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Corresponding author: Cristiane Xerez Barroso ( cristianexb@gmail.com ) Corresponding author: Tito Monteiro da Cruz Lotufo ( tmlotufo@usp.br ) Academic editor: Thierry Backeljau
© 2020 Cristiane Xerez Barroso, João Eduardo Pereira de Freitas, Helena Matthews-Cascon, Luis Ernesto Arruda Bezerra, Tito Monteiro da Cruz Lotufo.
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:
Barroso CX, Pereira de Freitas JE, Matthews-Cascon H, Arruda Bezerra LE, da Cruz Lotufo TM (2020) Molecular evidences confirm the taxonomic separation of two sympatric congeneric species (Mollusca, Gastropoda, Neritidae, Neritina). ZooKeys 904: 117-130. https://doi.org/10.3897/zookeys.904.46790
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A reliable taxonomy, together with more accurate knowledge of the geographical distribution of species, is a fundamental element for the study of biodiversity. Multiple studies on the gastropod family Neritidae record three species of the genus Neritina in the Brazilian Province: Neritina zebra (Bruguière, 1792), Neritina virginea (Linnaeus, 1758), and Neritina meleagris Lamarck, 1822. While N. zebra has a well-established taxonomic status and geographical distribution, the same cannot be said regarding its congeners. A widely cited reference for the group in Brazil considers N. meleagris a junior synonym of N. virginea. Using a molecular approach (phylogenetic, species delimitation, and statistical parsimony network analyses), based on two mitochondrial markers (COI and 16S), this study investigated if N. virginea and N. meleagris are distinct species. The molecular results confirmed the existence of two strongly supported distinct taxonomic entities in the Brazilian Province, which is consistent with the morphological descriptions previously proposed for N. virginea and N. meleagris. These species occur in sympatry in the intertidal sandstone formations of Northeastern Brazil. Despite the great variation in the colour patterns of the shells, the present study reinforced previous observations that allowed the differentiation of these two species based on these patterns. It also emphasized the importance of the separation of these two clades in future studies, especially those conducted in the Brazilian Province, since these species may cohabit.
Brazilian Province, Caribbean Province, geographic distribution, neritids, species delimitation
Molluscs from the gastropod family Neritidae are the most diverse members of Neritimorpha (
Several studies report three species of the genus Neritina on the Brazilian coast: Neritina zebra (Bruguière, 1792), Neritina virginea (Linnaeus, 1758), and Neritina meleagris Lamarck, 1822 (e.g.,
Since a reliable taxonomy, together with a more accurate knowledge about the geographical distribution of species, is fundamental to the study of biodiversity (
We collected specimens from Barra Grande beach (Piauí State) (2°54.125'S, 41°24.573'W) and Camocim beach (Ceará State) (02°51.778'S, 41°51.57'W), both located in Northeastern Brazil, and preserved them in 95% ethanol. We identified the species using the literature (
List of species included in the phylogenetic, species delimitation, and statistical parsimony network analyses. The voucher number of species collected in NE Brazil and the accession numbers of the sequences obtained in the present study and from GenBank are indicated. The numbers in parentheses next to the GenBank accession number correspond to each of the specimens analysed in the present study (see Figs
Family/Species | Locality | Voucher No. | Accession No. | Referencesb | |
---|---|---|---|---|---|
COI | 16Sa | ||||
Outgroups | |||||
Phenacolepadidae | |||||
Thalassonerita naticoidea (A. H. Clarke, 1989) | Gulf of Mexico | – | FJ977768 | FJ977721 | 1 |
Neritidae | |||||
Nerita fulgurans Gmelin, 1791 | Colombia | – | JX646664 | JX646655 | 2 |
Nerita tessellata Gmelin, 1791 | Colombia | – | JX646663 | JX646654 | 2 |
