Zoosyst. Evol. 96 (2) 2020, 449-454 | DO! 10.3897/zse.96.51350

yee
BERLIN

Genetic evidence for the recognition of two allopatric species of
Asian bronze featherback Notopterus (Teleoste1, Osteoglossomorpha,
Notopteridae)

Sébastien Lavoué!, Siti Zafirah Ghazali!*, Jamsari Amirul Firdaus Jamaluddin?,
Siti Azizah Mohd Nor’, Khaironizam Md. Zain!

1 School of Biological Sciences, Universiti Sains Malaysia, Penang, 11800, Malaysia
2 Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Nerus, 21030, Malaysia

http://zoobank.org/64287A82-6E8C-4A D0-9754-6C687D2AF15F

Corresponding author: Sébastien Lavoué (microceb@hotmail.com)

Academic editor: Nicolas Hubert # Received 22 February 2020 Accepted 20 April 2020 Published | July 2020

Abstract

The fish genus Notopterus Lacepede, 1800 (Notopteridae) currently includes only one species, the Asian bronze featherback No-
topterus notopterus (Pallas, 1769). This common freshwater species is widely distributed in the Oriental region, from the Indus basin
in the west, the Mekong basin in the east and Java Island in the south. To examine the phylogeographic structure of N. notopterus
across its range, we analysed 74 publicly available cytochrome oxidase I (COI) sequences, 72 of them determined from known-origin
specimens, along with four newly-determined sequences from Peninsular Malaysian specimens. We found that N. notopterus is a
complex of two allopatric species that diverge from each other by 7.5% mean p-distance. The first species is endemic to South Asia
(from Indus basin to Ganga-Brahmaputra system), whereas the distribution of the second species is restricted to Southeast Asia. The
exact limit between the distributions of these two species is not known, but it should fall somewhere between the Ganga-Brahmaputra
and Salween basins, a region already identified as a major faunal boundary in the Oriental region. The name N. notopterus is retained
for the Southeast Asian species, while the name Notopterus synurus (Bloch & Schneider, 1801) should be applied to the South Asian
species. A comparative morphological study is needed to reveal the degree of morphological differentiation between the two species.

Key Words

Orient, molecular taxonomy, biogeography, biodiversity, cryptic species

Introduction

The complex geological history of the Oriental region
caused a high degree of geographical genetic structure
within freshwater organisms and species, once consid-
ered widely distributed in this region, are often formed
by distinct genetic lineages (e.g. de Bruyn et al. 2013;
Dahruddin et al. 2017; Jamaluddin et al. 2019; Ruber et
al. 2020). The bronze featherback fish Notopterus no-
topterus, currently the only valid species of the genus
Notopterus, 1s one of such widely-distributed species,
occurring from the Indus basin (Pakistan and India) in

the west, to the Mekong region — slightly extending east
to the Annamite Range — (Cambodia, Laos, Thailand and
Vietnam) in the east and to Java (Indonesia) in the south.
Intriguingly, N. notopterus has not yet been recorded in
Borneo where it is most likely absent (Roberts 1989;
Christensen 1992; Roberts 1992; Kottelat 1995; Kottelat
and Widjanarti 2005; Parenti and Lim 2005).

Specimens of N. notopterus are identifiable from all
other Oriental freshwater fishes based on (amongst oth-
er features) their distinct tapered tail and the corners of
their mouth below eye (not behind as in the other Oriental
notopterid genus Chitala Fowler, 1934) (Fig. 1A). It is

Copyright Sébastien Lavoué et al. 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.

450

reported that this species reaches up to 60 cm in standard
length (Roberts 1992). A wealth of data is available for
this species, documenting its reproductive behaviour and
embryonic development (Yanwirsal et al. 2017), cytoge-
netics (Barby et al. 2019) and phylogeography (Gupta
et al. 2013; Takagi et al. 2010). However, none of these
studies simultaneously examined specimens sampled
across the whole distributional range of N. notopterus.

