Uploaded by Abdul Hameed

ZSC 12428 Rev1

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/341783617
Molecular phylogeny and diversity of penaeid shrimps (Crustacea: Decapoda)
from South‐East Asian waters
Article in Zoologica Scripta · May 2020
DOI: 10.1111/zsc.12428
5 authors, including:
Amirah Hurzaid
M. Nor Siti-Azizah
Universiti Sains Malaysia
Universiti Malaysia Terengganu
Zainal A. Muchlisin
Wei-Jen Chen
Syiah Kuala University
National Taiwan University
Some of the authors of this publication are also working on these related projects:
Species discrimination of Carangoides spp. complex using molecular and morphological approach View project
Assessing the nutritional value of selected prebiotics and probiotics with respect to growth and health status of Pangasianodon hypophthalmus juveniles. View project
All content following this page was uploaded by Amirah Hurzaid on 30 October 2020.
The user has requested enhancement of the downloaded file.
Received: 8 January 2020
DOI: 10.1111/zsc.12428
Revised: 15 March 2020
Accepted: 14 April 2020
Molecular phylogeny and diversity of penaeid shrimps
(Crustacea: Decapoda) from South-East Asian waters
Amirah Hurzaid1,2
| Tin-Yam Chan3 | Siti Azizah Mohd Nor4
Zainal Abidin Muchlisin5 | Wei-Jen Chen1
Institute of Oceanography, National
Taiwan University, Taipei, Taiwan
Biological Sciences Department, School
of Distance Education, Universiti Sains
Malaysia, Penang, Malaysia
Institute of Marine Biology and Center of
Excellence for the Oceans, National Taiwan
Ocean University, Keelung, Taiwan
Institute of Marine Biotechnology,
Universiti Malaysia Terengganu, Kuala
Terengganu, Malaysia
Faculty of Marine and Fisheries, Syiah
Kuala University, Banda Aceh, Indonesia
Wei-Jen Chen, Institute of Oceanography,
National Taiwan University, No. 1, Sec. 4,
Roosevelt Road, Taipei 10617, Taiwan.
Email: wjchen.actinops@gmail.com
Funding information
Ministry of Higher Education,
Malaysia, Grant/Award Number: 203/
PBIOLOGI/6711455; Ministry of Science
and Technology, Taiwan, Grant/Award
Number: MOST 107-2611-M-002-007,
108-2611-M-002-012-MY2 and MOST
The decapod family Penaeidae comprises most of the economically important marine
shrimp species. Its members are widespread throughout the world, with its highest
species diversity centred in the Indo-West Pacific region. Despite this importance,
their taxonomy, classification and phylogeny are not yet settled due in part to incongruence among hypotheses proposed from molecular versus morphological studies. In this study, using a thorough taxonomic sampling of especially the South-East
Asian species, we aim to (a) utilize a reconstructed phylogeny to test the monophyly
of the Penaeidae and its currently recognized genera and (b) explore its species diversity in South-East Asian waters. To infer the phylogeny, a combined gene data
set (including 109 ingroup and six outgroup taxa) of mitochondrial genes, COI and
16S rRNA, and two nuclear genes, NaK and PEPCK, was utilized. To explore its
diversity, another data set that included 371 COI gene sequences (231 newly generated and 140 retrieved from public sources) was compiled and subsequently analysed with two different tools (ABGD and bPTP) for species delimitation. Other
than supporting the non-monophyly of the Penaeidae with the Sicyoniidae nested
within the penaeid tribe Trachypenaeini, the genera Penaeus, Mierspenaeopsis and
Parapenaeopsis were also revealed to be polyphyletic. Our species delimitation analysis inferred that 94 putative species actually existed within the 71 morphospecies
reviewed, indicating an underestimated biodiversity in this family and the potential
presence of new species within the following morphospecies: Kishinouyepenaeopsis
cornuta, K. incisa, Mierspenaeopsis sculptilis, M hardwicki, Parapenaeopsis coromandelica and Penaeus monodon.
integrated approach in taxonomy, Penaeidae, phylogeny, South-East Asia, species delimitation
The shrimp species from the family Penaeidae RafinesqueSchmaltz, 1815, are among the most economically important
crustaceans (Chan, 1998; Dall, Hill, Rothlisberg, & Sharples,
1990; Holthuis, 1980; Pérez Farfante & Kensley, 1997). They
Zoologica Scripta. 2020;00:1–18.
are present in tropical and subtropical shallow and inshore
waters with their highest species diversity occurring in the
Indo-West Pacific (IWP) region. The family is considered a
‘primitive’ group of decapod crustaceans (Abele, 1991; Dall
et al., 1990; Schram, 1977, 1982). Dall et al. (1990) estimated there were 200 species of penaeids classified under
© 2020 Royal Swedish Academy of Sciences
17 genera. Later, Pérez Farfante and Kensley (1997) revised
the taxonomy and recognized 217 species, and split the previously defined genus Penaeus sensu lato Fabricius, 1798,
and genus Trachypenaeus sensu lato Alcock, 1901, into six
and four genera, respectively, to make 26 genera. In the most
recent revision, Sakai and Shinomiya (2011) delimited the
formerly defined genus Parapenaeopsis sensu lato (Alcock,
1901) into eight genera, thus introducing seven new genera.
The family Penaeidae now comprises 32 recognized genera with 224 known species (Chan, Cleva, & Chu, 2016;
De Grave & Fransen, 2011; Gusmão, Lazoski, Monteiro,
& Solé-Cava, 2006; Ma, Chan, & Chu, 2011; Tavares &
Gusmao, 2016; Timm et al., 2019; Tsoi et al., 2014; Yang,
Sha, Chan, & Liu, 2015). After compiling all the taxonomic
data available on the Penaeidae, a total of 96 species in 21
genera should occur in South-East Asian (SEA) waters. The
species number of SEA penaeids is probably underestimated,
as comprehensive surveys in the area are still rather limited
and cryptic or new species are likely present.
Based on morphology, two schemes have been proposed
for a higher-level classification of the Penaeidae. Kubo
(1949) divided the family into five groups (without proper
naming), but did not delve into details on their inter-relationships except to suggest that the group harbouring Penaeus
and Miyadiella was the most basal. Burkenroad (1983) divided the family into three different tribes, namely Penaeini,
Parapenaeini and Trachypenaeini, and placed Penaeini at the
basal position. In recent decades, several phylogenetic studies have been conducted to evaluate these two competing
‘phylogenetic’ hypotheses, with results that often supported
Burkenroad's three-tribe hypothesis (Quan, Zhuang, Deng,
Dai, & Zhang, 2004; Vazquez-Bader, Carrero, Garcia-Varela,
Grcia, & Laclette, 2004; Voloch, Freire, & Russo, 2005),
yet several uncertainties or conflicts still remained. For instance, in Chan, Tong, Tam, and Chu (2008), 16S rRNA sequences obtained from representative taxa from 20 of the
26 currently recognized genera phylogenetically inferred
that two members from the Trachypenaeini (Atypopenaeus
and Trachypenaeopsis) were grouped with Parapenaeini
and Penaeini, respectively, making the Trachypenaeini
polyphyletic. Ma, Chan, and Chu (2009) employed a new
data set of two nuclear protein-coding genes of phosphoenolpyruvate carboxykinase (PEPCK) and sodium–potassium ATPase a-subunit (NaK), revealing that Atypopenaeus
clustered with most of the sampled Trachypenaeini, in contrast to the findings of Chan et al. (2008). In addition, they
found that the genus Trachypenaeopsis was placed outside
the Trachypenaeini clade and was sister to Sicyoniidae with
moderate support, making the tribe Trachypenaeini and also
the family Penaeidae paraphyletic. Most recently, Cheng,
Chan, Zhang, Sun, and Sha (2018) employed mitochondrial
genome data for the same purpose, and their analytical results
also supported the non-monophyly of the Trachypenaeini
HURZAID et al.
and Penaeidae. Accordingly, the authors suggested elevating
the three penaeid tribes into familial status, with the family
Sicyoniidae being retained to resolve this taxonomic dispute.
For the generic classification, and following the suggestion of Pérez Farfante and Kensley (1997), by separating shrimps formerly classified under the long-accepted
genus Penaeus sensu lato into six genera (Penaeus sensu
stricto, Fenneropenaeus, Farfantepenaeus, Litopenaeus,
Marsupenaeus and Melicertus), much debate was devoted to
this new proposition. Since there was insufficient morphological evidence to support the monophyly of the proposed
genera (Davie, 2002; Flegel, 2008), many authors questioned
whether the proposed scheme reflected the evolutionary relationships among the 29 species classified under Penaeus
s.l. (Dall, 2007; Flegel, 2007, 2008; McLaughlin, Lemaitre,
Ferrari, Felder, & Bauer, 2008). To resolve this dilemma,
several advanced molecular phylogenetic studies were conducted over the past two decades.
The first molecular study to test this classification was
pioneered by Baldwin, Bass, Bowen, and Clark (1998).
