PLOS ONE

RESEARCH ARTICLE

DNA barcoding for the assessment of marine

and coastal fish diversity from the Coast of
Mozambique
Valdemiro Muhala ID1,2*, Aurycéia Guimarães-Costa1*, Isadola Eusébio Macate1,3, Luan
Pinto Rabelo ID1, Adam Rick Bessa-Silva1, Luciana Watanabe1, Gisele Damasceno dos
Santos1, Luı́sa Sambora4, Marcelo Vallinoto1, Iracilda Sampaio1
1 Laboratório de Evolução, Universidade Federal do Pará, Alameda Leandro Ribeiro, Aldeia, Bragança,
Pará, Brazil, 2 Divisão de Agricultura, Instituto Superior Politécnico de Gaza, Chókwè, Mozambique,
3 Departamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, Ilheus, BA,
a1111111111 Brazil, 4 Departamento de Produção Agrária, Escola Superior de Desenvolvimento Rural, Universidade
a1111111111 Eduardo Mondlane, Vilankulos, Moçambique
a1111111111
a1111111111 * valdemiro.muhala@ispg.ac.mz (VM); auryceia@yahoo.com.br (AG-C)
a1111111111

Abstract
The ichthyological provinces of Mozambique are understudied hotspots of global fish diver-
OPEN ACCESS
sity. In this study, we applied DNA barcoding to identify the composition of the fish fauna
Citation: Muhala V, Guimarães-Costa A, Macate IE, from the coast of Mozambique. A total of 143 species belonging to 104 genera, 59 families,
Rabelo LP, Bessa-Silva AR, Watanabe L, et al.
and 30 orders were identified. The overall K2P distance of the COI sequences within spe-
(2024) DNA barcoding for the assessment of
marine and coastal fish diversity from the Coast of cies ranged from 0.00% to 1.51%, while interspecific distances ranged from 3.64% to
Mozambique. PLoS ONE 19(2): e0293345. https:// 24.49%. Moreover, the study revealed 15 threatened species according to the IUCN Red
doi.org/10.1371/journal.pone.0293345
List of Threatened Species, with elasmobranchs being the most represented group. Addi-
Editor: Feng Zhang, Nanjing Agricultural University, tionally, the study also uncovered four new species that were not previously recorded in this
CHINA
geographic area, including Boleophthalmus dussumieri, Maculabatis gerrardi, Hippocam-
Received: May 1, 2023 pus kelloggi, and Lethrinus miniatus. This study represents the first instance of utilizing
Accepted: October 10, 2023 molecular references to explore the fish fauna along the Mozambican coast. Our results
indicate that DNA barcoding is a dependable technique for the identification and delineation
Published: February 6, 2024
of fish species in the waters of Mozambique. The DNA barcoding library established in this
Peer Review History: PLOS recognizes the
research will be an invaluable asset for advancing the understanding of fish diversity and
benefits of transparency in the peer review
process; therefore, we enable the publication of guiding future conservation initiatives.
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0293345

Copyright: © 2024 Muhala et al. This is an open

access article distributed under the terms of the 1. Introduction
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
The Mozambique Channel comprises an arm of the Indian Ocean located between the South-
reproduction in any medium, provided the original east African countries of Madagascar and Mozambique, stretching 1,600 km along the coast
author and source are credited. [1]. It is recognized as an important diversity hotspot due to a variety of coastal ecosystems
Data Availability Statement: All relevant data are
that distinguish it from other Western Indian Ocean (WIO) biogeographic provinces [2, 3].
within the paper and its Supporting Information These systems provide a range of habitats for both animals and plants, making the region
files. abundant in aquatic species [4–6]. The diverse habitats include a large rocky coastline in the

PLOS ONE | https://doi.org/10.1371/journal.pone.0293345 February 6, 2024 1 / 19

PLOS ONE DNA barcoding of the fish species from Mozambique

Funding: This research was financed by Conselho north with extensive coral reefs, a wider platform in the central part with river outflows and
Nacional de Desenvolvimento Cientı́fico e weaker ocean currents that form sandy banks, estuaries, and deltas, and a southern coast char-
Tecnológico (CNPq) through a research project
acterized by wide beaches, bays, and seagrass beds with numerous endemic species [7–9].
407536/2021-3, 309916/2021-6 and the APC was
funded by Pro-Reitoria de Pesquisa e Pós- Investigations into fish species diversity on the west coast of Africa remain in their early
Graduação of the Universidade Federal do Pará. stages, largely due to a scarcity of both traditional taxonomists and those employing molecular
The funders had no role in the study design, data tools for taxonomic inference within various fish groups [10]. Accurate classification and iden-
collection and analysis, decision to publish, or tification of fish species are important not only for taxonomists but also for various types of
preparation of the manuscript.
research, including fisheries, natural resource surveys, forensic studies, the discovery of cryptic
Competing interests: The authors have declared species, and the identification of species not previously known and their conservation status
that no competing interests exist. within a specific biogeographic region [11–15]. The impacts of lacking knowledge on ichthyo-
fauna may have even greater repercussions because research focused on fishing activity can be
seriously compromised [16].
Historically, surveys of fish fauna off the coast of Mozambique have primarily relied on tra-
ditional methods of identification, which use morphological features and meristic counts to
distinguish and define fish species [17, 18]. However, due to subtle morphological differences,
complex evolutionary patterns in certain groups, and the existence of cryptic species, these
methodologies may not always overcome the challenges of accurately identifying species,
resulting in an underestimation of the true diversity of ichthyofauna [19–21].
These concerns are particularly significant for international organizations focused on biodi-
versity conservation, which have traditionally relied on the rigid concept of species. This con-
cept has recently been widely challenged by the more comprehensive and integrated
approaches adopted by modern taxonomy for classifying living organisms. [22–24]. Although
there is consensus that species represent the smallest independent evolutionary unit, their rec-
ognition remains a subject of debate [25, 26], potentially impacting conservation efforts. Fur-
thermore, morphological identification of fish can prove challenging task if not conducted by
specialists, leading to inaccurate identifications [27, 28]. Additionally, previous genetic studies
have shown that molecular identification may not align with identifications based on morpho-
logical characteristics in some instances [29, 30]. This highlights the importance of carrying
out a systematic and comprehensive inventory of ichthyofauna using standardized DNA-
based methods.
In response to the limitations of classical taxonomy, DNA barcoding has emerged as a
widely adopted research method for accurately identifying species [31–33]. This technique,
which relies on the diversity of the Cytochrome C Oxidase I (COI) gene region within mito-
chondrial DNA, serves as an alternative to traditional taxonomic approaches. DNA barcoding
has proven crucial in enhancing the accuracy of species identification and is especially valuable
for differentiating between diverse and poorly understood flora and fauna that necessitate spe-
cies delimitation [31–37].
Large-scale investigations of ichthyofaunas across various ecosystems have demonstrated
that utilizing the cytochrome oxidase I gene captures a significant proportion of known diver-
sity [39–42]. However, it has also helped to reveal previously unknown diversity [43–46]. This
method is successful due to the availability of reliable DNA barcode reference libraries in the
BOLD Systems (Barcode of Life Data Systems) and GenBank [38]. New DNA barcodes can be
analysed with available data to check for potential taxonomic conflicts and improve taxonomic
resolution [46].
Considering the remarkable potential of DNA barcoding to identify fish species and consid-
ering the still unknown fish assemblage in the coast of Mozambique, the aim of this study is to
employ this approach to establish a DNA barcoding reference database for the composition of
the fish fauna off the coast of Mozambique. This will have significant implications for our