Inoue et al. (2009) reconstructed the molecular phy-
logeny of the family Notopteridae to discuss its evolution
and biogeography. These authors sequenced the complete
mitogenomes of two specimens of N. notopterus, one
from India and the other from Thailand. The comparison
of these two mitogenomic sequences revealed that these
two specimens diverged from each other about 25 million
years ago. This result was unexpected because no con-
sistent morphological variation was previously reported
within this species (see Roberts 1992).

We herein investigated the genetic diversity within the
genus Notopterus across its full range. For that, we anal-
ysed a dataset comprising 72 publicly available sequences
of the standard barcoding fragment (655 base pairs) of the
cytochrome oxidase I (COI) gene that were determined
from specimens of Notopterus with precise information
on their geographical collection plus two COI sequenc-
es extracted from two complete mitogenomes from two
specimens of Notopterus without precise localities (In-
oue et al. 2009) and four sequences we newly determined
from specimens collected in Peninsular Malaysia.

Material and methods

COI sequences mining and selection

Using the NCBI GenBank nucleotide database (https://
www.ncbi.nlm.nih.gov/), we searched for available cy-
tochrome oxidase subunit I sequences of N. notopterus
(search made on 02/12/2019) using the terms “Notopter-
us” and “oxidase.” This search retrieved 88 mitochon-
drial entries (excluding three entries related to whole
mitogenomes), from which we selected only those with
either latitude-longitude coordinates or geographic lo-
calities sufficiently precise that we can confidently esti-
mate their latitude-longitude coordinates. After this initial
screening, the dataset included partial COI sequences of
77 specimens of Notopterus. We then checked the length
and characteristics of these sequences using the software
Mesquite version 3.31 (Maddison and Maddison 2017).
Their quality was assessed in searching for stop codons,
indels and/or the presence of relatively higher number of
autapomorphic changes as indication of possible either
pseudogenes or sequencing/editing errors. Five out of 77
sequences were found below standards and were exclud-
ed (all 16 excluded sequences and the respective reasons
to exclude them are listed in Suppl. material 1: Table S1).
After this screening, the dataset comprised 72 COI se-
quences of specimens collected in India (40 specimens),

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Sébastien Lavoué et al.: Two living species of Notopterus

Bangladesh (two), Peninsular Malaysia (two), Indonesia
(six), Myanmar (16) and Thailand (six) (localities are
mapped in Fig. 1A and listed in the Suppl. material 1:
Table S1). We added to this dataset two COI sequences of
specimens (without precise locality) examined in Inoue et
al. (2009) along with the four sequences we newly deter-
mined from specimens collected in Peninsular Malaysia,
two from Kerian River (Penang State) and two from Bera
lake (Pahang State). We deposited these voucher spec-
imens in the ichthyological collection of the School of
Biological Sciences, Universiti Sains Malaysia under ac-
cession numbers USMFC (3) 00002-3.

DNA extraction, amplification and sequencing

To determine the COI sequences of these specimens,
we used the PCR technique to amplify them using the
following PCR primer pair: forward FishF1 (5’TCAAC-
CAACCACAAAGACATTGGCAC-3') and reverse
FishR1 (5’-TAGACTTCTGGGTGGCCAAAGAAT-
CA-3’) (Ward et al. 2005). Reactions were carried out
in 25 ul reaction volume containing 15.75 ul of sterile
distilled H,O, 5.5 wl of 5x MyTaq Red reaction buffer
(Bioline), 0.5 ul of each primer (10 uM), 0.25 ul of 1Taq
DNA polymerase (INtRON Biotechnology) and 2.5 ul
of template containing approximately 5 ng DNA. The
thermal cycle profile consisted of an initial 94 °C de-
naturation step for 4 min, 35 cycles of 94 °C for 30 sec,
annealing for 50 sec at 47.9 °C, extension at 72 °C for
1 min, followed by a final extension at 72 °C for 7 min.
PCR products were sent to First Base Sdn. Bhd. for se-
quencing analysis by the standard Sanger methodology.
Chromatograms were edited with the Molecular Evolu-
tionary Genetics Analysis X (MEGA X) (Stecher et al.
2020). Sequences are deposited in GenBank under ac-
cession numbers MT328860-3.