Based on the mitochondrial COI sequences of 13 Penaeus
s.l. species, the authors found evidence that challenged
the monophyly of Melicertus, Penaeus s.s., Litopenaeus
and Farfantepenaeus. The lineage containing taxa from
Melicertus and Marsupenaeus occupied the basal-most position in the inferred COI gene tree. These results were further
supported by a study of Gusmão, Lazoski, and Solé-Cava
(2000), with COI (mostly retrieved from Baldwin's study)
and new data from 11 isozyme loci. Conversely, another attempt on phylogenetic reconstruction using partial sequences
of 16S rRNA (Maggioni, Rogers, Maclean, & D'Incao, 2001)
showed opposing results that strongly supported the monophyly of Farfantepenaeus and Litopenaeus. However, a comprehensive view of interspecific relationships within each
genus was limited in these studies due to constraints in taxon
and character sampling. Later, Lavery, Chan, Tam, and Chu
(2004) analysed a concatenated data set of 16S rRNA and
COI sequences from 26 of the 28 Penaeus s.l. species and
confirmed the monophyly of all of the included genera with
strong bootstrap support except for Melicertus (paraphyletic
with respect to Marsupenaeus) and Penaeus s.s. (paraphyletic
with respect to Fenneropenaeus). Moreover, the division of
Penaeus s.l. into two clades (Melicertus + Marsupenaeus and
Fenneropenaeus + Farfantepenaeus + Litopenaeus + Penae
us s.s.) always had strong support. In conclusion, these molecular studies indicated that the six-genus scheme for classifying the Penaeus s.l. species as proposed by Pérez Farfante
and Kensley (1997) should be further revised. In the most
recent molecular study, Ma et al. (2011) conducted a comprehensive phylogenetic analysis of Penaeus s.l. using data
from three nuclear protein-coding genes (enolase, PEPCK
and NaK) and two mitochondrial ribosomal genes, 12S and
16S rRNA, from 18 sampled species of Penaeus s.l. and 13
HURZAID et al.
other penaeid species plus three outgroups from the families Aristeidae, Solenoceridae and Sicyoniidae. Their results
supported the hypothesis proposed by Lavery et al. (2004)
and advocated for the restoration of the originally defined
Penaeus genus (= Penaeus s.l.).
From a morphological point of view, Sakai and
Shinomiya (2011) delimited another formerly defined genus,
Parapenaeopsis s.l., into eight genera, thus introducing
seven new genera: Alcockpenaeopsis, Arafurapenaeopsis,
Batepenaeopsis, Ganjampenaeopsis, Holthuispenaeopsis,
Kishinouyepenaeopsis and Mierspenaeopsis. The revised
classification was based only on the structures of the male
petasma and male second pleopods, without considering any
characters from the female thelycum. Thus, Chanda (2016)
argued that the Sakai and Shinomiya (2011) classification
is impractical, and rearranged the various genera proposed
for Parapenaeopsis s.l., including replacing Mierspenaeopsis
with the new name Helleropenaeopsis (but this is invalid according to the International Code of Zoological Nomenclature
because Helleropenaeopsis is a synonym of Mieropenaeopsis
and they both have the same type species).
The first molecular work to test this classification was
based on data from COI and 16S rDNA sequences using
only six species from four of eight redefined Parapenaeopsis
s.l. genera collected from the East China Sea and the South
China Sea (Li, Xu, & Kou, 2014). Their results revealed with
high support that Kishinouyepenaeopsis comprising K. sinica, K. cornuta and K. incisa formed an independent and advanced lineage in the inferred tree while the other three species
(Batepenaeopsis tenella, Mierspenaeopsis cultirostris and
Alcockpenaeopsis hungerfordi) formed another clade with
moderate bootstrap support. In a recent study, Chowdhury,
Shanis, Chelath, Pavan-Kumar, and Krishna (2018) inferred
the phylogenetic relationships of five species in three genera
from Parapenaeopsis s.l. collected from Indian waters based
also on a combined data set of mitochondrial COI and 16S
rDNA sequences. Their results showed that Parapenaeopsis
(represented by P. coromandelica and P. stylifera) was monophyletic while Mierspenaeopsis was not. Mierspenaeopsis
sculptilis was resolved as a sister to Ganjampenaeopsis uncta
rather than M. hardwickii, with high bootstrap support. These
previous studies all indicated that the classification of the
Penaeidae at the generic level (and below) required further
The study of penaeid taxonomy is very challenging due to
the fact that accurate species identification in crustaceans in
general is difficult because conspecific individuals are phenotypically variable at different ontogenetic stages, between
genders, and sometimes in different environmental conditions
(Hebert, Ratnasingham, & de Waard, 2003). In spite of that,
many researchers still rely solely on the traditional method
(i.e. morphology) for recognition and identification of shrimp
and prawn species. Although morphology is the cornerstone
of taxonomic classification, the distinguishing traits in different taxa may vary with classification schemes, which are
not always homologous and provide limited phylogenetic
information (Avise, Nelson, & Sibley, 1994; Tassanakajon
et al., 2006). Consequently, taxonomic work on most shrimps
is sometimes unsuccessful and ambiguous, and species assignment may be erroneous (Hebert et al., 2003; Rajkumar,
Bhavan, Udayasuriyan, & Vadivalagan, 2015). These problems could be circumvented by using molecular characters
that not only provide complementary information for a more
precise means of taxonomic identification, but also valuable
information for studying phylogenetic, population genetics
and demography (Avise, 2012; Lo, Liu, Nor, & Chen, 2017).
In 2003, molecular identification for species was expedited by the ‘DNA barcoding’ technique introduced by
Hebert and his colleagues, which aimed to screen a single or
a few reference genes in order to assign unknown specimens
to known species and enhance the discovery of new species
(Hebert et al., 2003; Moritz & Cicero, 2004; Sharawy, Abbas,
Desouky, & Kato, 2016). Since then, DNA barcoding has
been used in various applications, including an assessment
of species occurrence in certain ecological systems (Page,
Humphreys, & Hughes, 2008) and unravelling the commercial fraud of peeled shrimp products where their original
morphology could not be ascertained (Galal-Khallaf, Ardura,
Borrell, & Garcia-Vazquez, 2016).
In the present study, molecular markers from mitochondrial 16S and COI as well as nuclear PEPCK and NaK genes
were used for phylogenetic inference in order to test both
the higher-level classification and phylogenetic hypotheses
of the Penaeidae with an extensive sampling from the SEA
taxa. In addition to phylogenetic exploration, we employed
an integrated approach in taxonomy by considering both
morphological and molecular evidences to resolve the taxonomic issues and subsequently the discovery of new species
of the Penaeidae. Here, a standard DNA barcoding procedure
incorporated with a more advanced analytical approach (i.e.
species delimitation) using COI gene was utilized to explore
the species diversity of SEA penaeids.
Sample collection
Sample collections in South-East Asia and Taiwan were
carried out between 2015 and 2018 either by direct trawling using research vessels in collaboration with the Fisheries
Research Institute, Malaysia, and Fisheries Research
Institute, Kinmen County, Taiwan, or by purchasing specimens from local fish landing sites. A total of 228 collected
shrimp specimens from 32 SEA localities (Figure S1; Table
S1) were examined. Individuals were identified to the species
HURZAID et al.
level wherever possible using morphological keys, notably Chan (1998) and Lovett (1981). Specimens that could
not be identified morphologically (juvenile specimens, unknown species, etc.) were preliminarily identified to the
genus level and then re-evaluated after COI gene analyses.
Freshly collected specimens were photographed and recorded per the DNA barcoding process, and muscle tissue
from specimen's pleopods was cut, preserved in 95% ethanol and stored at −20°C for subsequent molecular work. All
voucher specimens were preserved and deposited in invertebrate collections at the National Taiwan University Museums
(NTUM_INV), Taipei, Taiwan. In addition, tissue samples
from three specimens of Kishinouyepenaeposis cornuta from
India (deposited in the Department of Aquatic Biology &
Fisheries, University of Kerala, DABFUK) and the type locality from Japan (deposited in the Natural History Museum
and Institute, Chiba, CBM) were used to conduct molecular
Molecular data collection
Total genomic DNA was extracted from pleopod muscle
using the automated extractor LabTurbo 48 Compact System
and LGD 480-220 kits (Taigen Biosciences Corp.) following the manufacturer's protocol. Mitochondrial COI and 16S
genes and nuclear protein-coding genes NaK and PEPCK
were the gene markers targeted for molecular analyses in this
study. To amplify each gene fragment, a polymerase chain
reaction (PCR) was carried out in a 25 μl volume consisting
of 5X Green GoTaq® Flexi Buffer, 25 mM MgCl2 solution,
2.5 μM dNTP, 10 mM of each primer (see details below),
0.1 μl GoTaq® DNA Polymerase (Promega), 1–3 μl DNA
template (concentration of ~10 ng/μl) and deionized water
to volume. Cycling parameters consisted of an initial denaturation of 5 min at 95°C, followed by 35 cycles of denaturation (95°C, 1 min), annealing (53°C for COI and 16S rRNA
and 50°C for NaK and PEPCK, 1 min), 1.30 min at 72°C
and a final extension of 5 min at 72°C. PCR products were
then purified using the AMPure magnetic bead cleanup protocol (Agencourt Bioscience Corp). Purified PCR products
were Sanger-sequenced using dye-labelled terminators and
dye-labelled fragment read on ABI 3730 analysers (Applied
Biosystems) at Genomics BioSci and Tech (Taipei) and the
Center of Biotechnology (National Taiwan University).
Initially, we used the pair of universal primers for the COI
gene following Folmer, Hoeh, Black, and Vrijenhoek (1994),
but they failed to amplify certain species targeted in this study
even though we paired the reverse primer (HCO2198) with
one of the two forward primers, CRUSTF1 and CRUSTF2
as suggested in Costa et al. (2007). Therefore, we used the
second primer sets following Palumbi et al. (1991). However,
a problem arose when we compared the obtained sequences
with reference sequences from the GenBank database. We
noticed that the COI sequences deposited in GenBank for
penaeid species belonged to fragments from two different regions with limited overlap sequences, named ‘Folmer region’
and ‘Palumbi region’ (Figure S2). To amend this, we designed
a new set of COI primers for the Penaeidae named Penae_
GCA TCT-3′). The amplified fragment spanned both regions
and covered a nearly complete COI gene sequence. Universal
primers 16Sar and 16Sbr (Palumbi et al., 1991) were used for
16S. To amplify and sequence the two nuclear gene markers,
forward primer ‘NaK for- a’ and reverse primer ‘Nak rev 2’
were used for the NaK gene, and forward primer ‘PEPCK for’
and reverse primer ‘PEPCK rev’ were used for the PEPCK
gene (Tsang, Ma, Ahyong, Chan, & Chu, 2008).