PLOS ONE | https://doi.org/10.1371/journal.pone.0293345 February 6, 2024 2 / 19

PLOS ONE DNA barcoding of the fish species from Mozambique

understanding of diversity and could contribute to future conservation efforts for marine spe-
cies in Mozambique.

2. Material and methods

2.1 Ethical statement
The samples analyzed in this study were obtained in accordance with the requirements of envi-
ronmental legislation, approved by the Ministry of Fisheries (license no. 178MP11227B/20)
and the Instituto Superior Politécnico de Gaza—ISPG (license no. 409/GDG/ISPG/090), for
the collection and transportation of samples. The authorization also includes the use of anes-
thetic (5% Lidocaine) on the skin to minimize animal suffering, following the recommenda-
tions of the Herpetological Animal Care and Use Committee (HACC) of the American Society
of Ichthyologists and Herpetologists.

2.2 Sampling area and specimen collection

The Mozambican coast is in Eastern Africa (Fig 1), spanning over 2.700 km of coastline along
the Indian Ocean, second largest African coast [47, 48]. The coastal region showcases a range
of diverse landscapes, including sandy beaches, dunes, forests, mangrove swamps, seagrass,
and coral reefs. Three distinct ecological zones can be found along the coast, including the

Fig 1. Map of sample origin areas. All the yellow dots correspond to the total areas collected along the coast. The points marked with A to E Are Specific
illustrations of the different sampling areas.
https://doi.org/10.1371/journal.pone.0293345.g001

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PLOS ONE DNA barcoding of the fish species from Mozambique

Delagoa Basin in the south, the highly productive and rich Sofala Bank in the central region,
and the São Lazaro Bank further north [48, 49].
Sampling was conducted randomly across all coastal provinces of the country from Decem-
ber 2019 to April 2022, considering both dry and rainy seasons (Fig 1). Different fishing gear
(trawls, surface, and bottom gillnets) commonly used in the country’s artisanal fisheries were
used for specimen collection. The collected samples were stored in ice boxes and transported
to the sorting site, where a substantial portion of the specimens was photographed, and tissues
were removed. The specimens were identified to the lowest taxonomic level based on the
nomenclature used by the vendors and using species identification keys specific to South-east-
ern Africa [50, 51].
Approximately 50 mg of muscle tissue was collected from each specimen and preserved in
1.5 ml Eppendorf-type microtubes filled with 96% ethanol. The samples were stored in a
freezer set at -20˚C. After the initial collection of tissues, the specimens were transported to the
Evolution Laboratory at the Institute of Coastal Studies of the Federal University of Para,
located in Bragança, Pará, Brazil, for the purpose of generating genetic data.

2.3 Genomic DNA extraction, PCR amplification, and DNA sequencing

The extraction of DNA was carried out using the Wizard Genomic DNA Purification Kit (Pro-
mega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. Total geno-
mic DNA was extracted from muscle or fin tissue of fish specimens. To analyse intra- and
inter-variability, a partial COI gene from mitochondrial DNA was used as the molecular
marker. The 50 end of the COI gene was amplified using two pairs of universal primers for fish,
FishF1/FishR1 and FishF2/FishR2 [41], resulting in a maximum of 622 bp fragment. The 50
region of the COI gene was amplified by Polymerase Chain Reaction (PCR) in a 25 μL reaction
mixture that consisted of 4 μL of dNTPs (1.25 mM), 2.5 μL of 10X buffer solution, 1 μL of
MgCl2 (25 mM), 0.25 μL of each primer (200 ng/μL), 1–1.5 μL of genomic DNA (100 ng/μL),
1 U of Taq DNA polymerase (5 U/μL), and purified water. The amplification process was as
follows: an initial denaturation step at 94˚C for 3 minutes, followed by 35 cycles of denatur-
ation at 95˚C for 1 minute, annealing at 50˚C-58˚C for 45 seconds, and extension at 72˚C for
45 seconds. The process was concluded with a final extension at 72˚C for 5 minutes. The posi-
tive reactions were then sequenced using an ABI 3500 automatic sequencer (Applied
Biosystems).