Comparative analysis

The alignment of the 78 COI nucleotide sequences was
done by eye. Some sequences were shorter than oth-
ers (at the 5’ and 3’ ends) and the overall proportion of
missing data in the alignment was 4.6%. The alignment
comprised 655 positions of which 62 were parsimony-in-
formative. The alignment in Phylip format is provided as
Suppl. material 2: (“Notopterus_align 78COL phy”). We
did not use an outgroup and we present only an unrooted
haplotype network. Relationships amongst COI haplo-
types were inferred with an unrooted network constructed
with the programme PopArt (Leigh and Bryant 2015) us-
ing a median-joining algorithm (Bandelt et al. 1999) and
default settings. Uncorrected pairwise genetic distances
and numbers of nucleotide difference within and amongst
groups along with their respective standard errors (SE)
were calculated with MEGA X. SE were obtained by a
bootstrap procedure (500 replicates).

Zoosyst. Evol. 96 (2) 2020, 449-454 451

ue ae

Southeast Asia
(Notopterus notopterus)

South Asia
(Notopterus synurus)

10 specimens co
one specimen ©

Figure 1. A Map covering the Oriental biogeographic region and showing the distribution of localities (black and red circles) of
specimens of Notopterus examined in this study. Black star indicates the estimated type locality of Notopterus notopterus in Java (1.e.
Jakarta region) and red stars indicate the likely origin localities of the two syntypes of Notopterus synurus (i.e. coast of Malabar and
Coromandel coast at Tharangambadi [formerly Tranquebar]). Main Oriental river basins from West to East: 1. Indus basin; 2. Gan-
ga-Brahmaputra river system; 3. Irrawaddy basin; 4. Salween basin, 5. Mekong basin. Insert shows the left lateral view of a specimen
of Notopterus notopterus (Penang State, west Peninsular Malaysia; 20 cm in standard length, voucher specimen number USMFC (3)
00002, NO_1). B The unrooted network constructed with the software PopArt and a median-joining algorithm showing the COI hap-
lotype relationships within the genus Nofopterus. Branch lengths are not proportional to the number of changes. Red circles indicated
haplotypes of Notopterus synurus (South Asia) and black circles indicated haplotypes of Notopterus notopterus (Southeast Asia).

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452

Results

The haplotype network is shown Fig. 1B. Haplotypes
segregate into two main groups having distinct distribu-
tions. The first group includes all haplotypes from India
and Bangladesh (South Asia group). The second group
includes all haplotypes from Peninsular Malaysia, Thai-
land (Mekong River), Indonesia (Sumatra and Java) and
Myanmar (Lake Inle, Salween basin) (Southeast Asia
group). These two main groups diverge by 7.5% p-genetic
distance [min-max = 7.0—8.8%; SE = 1%] that represents,
on average, 45 differences [SE = 5.8] between any com-
bination of two specimens sampled from different groups.
In contrast, each of these two groups is genetically uni-
form with intra-group differentiation that does not ex-
ceed 1% (within the South Asia group, mean p-distance
= 0.4%; min-max = 0.0—1.6%; SE = 0.1%; and within the
Southeast Asia group, mean p-distance = 1%; min-max =
0.0—2.5%; SE= 0.2%). This represents six nucleotide dif-
ferences [SE = 1.5] on average within the Southeast Asian
group and only two nucleotide differences [SE = 0.6] on
average within the South Asian group. Furthermore, the
two specimens examined by Inoue et al. (2009) fell in
their corresponding geographic origin groups.