Data assembly
The obtained DNA sequences were assembled and edited
using CodonCode Aligner v.6.0.2 (CodonCode Corporation).
The sequences (usually at both ends) with low-quality or
problematic base calls (below phred quality value Q 20)
were verified by eye and manually trimmed. Edited sequences were compiled into two data sets (see below) and
manually aligned based on inferred amino acid translation
(for protein-coding genes) with Sequence Alignment Editor
(Se-Al) v2.0a11 (Rambaut, 2002). The resulting sequences
were blasted (Basic Local Alignment Search Tool, BLAST,
National Center for Biotechnology Information, NCBI)
against the reference data in GenBank to check for potential
contamination. Sequences of COI, NaK and PEPCK were
translated into corresponding amino acids by Se-Al to check
for the inclusion of pseudogenes (Song, Buhay, Whiting, &
Crandall, 2008). Obtained sequences were deposited at NCBI
(Nation Center for Biotechnology Information) GenBank
(Accession Nos: COI [MT178513–MT178743], 16S rRNA
[MT155937–MT155985], NaK [MT176243–MT176292],
PEPCK [MT176293–MT176342]), as shown in Table S2. An
additional 169 COI gene sequences, 57 16S gene sequences
and 103 nuclear gene sequences of penaeid taxa retrieved
from the GenBank were added into the data sets for further
molecular analyses (Table S3 and Table S4).
2.4 | Data sets and phylogenetic
Two types of data sets (combined and COI gene only) were
employed for subsequent analyses. The combined data set
included sequences from four genes (COI, 16S rRNA, NaK
and PEPCK) and was subject to higher-level phylogenetic
HURZAID et al.
inference carried out with 109 selected taxa from 98 species
of 26 penaeid genera (Tables S2 and S3). Examined taxa
were determined based on the phylogenetic results of the COI
gene data set, with one representative selected from each resolved genetic lineage from the inferred tree. The COI gene
data set consisted of 371 sequences of SEA shrimp samples
and was primarily used for evaluating the validity of penaeid
species and species diversity through phylogenetic and species delimitation analyses (see below). The sequences used
were from 71 morphospecies that were either retrieved from
GenBank or newly generated in this study (Tables S2 and
S4). PAUP* software, version 4.0 (Swofford, 2003), was
used to calculate descriptive statistics (sequence variations,
base composition, etc.) for the compared sequences of each
gene in the two data sets.
For phylogenetic inference, both partitioned maximum-likelihood (ML) method and Bayesian inference (BI)
were employed. The ML analysis was conducted by using
RAxML v. 8. 0 (Stamatakis, 2014) with the nucleotide
substitution model GTR + G. Partitions were set by gene
and protein-coding position. Each analysis was conducted
with ten independent runs, and the tree with the best ML
score was selected as the final tree. BI was performed using
MrBayes v. 3.2.6 (Ronquist et al., 2012) on the CIPRES
Science Gateway (Miller, Pfeiffer, & Schwartz, 2010).
Partition finder (Lanfear, Calcott, Ho, & Guindon, 2012)
was used to select the best partition scheme and accompanying substitution model according to the Bayesian information criterion (Table S5). Metropolis-coupled Monte
Carlo Markov chains (MCMCs) were run for 30 million
generations in two simultaneous runs, each with four differently chains. Convergence of the estimates was checked
by the standard deviation of split frequencies and by monitoring of the likelihood scores over time using Tracer v.1.6
(Rambaut, Suchard, Xie, & Drummond, 2014). Trees were
sampled every 1,000 generations, and the first 2,500 (25%)
were discarded as ‘burn-in’. The remaining sampled trees
were collected to construct a 50% majority-rule consensus
tree of BI. Nodal support of our phylogenetic inference was
assessed by bootstrapping (BP) (Felsenstein, 1981) with
the ML criterion, based on 1,000 pseudoreplicates and the
resulting a posteriori probabilities (PP) from BI.
Obtained trees were visualized and edited with FigTree
v1.4.0 (Rambaut, 2012). We tested the resulting Penaeidae
monophyly by including representative taxa from other
Penaeoidea families, namely Aristaeus mabahissae
(Aristeidae), Benthonectes filipes (Benthesicyimidae),
Sicyonia lancifer (Sicyonidae) and Solenocera crassicornis (Solenoceridae), in the combined gene data set
analyses. Two distantly related outgroups, Acetes sp. and
Robustosergia robusta, were chosen by following the phylogenetic results of Ma et al. (2009) to root the inferred
Species delimitation
In this study, analyses of the COI gene data set to delimit
species were performed using two different tools: Automatic
Barcode Gap Discovery (Puillandre, Lambert, Brouillet, &
Achaz, 2012) and Bayesian Poisson Tree Processes (Zhang,
Kapli, Pavlidis, & Stamatakis, 2013). Automatic Barcode
Gap Discovery (ABGD) is an exploratory tool based on pairwise distances of compared sequences to automatically detect
the significance between intra- and interspecific variation
(called barcode gap). The goal of ABGD is to improve and
redefine groups (putative species or operational taxonomic
units [OTUs]) based on sequence variation. The program first
partitions data into groups based on a statistically inferred
barcode gap and then the same procedure is recursively applied to the groups obtained in the first step. This analysis is
performed at a web interface (http://wwwabi.snv.jussi​eu.fr/
publi​c/abgd/) using a default value of relative gap width (X)
as 1.0, intraspecific divergence (P) values from 0.001 to 0.1
with 20 steps and the K2P model (Kimura, 1980) for pairwise
distance measurement.
The Bayesian Poisson Tree Processes (bPTP) model infers
putative species boundaries on a given phylogenetic tree. The
model delimits species based on a phylogenetic species criterion, assessing speciation or branching events in terms of the
number of mutations. bPTP analysis was conducted using a
phylogenetic input tree. This tree was reconstructed by using
a partitioned maximum-likelihood (ML) method based on
the COI data set (by removing the repeated sequences with
the same haplotype) with the GTR + G substitution model
as implemented in RAxML v.8.0 (Stamatakis, 2014). The
estimation of penaeid species delimitation with the bPTP
model was conducted on the bPTP web server (http:/speci​es.h-its.org/ptp/) with 500,000 MCMC generations and default
parameters. The topology of the inferred phylogenetic tree
based on the COI data set (i.e. resolved monophyletic groups)
and genetic similarity estimated by K2P genetic distances
(Kimura, 1980) using MEGA6 (Tamura, Stecher, Peterson,
Filipski, & Kumar, 2013) were used to check whether ABGD
and bPTP over- or underpredicted OTUs. Congruent results
between bPTP and ABGD were considered to be robust support of the resulting OTUs, thus providing final justification
for species inferences based on molecular data.
Due to the presence of two COI sequence regions in
GenBank with limited overlap, ABGD failed to compare
these two regions in the initial run. Therefore, two data sets
were used in the species delimitation analyses. The first data
set was comprised of the COI sequences generated in this
study plus the Folmer region from GenBank. The second
data set included sequences generated in this study plus the
GenBank Palumbi region. The number of OTUs generated
from both data sets was then compared, and final validation was made. Finally, as the establishment of reproductive
HURZAID et al.
Robustosergia robusta
Acetes chinensis
Benthonectes filipes
Aristeus mabahissae
Solenocera crassicornis
Pelagopenaeus balboae
Funchalia taaningi
Funchalia villosa
Heteropenaeus longimanus
Marsupenaeus japonicus
Marsupenaeus pulchricaudatus
Melicertus marginatus
Melicertus canaliculatus
Melicertus latisulcatus
Melicertus hathor
Melicertus plebejus
Melicertus longistylus
Melicertus kerathurus
Penaeus semisulcatus
Penaeus I
Farfantepenaeus aztecus
Farfantepenaeus californiensis
Farfantepenaeus paulensis
Litopenaeus stylirostris
Litopenaeus vannamei
Litopenaeus schmitti
Litopenaeus setiferus
Penaeus cf. monodon
Penaeus monodon I
Penaeus II
Penaeus monodon II
Penaeus esculentus
Penaeus III
Fenneropenaeus chinensis
Fenneropenaeus indicus
Fenneropenaeus penicillatus
Fenneropenaeus silasi
Fenneropenaeus merguiensis I
Fenneropenaeus merguiensis II
Metapenaeopsis provocatoria longirostris
Metapenaeopsis mogiensis mogiensis
Metapenaeopsis mogiensis intermedia
Metapenaeopsis toloensis
Metapenaeopsis palmensis
Metapenaeopsis sinica
Metapenaeopsis stridulans
Metapenaeopsis acclivis
Metapenaeopsis barbata
Metapenaeopsis coniger
Metapenaeopsis liui
Metapenaeopsis andamensis
Metapenaeopsis commensalis
Metapenaeopsis evermanni
Metapenaeopsis lamellata
Metapenaeopsis dalei
Metapenaeopsis gaillardi
Artemesia longinaris
Penaeopsis eduardoi
Parapenaeus longipes
Parapenaeus investigoris
Parapenaeus murrayi
Parapenaeus politus
Parapenaeus longirostris
Parapenaeus perezfarfante
70 Parapenaeus sextubercultus
Parapenaeus ruberoculatus
Parapenaeus fissurus
Parapenaeus indicus
Parapenaeus lanceolatus
Parapenaeus kensleyi
Parapenaeus australiensis
Parapenaeus fissuroides
Parapenaeus americanus
Parapenaeus cayrei
Sicyonia lancifer
Trachypenaeopsis mobilispinis
Atypopenaeus stenodactylus
Atypopenaeus dearmatus
Metapenaeus dobsoni
Metapenaeus lysianassa
Metapenaeus joyneri
Metapenaeus brevicornis
Metapenaeus tenuipes
Metapenaeus elegans
Metapenaeus intermedius
94 Metapenaeus stebbingi
Metapenaeus ensis
Metapenaeus affinis
Metapenaeus anchistus
Metapenaeus conjunctus
Xiphopenaeus kroyeri
Rimapenaeus constrictus
Megokris pescadoreensis
Megokris granulosus
Megokris sedili
Trachysalambria malaiana
Trachysalambria starobogatovi
Trachysalambria curvirostris
Trachysalambria longipes
Parapenaeopsis gracillima
Parapenaeopsis I
100 Mierspenaeopsis sculptilis I
Mierspenaeopsis I
Mierspenaeopsis hardwickii I
Mierspenaeopsis II
Mierspenaeopsis hardwickii II
Ganjampenaeopsis uncta
Alcockpenaeopsis hungerfordi
Batepenaeopsis tenella
Parapenaeopsis stylifera
Parapenaeopsis coromandelica I
Parapenaeopsis II
Parapenaeopsis coromandelica II
Kishinouyepenaeopsis amicus
Kishinouyepenaeopsis cf. cornuta I
Kishinouyepenaeopsis cornuta
Kishinouyepenaeopsis cf. cornuta II
Kishinouyepenaeopsis cf. cornuta III
Kishinouyepenaeopsis cf. cornuta IV
Kishinouyepenaeopsis incisa
Kishinouyepenaeopsis cf. incisa
(= Penaeidae)
(= Parapenaeidae)
HURZAID et al.
