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Genetic diversity and characterization of the complete mitochondrial genome of the Atlantic
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mudskipper (Periophthalmus barbarus)
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Olosula Sokefun1, Han Ming Gan2, Min Pau Tan3 (not yet finalized)
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Terengganu, Malaysia
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus,
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Corresponding author:
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Abstract
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Introduction
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Mudskippers are distributed in the seven, disjunct mangrove divisions of the world, namely
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West Africa, East Africa, the Arabian gulf, Indo-Malaya, Australia/New Guinea, the China sea
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and the Oceania (Murdy, 1989). Oxudercine gobies are a common sight in the several markets
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around the coastal areas of Nigeria. Even though their origin is established as being the Indo-West
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Pacific and the tropical western parts of Africa, they are naturally occurring in several muddy flood
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plains from Lagos to Port Harcourt in Rivers State, Nigeria. Mudskippers are often one of the
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dominant ecological components on tidal flats and they play an important role as carnivores and
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the preferred prey for avian predators (Clayton 1993). They are also major contributor to the
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maintenance of the integrity and health of mudflat ecosystems, which are usually vulnerable to the
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negative anthropocentric activities of man. They are frequently called are part of the menu of
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riverine areas providing the needed animal protein. There distribution is largely along that of
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modern mangrove habitats (Irvine,1974; FAO, 1990). Several revisions and classifications of the
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group based on updated distributional and taxonomic data has left out the Nigerian group.
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One major controversy about the Nigerian species of Mudskippers is its systematics.
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Severally, they are generally called Atlantic, blue spotted and silver line mudskippers with the
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basis of these name being passed on across generations. In some other works that documents their
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morphometry and meristic characteristics, for instance Lawson (2010), he referred to them as
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Periphthalmus papilio. The basis of this nomenclature is not stated. Most of the research works
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have focused aspects of Mudskipper’s biology (Udo, 2002), patterns of consumption in selected
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communities (Edun,2010), associated parasite (Elele and Aziaka,2019) and the biology of
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Periphthalmus barbarus in Jaja Creek of the Nigerian Niger Delta (Nsikak, 2021). Of the thirty-
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one species that the Food and Agricultural Organization of the United Nations (FAO) mentioned,
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only one has been principally featuring in researches in Nigeria.
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Several other studies from different parts of the world and by researchers have reported
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mudskippers as being generally cryptic, exhibiting morphological plasticity and convergent
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evolution (Thacker 2003, Vanhove et.al. 2012, Winterbottom et. al. 2014). Cryptic species are
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usually so similar that they are difficult or impossible to be distinguished based on morphological
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characters alone. This suggests that clear identification within these group in poorly known.
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Therefore, the aim of this study is to take advantage of molecular genetic data using the
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cytochrome c oxidase subunit 1 (CO1) mitochondrial gene which is a DNA barcoding tool to
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identify the mudskippers collected at the central market in Port Harcourt, Nigeria due to the unclear
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nature of the basis of the species into which experts and non-experts classify then into and also see
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if there is any congruence in this classification.
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Materials and methods
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Field sampling and samples preservation
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Sampling was carried out in conjunction with Dr Akinrotimi of the Institute of
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Oceanography and Marine Research, Port Harcourt outstation in Port Harcourt, Nigeria. These
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mudskippers were purchased at the open market and were identified at the species level as the
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Atlantic mudskipper, blue spotted and the silverline mudskippers. The market women noted that
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they also catch them at Buguma in the sub-urban areas of Port Harcourt. They noted that these
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were the names that these species have been called from time immemorial and that the
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morphological basis was unclear. Five specimen each group as specified by the expert and market
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women were purchased. They are the Atlantic (AM), the blue spotted (BM) and the silverline (SM)
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mudskippers and another five from a seller who got her from Abonema Waterside, also in Port
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Harcourt were simply labelled as GM. These specimens were quickly taken to the laboratory in
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ice where tissue samples were collected. 50mg to 100mg of muscle tissue was dissected with a
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sterilized surgical scissors, placed into 1.5ML screw top cryogenic vials and preserved in 95%
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Ethanol. Fish tissue samples were then shipped to the Institute of Marine Biotechnology, Universiti
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Malaysia Terengganu for laboratory processes.
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DNA extraction and polymerase chain reaction (PCR) amplification
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Han Ming
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Data analysis for population genetics
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Multiple sequences were aligned and trimmed using... with final alignment truncated to
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657 base pairs (bp). Seventeen individuals were successfully amplified. To ensure that the
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sequences were aligned correctly, they were first translated into a protein sequence (no stop codon
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or indel was found). Then, the sequence identity was checked against GenBank database by using
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Basic Local Alignment Search Tool (BLAST), National Centre for Biotechnology Information
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(NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). It is worth noting that to date, only a single COI
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sequence of P. barbarus was available in the GenBank database (AF391339, sample from Nancy
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Aguilar, Nigeria; Thacker, 2003). All haplotype sequences obtained in this study were deposited
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in the GenBank database with accession numbers ..
