Vietnam Journal of Biotechnology 21(2): 347-363, 2023
347

SPECIES DIVERSITY AND PHYLOGENETIC RELATIONSHIPS OF
SNAPPER (LUTJANIDAE: LUTJANUS) INFERRED FROM
MITOCHONDRIAL DNA MARKERS

Dinh Thi Hai Yen
1,
, Hoang Ngoc Lam
1
, Vo Thi Ha
1
, Truong Thi Oanh
2
, Tran Quang
Sang
2
, Dang Thuy Binh
2


1
Coastal Branch of Joint Vietnam - Russia Tropical Science and Technology Research
Center, 30 Nguyen Thien Thuat Street, Nha Trang City, Khanh Hoa Province, Vietnam
2
Institute of Biotechnology and Environment, Nha Trang University, 02 Nguyen Dinh Chieu
Street, Nha Trang City, Khanh Hoa Province, Vietnam

To whom correspondence should be addressed. E-mail: [email protected]
Received: 17.01.2023
Accepted: 25.4.2023
SUMMARY
Snappers (Lutjaniformes: Lutjanidae) are commercially important fishes in tropical and
subtropical waters. However, species complexes still exist due to low-level morphological
differences. Additionally, current availability of molecular sequences has led to significant
changes in fish taxonomy. Therefore, species diversity of lutjanids is still unclear. In this
study, we applied two mitochondrial markers (16S rRNA and Cytochrome c oxidase subunit
I, COI mtDNA) to investigate species diversity and phylogenetic relationships of lutjanid
species collected from the coastal waters of Nghe An – Ha Tinh provinces, Northern Central,
Vietnam. A total of 17 Lutjanus species have been identified using morphological and
molecular methods. Combined with Genbank sequences, the phylogenetic tree was
constructed using Neighbor Joining and Maximum Likelihood approaches based on 16S
rRNA and COI mtDNA data set. Two main lineages have been detected with inconsistent
basal clades between two topologies. Members of species complexes also showed a certain
degree of closely relationships; however, conflicts between two topologies have also been
recorded. These data contribute to the assessment of lutjanid biodiversity in Vietnam, and
for resource management and conservation.
Keywords: Lutjanus, mitochondrial markers, phylogenetic relationships, species
composition.
INTRODUCTION
Snappers (Lutjaniformes: Lutjanidae) are
commercially important fishes in tropical
and subtropical waters, consisting of 135
species, 21 genera. Etelinae, Apsilinae,
Paradicichthyinae and Lutjaninae are
currently recognized as four subfamilies of
the family Lutjanidae (Betancur-R et al.,
2017). These fishes are considered long-
lived, slow-growing and capable of
occupying a wide range of habitats such as
coral reefs or other associated structures.
During early life cycle, some species, such as