F I G U R E 1 Phylogenetic tree of penaeid shrimps and their allies based on the combined gene data set inferred with the maximum-likelihood
method using the GTR + G model. Branch lengths are proportional to inferred nucleotide substitutions. Numbers at nodes represent bootstrap
values in percentages (BP); values below 60% are not shown. Bold branches on the tree indicate statistically robust nodes with a posterior
probabilities (PP) from partitioned Bayesian inference ≥ 0.95
isolation between putative species in the field can only be
tested in sympatric species (Coyne & Orr, 2004), and thus,
where incongruence was found between the clusters recognized by ABGD and bPTP analyses, we took the sympatry
of the sister OTUs into consideration and merged allopatric
sister OTUs (Kekkonen & Hebert, 2014). Additionally, further checking with morphological characteristics and/or photographs of specimens and other criteria/methods (reciprocal
monophyly of the COI trees, biogeography and genetic diversity) (Hung, Russell, & Chen, 2017; Lo et al., 2017) was
done to minimize the bias from these two analyses for the
final validation.
Characteristics of sequence data
A total of 3,375 and 1,489 bp nucleotides were present from
the aligned sequences of the targeted genes compiled into
two operational data sets (combined [with 109 ingroup taxa]
and COI [with 371 ingroup taxa]), respectively. No nucleotide insertions/deletions or stop codons were found in our
aligned sequences. The lengths of the aligned sequences and
other descriptive statistics for each gene and each data set are
summarized in Table S6. Most of the variable sites are found
at the third codon position of the protein-coding genes. Tests
of base composition stationarity revealed no significant base
composition bias across taxa at each codon position of each
protein-coding gene. The subsequent phylogenetic analyses
were thus performed on a matrix without an application of
a down-weighted (or RY coding) strategy on the sites at the
third codon position, an analytical scheme suggested by Chen
and Mayden (2009) to avoid potential noise in phylogenetic
Combined gene tree
The results from the partitioned ML analysis and BI conducted with the combined data set are largely congruent.
Here, only the ML tree was presented with support values
from both ML and BI shown (Figure 1). In both resulting phylogenies, the traditionally defined Penaeidae is revealed as a
paraphyletic group with respect to the Sicyoniidae. Among
26 penaeid genera examined in this study, 13 are resolved
as monophyletic groups with high support values (BP ≥ 80;
PP ≥ 0.95), except Funchalia (BP = 50; PP = 0.81),
Melicertus (BP = 74; PP = 0.98), Atypopenaeus (BP = 56;
PP = 0.98) and Trachysalambria (BP = 67; PP = 0.98). Six
others were monotypic genera: Alcockpenaeopsis, Artemesia,
Ganjampenaeopsis, Heteropenaeus, Pelagopenaeus and
Xiphopenaeus. Three others (Penaeus, Parapenaeopsis and
Mierspenaeopsis) are not monophyletic. Due to only a single sample being represented for the genera Batepenaeopsis,
Penaeopsis, Rimapenaeus and Trachypenaeopsis, their
monophyly was not tested in this study.
Within the ‘Penaeidae’ + Sicyoniidae clade, three major
clades were found with moderate support (BP = 87, 62 and
69; PP = 1.00, 1.00 and 0.99), which largely corroborated
the traditional three-tribe classification scheme proposed by
Burkenroad (1983): the Penaeini (Clade A), Parapenaeini
(Clade B) and ‘Trachypenaeini’ (Clade C). Within Clade A,
four subclades can be identified (ML bootstrap support values ranging from 81 to 100; PP ranging from 0.94 to 1.00),
with the first subclade consisting of the genera Funchalia and
Pelagopenaeus. These two genera together are sister to the
rest of the members included in Clade A. The second subclade contains a single genus, Heteropenaeus, and is a sister
group of Penaeus’ s.l., which is composed of two reciprocally
monophyletic groups (third and fourth subclades). However,
none of these higher-level sister-group relationships are supported (Figure 1). Within the third subclade, Marsupenaeus
appears to be sister to Melicertus with high support. The
fourth subclade includes Penaeus’ s.s. and three other genera,
Farfantepenaeus, Litopenaeus and Fenneropenaeus. Here,
Penaeus s.s. is found to be polyphyletic with at least three
different lineages. Lineage I is sister to the rest of the members included in the subclade with high support. Lineages II,
III and the monophyletic Fenneropenaeus form a strongly
supported monophyletic group (Figure 1).
Clade B can be further divided into three subclades with
ML bootstrap support values ranging from 64 to 99 and with
PP ≥ 0.95 (Figure 1). The first subclade contains all of the examined species from the genus Metapenaeopsis. The second
subclade includes two genera, Artemesia and Penaeopsis,
which together are sister to the third subclade containing all
the examined species from the genus Parapenaeus.
Clade C is composed of three main and well-supported
(BP = 89, 94 and 93; PP = 1.00 for all) subclades and the
two represented species from the genus Atypopenaeus
(Figure 1). The first subclade includes a non-penaeid shrimp
(Sicyonia lancifer) and a penaeid shrimp, Trachypenaeopsis
mobilispinis, from the Trachypenaeini (BP = 89; PP = 1.00).
The second well-supported subclade comprises all the
examined species from the genus Metapenaeus. Within
HURZAID et al.
the third supported subclade, several strongly supported
clades or sister-group relationships are resolved (Figure 1).
These results confirm the monophyly of some recognized genera (Megokris and Kishinouyepenaeopsis), but
reject others (Parapenaeopsis and Mierspenaeopsis).
Parapenaeopsis contains two independent lineages with
Parapenaeopsis II sister to Kishinouyepenaeopsis (BP = 95;
PP = 1.00). Mierspenaeopsis is paraphyletic with respect
to Ganjampenaeopsis, and these taxa together are sister to
Parapenaeopsis I (= P. gracillima) but with only moderate
support (BP = 61; PP = 0.98) (Figure 1). It is also noteworthy that the traditionally defined, Parapenaeopsis s.l., appears to be monophyletic, whereas Trachypenaeus s.l. is not
(Figure 1).
COI tree of SEA penaeids
The inferred ML COI tree of SEA penaeids is presented in
Figure S3. The interspecific relationships of each genus along
with results from the species delimitation are illustrated and
summarized in Figures 2 and 3. Despite its low phylogenetic
resolution in terms of bootstrap support on inter-relationships
among the genera of SEA penaeids, the result is congruent
with the combined data set analysis (Figure 1 and Figure
S3). Most of the highly supported nodes in the COI gene
tree occur within the genus on shallow branches depicting
sister-group relationships among closely related species and
groupings of currently recognized species (Figures 2 and 3).
When multiple samples were used in this study to represent
a species, all but two of the known species were confirmed
to be monophyletic with high bootstrap support. Melicertus
canaliculatus and Marsupenaeus japonicus appear to be nonmonophyletic, because one of the individuals in Melicertus
canaliculatus collected from the South China Sea (GenBank
no. AY264893) is nested within the Marsupenaeus japonicus
samples (Figure 2). This result is likely due to misidentification, especially for sources such as GenBank and BOLD
databases. To assign a ‘true’ species name to each monophyletic clade, the identity of the species was reconfirmed
by examining the deposited specimens in the NTUM_INV
collection or validated by the clade that included samples collected from the type locality.
Species delimitation
The collected COI sequences from 71 recognized SEA penaeid species (371 specimens) were included for species
delimitation analyses using ABGD and bPTP analysis tools.
The ABGD analysis conducted by partitioning all of the
individuals in the Folmer region (prior maximal distance,
P = 0.0215) resulted in 87 OTUs by both distance metrics, JC69 and K2P. The sequences from the second data
set aligned at the Palumbi region (prior maximal distance,
P = 0.0215) generated 56 OTUs. The incongruent number
of OTUs between these two data sets is due to the non-availability of common COI data. In fact, the number of representative sequences from the Folmer region (140 sequences)
is higher than that from the Palumbi region (27 sequences)
in GenBank. In summary, 91 OTUs were determined for
ABGD analyses from the COI data sets, whereas the bPTP
analysis predicts a higher number (96) of OTUs. Finally, 94
congruent OTUs were determined (Figures 2 and 3). Species
recognition and disagreement between the obtained results
are further explored below.
4.1 | Phylogeny and taxonomy of the
With an extensive sampling from the SEA, this study represents the most comprehensive investigation to date on the
molecular phylogeny of the Penaeidae. The data were generated from four gene markers in both mitochondrial and nuclear genomes from 98 of 224 known morphospecies. One of
the striking results (already known from previous studies) is
the paraphyly of the Penaeidae with respect to Sicyoniidae.
