuela |
|
401 | JQ060584 | Venezuela |
|
403 | JQ060585 | Venezuela |
|
414 | JQ060593 | Venezuela |
|
408 | JQ060590 | Brazil |
|
411 | JQ060591 | Guadeloupe |
|
419 | JQ060594 | Belize |
|
404 | JQ060586 | Eastern USA |
|
407 | JQ060589 | Eastern Mexico |
|
417 | JQ060605 | Uruguay |
|
418 | JQ060606 | Uruguay |
|
405 | JQ060587 | Honduras |
|
6435 | JX559534 | Galapagos Is. |
|
6445 | JX559535 | Galapagos Is. |
|
299 | JQ060609 | French Guyana |
|
302 | JQ060608 | French Guyana |
|
780 | HQ285928 | Venezuela |
|
782 | HQ285929 | Venezuela |
|
785 | HQ285930 | Venezuela |
|
786 | HQ285931 | Venezuela |
|
788 | HQ285927 | Venezuela |
|
A 546 base-pair (bp) fragment of the mitochondrial cytochrome oxidase subunit I gene (COI) was amplified by PCR using primers FishF1 and FishR2 (
Tree reconstructions were conducted using neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian inference (BI) analyses. The NJ and ML analyses (1000 bootstrap replicates) were performed using MEGA5 and PhyML v3.0 (
Cell suspensions were obtained from the cephalic kidney, following the procedure reported by
Fluorescence in situ hybridization (FISH) was accomplished according to
The mitotic figures were photographed using an Olympus BX61 photomicroscope equipped with the appropriate selective filters for FISH and with a DP70 digital camera. The images were digitally edited with Adobe Photoshop CS6 Extended.
Similarity searching of the obtained COI sequences in the GenBank database, using the BLAST function, provided 99.6–100% similarity with those obtained by
Neighbor-joining tree based on COI sequences. At each node, bootstrap values > 70% (NJ and ML) and posterior probabilities > 0.9 (BI) are shown. Stars indicate sequences obtained in this study; the remaining sequences are from
In all the individuals, the karyotype is composed of 46 chromosomes, 2 metacentric and 44 subtelocentric/acrocentric, with a fundamental number (FN) of 48 (Fig.
Conventional Giemsa-stained karyotype of the Pacific white mullet. In the inset, the acrocentric chromosome pair n. 15 sequentially Ag-stained; m: metacentric chromosomes; st/a: subtelocentric/acrocentric chromosomes. Scale bar: 10 μm.
C-banding (Fig.
Dual FISH (Fig.
Mapping of the (TTAGGG)n telomeric repeats showed the presence of positive signals on both telomeres of all chromosomes. No additional, interstitial or centromeric (TTAGGG)n positive signals were detected (Fig.
Somatic C-banded metaphases of the Pacific white mullets. Arrowheads indicate chromosome pair number one. Arrows indicate terminal heterochromatic blocks on chromosome pair 15. Scale bar: 10 μm.
Somatic metaphases of the Pacific white mullet showing positive sites after FISH (a) with 18S rDNA (arrows) and 5S rDNA (asterisks) probes and (c) with telomeric repeats. Arrowheads indicate chromosome pair number one. In (b) enlargement of selected samples of chromosome pairs 15 and 20, after DAPI staining and FISH with rDNA probes, showing 18S (above) and 5S (below) positive sites, respectively. Scale bar: 10 μm.
Most of the approximately 20 species of Mugilidae cytogenetically investigated so far (see
The specimens analyzed in this study, molecularly assigned to the Pacific Mugil sp. O (
The Mugil sp. O described in this study shows the presence of NORs on a single chromosome pair, as well as minor ribosomal genes carried by a single chromosome pair. These features are common to most of the mugilids, including all the Mugil species (
Further analyses are required to draw a comprehensive picture of the chromosomal evolution within the M. curema species complex. Data on the karyotype of M. thoburniand of the white mullet Mugil sp. M from the East Atlantic (
Data, although preliminary, strongly suggest that each of the “Mugil curema” lineages within the species complex has its own karyotype. This evidence, and the absence of intermediate karyotypes in the geographic area where different lineages/cytotypes are in sympatry, supports Durand and Borsa’s hypothesis (2015) that chromosomal differences probably prevent interbreeding and indicate the actual reproductive isolation of cryptic species. In this context, a morphological analysis is now needed to assign a species name to the here-examined Pacific Mugil sp. O and possibly to the remaining allopatric East Atlantic Mugil sp. M.
Finally, it needs to be verified whether the karyotype observed in the specimens from Ecuador is also shared by specimens belonging to the Mugil sp. O. from other sampling sites along the American Pacific coast. In particular, a karyotypic analysis is needed for the western Mexican coast, because in the phylogenetic trees two individuals from this region are grouped in a subcluster that is highly divergent from the one that includes the remaining Pacific specimens.
This work was supported by Centro de Investigación, Universidad Técnica de Machala -UTMACH-, Ecuador (to MN); Sapienza University, Rome, Italy – Progetto di Università 2016 (to LS, VM, AR); Fundação de Amparo à Pesquisa do Estado de São Paulo -FAPESP-, Brazil and Conselho Nacional de DesenvolvimentoCientífico e Tecnológico -CNPq-, Brazil (to CO).
The authors wish to thank Dr Omar Rogerio Sánchez Romero (UTMACH) and Dr Ricardo Britzke (UTMACH) for their assistance in sampling.