Dinh Thi Hai Yen et al.
348
Lutjanus argentimaculatus, L. griseus, may
also occur in estuarine mangroves. Adult
snapper plays an ecologically important role,
acting as predators of a variety of food
sources, which can modify the structure of
their living environment (Nelson, 2006).
Among the family Lutjanidae, Lutjanus
is by far the most speciose genus, with 73
known species (Andriyono et al., 2019).
Following the external coloration and
diagnostic characters, lutjanids species were
divided into several species groups, such as
the black spot complex (6 species, Miller,
Cribb (2007)), blue-lined complex (6
species, Barman (2014)), yellow-lined
complex (7 species, Iwatsuki et al. (2015)),
and red snapper (12 species, Rivas (1996)).
Like other fish species, snappers are
threatened by overfishing, habitat
degradation, etc. (Gold, 2015). Thus,
accurate species identification is
fundamental for conservation efforts and
the management of these valuable fish
species.
Fish species identification is
traditionally based on external
morphological features, including body
shape, color pattern, number of fin rays and
spines, or various relative measurements of
body parts (Allen, 1985). However,
taxonomic identification of snapper species
is difficult because of the similarities in
their external morphology and overlap of
diagnostic characters, as in case of L.
erythropterus and L. malabaricus (Halim
et al., 2022). Additionally, species
hybridization probably occurred (L.
erythropterus×L. sebae) (Chen, 2006),
leading to species misidentification.
Presently, molecular markers provide
useful and powerful tools to discriminate
species. The generating genetic data can
provide a valuable source of information
for studies on phylogeny, phylogeographic,
and evolutionary history (Afriyie, 2020). In
the last few decade, molecular markers
such as mitochondrial and nuclear genes
(Miller, Cribb, 2007; Gold et al., 2011;
Chu et al., 2013; Gold et al., 2015; Wang
et al., 2015), and mitogenomes (Andriyono
et al., 2019) have been increasingly applied
to investigate the diversity and
phylogenetic relationships of the snapper
species.
In Vietnam, based on 27 documents
published during 1978-2010, Le (2013)
confirmed 40 species belonging to 10 genera
of family Lutjanidae, of which 26 species
belong to the genus Lutjanus. Recently,
Nguyen and Mai (2020) reported 26 species
(4 genera), and 10 species (2 genera) of the
families Lutjanidae and Caesionidae,
respectively. Besides, phylogenetic
relationships of lutjanids species have been
explored using molecular data. Truong et al.
(2015) used the 16S rRNA marker to
investigate the molecular relationships of 12
species (Lutjanus and Paracaesio) collected
from Kien Giang, Vung Tau, Khanh Hoa and
Da Nang provinces. Their analyses showed
that Lutjanus spp. species are monophyletic,
and sister groups to Paracaesio xanthura.
Pham et al. (2019) found 18 snapper species
belonging to 6 genera. A phylogram from the
barcode of COI mtDNA showed the
paraphyly of Lutjanus species.
The research aims to examine the
morphological and molecular characteristics
of Lutjanus species collected from the
coastal waters of Nghe An – Ha Tinh
provinces based on 16S rRNA and COI
mtDNA genes. The generated data will serve
as scientific information, which could be
applied for improving the monitoring
program and conservation of fisheries
resources.

Vietnam Journal of Biotechnology 21(2): 347-363, 2023
349
MATERIAL AND METHODS
Study sites and fish sampling
A total of 55 snapper specimens were
collected from local fishermen of Nghe An –
Ha Tinh coastal waters (Lat.
18°06’33.24’’N-19°14’00.94’’N and Long.
105°44’49.31’’E -106°21’13.68’’E, Figure
1) during 2020-2022. All fishermen were
interviewed to confirm that fish were caught
within 10 nautical miles and no more than 12
hours. The fish were caught from a depth
range of 5 to 50 m using traditional fishing
gears, including gill nets, trawl nets (mesh
size 40-60 mm), fishing fish, or fish traps. On
the field, all specimens were photographed,
and their body, fin coloration, and diagnostic
features (spot, stripes or bars) were recorded.

Figure 1. Sampling sites for the snapper specimens in Nghe An – Ha Tinh.

Muscle tissue samples (~1 cm
2
) were
excised from the right side of the fish
specimens and put into 2 ml cap-screw tubes
filled with 95% ethanol. All samples were
kept on ice and transported to the Molecular
Biology Laboratory of Nha Trang University
for DNA analysis. Specimens were
transported in an ice box to the Laboratory of
the Coastal Branch of Joint Vietnam - Russia
Tropical Science and Technology Research
and kept at –20ºC until further study.
Morphological identification and voucher
preservation
At the laboratory, specimens were
defrosted under running water. All

Dinh Thi Hai Yen et al.
350
specimens were identified based on
taxonomic characteristics such as the
coloration patterns on the head, body, and
fins; presence of diagnostic features (spot,
stripes or bars) on the body following the
identification keys of Allen (1985). The
number of spines and rays of the dorsal fin
(D), anal fin (A), and pectoral fin (P) were
counted (Carpenter, Allen, 1989) (Figure 2).
After the morphological analysis, one to
three representative specimens of each
species were preserved following Motomura,
Ishikawa (2013). For fixation, the specimens
were injected through the vent and dorsal
musculature and completely soaked with
10% formalin. After 30 - 40 days, the
specimens were transferred to a 3 – 5L wide-
mouth glass jar filled with 99% ethanol for
long-term preservation. All voucher
specimens were labeled and kept at Coastal
Branch of Joint Vietnam - Russia Tropical
Science and Technology Research.