Based on morphology, the Sicyoniidae has many distinctive
characteristics not found in other penaeoid members; for
F I G U R E 2 Partial phylogenetic tree of South-East Asian penaeids inferred by the partitioned maximum-likelihood method with
GTR + G nucleotide substitution model based on the COI gene data set and results from species delimitation analyses and three additional criteria.
Branch lengths are proportional to inferred nucleotide substitutions. Numbers at nodes represent bootstrap values in percentages. Values below 70%
are not shown; * indicates 100%. Grey diamond indicates the specimen(s) from type locality for the morphospecies. Taxa name in bold indicates
probable misidentification samples. L.vannamei is an introduced species in SEA waters. Numbers within the parentheses shown after taxon names
indicate the number of sequences within each collapsed clade or lineage. The tree is rooted with Solenocera crassicornis. A summary of the
determined OTUs (vertical bars) is presented on the right side of the phylogenetic tree. The white bar indicates congruent OTUs as suggested by
both species delimitation methods (i.e. robust results). The black bar indicates the presence of multiple OTUs determined by bPTP in the particular
genetic lineage that is incongruent to ABGD results. The white bar with a question mark indicates the missing region from the GenBank database.
Missing data are marked by black crosses. Abbreviations for sequences collected: AS, Andaman Sea; CS, Celebes Sea; ECS, East China Sea; IO,
Indian Ocean; MS, Mediterranean Sea; SAO, South Atlantic Ocean; SCS, South China Sea; SOM, Strait of Malacca
HURZAID et al.
Solenocera crassicornis
Atypopenaeus dearmatus
M. longistylus (3): IO
* M. latisulcatus (5): SCS + AS + MS
M. hathor: MS
M. canaliculatus: AS
M. canaliculatus: SCS
* M. japonicus: ECS
M. japonicus: ECS
M. japonicus: MS
* M. pulchricaudatus (7): SOM + CS
L. vannamei: IO
* 83L. vannamei:
L. vannamei: MS
L. vannamei: ECS
88 L. vannamei: SOM
63 L. vannamei: IO
L. vannamei: IO
F. indicus (2): SOM + IO
* F. chinensis (5): ECS + SCS
99 * F. penicillatus (2): SCS + SOM
87 F. silasi (10): SCS + SOM + CS
* F. merguiensis (6): SOM + AS
F. merguiensis (8): SOM + AS + ECS
P. semisulcatus: IO
P. semisulcatus: IO
98 85 P. semisulcatus: IO
* P. semisulcatus (8): SOM + SCS
* P. cf. monodon (8): IO Pmon sp. I
P. monodon: IO
P. monodon: IO
* 82P. monodon: SCS
70 P. monodon: SOM
Pmon sp. II
P. monodon: SCS
P. monodon: IO
P. monodon: IO
Pmon sp. III
P. monodon: IO
Pmon sp. IV
P. monodon: IO
P. monodon (10): SOM +SCS + CS+ AS
* P. longipes (3): ECS
P. murrayi (2): ECS
* P. investigoris (2): IO
91 * P. investigoris (3): ECS
P. perezfarfante: ECS
P. fissurus: ECS
* P. lanceolotus (2): ECS
99 P. fissuroides (2): ECS
97 P. indicus (3): ECS
98 P. kensleyi: ECS
* P. cayrei (2): ECS
99 P. sextuberculatus (3): ECS
78 P. ruberoculatus (2): ECS
72 P. australiensis (3): ECS
M. commensalis: ECS
M. liui: ECS
M. lamellata: ECS
M. evermanni: ECS
M. dalei: ECS
* M. mogiensis intermedia (2): ECS
M. gaillardi: ECS
* M. mogiensis mogiensis: ECS
* M. provocotaria longirostris (2): ECS
* M. sinica (2): ECS
M. palmensis: SCS
toloensis: ECS
* * M.
M. palmensis (2): ECS
M. barbata (7): SOM + SCS
M. stridulans (11): SOM + SCS + MS + IO
* T. mobilispinis (3): CS
M. conjunctus: SOM
* M. anchistus (2): SOM
M. affinis: AO
M. affinis (6): SOM + SCS
M. elegans: AS
M. intermedius (3): SCS
* M. ensis (7): SCS + CS
* 99 M.M.dobsoni:
dobsoni (4): SOM + SAO
* M. lysianassa (8): SCS + SOM
M. joyneri (3): SCS
* M. brevicornis (4): SCS
99 M. tenuipes (5): SCS
Parapenaeopsis s.l.
& Trachypenaeus s.l.
P. monodon
AB i A
er mb P DNA rph al
T u
o n
lm u
Fo Pal bP n M Fi
HURZAID et al.
Solenocera crassicornis
Atypopenaeus dearmatus
Penaeus s.l.
(2): IO
*** P.P.stylifera
stylifera (3): IO
* P. coromandelica (10): IO
90 P. coromandelica (8): SOM
K. amicus (6): ECS
* K. incisa (3): ECS
70 P. coromandelica (2): IO
80 P. coromandelica (2): IO
94 P. coromandelica (3): IO
K. cf. incisa (5): SOM
K. cf. cornuta: IO Kcor sp. I
* K. cf. cornuta (4): SOM Kcor sp. II
* K. cf. cornuta (8): SOM + AS Kcor sp. III
cornuta (6): ECS + IO Kcor sp. IV
* K. cf. cornuta (7): SCS Kcor sp. V
* B. tenella (2): SOM
* A. hungerfordi (7): SOM + SCS + SS
T. malaiana: AS
* T. curvirostris (2): SCS
* T. longipes (6): SCS
* M. pescadoreensis (5): SOM
M. sedili: AS
* M. granulosus (7): SCS + GOT
* P. gracillima (5): SOM + SCS
* G. uncta (6): SCS + IO
90 M. sculptilis (5): SOM
* * M. sculptilis (4): IO
* * M. sculptilis (7): SCS
* M. hardwickii: IO
* 98 M. hardwickii (7): SOM
M. hardwickii (18): SCS
AB bi A
P N p l
lm lum PT uD or na
Fo Pa b n M Fi
P. coromandelica
K. incisa
K. cornuta
F I G U R E 3 Partial phylogenetic tree of South-East Asian penaeids inferred by the partitioned maximum-likelihood method with
GTR + G nucleotide substitution model, based on the COI gene data set, species delimitation analyses and three additional criteria. Branch lengths
are proportional to inferred nucleotide substitutions. Numbers at nodes represent bootstrap values in percentage. Values below 70% are not shown;
* indicates 100%. Grey diamond indicates the specimen(s) from type locality for the morphospecies. Numbers within the parentheses shown after
the taxon names indicate the number of sequences within each collapsed clade or lineage. The tree is rooted with Solenocera crassicornis. A
summary of determined OTUs (vertical bars) is presented on the right side of the phylogenetic tree. The white bar indicates congruent OTUs as
suggested by both species delimitation methods (i.e. robust results). A black bar indicates the presence of multiple OTUs as determined by bPTP in
the particular genetic lineage that is incongruent to ABGD result. A white bar with a question mark indicates the missing region from the GenBank
database. Missing data are marked by black crosses. Abbreviations for sequences collected: AS, Andaman Sea; ECS, East China Sea; GOT, Gulf of
Thailand; IO, Indian Ocean; MS, Mediterranean Sea; SCS, South China Sea; SOM, Strait of Malacca; SS, Sulu Sea
instance, their three posterior pleopods are uniramous (versus.
normally biramous in other penaeoids) (Burkenroad, 1983;
Crosnier, 2003; Pérez Farfante & Kensley, 1997). Another
characteristic often used to differentiate Sicyoniidae from
other penaeoids is their rigid and stony integument (Pérez
Farfante & Kensley, 1997). However, this character is problematic, as a few Sicyonia species have non-rigid integument
(Crosnier, 2003; Yu & Chan, 1986). Also, the shape of the
genitalia of sicyoniid shrimps, particularly the very rigid
and strongly ridged petasma of males, is similar to that of
those found in many penaeid genera of Trachypenaeini and
some Parapenaeini. An inference from our phylogenetic results (Figure 1) suggests that this feature might derive from
the common ancestor of Clades B and C, which includes the
Parapenaeini, Trachypenaeini and Sicyoniidae, but was secondarily lost in some lineages. Robalino, Wilkins, BrackenGrissom, Chan, and O'Leary (2016) also observed four
synapomorphic characters for Sicyonia based on a limited
taxon sampling. These characters should thus be autapomorphic within the Clade C. Finally, given the present phylogenetic results, it is reasonable to synonymize Sicyoniidae with
Trachypenaeidae (Figure 1) as suggested by other studies
HURZAID et al.
(Cheng et al., 2018; Ma et al., 2009; Robalino et al., 2016). The
putative synapomorphic characters for the taxonomic groups
resolved in this study and potential autapomorphic characters
to ‘sicyoniids’ are shown in Table S7 for reference. Further
morphological investigation is required to identify the ‘true’
synapomorphic characters for the Trachypenaeidae.
As for the generic level, Pérez Farfante and Kensley
(1997) separated Penaeus s.l., into six genera. This classification was based on characters such as grooves on the carapace and the last abdominal somite, hepatic ridges, thelycum
shape and geographic distribution (Lavery et al., 2004). Our
phylogenetic results confirm the monophyly of five proposed
genera (Marsupenaeus, Litopenaeus, Farfantepenaeus,
Fenneropenaeus and Melicertus), but reveal a polyphyletic
Penaeus s.s. (Figure 1). These results are mostly congruent
to the previous 16S and COI data molecular studies conducted by Lavery et al. (2004) and Ma et al. (2011) with
combined data from both mitochondrial and nuclear genes.
In general, the division of Penaeus s.l., into two clades
(Melicertus + Marsupenaeus and Fenneropenaeus + Farfan
tepenaeus + Litopenaeus + Penaeus s.s.) and non-monophyletic status for Penaeus s. s. are always supported. However,
we did not find any shared morphological characters that supported the grouping of these two clades.