2.4 Molecular data analysis

The final sequences were aligned using ClustalW [52], implemented in GENEIOUS 9.0.5
(https://www.geneious.com), and then translated into amino acids to check for potential stop
codons using the MEGA 11 software. All the generated sequences were submitted to BOLD
database. The high-quality sequences were compared using the National Canter for Biotech-
nology Information (NCBI) BLAST search engine and the Barcode of Life Data Systems
(BOLD) database. The Eschmeyer’s Catalog of Fishes repositor was used to compile the taxo-
nomic information of the species, and the classifications of the families and orders followed
the study by [53]. After analysing the sequences, parameters such as the presence of barcode
gap, sequence length, GC content, and intra/inter genetic distances within and between fami-
lies, genera, species, and minimum distances to the nearest neighbour were calculated using
tools in the BOLD Systems platform using Kimura’s two-parameter model and bootstrap of
1000 replicates [54]. Phylogenetic trees were constructed in MEGA 11 using the NJ method
based on the Kimura 2-Parameter model with 1000 bootstrap pseudoreplicates [54, 55] and
the online tool Interactive Tree of Life (iTOL) [56] were used to visualize and edit the tree.

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PLOS ONE DNA barcoding of the fish species from Mozambique

Analysis of Barcode Index Numbers (BIN) [57] was conducted for species delimitation,
based on uncorrected p-distances, which provide a single BIN for each Operational Taxo-
nomic Unit (OTU) obtained from the COI sequences. The analysis of BINs reveals the maxi-
mum intra-specific distance and the minimum inter-specific distance that overlap and allow
for species identification. The aligned sequences, primer pairs, trace files, taxonomic informa-
tion, and collection data were deposited in the Barcode of Life Data Systems under the project
MOZFH [57]. All Barcode Index Numbers (BIN) and GenBank accession number are
included in the Table 1.

3. Results
3.1 DNA barcoding results
In this study, we generated a final alignment with 419 sequences of the barcode region of COI
gene with a length of 622 bp. Of these, we only used sequences with a length of 400 up to 622
bp for genetic analysis. The data correspond to 143 species, 104 genera, 59 families, 30 orders,
and 4 unidentified taxa (Table 1 and S1 Table). All samples were assessed morphologically and
then subjected to DNA barcoding evaluation to confirm their identification. There were two
classes (Teleost and Elasmobranch) of fish samples, including 392 and 27 respectively, which
were acquired from local artisanal fishermen at landing sites and fish markets. For more details
of the NJ tree clustering and composition see (S1 Fig).
The species composition was represented by Carangidae (16 species), Sparidae (10 species),
Serranidae (9 species), Haemulidae and Lutjanidae (7 species each), Scombridae and Mugili-
dae (6 species each). The remaining families were composed of fewer than 5 species (Table 1).
The sequences generated in this study were all submitted to the BOLD Systems and the species
list details are provided in (S1 Table).
The final analysed database had no insertion, deletion, or stop codon, indicating that it
accurately represents the mitochondrial COI fragment. Sequences with poor quality DNA
were excluded from the analysis. The nucleotide content showed the following average fre-
quency: G = 17.77%, C = 28.74%, A = 23.88% and T = 29.61% with an average GC content of
46.51%.
The NJ tree included 143 distinct OTUs with high bootstrap support of 70/99 (Fig 2). The
specimens belonging to the same species exhibited clustering based on both morphological

Table 1. List of the 143 fish species, from Mozambican coast waters, which were DNA barcoded.
Order Family Species Authorship Status Conservation BIN Accession
(IUCN)
Carcharhiniformes Carcharhinidae Carcharhinus leucas (Müller & Henle, 1839) VU BOLD:AAA6060 OR284674
Loxodon macrorhinus (Müller & Henle, 1839) NT BOLD:AAA6751 OR284672
Sphyrnidae Sphyrna lewini (Griffith & Smith, 1834) CR BOLD:AAA2402 OR284675
Myliobatiformes Dasyatidae Himantura uarnak (Gmelin, 1789) EN BOLD:AAB7830 OR284662
Maculabatis gerrardi (Gray, 1851) EN BOLD:AAA8673 OR284658
Pastinachus ater (Macleay, 1883) VU BOLD:AAC1400 OR284664
Myliobatidae Rhinoptera sp. (Cuvier, 1829) - BOLD:AAC7667 OR284652
Plesiobatidae Plesiobatis daviesi (Wallace, 1967) LC BOLD:AAF2578 OR284653
Acanthuriformes Acanthuridae Acanthurus mata (Cuvier, 1829) LC BOLD:AAE4025 OR284615
Naso brevirostris (Cuvier, 1829) LC BOLD:AAC1635 OR284418
Anguilliformes Muraenesocidae Muraenesox bagio (Hamilton, 1822) LC BOLD:ACK7558 OR284694
Beloniformes Hemiramphidae Hemiramphus far (Forsskål, 1775) - BOLD:AAC0565 OR284496
(Continued )

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PLOS ONE DNA barcoding of the fish species from Mozambique

Table 1. (Continued)