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The aligned sequences were screened for nucleotide variable sites, parsimony informative
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sites, the number of haplotypes (nh), and amino acid substitutions in MEGA 6.0 (Tamura et al.,
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2013). The genetic diversity indices, namely haplotype diversity (H) and nucleotide diversity (π),
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were calculated in DnaSP v6 (Rozas et al., 2017).
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The best nucleotide substitution models with the lowest BIC (Bayesian Information
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Criterion) score for the dataset was the Kimura 2-parameter (K2P) model (Kimura, 1980). The
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phylogenetic relationships of P. barbarus were assessed by constructing a maximum likelihood
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(ML) tree in MEGA 6.0. The robustness of the statistical support for the ML tree branch was
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determined by 1,000 bootstrap replicates (Felsenstein, 1985). The Silverline mudskipper (P.
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argentilineatus) (KU692746), the Pearse’s mudskipper (P. novemradiatus) (KU692758) and the
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Blue-spotted mudskipper (Boleophthalmus boddarti) (MK572067) were used as outgroup taxa.
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The genetic distance among samples was calculated by employing K2P model in MEGA 6.0.
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Data analysis for characterization of the complete mitochondrial genome
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Han Ming
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Results and discussions
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Population genetics of Periophthalmus barbarus
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Final alignment of the COI sequences revealed a total of eight variable sites, resulting in a
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total of seven putative haplotypes. The overall haplotype and nucleotide diversity was 0.809 and
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0.0046, respectively. Pairwise genetic distance between samples from this study ranges from 0.000
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to 0.008, while pairwise genetic distance of interspecies ranges from 0.124 to 0.203 (Table 1).
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Maximum likelihood gene tree clusters all samples obtained in this study into a single clade with
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99% bootstrap support value (Figure 1). These analyses strongly suggest that the mudskipper
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samples belong to a single taxon despite being identified as different species by the local residents.
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Table 1: Pairwise genetic distance of the intra- and interspecies of Periophthalmus barbarus inferred from the COI sequences
AM15
AM17
BM1
BM2
BM3
BM4
BM5
GM2
GM3
GM4
GM5
SM1
SM2
SM3
SM4
SM5
BB
PA
PN
AM14
0.002
0.003
0.000
0.000
0.003
0.002
0.003
0.005
0.003
0.003
0.000
0.000
0.002
0.003
0.003
0.005
0.197
0.130
0.161
AM15
AM17
BM1
BM2
BM3
BM4
BM5
GM2
GM3
GM4
GM5
SM1
SM2
SM3
SM4
SM5
BB
PA
0.002
0.002
0.002
0.002
0.000
0.002
0.006
0.002
0.002
0.002
0.002
0.003
0.002
0.002
0.003
0.199
0.128
0.159
0.003
0.003
0.000
0.002
0.000
0.008
0.000
0.000
0.003
0.003
0.005
0.000
0.003
0.002
0.199
0.126
0.157
0.000
0.003
0.002
0.003
0.005
0.003
0.003
0.000
0.000
0.002
0.003
0.003
0.005
0.197
0.130
0.161
0.003
0.002
0.003
0.005
0.003
0.003
0.000
0.000
0.002
0.003
0.003
0.005
0.197
0.130
0.161
0.002
0.000
0.008
0.000
0.000
0.003
0.003
0.005
0.000
0.003
0.002
0.199
0.126
0.157
0.002
0.006
0.002
0.002
0.002
0.002
0.003
0.002
0.002
0.003
0.199
0.128
0.159
0.008
0.000
0.000
0.003
0.003
0.005
0.000
0.003
0.002
0.199
0.126
0.157
0.008
0.008
0.005
0.005
0.006
0.008
0.008
0.009
0.199
0.130
0.159
0.000
0.003
0.003
0.005
0.000
0.003
0.002
0.199
0.126
0.157
0.003
0.003
0.005
0.000
0.003
0.002
0.199
0.126
0.157
0.000
0.002
0.003
0.003
0.005
0.197
0.130
0.161
0.002
0.003
0.003
0.005
0.197
0.130
0.161
0.005
0.005
0.006
0.197
0.131
0.163
0.003
0.002
0.199
0.126
0.157
0.005
0.201
0.126
0.157
0.197
0.124
0.155
0.194
0.203
0.161
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Note: BB: Boleophthalmus boddarti (MK572067); PA: Periophthalmus argentilineatus (KU692746); PN: Periophthalmus
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novemradiatus (KU692758)
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GM5
SM1
BM2
BM1
AM14
SM2
GM2
AM15
BM4
SM4
AM17
BM3
BM5
GM3
GM4
SM3
SM5
KU692746.1 Periophthalmus argentilineatus
KU692758.1 Periophthalmus novemradiatus
MK572067.1 Boleophthalmus boddarti
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Figure 1: ML gene tree of Periophthalmus barbarus inferred from the CO1 sequences, constructed
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in MEGA 6.0 (https://www.megasoftware.net/resources). Branches were drawn to scale and
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bootstrap values < 50% were not shown.
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Complete mitochondrial genome of Periophthalmus barbarus
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Conclusion
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Acknowledgements
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