Metapenaeopsis is the most diverse penaeid shrimp
genus. Based on morphology, Crosnier (1987, 1991, 1994a,
1994b) proposed that the species in Metapenaeopsis can
be separated into two groups according to the shape of the
genitalia. The first group contains species with one valve in
the petasma, including one IWP distributed species, M. commensalis, and 10 other species distributed in the Atlantic and
East Pacific. The second group is composed of the remaining Metapenaeopsis species characterized by a two-valved
petasma, including at least 58 species that are all found in the
Indo-West Pacific. This latter group can be further divided
into two subgroups, one with a stridulating organ and the
other without. A previous phylogenetic study by Tong, Chan,
and Chu (2000) with mtDNA 16S and COI gene data from
seven Metapenaeopsis species distributed in the Indo-West
Pacific showed that M. commensalis was separated from
other two-valved petasma species with high support. Within
the two-valved petasma group, two clades were found, each
with and without a stridulating organ, with high and low
bootstrap support, respectively (Tong et al., 2000). Cheng,
Sha, and Liu (2015), using 16S, NaK and PEPCK gene data
on 14 Metapenaeopsis species, also found that the two species having only one petasma valve (M. commensalis and M.
sp.) were separated from the other species having two valves.
However, the remaining Metapenaeopsis species with twovalved petasmas formed a monophyletic group but with weak
support. Similar to the previous study, species with the stridulating organ constituted a strongly supported clade. On the
other hand, species without the stridulating organ formed a
paraphyletic group. In our inferred phylogeny, even though
we used a higher number of taxa (in 17 species) and more
markers than in previous studies, groupings suggested by
morphology are not clear due to low phylogenetic resolution.
Nevertheless, the major pattern of relationships among all
studied taxa was similar across all these studies, which consistently reinforce the monophyly of Metapenaeopsis with
high support values.
Parapenaeus shrimps are generally found in deeper waters (usually >100 m in depth). At present, 16 extant species
are known from this genus (Crosnier, 2005; Pérez Farfante &
Kensley, 1997), with most of them (13/16) found in the IndoWest Pacific. No fossilized Parapenaeus shrimp has ever
been found (Schweitzer, Feldmann, Garassino, Karasawa, &
Schweigert, 2010). The Atlantic species are P. americanus,
P. longirostris and P. politus. Crosnier (1986), Crosnier
(2005) proposed that the IWP species form four groups based
on genital organ similarities. The first group, namely the ‘longipes’ group, includes only P. longipes; the second group,
namely the ‘investigoris’ group, contains P. Pérezfarfante,
P. investigatoris and P. murrayi; and the third ‘fissurus’ group
includes all the remaining species, while the fourth group includes three species within the ‘fissurus’ group, later named
the ‘sextuberculatus’ group. In the present study, even though
all species in this genus are included, the grouping suggested
by morphology is still ambiguous due to the absence of significant phylogenetic support. Yet, the monophyly of this
genus is well supported, in congruence with a previous study
(Yang et al., 2015).
Within Metapenaeus, our phylogenetic results show that
this genus is bifurcated into two major clades with high
and moderate bootstrap support. The first clade consists
of M. dobsoni, M. lysianassa, M. joyneri, M. brevicornis
and M. tenuipes, while the second clade contains M. elegans, M. intermedius, M. ensis, M. affinis, M. anchistus and
M. conjuctus. Our morphological examinations found that the
rostrums of the species from the first clade are more or less
crest-like and with the anterior 1/3 or even 1/2 unarmed. The
species from the second clade have nearly straight rostrums
armed with teeth almost along the entire length. Moreover, of
the species examined, only M. intermedius and M. anchistus
present movable lateral teeth on the telson but these two species do not form a clade.
In 1997, Pèrez Farfante and Kensley separated the traditionally defined genus Trachypenaeus s.l. into four genera
and added three more genera: Megokris, Rimapenaeus and
Trachysalambria. This proposition was mainly based on the
different shapes of the genitalia, particularly the thelycum.
Our inferred phylogenetic tree confirms the monophyly of
the two proposed genera Megokris and Trachysalambria with
high and moderate support, respectively. However, due to
only one species of Rimapenaeus being sampled, the monophyletic status of this genus cannot be tested. This revised
classification has also been supported by previous molecular
analyses, which revealed that Trachypenaeus s.l. was polyphyletic (Chan et al., 2008; Ma et al., 2009).
Sakai and Shinomiya (2011) split the genus Parapenaeopsis
s.l. into eight genera. Our present molecular analyses included taxa from all these genera except Arafurapenaeopsis
and Holthuispenaeopsis. Referring to the classification based
on petasma morphology proposed by Sakai and Shinomiya
(2011), our study confirms the monophyletic status of
Kishinouyepenaeopsis with high support, similar to the findings of Li et al. (2014). The genus Mierspenaeopsis, which
is unique in having leaf-shaped distomedian projections of
the petasma unlike other members of Parapenaeopsis s.l.,
was found to be paraphyletic with the monotypic genus
Ganjampenaeopsis nested within it but with low support.
Here, we resolve the genus Parapenaeopsis s.l. to be paraphyletic, with P. gracillima grouped with Mierspenaeopsis
and Ganjampenaeopsis with moderate support. The other
two congenic species, P. stylifera and P. coromandelica, appear to be sister to each other with high support. The morphological characters used for diagnosing Parapenaeopsis
s.l. from other genera should be symplesiomorphic. Finally,
according to conventional classification, P. coromandelica
Alcock, 1906, was considered a synonym of P. stylifera because of their similarly shaped petasmas (Chanda, 2016;
George, 1973; Pathan & Jalihal, 1998; Racek & Dall, 1965;
Ravindranath, 1989; Sakai & Shinomiya, 2011). However,
our molecular results show that these two species are genetically distinct and each also contains cryptic species. Similar
results were found by Chowdhury et al. (2018) using COI
and 16S rRNA genes. Therefore, both should be regarded as
valid species.
Overall, the current inclusive phylogenetic analyses of
the Penaeidae have re-evaluated several aspects of the ambiguous evolutionary relationships among its genera, in
particular the newly proposed classification of the genus
Parapenaeopsis (Sakai & Shinomiya, 2011), which has not
been properly addressed until now. Previously, phylogenetic
studies on the Penaeidae have focused solely on mtDNA
(Chan et al., 2008; Cheng et al., 2018; Quan et al., 2004;
Vazquez-Bader et al., 2004; Voloch et al., 2005) or nuclear
DNA (Ma et al., 2009; Robalino et al., 2016), with few studies combining both markers, but emphasized only on selected genera within the Penaeidae (Cheng et al., 2015; Ma
et al., 2011; Yang et al., 2015). Generally, mitochondrial
DNA sequences are susceptible to obscure phylogenetic signals because mitochondrial genes have characteristics that
tend to lead to high levels of homoplasy resulting from extreme compositional biases, rapid nucleotide substitution
and highly asymmetrical transformation rate matrices (Chen,
Lheknim, & Mayden, 2009; Johnson & Clayton, 2000;
Lin & Danforth, 2004). In contrast, nuclear protein-coding
genes exhibit better performance in recovering phylogenetic
HURZAID et al.
relationships at deeper taxonomic levels and provide greater
bootstrap support (Ma et al., 2009). Therefore, an analysis
combining both mitochondrial and nuclear genes to resolve
the phylogeny of the Penaeidae and its allies should provide
a better insight into their systematics because the two types
of data are unlinked and evolved under different evolutionary
constraints. However, it is important to note that even when
using the combined sequences of four markers (COI, 16S
rRNA, NaK and PEPCK) and a comprehensive set of samples
(98 penaeid species), some phylogenetic relationships within
the family remain uncertain. Obviously, more genes and taxa
must be sequenced in the future to clarify the taxonomic issues of the genera in the Penaeidae and its relatives.
4.2 | Species diversity and taxonomy in
SEA Penaeidae
Species diversity and taxonomy were further explored in this
study with supplementary species delimitation analyses using
ABGD and bPTP. With an input of 71 morphospecies, 91
and 96 putative species (OTUs) were detected from ABGD
and bPTP, respectively (Figures 2 and 3); 94 congruent and
robust OTUs were found in both analyses. These results suggest that the species diversity of the Penaeidae is underestimated. In general, bPTP analysis tends to generate more
OTUs than ABGD due to the different algorithms applied
in each (Hung et al., 2017). ABGD is based on the genetic
distance of compared sequences, whereas bPTP assess speciation events in terms of the number of mutations along the
gene genealogy. In this study, further evaluation of discordant OTUs was made by considering the combined evidence
from monophyletic groups of the COI gene tree, geographic
distribution and morphology. Below, we first discuss the discordant results between the two species delimitation analyses
performed in this study.
In our research, bPTP tends to merge sister OTUs, while
ABGD does not do so for Parapenaeus fissuroides and P. indicus, and P. ruberoculatus and P. sextuberculatus (Figure 2).
However, these taxa belong to the four respected morphospecies and are genetically distinct (Figures 1 and 2). Thus, we
consider that they are four distinct species regardless of the
bPTP results. In some other cases, ABGD tends to merge sister OTUs that are determined by bPTP. These are L. vannamei,
P. monodon, P. stylifera, K. incisa (ECS lineage) and K. cf. cornuta (Strait of Malacca and Andaman Sea lineage) (Figures 2
and 3). In most of the cases mentioned above, further examination of morphology was not possible due to the non-availability of specimens. Because of the short genetic distance
(P < 3%), the emergence of those sister clusters into single
inferred species is tentatively suggested except for L. vannamei, P. monodon and P. stylifera. In L. vannamei, the GenBank
sequence from Brazil (AY344218) could be a putative species
HURZAID et al.
due to the large genetic distance (K2P = 4.1%–6.3%) with the
other L. vannamei sequences (Figure 2). Similarly, in P. monodon, the GenBank sequence from Mumbai (KC409381) also
shows a high degree of genetic divergence (K2P = 4.1%–5%)
in comparison with other P. monodon sequences (Figure 2).