Order Family Species Authorship Status Conservation BIN Accession

(IUCN)
Carangiformes Carangidae Alepes djedaba (Forsskål, 1775) LC BOLD:AAB5772 OR284371
Caranx ignobilis (Forsskål, 1775) LC BOLD:AAB0587 OR284379
Caranx papuensis (Alleyne & Macleay, 1877) LC BOLD:ACF4541 OR284388
Caranx sexfasciatus (Quoy & Gaimard, 1825) LC BOLD:AAB0584 OR284383
Decapterus macarellus (Cuvier, 1833) LC BOLD:AAB6796 OR284404
Gnathanodon speciosus (Forsskål, 1775) LC BOLD:AAB7462 OR284373
Megalaspis cordyla (Linnaeus, 1758) LC BOLD:AAB5271 OR284374
Parastromateus niger (Bloch, 1795) LC BOLD:AAB3884 OR284400
Platycaranx chrysophrys (Kimura, Takeuchi & Yadome, - BOLD:AAB2977 OR284393
2022)
Platycaranx malabaricus (Kimura, Takeuchi & Yadome, - BOLD:AAB3474 OR284391
2022)
Scomberoides (Lacepède, 1801) LC BOLD:AAB6417 OR284447
commersonnianus
Scomberoides tol (Cuvier, 1832) LC BOLD:AAB6418 OR284444
Scyris indica (Cuvier & Valenciennes, 1833) - BOLD:AAB7826 OR272032
Selar crumenophthalmus (Bloch, 1793) LC BOLD:AAB0870 OR284408
Turrum coeruleopinnatum (Whitley, 1932) - BOLD:AAD2297 OR284399
Turrum fulvoguttatum (Whitley, 1932) - BOLD:AAC2745 OR284396
Coryphaenidae Coryphaena hippurus (Linnaeus, 1758) LC BOLD:AAA5277 OR284696
Rachycentridae Rachycentron canadum (Linnaeus, 1766) LC BOLD:AAB2939 OR284680
Centrarchiformes Terapontidae Terapon jarbua (Forsskål, 1775) LC BOLD:AAA9351 OR284426
Cichliformes Cichlidae Oreochromis mossambicus (Peters, 1852) VU BOLD:AAA8511 OR284549
Oreochromis niloticus (Linnaeus, 1758) LC BOLD:ADI0792 OR284554
Oreochromis aureus (Steindachner, 1864) - BOLD:AAC9904 OR284556
Tilapia sp. (Smith, 1840) - BOLD:ABZ6465 OR284560
Chaetodontiformes Leiognathidae Leiognathus equulus (Forsskål, 1775) LC BOLD:AAB2487 OR284620
Clupeiformes Chirocentridae Chirocentrus dorab (Forsskål, 1775) LC BOLD:AAC2273 OR284640
Clupeidae Hilsa kelee (Cuvier, 1829) LC BOLD:AAC0856 OR284533
Sardinella gibbosa (Bleeker, 1849) LC BOLD:AAB7263 OR284538
Sardinella jussieu (Lacepède, 1803) DD BOLD:AEW0759 OR284540
Pristigasteridae Pellona ditchela (Valenciennes, 1847) LC BOLD:AAD4543 OR284631
Engraulidae Thryssa vitrirostris (Gilchrist & Thompson, 1908) LC BOLD:AAE3536 OR284635
Cypriniformes Cyprinidae Hypophthalmichthys molitrix (Valenciennes, 1844) NT BOLD:AAF6633 OR284617
Ephippiformes Ephippidae Platax teira (Forsskål, 1775) LC BOLD:AAC5812 OR284532
Tripterodon orbis (Playfair, 1867) - BOLD:AAD7693 OR284531
Gadiformes Merlucciidae Merluccius paradoxus (Franca, 1960) - BOLD:AAC4936 OR284610
Gerreiformes Gerreidae Gerres longirostris (Lacepède, 1801) LC BOLD:AAE6359 OR284518
Gerres oyena (Forsskål, 1775) LC BOLD:AAC1291 OR284515
Gerres methueni (Regan, 1920) - BOLD:AAF8786 OR284523
Gobiiformes Gobiidae Boleophthalmus dussumieri (Valenciennes, 1837) LC BOLD:ABZ8637 OR284562
Periophthalmus (Valenciennes, 1837) LC BOLD:AAF8789 OR284567
argentilineatus
Istiophoriformes Istiophoridae Istiompax indica (Cuvier, 1832) DD BOLD:ACF5077 OR284572
Labriformes Labridae Cheilinus trilobatus (Lacepède, 1801) LC BOLD:AAB4188 OR284596
Cheilio inermis (Forsskål, 1775) LC BOLD:AAA6101 OR284546
Lobotiformes Lobotidae Lobotes surinamensis (Bloch, 1790) LC BOLD:AAC1878 OR284651
(Continued )

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PLOS ONE DNA barcoding of the fish species from Mozambique

Table 1. (Continued)

Order Family Species Authorship Status Conservation BIN Accession

(IUCN)
Lutjaniformes Haemulidae Diagramma picta (Thunberg, 1792) - BOLD:AAD4477 OR284508
Plectorhinchus gaterinus (Forsskål, 1775) LC BOLD:AAH9156 OR284504
Plectorhinchus gibbosus (Lacepède, 1802) LC BOLD:ACK7983 OR284512
Plectorhinchus (Cuvier, 1830) - BOLD:AAC4020 OR284499
flavomaculatus
Pomadasys kaakan (Cuvier, 1830) LC BOLD:AAD6593 OR284369
Pomadasys maculatus (Bloch, 1793) LC BOLD:AAB3687 OR284367
Lutjanidae Aprion virescens (Valenciennes, 1830) LC BOLD:AAB8692 OR284415
Lutjanus argentimaculatus (Forsskål, 1775) LC BOLD:AAB2440 OR284348
Lutjanus bengalensis (Bloch, 1790) LC BOLD:AAB7901 OR284352
Lutjanus erythropterus (Bloch, 1790) LC BOLD:AAB3276 OR284351
Lutjanus fulviflamma (Forsskål, 1775) LC BOLD:ADF5681 OR284341
Lutjanus lutjanus (Bloch, 1790) LC BOLD:AAA8168 OR284345
Pristipomoides filamentosus (Valenciennes, 1830) LC BOLD:AAB4096 OR284363

Mugiliformes Mugilidae Chelon dumerili (Steindachner, 1870) DD BOLD:AAI6001 OR284442