Nerita peloronta Linnaeus, 1758 | Colombia | – | JX646665 | JX646656 | 2 |
Nerita versicolor Gmelin, 1791 | Colombia | – | JX646666 | JX646658 | 2 |
Neritina piratica Russell, 1940 | Colombia | – | JX646669 | JX646660 | 2 |
Neritina usnea (Röding, 1798) | Colombia | – | JX646670 | JX646661 | 2 |
Neritina punctulata Lamarck, 1816 | Colombia | – | JX646667 | JX646657 | 2 |
Ingroup | |||||
Neritina meleagris Lamarck, 1822 | Colombia | – | JX646671 | JX646662 | 2 |
Camocim, Ceará, Brazil | CMPHRM 6408B | MK628548 (1) | MK628556 (1) | 3 | |
Barra Grande, Piauí, Brazil | CMPHRM 6409B | MK628549 (2), MK628550, (3) MK628551 (4) | MK628557 (2), MK628558 (3), MK628559 (4) | 3 | |
Neritina virginea (Linnaeus, 1758) | Colombia | – | JX646668 | JX646659 | 2 |
Panama | – | JF810998 to JF811004c and FJ977766 | – | 1, 4 | |
Puerto Rico | – | FJ348932 to FJ348975 | – | 5 | |
Camocim, Ceará, Brazil | CMPHRM 6410B | MK628552 (1) | MK628560 (1) | 3 | |
Barra Grande, Piauí, Brazil | CMPHRM 6411B |
MK628553 (2), MK628554 (3), MK628555 (4) | MK628561 (2), MK628562 (3), MK628563 (4) | 3 |
We extracted whole genomic DNA from the foot muscle of specimens, using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). The quality and integrity of the DNA obtained were evaluated in a micro-volume spectrophotometer. Amplification of double-stranded fragments from the cytochrome c oxidase I (COI) and 16S mitochondrial genes was achieved by polymerase chain reaction (PCR) using newly developed neritid-specific custom primers for the 16S gene [(16SNer_F 5’ACTACTCCGCCTGTTTATCAAA3’) and (16SNer_R 5’GGGCTTAAACCTAATGCACTT3’)] and modified versions of
The forward and reverse sequences for each gene fragment were edited using Geneious v. 7.1 (Biomatters). The concatenated alignments of COI and 16S were conducted using the MAFFT program with the G-INS-I algorithm (
For the species delimitation analyses, we initially constructed a distance matrix based on the Kimura 2-parameter (K2P) model, using the COI sequences, in the MEGA 6.0.6 software (
A statistical parsimony network analysis was conducted with COI sequences (347 bp), using the TCS algorithm (
The shells and opercula of the specimens of N. virginea and N. meleagris submitted to molecular analyses were observed and photographed under a stereomicroscope. A scanning electron microscope (SEM) was used to view their radulae (two females of N. virginea and two males and one female of N. meleagris) in the Analytical Facility of
The results of our molecular analyses (phylogeny, species delimitation, and statistical parsimony network) confirmed the existence of two strongly supported clades living in sympatry in the intertidal beachrocks of Northeastern Brazil (Brazilian Province). The Bayesian and maximum likelihood trees showed the same topology, with the formation of four clades within Neritina: Group I (Neritina piratica + Neritina usnea), Group II (Neritina meleagris, collected in NE Brazil), Group III (Neritina virginea, collected in NE Brazil and Colombia, + “Neritina meleagris”, from Colombia), and Group IV (Neritina punctulata) (Fig.
Molecular phylogenetic hypothesis (Bayesian tree) of some species of Neritidae of the Western Atlantic. The Bayesian tree was based on partial mitochondrial COI and 16S sequences. The Neritina meleagris and Neritina virginea clades (ingroup) are highlighted. The other taxa were used as outgroup. Numbers on and below the main branches represent the posterior Bayesian probabilities (BP) (>0.90) and bootstrap values for maximum likelihood (ML) (>70%), respectively. Specimens with the number “1” are from Camocim beach (Ceará State, NE Brazil) and those with numbers “2”, “3”, and “4” are from Barra Grande beach (Piauí State, NE Brazil). The numbered specimens of N. virginea (1, 2, 3, and 4) and N. meleagris (1, 2, 3, and 4) are the same specimens shown in Figure
Species delimitation results from the Bayesian concatenated and Neighbor-Joining trees. These analyses were performed with the Species Delimitation plugin for Geneious.