Figure 2. Morphometric and meristic characters (Carpenter, Allen, 1989).

Molecular identification
Ethanol-preserved tissue samples were
extracted using Wizard® Genomic DNA
Purification Kit (Promega, USA) following
to the manufacturer's instructions. The
extracted DNA samples (5 µl) were PCR
amplified at partial fragments of the 16S
rRNA and COI mtDNA genes using the
primers 16Sar, 16Sbr (Palumbi, 1996) and
FishF1, FishR1 (Ward et al., 2005),
respectively. The components and thermal
cycle of the PCR reaction were performed
following Vu et al. (2018). Successful PCR
products were purified with the Promega
PCR Purification Kit following the
manufacturer’s protocol. Sequences of
both strands were performed at 1
st
Base
Company (Malaysia) using ABI PRISM
3100 Genetic Analyzer (Applied
Biosystems, USA) with the amplification
primers.

Vietnam Journal of Biotechnology 21(2): 347-363, 2023
351
Forward and reverse sequences were
assembled using Geneious Pro 5.5.7 (Kearse
et al., 2012). The Basic Local Alignment
Search Tool (BLAST,
https://blast.ncbi.nlm.nih.gov/Blast.cgi) was
used to search for identical sequences. The
obtained sequences were aligned, analysed
using BioEdit 7.0.5.3 (Hall, 1999), and
deposited to GenBank under the accession
numbers OP316912-OP316928 (16S rRNA)
and OP316932-OP316948 (COI mtDNA).
Phylogenetic relationships
Each sequence set of 16S rRNA and COI
mtDNA (17 sequences from each gene) was
separately tested for substitution
oversaturation based on the concept of
entropy information theory using DAMBE
6.4.101. Following Gontcharov et al. (2004),
combined gene analysis e nhanced
phylogenetic resolution, 2 current sequence
sets were combined with 8 available
Genbank sequences using Geneious Pro
5.5.7. An incongruence-length difference
(ILD) test (Farris et al., 1995) was performed
in PAUP*4.0b10 (Swofford, 2003) with
1,000 randomized replicates to estimate any
difference in phylogenetic signal among the
different molecular sections. ILD test (p-
value = 0.01) indicating significant
incongruence between the two data sets,
therefore combination was not applied for
further analysis.
Phylogenetic trees were constructed for
16S rRNA and COI mtDNA sequence
alignments using the Neighbor-joining (NJ)
and Maximum likelihood (ML) approaches in
MEGA 11.0.11 (Kumar et al., 2018). NJ
analysis was conducted to determine the
evolutionary relationships of all samples
based on the Kimura 2-parameter model
under 1,000 replicates. Prior to ML analysis,
the best-fit model of nucleotide substitution
was selected by the Akaike Information
Criterion as implemented by Modeltest 3.7
(Posada, Crandall, 1998). General Time
Reversible (GTR) and Hasegawa–Kishono–
Yano (HKY) models with a proportion of
invariable sites (+I) and rate of variation
across sites (+G) were selected for 16S rRNA
and COI mtDNA datasets, respectively. ML
tree was applied under the selected best-fit
models with 1,000 replicates. Pristipomoides
multidens (Lutjaniformes: Lutjanidae) was
chosen to root the ML and NJ tree
constructions in this study.
RESULTS AND DISCUSSION
Morphological and molecular
identification
Morphological identification
A total of 17 Lutjanus species were
morphologically identified, and tentatively
divided into four complexes (Rivas, 1996;
Miller, Cribb, 2007). Their morphologic
characters were presented in Table 1.
Black-spot snapper complex
Four species have been identified, the main
taxonomic character is a black spot on the
lateral line and below the soft part of the dorsal
fin (Figure 3.A1-D1). L. fulviflamma is the most
distinctive in possessing a series of six thin
yellow stripes on the side (Figure 3.A2). L.
russellii is quite similar to L. monostigma,
however, only the dorsal and anal fins of L.
russellii are yellow (Figure 3.B2), while all fins
of L. monostigma are yellow (Figure 3.C2). L.
johnii was distinguished from the others by the
largest black spot and each scale with a black
spot forming horizontal lines on the body
(Figure 3.D2).