Crenimugil buchanani (Bleeker, 1853) - BOLD:AAE3561 OR284437
Moolgarda sp1 (Moolgarda) - BOLD:AAC4146 OR284440
Moolgarda sp2 (Moolgarda) - BOLD:AAG6597 -
Planiliza melinoptera (Valenciennes, 1836) - BOLD:AAD7507 -
Planiliza macrolepis (Smith, 1846) LC BOLD:AAC0698 OR284431
Pempheriformes Pempheridae Pempheris connelli (Randall & Victor, 2015) - BOLD:AAC6084 OR284691
Perciformes Caesionidae Caesio caerulaurea (Lacepède, 1801) LC BOLD:AAB4823 OR284336
Caesio xanthonota (Bleeker, 1853) LC BOLD:AAE8330 OR284339
Caesio sp. (Lacepède, 1801) - BOLD:AAB4822 OR284335
Pterocaesio tile (Cuvier, 1830) LC BOLD:AAB4821 OR284331
Dinopercidae Dinoperca petersi (Day, 1875) - BOLD:AAD0006 OR284361
Menidae Mene maculata (Bloch & Schneider, 1801) - BOLD:AAB4862 OR284410
Polynemidae Polydactylus malagasyensis (Motomura & Iwatsuki, 2001) - BOLD:AAB7311 OR284649
Scaridae Chlorurus atrilunula (Randall & Bruce, 1983) LC BOLD:AAE8961 OR284527
Chlorurus sordidus (Forsskål, 1775) LC BOLD:AAB6670 OR284526
Scarus psittacus (Forsskål, 1775) LC BOLD:AAB8901 OR284525
Scarus quoyi (Valenciennes, 1840) LC BOLD:AAD0849 OR284524
Serranidae Variola louti (Forsskål, 1775) LC BOLD:AAC5719 OR284591
Cephalopholis argus (Schneider, 1801) LC BOLD:AAC4474 OR284493
Cephalopholis boenak (Bloch, 1790) LC BOLD:AAB3684 OR284494
Epinephelus rivulatus (Valenciennes, 1830) LC BOLD:ACZ9919 OR284480
Epinephelus coioides (Hamilton, 1822) LC BOLD:AAB8391 OR284486
Epinephelus areolatus (Forsskål, 1775) LC BOLD:AAA9822 OR284490
Epinephelus macrospilos (Bloch, 1793) - BOLD:AAE1882 OR284479
Epinephelus malabaricus (Bloch & Schneider, 1801) LC BOLD:AAB8389 OR284481
Epinephelus merra (Bloch, 1793) LC BOLD:AAB8387 OR284492
Siganidae Siganus stellatus (Forsskål, 1775) LC BOLD:AAB2341 OR284356
Siganus sutor (Valenciennes, 1835) LC BOLD:AAB6556 OR284353
Sillaginidae Sillago sihama (Forsskål, 1775) LC BOLD:AAA7600 OR284541
Platycephalidae Cociella heemstrai (Knapp, 1996) LC BOLD:AAD7871 OR284627
(Continued )

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PLOS ONE DNA barcoding of the fish species from Mozambique

Table 1. (Continued)

Order Family Species Authorship Status Conservation BIN Accession

(IUCN)
Pleuronectiformes Citharidae Citharoides macrolepis (Gilchrist, 1904) DD BOLD:AAB1336 OR284684
Cynoglossidae Cynoglossus attenuatus (Gilchrist, 1904) LC - OR272033
Paralichthyidae Pseudorhombus arsius (Hamilton, 1822) LC BOLD:ACX7474 OR284688
Psettodidae Psettodes erumei (Bloch & Schneider, 1801) DD BOLD:AAB6707 OR284573
Priacanthiformes Priacanthidae Priacanthus hamrur (Forsskål, 1775) LC BOLD:AAB1643 OR284626
Priacanthus arenatus (Cuvier, 1829) LC BOLD:AEV2303 OR284623
Rhinopristiformes Rhinidae Rhynchobatus djiddensis (Forsskål, 1775) CR BOLD:AAC4065 OR284670
Rhynchobatus australiae (Whitley, 1939) CR BOLD:AAC4063 OR284668
Rhinobatidae Acroteriobatus leucospilus (Giltay, 1928) - BOLD:AAI0445 OR284671
Scombriformes Nomeidae Cubiceps whiteleggii (Waite, 1894) - BOLD:AAB5183 OR270958
Scombridae Acanthocybium solandri (Cuvier, 1832) LC BOLD:AEZ9639 OR270954
Euthynnus affinis (Cantor, 1849) LC BOLD:AAB2798 OR270940
Katsuwonus pelamis (Linnaeus, 1758) LC BOLD:AAA5421 OR270939
Rastrelliger kanagurta (Cuvier, 1816) DD BOLD:AAA9666 OR284326
Scomberomorus plurilineatus (Fourmanoir, 1966) DD BOLD:AAE9334 OR270952
Scomberomorus commerson (Lacepède, 1800) NT BOLD:AAB5513 OR270947
Thunnus albacares (Bonnaterre, 1788) LC BOLD:AAA7352 OR270942
Trichiuridae Trichiurus sp. (Linnaeus, 1758) - BOLD:AAB0164 OR284528
Ariommatidae Ariomma indicum (Day, 1871) - BOLD:AAB5940 OR270959
Scorpaeniformes Platycephalidae Platycephalus indicus (Linnaeus, 1758) DD BOLD:AAB2371 OR284576
Siluriformes Pangasiidae Pangasius djambal (Bleeker, 1846) LC BOLD:AAE3237 OR284586
Ariidae Plicofollis polystaphylodon (Bleeker, 1846) - BOLD:AEW1876 OR284581
Spariformes Lethrinidae Lethrinus nebulosus (Forsskål, 1775) LC BOLD:ABY6363 OR284420
Lethrinus miniatus (Forster, 1801) LC BOLD:AAC8078 OR284423
Lethrinus borbonicus (Valenciennes, 1830) LC BOLD:AAB0511 OR284419
Sparidae Acanthopagrus vagus (Peters, 1852) VU BOLD:AAD8720 OR284460
Acanthopagrus berda (Forsskål, 1775) LC BOLD:AAC4120 OR284456
Chrysoblephus puniceus (Gilchrist & Thompson, 1908) LC BOLD:AAD8479 OR284471
Crenidens crenidens (Forsskål, 1775) LC BOLD:AAE4408 OR284468
Dentex macrophthalmus (Bloch, 1791) LC BOLD:AAL1497 OR284477
Lithognathus mormyrus (Linnaeus, 1758) LC BOLD:AAD3990 OR284465
Pagellus natalensis (Steindachner, 1903) LC BOLD:AAF8829 OR284474
Rhabdosargus thorpei (Smith, 1979) LC BOLD:AAE3481 OR284449
Rhabdosargus sarba (Forsskål, 1775) LC BOLD:AAB4844 OR284454
Nemipteridae Scolopsis bimaculata (Rüppell, 1828) LC BOLD:AAD6249 OR284619
Syngnathiformes Mullidae Parupeneus indicus (Shaw, 1803) LC BOLD:AAB0334 OR284513
Upeneus vittatus (Forsskål, 1775) LC BOLD:ABZ7416 OR284514
Fistulariidae Fistularia petimba (Lacepède, 1803) LC BOLD:AAB8439 OR284590
Syngnathidae Hippocampus kelloggi (Jordan & Snyder, 1901) VU BOLD:AAF0667 OR284677
Incertae sedis Sciaenidae Argyrosomus japonicus (Temminck & Schlegel, 1843) EN BOLD:AAC2818 OR284602
Argyrosomus thorpei (Smith, 1977) EN BOLD:AAD8716 OR284597
Otolithes ruber (Bloch & Schneider, 1801) LC BOLD:AAB6481 OR284605