We therefore tentatively consider Brazil's L. vannamei and
Mumbai's P. monodon as separate species from their original
status. However, further verification is necessary by including additional sequences (to check the sequence accuracy)
and/or by detailed morphological examination of specimens
when they become available. Five samples of P. stylifera from
the Indian Ocean were included in the analyses, resulting in
two reciprocal monophyletic groups in the COI tree that corresponded to two bPTP OTUs (Figure 3). We thus took the
sympatry of the sister OTUs into consideration and validated
them as two putative species.
A total of 94 species are inferred within the SEA
Penaeidae, of which 71 match the current definition of morphospecies (Figures 2 and 3), revealing an underestimated
biodiversity. Multiple inferred species clusters were observed
in the following morphospecies: Fenneropenaeus merguiensis (2 species), introduced Litopenaeus vannamei (2 species), Penaeus semisulcatus (4 species), Penaeus monodon
(4 species), Metapenaeus affinis (2 species), Metapenaeus
dobsoni (2 species), Mierspenaeopsis sculptilis (3 species),
Mierspenaeopsis hardwickii (3 species), Parapenaeopsis coromandelica (2 species), Kishinouyepenaeopsis incisa (2 species) and Kishinouyepenaeopsis cornuta (5 species). These
inferred species clusters include some questionable taxa that
are either undescribed or simply not well identified, and some
cryptic species. However, for those sequences downloaded
from GenBank or BOLD (Fenneropenaeus merguiensis,
Litopenaeus vannamei, Metapenaeus affinis, Metapenaeus
dobsoni, Penaeus semisulcatus and Parapenaeopsis stylifera), further morphological examination was not possible due
to there being no access to specimens. One specimen, identified as Melicertus canaliculatus (sequence retrieved from
GenBank, accession no. AY264893) from the South China
Sea, clustered tightly with specimens of Marsupenaeus japonicus and is likely a misidentification (Figure 2). According to
Chan (1998), the misidentification between these two known
species often occurs due to their similar morphological appearance. We discuss below some cases that need further taxonomic consideration.
Mierspenaeopsis spp.
Our study highlights the inferred existence of three cryptic
species in M. hardwickii, the first being represented by a single sample from India (Indian Ocean) that is sister to a second collected from the Strait of Malacca, Malaysia. The third
species is indicated by samples from the South China Sea
(Malaysia) and GenBank sequences from China and Taiwan.
The biogeographic pattern of genetic structuring observed
in the third species is further supported by both ABGD and
bPTP analyses and suggests the hypothesis of a South China
Sea origin with later invasion to other regions (Figure 3).
However, a contrasting biogeographic pattern of genetic
structuring is detected in another Mierspenaeopsis species,
M. sculptilis. Our specimens collected from the South China
Sea reveal a sister relationship to the GenBank sequences
from India with strong support, whereas specimens from the
Strait of Malacca are sister to them. The oceanographic features that could enable the biogeographic patterns observed
in this and other Mierspenaeopsis species clusters require
more investigation. A higher number of samples encompassing a wider sampling area across species distribution ranges
and detailed analyses on population genetics would provide
a better insight into the genetic structuring (and the influencing factors) observed in these species.
4.2.2 | Parapenaeopsis
coromandelica complex
A pattern of biogeographic structuring was also observed
among samples within the P. coromandelica complex, with
two cryptic species being detected. The first inferred species
included our samples collected from West Aceh, Indonesia
(Indian Ocean). This species is sister to the samples collected
from the Strait of Malacca, Malaysia and the GenBank sequences from India and Sri Lanka. Both ABGD and bPTP
analyses recognized these two inferred species as distinct
species, and this is further confirmed by the topology of individual and combined gene trees (Figures 1 and 3). In this
study, all individuals were examined morphologically (except for sequences downloaded from GenBank). However,
we cannot find any morphological characters to distinguish
them from each other. The presence of at least two cryptic
species in this morphospecies is tentatively suggested.
Kishinouyepenaeopsis spp.
Kishinouyepenaeopsis, namely K. amicus, K. incisa and
K. cornuta. From our molecular analyses, however, eight inferred species were revealed in this genus (Figure 3). Our morphological examination of the collected specimens revealed
that the three morphospecies shared similarly shaped petasmas,
but their thelycums were different from each other. The female
K. amicus thelycum lacks a median lobe but has a longitudinal
median grove, pereiopod III is without a basial spine, and male
pleopod II is normal (Figure S4). The thelycum of K. incisa
lacks a median lobe and a longitudinal median groove, male
HURZAID et al.
pereiopod III is without a basial spine, and pleopod II of the
male is abnormal. In K. cornuta, the thelycum is characterized
by a large median lobe, pereiopod III has a basial spine, and
pleopod II is abnormal in the male.
Our molecular results suggest that ‘K. cornuta’ contains five inferred species (assigned as Kcor sp. I to Kcor
sp. V supported by ABGD, bPTP and phylogenetic analyses; Figure 1, Figure S3 and S4). Geographic factors tend
to be important in their species diversification. Kcor sp. I is
represented by a single individual collected from Indonesia
(Indian Ocean); Kcor sp. II includes samples collected only
from Indonesia (Strait of Malacca); Kcor sp. III contains
samples from Malaysia and Thailand (Strait of Malacca and
Andaman Sea); Kcor sp. IV exhibits no obvious geographic
structuring and comprises the specimen from the type locality from Japan and others from India and China; and Kcor sp.
V includes samples collected only from Malaysia's coast off
the South China Sea. Among the different inferred species
in ‘K. cornuta’, we cannot detect significant morphological
differences in the shape of their genital organs, which is considered one of the most useful diagnostic characters for species identification in the Penaeidae (Chan, 1998). However,
we observe that their body colour patterns are different and
may be used for species identification (Figure S5). Kcor sp.
I: the first through third abdominal segments are banded
reaching to the ventral end; bands are absent on the fourth
to sixth abdominal segments but replaced with conspicuous
dots in the middle of the body; the rostrum has no stripe; a
blotch is absent on the dorsal crest; and the distal part of the
uropod has a reddish margin. Kcor sp. II: body is greyish in
colour, and discontinuous bands are on the first to third body
segments; rostrum has no stripe, and blotch is absent on the
dorsal crest; and the distal part of the uropod is greyish in
colour. Kcor sp. III: distinct bands extending to the end of the
body were observed on all body segments; rostrum has no
stripe; dark and distinct blotch is on the dorsal crest; and the
distal part of uropods is with a patch of striking yellow. Kcor
sp. IV: we are not able to describe the colour pattern since the
specimens examined have been preserved in alcohol. Kcor
sp. V: all six abdominal segments have a discontinuous band
not reaching to the end of the body; indistinct blotch is on the
dorsal crest; conspicuous stripe is on rostrum; and the distal
part of the uropod has patch of bright yellow (Figure S5).
In ‘K. incisa’, we are unable to find any morphological
characters or body colour patterns to differentiate specimens
from one inferred species to another (SOM versus. ECS species) (Figure 3).
Penaeus monodon complex
Penaeus monodon was thought to be a widespread species
with its distribution ranging from south-east Africa to eastern
Australia to Japan (Dore & Frimodt, 1987). However, numerous instances of cryptic diversity within Penaeus monodon
have been documented (Alam, Westfall, & Pálsson, 2016;
Benzie, Ballment, & Frusher, 1993; Klinbunga, Penman,
McAndrew, & Tassanakajon, 1999; Klinbunga et al., 2001;
Palumbi & Benzie, 1991; Sodsuk, 1996; Tassanakajon
et al., 2006). Based on the available samples examined in
the present study, four cryptic species can be determined
in P. monodon, which is supported by species delimitation
analyses and phylogenetic analyses. The first inferred species (Pmon sp. I; n = 8) contains samples collected only from
western Aceh and is sister to the other three species. The
second inferred species (Pmon sp. II; n = 8) is widespread,
including samples collected from the Strait of Malacca and
South China Sea, and GenBank sequences from China, India
and Sri Lanka, and is paraphyletic with respect to Pmon sp.
III (GenBank sequence from Mumbai, western India). The
individuals from Pmon sp. II and Pmon sp. III together form
a sister clade to Pmon sp. IV (n = 10, samples collected from
the Strait of Malacca, South China Sea, Celebes Sea and
Andaman Sea) (Figure 2).
Upon closer inspection of ‘P. monodon’ specimens, we
find that Pmon sp. I individuals possess four distinct morphological characters that are not shared with Pmon species
II to IV (Figure S6). The first character is the length of the
rostrum, which is slightly longer and more pointed than other
‘P. monodon’ species. The second character is the body colour, which is greyish greenish or dark greenish-blue without
transverse banding as seen in other species. The third character is the uropod lacking the mud-yellow median transverse band present in other ‘P. monodon’ species. The fourth
character is the antennal flagella with a light yellow stripe,
whereas this structure is uniformly greenish-brown in other
species. However, no obvious differences in morphology
or body colour pattern were observed among specimens of
Pmon sp. II, Pmon sp. III and Pmon sp. IV.
4.3 | Recommendation of a new set DNA
barcoding primers for shrimps
The idea of DNA barcoding is the utilization of a short fragment of mitochondrial COI gene as a ‘barcode’ to identify different species and assess cryptic diversity (Hebert, Stoeckle,
Zemlak, & Francis, 2004; Ward, Zemlak, Innes, Last, &
Hebert, 2005). The advantage of using COI is that it is short
enough to be sequenced quickly and cheaply yet long enough
to identify variations among species. Decapoda DNA barcoding is primarily based on a specific region of the COI gene
that is PCR-amplified by primers HCO2198 and LCO1490
(‘Folmer primers’) designed by Folmer et al. (1994). Based
on our own experience and discussions with colleagues, these
primers are not ‘universal’ enough and often perform poorly
HURZAID et al.
or fail altogether, producing vague products despite attempts
at optimization. Similarly, this problem was also reported by
Geller, Meyer, Parker, and Hawk (2013). Until now, several
studies have attempted to design new universal primers for
Decapoda (Fernandes, Silva, Costa, Oliveira, & Mafra, 2017;
Geller et al., 2013; Mantelatto et al., 2016). However, the
presence of two different COI-amplified regions with limited
overlap in the GenBank database has not only led to confusion among taxonomists but also obscured the advancement
of taxonomic work (Figure S2).