Dinh Thi Hai Yen et al.
352

Figure 3. Images of four species belonging to black-spot snapper complex (Scale bar = 5 cm). (A) L.
fulviflamma; (B) L. russellii; (C) L. monostigma; (D) L. johnii.

Blued-lined snapper complex
Two species have been found, and
characterized in having conspicuous blue
lateral bands on the body (Figure 4.A1-B1).
L. quinquelineatus has two characters to
distinguish it from L. bengalensis. Instead of
four blue stripes on each side (Figure 4.A), it
has five (Figure 4.B1). It also has a dark spot
above lateral line and below soft dorsal fin
(Figure 4.B2).
Yellowed-lined snapper complex
Three species have been recorded, the
diagnosis character is numerous thin yellow
to brownish stripes on the body and with a
more prominent (usually wider or darker)
yellow or brown stripe mid-lateral running
along the side (Figure 4.C1-E1).
They can be differentiated by the
coloration and size of the mid-lateral stripe.
L. vitta most differs in having thin brown
stripes plus a broad dark brown mid-lateral
stripe (Figure 4.C2), whereas L. lutjanus and
L. xanthopinnis have thin yellow stripes with
a broad yellow mid-lateral stripe (Figure
4.D2-E2). L. lutjanus can be distinguished
from L. xanthopinnis in having a broader
mid-lateral stripe twice as wide as others vs.
a darker mid-lateral stripe as others.
Red snappers
Five species have been reported, which
possessing the reddish to pink body (Figure
5). Among them, L. gibbus, L. sebae and L.
timoriensis have distinct characteristics
from the other species. L. gibbus is
characterized by a forked caudal fin with
rounded lobes (Figure 5.A1) compare to
slightly emarginated in L. sebae (Figure
5.B) or truncate in L. malabaricus, L.
erythropterus and L. timoriensis (Figure
5.C-E). L. sebae differs in having three dark
red bands on the body (Figure 5.B1) and
15–16 soft rays of dorsal fin and 10 soft
rays of anal fin vs. 12–14 and 8–9 soft rays,
respectively as others (Table 1). L.
timoriensis is defined by elongation of
posterior dorsal- and anal-fin rays (Figure
5.E1). L. malabaricus look very similar to
L. erythropterus, and can be distinguished
by its larger head and mouth (Figure 5.D2).

Vietnam Journal of Biotechnology 21(2): 347-363, 2023
353

Figure 4. Images of two species belonging to blued-lined complex ((A) L. quinquelineatus, and (B) L.
bengalensis), and three species belonging to yellowed-lined complex ((C) L. vitta, (D) L. lutjanus, and
(E) L. xanthopinnis) (Scale bar = 5 cm).

Figure 5. Images of five species belonging to red snapper (Scale bar = 5 cm). (A) L. erythropterus,
(B) L. malabaricus, (C) L. gibbus, (D) L. sebae, (E) L. timoriensis.

Dinh Thi Hai Yen et al.
354
Other snapper species
Three species have been classified. L.
argentimaculatus differs most notably from
others by scale rows on the back parallel to
lateral line (Figure 6.A1). L. bohar is
characterized by two whitish spots on upper
back (Figure 6.B1). L. fulvus has a caudal fin
and outer of soft dorsal fin blackish (Figure
6.C1).