Sphyraenidae Sphyraena jello (Cuvier, 1829) - BOLD:AAA6102 OR284644

Sphyraena chrysotaenia (Klunzinger, 1884) - BOLD:AAD0400 OR284690
https://doi.org/10.1371/journal.pone.0293345.t001

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PLOS ONE DNA barcoding of the fish species from Mozambique

Fig 2. NJ tree illustrating the relationships among Mozambican fish species based on DNA barcode sequences. The NJ
tree includes both teleost and elasmobranch fish species, constructed using K2P distances from 400 pb barcode sequences
(COI). Bootstrap support values are represented by dots at the nodes. Images credits are given to i-iii, vii, xii—(Pollerspöck,
J. & Straube, N. 2022, www.shark-references.com; iv - https://link.springer.com/article/10.1007/s12526-021-01208-6; v—
Infofish Austrália; vi–FishBase; viii-https://indiabiodiversity.org/; ix-FishBase; xi - https://fishesofaustralia.net.au; xiii -
https://www.fishesoftexas.org/; xiv -https://animaldiversity.org/; xv - https://fishider.org; xvi - https://fishesofaustralia.net.
au.
https://doi.org/10.1371/journal.pone.0293345.g002

characteristics and genetic similarities, demonstrating a coherent relationship between the two
aspects. No evidence of taxonomic deviation was found in any of the species’ group, indicating
that the DNA barcoding approach accurately identified the analysed species.

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PLOS ONE DNA barcoding of the fish species from Mozambique

Table 2. Summary of the genetic divergence based on (K2P percentage) at each taxonomic level.
Label n Taxa Comparisons Min Dist (%) Mean Dist (%) Max Dist (%) SE Dist (%)
Within Species 352 101 504 0.00 0.21 1.51 0.00
Within Genus 166 25 399 3.64 12.53 23.43 0.01
Within Family 246 19 2002 8.27 17.67 25.52 0.00
https://doi.org/10.1371/journal.pone.0293345.t002

3.2 Genetic distances and barcoding gap

The Kimura-2-parameter model (K2P) was used to calculate the genetic distance within and
between species and the presence of barcode gap. The overall K2P distance of COI sequences
are shown in (Table 2). Intraspecific distances ranged from 0.00% to 1.51% (within Species),
while interspecific distances ranged from 3.64% to 23.43% (within Genus), which is greater
than the minimum intraspecific distance of 2%, the threshold for the identification of fish spe-
cies by DNA barcoding [58, 59] (Table 2 and Fig 3A and 3B).
This distance increased with increasing taxonomic level, with distance within families vary-
ing from a minimum of 8.27 to a maximum of 25.52 (within Family). The genetic distance of
all species did not exceed the 2%, with the highest intraspecific distance belonging to
Acanthurus mata OTU-4, with a genetic distance of 1.51% and a barcode gap. The remaining
species had distances less than 1%. The minimum nearest neighbor genetic distance was 3.64%
from Epinephelus coioides OTU-42 to Epinephelus malabaricus OTU-41, and the maximum
was detected between Hippocampus kelloggi OTU-52 and Carcharhinus leucas OTU-19, with a
genetic distance of 22.08%. All details regarding the species distance are summarized on the
(S3 Table).