Discussion

The minimal genetic distance separating the genus No-
topterus into two main groups is well above 3% (us-
ing COI marker) which is considered as a conservative
threshold between population and species levels in verte-
brates (Ward et al. 2009). Furthermore, the existence of
a so-called barcode gap (= intergroup distance/intragroup
distance) of magnitude ~7X between these two groups,
along with the fixation of more than 40 diagnostic nucle-
otide changes in COI, strongly indicate that Notopterus is
formed by two species (Meyer and Paulay 2005; Meier et
al. 2008). One species is distributed in South Asia (from
the Indus basin to Ganga-Brahmaputra basin) and the oth-
er in Southeast Asia (from the Salween basin to Mekong
basin plus Malay Peninsula, Sumatra and Java).

Roberts (1992) examined the variation of several mer-
istic characters of N. notopterus throughout its entire
range. He did not report any significant intraspecific vari-
ability that could be additional evidence for the recogni-
tion of more than one species in the genus Notopterus.
The low amount of morphological variability within the
genus Notopterus, however, is not surprising given that
the family Notopteridae is known for its morphological
stasis. Several valid species of Notopteridae are morpho-
logically similar. For example, species of the genus Chi-
tala are only distinguishable based on their colour pattern
(Roberts 1992) and more than one species is suspected
to occur within Chitala lopis (Bleeker, 1851) (Kottelat
and Widjanarti 2005; personal observation). Barby et al.
(2019) and earlier cytogenetic studies cited in this work,
reported the same karyotype formula in N. notopterus,

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Sébastien Lavoué et al.: Two living species of Notopterus

regardless to their origins, with 2n = 42 and all chromo-
somes acrocentric.

Our COI-based results strongly support the presence of
two allopatric species of Notopterus which need names.
There are several nominal species of Notopterus which
have been described from Southeast Asia and South Asia
and several of these names are available. To determine
which name should be applied to each of our two species,
we examined the synonym lists of Fricke et al. (2020) and
Kottelat (2013) and checked the date of description and
type locality of these synonyms.

Notopterus notopterus was described and illustrated by
Pallas in 1769 as Gymnotus notopterus from a specimen
said to have been collected nearby Ambon [Ambon Island],
Indian Ocean, a region where this species has never been
recorded since. Kottelat (2013) and Fricke et al. (2020)
suggested that the type locality given in the description of
Pallas (1769) is an error because this species does not oc-
cur on the Island of Ambon which lies east of the Wallace
Line, in a different biogeographical region. Furthermore,
the local vernacular name of this species “Ikan Pangaio”,
reported by Pallas, is in the Malay language which was
not used in Ambon at that time. For these reasons, Kottelat
(2013) suggested that the specimen used by Pallas (1769)
for the description of NV. notopterus should have been col-
lected in Java where the Dutch established their main col-
ony in Indonesia. Consequently, the name N. notopterus
should be retained for the species occurring in Southeast
Asia with Java as its type locality. According to Fricke et
al. (2020), there is no type specimen known.

Bloch and Schneider (1801) described Clupea synu-
ra Bloch & Schneider, 1801 from two syntypes, a name
which was soon considered as a junior synonym of N. no-
topterus (e.g. as in Cuvier and Valenciennes 1847). Bloch
and Schneider (1801) first indicated that these specimens
are from the coast of Malabar, India (“Habitat ad oram
Malabaricam’” in Bloch and Schneider [1801]). Howev-
er, in a following remark signed only by J.G. Schneider,
China and Tranquebar (now known as Tharangamba-
di, India) were listed as the localities of the syntypes.
Notopterus, however, does not seem to occur in China
and Paepke (1999) found convincing explanations why
Schneider could have mistaken China with the coast of
Malabar, India (see Paepke 1999; Kottelat 2013; Fricke
et al. 2020). According to Paepke (1999), the localities of
the dry right skins of the two syntypes of Clupea synura
that are housed in the Berlin Museum under catalogue
numbers ZMB 8806 and ZMB 32057 are coast of Mal-
abar and Tranquebar, respectively. We suggest the reval-
idation of Clupea synura for the species of Notopterus
occurring in South Asia which should be recognised as
Notopterus synurus.