Figure 6. Images of other snapper species (Scale bar = 5 cm). (A) L. argentimaculatus, (B) L. bohar,
and (C) L. fulvus.

Table 1. Morphological and meristic characteristics of 17 Lutjanus species in Nghe An – Ha Tinh
coast water (main taxonomic characters were bold, N: number of individuals)
No. Species N
Meristic characteristics
Morphological characteristics
Dorsal-
fin rays
Anal-fin
rays
Pectoral
-fin rays
Black-spot complex
1 L. fulviflamma 5 X, 13 III, 8 16-17
Color of back and upper sides brown, lower
sides whitish or light brown, a black spot on
the lateral line below the soft part of the
dorsal fin (Figure 3.A1), 6 thin yellow
stripes on the sides (Figure 3.A2), and
longest stripe continuing through eye to
snout (Figure 3.A3). All fins are yellow.
2 L. russellii 3 X, 14 III, 8 16
Body color is grey. There is a black spot on
the lateral line below the soft part of the
dorsal fin (Figure 3.B1). Dorsal and caudal
fins are dark maroon, and other fins are
yellow (Figure 3.B2).
3 L. monostigma 3
X,
13-14
III, 8 16
Grey body on the upper sides and yellow
ventrally. A black spot on the lateral line
below the soft part of the dorsal fin (Figure
3.C1). All fins are yellow (Figure 3.C2).

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355
4 L. johnii 3
X,
13-14
III, 8 17
Body slight light brown, sides and below
whitish. A large black spot on the lateral
line below the soft part of the dorsal fin
(Figure 3.D1). Center of each scale with a
black spot forming horizontal lines on the
body (Figure 3.D2).
Blue-lined complex
5 L. bengalensis 3
X,
13-14
III, 8 16-17
Bright yellow with 4 blue stripes on sides (1
below eye) (Figure 4.A1).
6 L. quinquelineatus 3
X,
13-14
III, 8 16-17
Generally bright yellow, including fins. Five
longitudinal blue stripes on sides (Figure
4.B1), and large black spot mostly above
lateral line below soft dorsal fin (Figure 4.B2).
Yellow-lined complex
7 L. vitta 3
X,
12-14
III, 8-9 16-17
Body color is pinkish. A series of thin brown
horizontal stripes below lateral line and
oblique above lateral line (Figure 4.C1), with
a broad dark brown mid -lateral stripe
running along the side from the eye to the
caudal fin base (Figure 4.C2). All fins are
yellow except pelvic whitish.
8 L. lutjanus 3 X-XII, 12 III, 8 16-17
Body color is silvery white. A series of yellow
horizontal lines below lateral line and oblique
above lateral line (Figure 4.D1), with a broad
yellow stripe running along the side from the
eye to the caudal fin base (Figure 4.D2). All
fins are yellow except pelvic whitish.
9 L. xanthopinnis 3 X, 13 III, 8 15-16
Color pinkish to silvery-grey on the dorsum with
the whitish abdomen. A series of yellow
stripes along sides (obliquely above lateral line,
and horizontally below the lateral line) (Figure
4.E1), and mid-lateral stripe slightly wider
than others (Figure 4.E2). All fins yellow except
pelvic whitish.
Red snapper
10 L. gibbus 3
X,
13-14
III, 8 17
Body red to greyish, fins red to dusky, narrow
white margin to soft dorsal, caudal and anal
fins. Caudal fin forked (Figure 5.A1). An
orange hue to lower part of the opercle
and the pectoral fin axil (Figure 5.A2).
11 L. sebae 3
XI,
15-16
III, 10 17
Body red or pink, fins are red except the
pectorals pink. Three dark red bands (from
first dorsal spine through eye to tip of snout;
from mid-dorsal fin to pelvic fin; from base of
last dorsal spine to caudal peduncle) (Figure
5.B1).
12 L. malabaricus 5
XI,
12-14
III, 8-9 16-17
Body pinkish red, a black band across
caudal peduncle (Figure 5.C1). A large
mouth (length of upper jaw equal to distance