3.3 Revealing hidden fish diversity

Four species were unexpectedly detected for the first time in this geographical area, whose
presence was not expected, including: Boleophthamus dussumieri, Hippocampus kelloggi,
Lethrinus miniatus and Maculabatis gerrardi. The specie Boleophthalmus dussumieri is known
to be endemic from the Persian Gulf region down to the Indian region [2], and Lethrinus min-
iatus from the Western Pacific [60]. For B. dussumieri, our molecular analysis has shown that

Fig 3. Barcoding gap: Maximum intraspecific Kimura 2-parameter (K2P) distances compared with the minimum
interspecific K2P distances recorded in fish species from the coast of Mozambique. The graphs show the overlap of the
maximum and mean intra-specific distances with the inter-specific (NN = nearest neighbour) distances.
https://doi.org/10.1371/journal.pone.0293345.g003

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PLOS ONE DNA barcoding of the fish species from Mozambique

there is more than one lineage that has not been discussed previously. On the other hand, the
data also showed the presence of the Great Seahorse (Hippocampus kellogi) in the Central
Province of Mozambique. This specie is listed as Vulnerable in the IUCN Red List, and its
occurrence is known only from the Indo-Pacific region of Southeast Asia.
The other significant finding from our study was the presence of one of the most invasive
fish species, the silver carp (Hypophthalmichthys molitrix), which is native to East Asia. This
species is classified as "Near Threatened" and has spread globally, including in the Limpopo
River in southern Mozambique. Rhinoptera sp was also recorded in this study. However, the
occurrence of this species in Mozambique is not officially listed in the global fisheries database.
Our molecular data also allowed us to detect for the first time, the presence of Pangasius djam-
bal [61], which is known from Southeast Asia, Dentex macrophthalmus, which is distributed
from the Mediterranean Sea to the eastern Atlantic [62], and Fistularia petimba, which is
found in the western Atlantic [63].

3.4 Keystones species and conservation status

Sharks, stingrays, and seahorses are the most threatened species in the world due to their
importance in commercial trade and use for various purposes, and therefore, the conservation
and management of these species must be carefully ensured. In our study, we recorded four-
teen endangered species (Table 1, Fig 2). Most of them were classified as elasmobranchs: Acro-
teriobatus leucospilus, Endangered (EN); Carcharhinus leucas, Vulnerable (VU); Himantura
uarnak, Endangered (EN); Loxodon macrorhinus, Near Threatened (NT); Maculabatis ger-
rardi, Endangered (EN); Pastinachus ater, Vulnerable (VU); Rhynchobatus djiddensis and
Rhynchobatus australiae, Critically Endangered (CR); Sphyrna lewini, Critically Endangered
(CR); and one Syngnathiformes, Hippocampus kellogi, Vulnerable (VU).
Most of the teleost species identified in this study were classified as Least Concern in the
IUCN Red List. However, some of them presented a certain level of threat: Acanthopagrus
vagus, Vulnerable (VU); Argyrosomus japonicus, Endangered (EN), Argyrosomus thorpei,
Endangered (EN), Hypophthalmichthys molitrix, Near Threatened (NT) and Scomberomorus
commerson, Near Threatened (NT).

4. Discussion
This study has demonstrated the utility of DNA barcoding to supplement morphological iden-
tification. These results are particularly important because, while the DNA barcoding method
has been applied globally to identify fish species, this study provides, for the first time, an over-
view of the marine fish diversity in the coastal region of Mozambique. This will make a signifi-
cant contribution to our understanding of the understudied diversity in the Indo-West Pacific
biogeographic region.
This study substantially improves our comprehension of fish species and families inhabiting
the Mozambique Coast. The prominent occurrence of species within particular families is
attributable to both the availability of specimens in the examined areas and the presence of
economically important fish species. For instance, the Labridae family is especially abundant
in southern Mozambique [64, 65]. In terms of family representation, this research ranks
fourth, identifying 60 distinct families. This number is lower than those reported in previous
studies, which recorded 90 and 94 families respectively, all using morphological identification
methods [65]. Since morphological identification is prone to errors that can lead to misclassifi-
cation of species, this study presents the most accurate and reliable assessment of fish diversity
along Mozambique’s coastline to date.

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PLOS ONE DNA barcoding of the fish species from Mozambique

Accurate DNA barcoding species identification relies on their intra- and interspecific
genetic distances. Consequently, closely related species may be readily detected in nearby geo-
graphic regions of occurrence. To be considered as species divergence, intraspecific genetic
distance values must exceed 2%, which is the established threshold for species delimitation
[41]. However, no specific threshold has been defined for interspecific or interfamily genetic
distances. In the current study, the mean K2P genetic distances for the barcode region of the
COI gene at the species, genera, and family levels are: 0.21±0.00%, 12.57±0.01%, and 17.29
±0.00%, respectively (Table 2). The average of K2P genetic distances among species, genera,
and families in this study was analogous to findings reported in other studies [31, 66, 67].
However, the minimum interspecific distance observed was substantially greater than that
reported in [68–70] for fish in the China Sea, which can be ascribed to the increased taxonomic
diversity of fish species in the present study.
In the present study, six species had low genetic distance from each other and clustered
closely and two of them were not identified at specie level. The nearest neighbour of Rhyncho-
batus australiae is R. djadensis with 3.72%, Oreochromis mossambicus X O. niloticus with
3.68%, Epinephelus malabaricus X E. coioides, Selar crumenophthalmus X Selar sp and Caesio
caerulaurea X Caeseo sp (Fig 2). These species, which we could not identify at species level,
suggest that they belong to the same genus. Therefore, it is very likely that the sequence of
these two morphospecies belongs to the same genus [71].
The barcode gap method was highly effective in distinguishing and delimiting all possible
species. Despite the identification of 143 species represented by their respective operational
taxonomic units (OTUs), some of these OTUs had remarkably low intraspecific distance and
were treated as single species, yet were grouped into separate OTUs, such as Molgarda sp1
(OTU-72), Molgarda sp2 (OTU-73), and Selar sp. (OTU-138), and Selar crumenophthalmus
(OTU-139) (S2 Table). This scenario arises when there is insufficient data in public databases
to match the analysed specimens.