Bayesian concatenated tree (COI + 16S) | ||||||
Species | Monophyly | Intra Dist | Inter Dist-closest | Intra/Inter | P ID(Strict) | P ID(Liberal) |
Neritina virginea | yes | 0.006 | 0.073 | 0.08 | 0.88 (0.76, 1.0) | 0.97 (0.87, 1.0) |
Neritina meleagris | yes | 0.004 | 0.088 | 0.04 | 0.84 (0.70, 0.98) | 0.97 (0.86, 1.0) |
Neighbor-Joining K2P COI tree | ||||||
Species | Monophyly | Intra dist | Inter Dist-closest | Intra/Inter | P ID(Strict) | P ID(Liberal) |
Neritina virginea | yes | 0.008 | 0.084 | 0.09 | 0.87 (0.75, 1.00) | 0.97 (0.87, 1.0) |
Neritina meleagris | yes | 0.002 | 0.096 | 0.02 | 0.86 (0.72, 1.00) | 0.98 (0.87, 1.0) |
Although the distinction between clades showed high support values, the phylogenetic relationship between them could not be recovered. As we did not have access to the specimens, it was not possible to check the shell colour patterns of the Neritina meleagris from Colombia (obtained from GenBank) that was included in the same clade as Neritina virginea. Thus, we suspect that an error may have occurred at the time of submission of the sequences to GenBank, since, in the study of
Despite the geographical distance, all N. virginea sequences from Brazil and the Caribbean were very similar, with all haplotypes grouped within a few mutational steps (Fig.
Statistical parsimony network analysis (TCS algorithm) based on 64 partial mitochondrial COI sequences (347 bp). This analysis included specimens of Neritina meleagris and Neritina virginea from the Caribbean and Brazilian Provinces. Size of the circle is proportional to frequency of the haplotype and colours inside the circles designate geographical locations to which the samples belong. Black circles correspond to hypothetical haplotypes. The number of mutational steps is indicated by dashes on branches. We highlighted the 36 mutational steps that separate the two species haplotypes.
Figure
Colour patterns of shells, opercula, and radulae of the Neritina virginea and Neritina meleagris analysed. The red arrows highlight the differences between the leading edges of colour patterns of both species: N. virginea has the leading edges outlined in heavy black, while N. meleagris has the leading edge outlined in white or black and white. A Neritina virginea_1 B Neritina virginea_2 C Neritina virginea_3 D Neritina virginea_4 E Neritina meleagris_1 F Neritina meleagris_2 G Neritina meleagris_3 H Neritina meleagris_4 I ventral view of shell of Neritina virginea J operculum (outer and inner views) of Neritina virginea K ventral view of shell of Neritina meleagris L operculum (outer and inner views) of Neritina meleagris M radula of Neritina virginea (SEM), with rachidian tooth enlarged in the upper left quadrant N radula of Neritina meleagris (SEM), with rachidian tooth enlarged in the upper left quadrant. Abbreviations: l1 first lateral tooth, l4 fourth lateral tooth, m marginal teeth, r rachidian tooth. The specimens with the number “1” are from Camocim beach (Ceará State, NE Brazil) and those with numbers “2”, “3”, and “4” are from Barra Grande beach (Piauí State, NE Brazil). The numbered specimens of N. virginea (1, 2, 3, and 4) and N. meleagris (1, 2, 3, and 4) are the same specimens used in the phylogenetic analysis of Figure
Besides the shell colour patterns, N. virginea and N. meleagris differ from each other in subtle ways. The inner lips of the shells of the two species are denticulated. However, in N. virginea there are several small denticles interspersed by two larger teeth, while in N. meleagris the teeth are larger, more prominent in the central region, and less numerous when compared to N. virginea (Fig.
Our molecular data show that N. virginea and N. meleagris are two distinct species, thus confirming the N. meleagris record for the Brazilian coast. In summary, our results, along with the already well-established record of Neritina zebra (
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) provided a postdoctoral fellowship to C.X. Barroso (PNPD process number 88882.306440/2018-01) and J.E.P Freitas (PNPD process number 88887.466769/2019-00). H. Matthews-Cascon and T.M.C. Lotufo are research fellows from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was partially funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), projects # 2015/17177-6 and 2017/11948-6. The authors would like to thank the Central Analítica-
Alignments used to construct the phylogenetic trees and statistical parsimony network analysis
Radulae of the Neritina meleagris (A) and Neritina virginea (B) analysed