Dinh Thi Hai Yen et al.
356
between bases of last dorsal- and anal-fin
rays) (Figure 5.C2).
13 L. erythropterus 3
XI,
12-13
III, 8-9 16-17
Body pinkish red. Longitudinal scale rows
above lateral line obliquely positioned. A
large black spot at the caudal peduncle
(Figure 5.D1). A small mouth (length of
upper jaw smaller than distance between
base of last dorsal and anal rays) (Figure
5.D2).
14 L. timoriensis 3
XI,
14-15
III, 8 17
Body reddish. All fins reddish, and axil of
pectoral fin black. Posterior dorsal- and anal-
fin rays elongate (Figure 5.E1).
Other snapper
15 L. argentimaculatus 3
X,
13-14
III, 8 16-17
Color of the body dark reddish-brown on
back, grading to a reddish belly. Scale rows
on the back parallel to lateral line (Figure
6.A1).
16 L. bohar 3
X,
13-14
III, 8 16-17
Color of body dark brown with two whitish
spots on upper back (one below last four
dorsal spines and one under last six dorsal
rays) (Figure 6.B1). Scale rows on the back
rising obliquely above lateral line.
17 L. fulvus 3 X, 14 III, 8 16
Light yellow body. Caudal fin and outer of
soft dorsal fin blackish (Figure 6.C1) and
other fins are yellow.

Molecular identification
In this study, sequences were
successfully generated from each gene
region of 17 morphologically determined
Lutjanus species. Most sequences
exhibited more than 99% identity to the
sequences of the same species available on
Genbank database. Among that, 16S rRNA
reference sequences are not available for
three species (L. timoriensis, L.
xanthopinnis, and L. lutjanus), however,
the COI mtDNA sequence showed 100 %
matching (Table 2).
In this study, genetic characteristics are
very effective to verify the morphological
identification of 17 Lutjanus species
distributed in Nghe An - Ha Tinh provinces,
Northern Central, Vietnam. Previous studies
were mainly conducted in the Central and
Southern regions of Vietnam. Using 16S
rRNA molecular markers, Truong et al.
(2015) also verified the morphological
identification of 12 species belonging to 2
genera (Lutjanus and Paracaesio) at Kien
Giang, Vung Tau, Khanh Hoa and Da Nang.
Meanwhile, Pham et al. (2019) used COI
barcode to identify 18 species (6 genera) in
Ninh Thuan. Nguyen, Xuan (2020)
investigated the Lutjanus species composition
in the Central region (Quang Tri - Binh
Thuan) based on morphological
characteristics.

Vietnam Journal of Biotechnology 21(2): 347-363, 2023
357
Table 2. Species composition and the comparison of sequences (16S rRNA and COI mtDNA) with
Genbank database.
No. Species studied
16S rRNA COI mtDNA
Species
identification
%
similarit
y
Accession
no.
Species
identification
%
similarity
Accession
no.
1 L. fulviflamma L. fulviflamma 100 DQ784731 L. fulviflamma 100 EU502683
2 L. erythropterus L. erythropterus 99.7 NC_031331 L. erythropterus 100 GU673841
3 L. gibbus L. gibbus 100 DQ784733 L. gibbus 100 OQ387116
4 L. malabaricus L. malabaricus 100 NC_012736 L. malabaricus 99.9 ON394557
5 L. sebae L. sebae 100 DQ784738 L. sebae 100 MN870188
6 L. johnii L. johnii 100 NC_024572 L. johnii 100 KJ013052
7 L. argentimaculatus L. argentimaculatus 100 LC508391 L. argentimaculatus 100 MN243478
8 L. bohar L. bohar 100 DQ784729 L. bohar 99.7 GU673902
9 L. russelli L. russelli 100 DQ784737 L. russelli 100 OQ387794
10 L. fulvus L. fulvus 100 DQ784732 L. fulvus 99.7 KU176437
11 L. vitta L. vitta 100 NC_042930 L. vitta 99.6 OQ385788
12 L. timoriensis
L. malabaricus 96.4 NC_012736 L. timoriensis 100 OQ387106
L. sebae 95.5 NC_012737
13 L. xanthopinnis
L. ophuysenii 99.5 NC_056806 L. xanthopinnis 100 JN311964
L. ehrenbergii 98.6 OR123973
14 L. quinquelineatus L. quinquelineatus 100 DQ784736 L. quinquelineatus 100 KC970484
15 L. lutjanus
L. ophuysenii 98.1 NC_056806 L. lutjanus 100 MN870571
L. carponotatus 97.6 NC_044104
16 L. monostigma L. monostigma 100 LC508471 L. monostigma 100 MN562555
17 L. bengalensis L. bengalensis 100 NC_011275 L. bengalensis 100 OL512911