Whereas the genetic evidence presented in this work
supports the recognition of two valid living species of No-
topterus, a detailed morphological comparison of the two
species is lacking. Such morphological study is needed to
identify possible diagnostic characters (in addition to the
molecular diagnostic characters presented in this study)

Zoosyst. Evol. 96 (2) 2020, 449-454

and to document the early diversification of the genus No-
topterus in the Orient. In this respect, a fossil of Notopter-
us, morphological similar to living species, indicates that
this genus was already present in Sumatra at least 33 mil-
lion years ago (the Eocene-Oligocene boundary) (Sanders
1934). In addition, because of the cryptic diversity occur-
ring in the genus Notopterus and the difficulty to identi-
fy the type localities of species of this genus, it will be
important to designate a neotype for N. notopterus and a
lectotype for N. synurus. Finally, the geographic coverage
needs to be expanded with the study of specimens collect-
ed from the Mekong basin, Indus basin and, especially,
from the region comprising the Irrawaddy basin situat-
ed between the Ganga-Brahmaputra river system and the
Salween basin to determine the location of the exact dis-
tributional limit between these two species.

Acknowledgements

We conducted fieldwork in Tasek Bera (Pahang, Pen-
insular Malaysia) under permit reference number 100-
34/1.24Jld14(67) delivered by the Department of Wild-
life and National Parks (DWNP). We thank the staff of
Tasek Bera Reserve and Siti Zuliana Ahmad (USM) for
their assistance in the field. We express our gratitude to
Richard van der Laan and Ron Fricke (Eschmeyer’s Cata-
log of Fishes) for helpful discussions on the nomenclature
of these fishes. We also thank Nicolas Hubert (editor) for
handling our manuscript and Erick Guimaraes and Ru-
pert Collins (reviewers) for their constructive comments
on the submitted version of this work. This study was
supported by a short-term research grant 304/PBIOLO-
GI/6315400 from Universiti Sains Malaysia to SL.

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Sébastien Lavoué et al.: Two living species of Notopterus

Supplementary material |

Table SI

Authors: Sébastien Lavoué, Siti Zafirah Ghazali, Jamsa-
ri Amirul Firdaus Jamaluddin, Siti Azizah Mohd Nor,
Khaironizam Md. Zain

Data type: Genetic data information

Explanation note: List of COI sequences of specimens
of Notopterus mined from Genbank and examined
in this study along with their Genbank accession
numbers, the geographical origins with geographical
coordinates (in Decimal Degrees) of specimens and
the published references where sequences were pub-
lished.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacom-
mons.org/licenses/odbl/1.0/). The Open Database
License (ODbL) is a license agreement intended to
allow users to freely share, modify, and use this Data-
set while maintaining this same freedom for others,
provided that the original source and author(s) are
credited.

Link: https://doi.org/10.3897/zse.96.51350.suppl1

Supplementary material 2

DNA Data matrice

Authors: Sébastien Lavoué, Siti Zafirah Ghazali, Jamsa-
ri Amirul Firdaus Jamaluddin, Siti Azizah Mohd Nor,
Khaironizam Md. Zain

Data type: Phylogenetic

Explanation note: Phylogenetic matrice used in the com-
parative part of this work.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacom-
mons.org/licenses/odbl/1.0/). The Open Database
License (ODbL) is a license agreement intended to
allow users to freely share, modify, and use this Data-
set while maintaining this same freedom for others,
provided that the original source and author(s) are
credited.

Link: https://doi.org/10.3897/zse.96.51350.suppl2