4.1 Revealing hidden fish diversity

The Indo-West Pacific region is renowned for its high diversity of fish species; however, there
have been limited or inadequate studies conducted on the composition of fish fauna in the
western Indian Ocean. This lack of information can result in an overestimation or underrepre-
sentation of the biodiversity in this region. As an example, this study provides valuable data on
the enigmatic presence of the amphibious fish species B. dussumieri, whose distribution was
previously known to be restricted to the coast of Iran, across the Arabian Sea, and into the
Indian region [72, 73].
This study also reports the presence of the stingray species Maculabatis gerrardi for the first
time. This species is recognized as a complex of species within the Indo Pacific region, with
three distinct lineages distributed along its geographic range [74]. Through the analysis of our
COI gene sample and available COI gene sequences from GenBank and the BOLD System,
our samples were found to cluster with M. gerrardi samples from Kwazulu-Natal (JF493650.1,
JF493649.1) and Durban (JF493648.1) in South Africa, and the detection of an additional pos-
sible lineage in southern Africa suggests the need for further taxonomic examination of this
group of stingrays.
Our data has demonstrated the occurrence of Hippocampus kelloggi, which significantly
expands the known distribution range of the species, previously reported in the regions of
China, India, Indonesia, Japan, Malaysia, Philippines, Thailand, Vietnam, and Tanzania [75].
The study confirms the presence of silver carp Hypophthalmichthys molitrix in the Limpopo
estuary of southern Mozambique, which is widely regarded as one of the most introduced fish

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PLOS ONE DNA barcoding of the fish species from Mozambique

species worldwide [76]. The exact circumstances surrounding its introduction to Mozambique
for aquaculture purposes, remain unclear due to the inexistence in the list of produced specie
in Aquaculture. It is possible that the presence of this species in the region may be attributed to
flood runoff from South Africa [77].
The presence of Lethrinus miniatus in the western Indian Ocean is surprising, as it is gener-
ally found in the Western Pacific, the Ryukyu Islands, eastern Philippines, northern Australia,
and New Caledonia [78]. Previous records of its occurrence outside of this range are misidenti-
fications with another species of the same genus, Lethrinus olivaceus. However, our molecular
data, when compared with studies conducted by [79–81], affirms the existence of Lethrinus
miniatus in Mozambican waters.
Our study also documented the presence of Fistularia petimba. This species has been
reported in several studies as spreading into new areas, including the archipelago of the
Azores, Portugal [82], the Aegean Sea [83], the southern Iberian Peninsula [84], the Mediterra-
nean Sea [85, 86], and the Syrian coast [87]. Notably, Fistularia petimba has also been recorded
in the Gulf of Carpentaria in Australia [88]. Up until now, there have been no previous con-
firmed reports of its presence in the waters of Mozambique.
In summary, we provide strong evidence of the effectiveness of using DNA barcoding to
accurately discriminate and identify many marine and coastal fish species examined thus far.
The COI divergence patterns corresponded with morphologically recognized species; how-
ever, in some cases, the molecular data revealed previously undetected genetic divergence
within a group and instances of low interspecific variation. The presence of cryptic taxa is rela-
tively common among marine animals, emphasizing the need to consider the possibility of
neglected diversity and the occurrence of species complexes. The more comprehensive the bar-
code library, the more useful it will be for the Barcode of Life Initiative [89]. This study repre-
sents a significant contribution towards consolidating DNA barcoding as a global system for
identifying life forms and enhancing our understanding of the genetic diversity of Mozambi-
can marine fish.
However, even among experienced taxonomists, consistent application of species names
remains a challenge, especially when cryptic diversity is present. This is reflected in the conflict-
ing names applied to specimens within the same BIN by different research groups. As the collec-
tion of global data increases, barcodes and BINs will play a vital role in integrating taxonomic
feedback and significantly contributing to the standardization of names at the international
level. This standardization is crucial for the sustainable management of the world’s fisheries.

Supporting information
S1 Table. List of the identified species with detailed taxonomic information. The list of spe-
cies is from Eschmeyer’s Catalog of Fishes and GBIF.
(XLSX)
S2 Table. Cluster sequence result. The distribution of sequence divergence at each taxonomic
level based on the Pairwise Distance model.
(DOCX)
S3 Table. Summary of the distance based on the K2P model. The Distance Summary reports
the sequence divergence between barcode sequences at the species, genus, and family level,
and contrasts the distribution of within-species divergence to between-species divergence.
(PDF)
S1 Fig. Phylogenetic tree (NJ) of all studied species.
(EPS)

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PLOS ONE DNA barcoding of the fish species from Mozambique

Acknowledgments
We thank Manuel Carlos Lopes, Arnaldo Rumieque, Eusébio Fiquisse, Lucinda Cuamba,
Afonso Muhala, Celia Cassamo, Osvaldo Muhala, Graça Malavi, Iolanda Lameira and Ger-
trudes Ussene for their contribution in acquiring and systematically identifying part of the
samples.

Author Contributions
Conceptualization: Valdemiro Muhala, Aurycéia Guimarães-Costa, Adam Rick Bessa-Silva,
Iracilda Sampaio.
Data curation: Valdemiro Muhala, Luan Pinto Rabelo, Luciana Watanabe, Gisele Damasceno
dos Santos.
Formal analysis: Valdemiro Muhala, Aurycéia Guimarães-Costa, Luan Pinto Rabelo, Luciana
Watanabe, Gisele Damasceno dos Santos.
Investigation: Valdemiro Muhala, Isadola Eusébio Macate, Luı́sa Sambora.
Methodology: Valdemiro Muhala, Isadola Eusébio Macate, Luciana Watanabe, Luı́sa
Sambora.
Project administration: Iracilda Sampaio.
Resources: Marcelo Vallinoto, Iracilda Sampaio.
Software: Valdemiro Muhala, Luan Pinto Rabelo.
Supervision: Aurycéia Guimarães-Costa, Adam Rick Bessa-Silva, Marcelo Vallinoto, Iracilda
Sampaio.
Validation: Valdemiro Muhala, Marcelo Vallinoto, Iracilda Sampaio.
Visualization: Iracilda Sampaio.
Writing – original draft: Valdemiro Muhala.
Writing – review & editing: Valdemiro Muhala, Aurycéia Guimarães-Costa, Adam Rick
Bessa-Silva.

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