Although taxonomic literatures
available for Lutjanus species (Allen,
1985; Nelson, 2006) misidentification was
reported due to overlapping of
morphometric and meristic characteristics,
or the change during its development
stages (Allen, 1985). For instance,
Lutjanus malabaricus is often
misidentified as L. sanguineus or L.
erythropterus (Guo et al., 2007; Halim et

Dinh Thi Hai Yen et al.
358
al., 2022), L. xanthopinnis as L. ophuysenii
(Iwatsuki et al., 2015), L. alexandrei as
either L. griseus or L. apodus (Moura,
Lindeman, 2007). In our study, it is
possible that the two species (L.
malabaricus and L. erythropterus) may be
morphologically confused because they
both belong to the "red snapper" group.
The outstanding feature to distinguish
these two species is that species (L.
erythropterus) has a "back spot", while
species (L. malabaricus) has a "back band"
at the caudal peduncle (Figure 5.C1&5D1).
This characteristic can be easily
distinguished when the sample is fresh, but
can cause confusion in preserved samples.
Fortunately, the sequence of the two genes
has supported the identification of these
two species with up to 99.7-100% sequence
identity (Table 2). Clearly, DNA-based
molecular markers are proved as effective
tool to overcome the issue of
morphological identification, and provided
insight into the evolutionary relationship of
species (Avise, 1994).
Phylogenetic relationships
The length of the aligned sequences was
627 bp and 652 bp for 16S rRNA and COI
genes, respectively. All of the datasets
passed the Xia test, indicating that there
was no substantial substitution saturation.
In both datasets, the topologies of NJ and
ML were similar and thereby we only show
the NJ topology. Only bootstrap values of
two approaches above 70% are displayed at
the nodes (Figure 7).
For the 16S rRNA gene, the
phylogenetic analysis displayed two main
lineages (Figure 7A). The first lineage
included basal clade of three blue-lined
species (L. quinquelineatus, L.
bengalensis, and L. kasmira). The second
lineage was divided into two groups.
Group 2.1 containing four species (L.
fulviflamma, L. monostigma, L. russelli,
and L. carponotatus), which clustered as a
sister group of three yellow-lined species
(L. lutjanus, L. vitta, and L. xanthopinnis).
In Group 2.2, three species (L.
erythropterus, L. gibbus and L. bohar)
were clustered together, as a sister group to
four species (L. argentimaculatus, L.
sebae, L. malabaricus and L. timoriensis).
Two species (L. fulvus and L. johnii) and L.
stellatus from Genbank (Accession number
NC-057609) performed the unidentified
position in the phylogenetic tree.
In the COI phylogram, two main
lineages were detected (Figure 7B). A
basal clade includes four red snapper
species (L. erythropterus, L. timoriensis, L.
malabaricus, and L. sebae) (first lineage).
In second lineage, the remaining lutjanids
species were clustered into three groups.
Members of Group 2.1 are similar as in
16S rRNA phylogenetic tree. Group 2.2
consisted of two species ( L.
argentimaculatus, and L. stellatus), and
placed a sister group to four species (L.
fulvus, and L. quinquelineatus, L.
bengalensis, and L. kasmira). Group 2.3 is
composed of two species (L. bohar and L.
gibbus). The unidentified position of L.
johnii also was shown.

Vietnam Journal of Biotechnology 21(2): 347-363, 2023
359

Figure 7. Phylogenetic tree of Lutjanus spp. species resulting from the Neighbor Joining analysis of
16S rRNA (A) and COI mtDNA (B) datasets. Bootstrap values (≥70%) from NJ/ ML along the branch
(*sequences from Genbank).


In comparation, two phylogenetic trees
showed the inconsistent basal clade, either
three blue-lined species on the 16S tree
(Figure 7A) or four red snapper species from
the COI topology (Figure 7B).
Morphologically, the red snapper species
consisted of five species. In COI
phylogenies, L. gibbus is sister species to L.
bohar (Group 2.3), while in 16S topology,
they clustered in the Group 2.2, and also
displayed the sister relationship to L. bohar
and L. argentimaculatus.
In both analyses, L. johnii showed the
unidentified position. This species has a
black spot above the lateral line and below
the anterior portion of the soft dorsal fin
(Table 1, Figure 3.D). Based on those
taxonomic characters, L. johnii should be
subsumed within Group 2.1 of the black-
spot species. This unknown taxonomic
position of L. johnii was also recorded in
previous studies using the COI mtDNA
(Velamala et al., 2019) and 16S rRNA
(Ramadan et al., 2023) genes. Additionally,

Dinh Thi Hai Yen et al.
360
L. fulvus also could not be determined on the
16S tree, while COI phylogeny placed it in
the three blue-lined species group, which is
consistent with other studies (Gold et al.,
2015; Frédérich, Santini, 2017; Velamala et
al., 2019).
Group 2.1 - contained two sister clades:
i) three black spot species (L. fulviflamma, L.
monostigma, and L. russelli) and L.
carponotatus - yellow-lined species), and ii)
three yellow-lined species (L. lutjanus, L.
vitta, and L. xanthopinnis) - is consistent in
both analyses. In this group, L. carponotatus
has a series of eight or nine orange or yellow
stripes on the sides, that is the diagnostic
feature of the yellow-lined species complex
(Allen, 1985). However, L. carponotatus
performed closely related relationship to the
black-spot species. This result has already
been reported by previous studies based on
16S rRNA and cytb mitochondrial genes
(Miller, Cribb, 2007) or mitogenomes (Kim
et al., 2019). Miller, Cribb (2007) stated that
the lack of the black spot in L. carponotatus
may be a secondarily derived loss of this
character.
In conclusion, based on the two
phylogenetic trees, no species complex
relationship was completely resolved.
Members of species complexes also showed
a certain degree of closeness; however,
conflicts between two topologies have also
been recorded. Thereby, we can see the
necessity of applying multiple markers or
complete genomes in phylogenetic analysis,
along with investigate diagnostic
characteristics in species identification,
or/and determined species complexes.
CONCLUSION
In this study, 17 Lutjanus species
distributed in Nghe An - Ha Tinh
provinces, Northern Central, Vietnam were
morphologically and molecular identified.
Based on 16S rRNA and COI mtDNA
markers, both phylogenetic trees detected
two main lineages, and showed
inconsistent basal groups. The
morphologic species complex was not
completely resolve, despite of their
members showed a certain degree of
closely relationships. Thereby, we
highlight the necessity of applying multiple
markers or complete genomes in
phylogenetic analysis, along with
investigate diagnostic characteristics in
species identification, or/and determined
species complexes.
Acknowledgements. This research was
funded by Coastal Branch of Joint Vietnam -
Russia Tropical Science and Technology
Research Center through project E 3.1-15
“The study of the reproductive biology of
Malabar blood snapper Lutjanus
malabaricus in the coastal waters of Central
Vietnam for the conservation, reproduction
and sustainable use”.
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