In this study, we designed a new pair of barcoding primers
for Penaeidae. These novel primers, namely Penae_COIF and
Penae_COIR, span both the Folmer and Palumbi regions and
can successfully amplify a nearly complete COI gene from
all of the penaeid taxa tested (1,425 bp). With a longer region being sequenced, phylogenetic resolution is improved
as seen by a great proportion of nodes being supported by
80% and higher bootstrap values on the inferred COI tree. We
also tested this new pair of primers on the samples of other
families within the Superfamily Penaeoidea, and it proved to
work well on PCR and sequencing.
We would like to thank our colleagues from the Marine
Biodiversity and Phylogenomics Laboratory at National
Taiwan University for stimulating discussions and insightful
comments; the Molecular Ecology and Evolution Laboratory
at Universiti Sains Malaysia, Syiah Kuala University in
Aceh and Can Tho University in Vietnam for assistance in
sample collection; and T. Komai (CBM, Japan) and A. B.
Kumar (DABFUK, India) for sharing us valuable material.
This work was supported, in part, by research grants from the
Ministry of Science and Technology, Taiwan (MOST 1072611-M-002-007, 108-2611- M-002-012-MY2 to W.-J. C.,
MOST 107-2119-M-001-048 [SCSMART]), and Ministry
of Higher Education, Malaysia (203/PBIOLOGI/6711455
to S.A.M.N.). T.-Y. C. would like to thank the Featured
Areas Research Center Program within the framework
of the Higher Education Sprout Project by the Ministry of
Education, Taiwan.
The authors declare that they have no competing interests.
A. H. collected materials in the field, conducted the experiments, performed the data analysis and wrote the manuscript;
S. A. M. N., Z. A. M. and W.-J. C. made this project possible by facilitating sampling and contributing ideas; T.-Y.
C. provided several materials and provided reassurance on
taxonomic identification and advised the work; W.-J. C. advised on data analysis and co-wrote the manuscript; and S. A.
M. N. and W.-J. C. provided laboratory and funding support.
Amirah Hurzaid
Wei-Jen Chen
Abele, L. G. (1991). Comparison of morphological and molecular phylogeny of the Decapoda. Memoirs of the Queensland Museum, 31,
Alam, M., Westfall, K., & Pálsson, S. (2016). Mitogenomic variation
of Bangladesh Penaeus monodon (Decapoda, Penaeidae) and reassessment of its phylogeography in the Indo-West Pacific region.
Hydrobiologia, 763(1), 249–265. https://doi.org/10.1007/s1075​
Avise, J. C. (2012). Molecular markers, natural history and evolution.
Berlin, Germany: Springer Science & Business Media.
Avise, J. C., Nelson, W. S., & Sibley, C. G. (1994). DNA sequence support
for a close phylogenetic relationship between some storks and New
World vultures. Proceedings of the National Academy of Sciences,
91(11), 5173–5177. https://doi.org/10.1073/pnas.91.11.5173
Baldwin, J. D., Bass, A. L., Bowen, B. W., & Clark, W. H. Jr (1998).
Molecular phylogeny and biogeography of the marine shrimp
Penaeus. Molecular Phylogenetics and Evolution, 10(3), 399–407.
Benzie, J., Ballment, E., & Frusher, S. (1993). Genetic structure of
Penaeus monodon in Australia: Concordant results from mtDNA
and allozymes. In G. A. E. Gall, & H. Chen (Eds.), Genetics in
aquaculture (pp. 89–93). Amsterdam, the Netherlands: Elsevier.
Burkenroad, M. (1983). A natural classification of the Dendrobranchiata,
with a key to recent genera. Crustacean Phylogeny, Crustacean
Issues, 1, 279–290.
Chan, T.-Y. (1998). Shrimps and prawns. FAO species identification
guide for fishery purposes. In K. E. Carpenter, & V. H. Niem (Eds.),
The living marine resources of the Western Central Pacific, (vol. 2,
pp. 851–972). Rome, Italy: Food and Agriculture Organization of
the United Nations.
Chan, T.-Y., Cleva, R., & Chu, K. H. (2016). On the genus Trachysalambria
Burkenroad, 1934 (Crustacea, Decapoda, Penaeidae), with descriptions of three new species. Zootaxa, 4150(3), 201–254. https://doi.
Chan, T.-Y., Tong, J., Tam, Y. K., & Chu, K. H. (2008). Phylogenetic
relationships among the genera of the Penaeidae (Crustacea:
Decapoda) revealed by mitochondrial 16S rRNA gene sequences.
Zootaxa, 1694(3), 38. https://doi.org/10.11646​/zoota​xa.1694.1.2
Chanda, A. (2016). A study on newly described genera Alcockpenaeopsis,
Batepenaeopsis, Helleropenaeopsis, Kishinouyepenaeopsis and
Parapenaeopsis from Indian water. Poultry, Fisheries & Wildlife
Sciences, 4(147), 2. https://doi.org/10.4172/2375-446X.1000147
Chen, W.-J., Lheknim, V., & Mayden, R. L. (2009). Molecular phylogeny of the Cobitoidea (Teleostei: Cypriniformes) revisited:
Position of enigmatic loach Ellopostoma resolved with six nuclear
genes. Journal of Fish Biology, 75(9), 2197–2208. https://doi.
Chen, W.-J., & Mayden, R. L. (2009). Molecular systematics of the
Cyprinoidea (Teleostei: Cypriniformes), the World's largest clade
of freshwater fishes: Further evidence from six nuclear genes.
Molecular Phylogenetics and Evolution, 52(2), 544–549. https://doi.
Cheng, J., Chan, T. Y., Zhang, N., Sun, S., & Sha, Z. L. (2018).
Mitochondrial phylogenomics reveals insights into taxonomy and
evolution of Penaeoidea (Crustacea: Decapoda). Zoologica Scripta,
47(5), 582–594. https://doi.org/10.1111/zsc.12298
Cheng, J., Sha, Z. L., & Liu, R. Y. (2015). DNA barcoding of genus
Metapenaeopsis (Decapoda: Penaeidae) and molecular phylogeny inferred from mitochondrial and nuclear DNA sequences.
Biochemical Systematics and Ecology, 61, 376–384. https://doi.
Chowdhury, L. M., Shanis, R., Chelath, M., Pavan-Kumar, A., &
Krishna, G. (2019). Molecular identification and phylogenetic assessment of species under genus Parapenaeopsis Alcock, 1901,
from Indian waters. Mitochondrial DNA Part A, 30, 191–200.
Costa, F. O., DeWaard, J. R., Boutillier, J., Ratnasingham, S., Dooh, R.
T., Hajibabaei, M., & Hebert, P. D. (2007). Biological identifications through DNA barcodes: The case of the Crustacea. Canadian
Journal of Fisheries and Aquatic Sciences, 64(2), 272–295. https://
Coyne, J. A., & Orr, H. A. (2004). Speciation. Sunderland, MA: Sinauer
Associates Inc.
Crosnier, A. (1986). Crustacés Décapodes: Penaeidae. Les espèces indo-ouest pacifiques du genre Parapenaeus. In: Crosnier, A. (ed.),
Résultats des Campagnes MUSORSTOM I et II—Philippines (1976,
1980), (vol. 2, pp. 303- 353). Mémoires du Muséum national d’Histoire naturelle (A) Zoologie.
Crosnier, A. (1987). Les espèces indo-ouest-pacifiques d'eau profonde
du genre Metapenaeopsis (Crustacea Decapoda Penaeidae). Bulletin
du Muséum national d'Histoire naturelle. Section A, Zoologie,
Biologie et Écologie Animales, 9(2), 409–453.
Crosnier, A. (1991). Crustacea Decapoda: Les Metapenaeopsis
indo-ouest-pacifiques sans appareil stridulant (Penaeidae).
Deuxième Partie. InA. Crosnier (Ed.), Résultats Des Campagnes
MUSORSTOM, (vol. 9, pp. 155–297). Paris, France: Mémoires du
Muséum national d'Histoire naturelle.
Crosnier, A. (1994a). Crustacea Decapoda: Les Metapenaeopsis indoouest-pacifiques avec un appareil stridulant (Penaeidae). Deuxième
partie. In A. Crosnier (Ed.), Résultats des Campagnes MUSORSTOM,
(A) (vol. 161, pp. 255–337). Paris, France: Mémoires du Muséum
national d'Histoire naturelle.
Crosnier, A. (1994b). Crustacea Decapoda: Les Metapenaeopsis indoouest-pacifiques sans appareil stridulant (Penaeidae). Descrition
de deux espèces nouvelles. In A. Crosnier (Ed.), Résultats des
Campagnes MUSORSTOM, (A) (vol. 161, pp. 339–349). Paris,
France: Mémoires du Muséum national d'Histoire naturelle.
Crosnier, A. (2003). Sicyonia (Crustacea, Decapoda, Penaeoidea,
Sicyoniidae) de l'Indo-ouest Pacifique. Zoosystema, 25, 197–350.
Crosnier, A. (2005). Deux Parapenaeus nouveaux (Crustacea,
Decapoda, Penaeidae) du Sud-Ouest Pacifique. Zoosystema, 27,
Dall, W. (2007). Recent molecular research on Penaeus sensu lato.
Journal of Crustacean Biology, 27(2), 380–382. https://doi.
Dall, W., Hill, B., Rothlisberg, P., & Sharples, D. (1990). The biology of
the Penaeidae (vol. 27). Cambridge, MA: Academic Press.
Davie, P. J. (2002). Crustacea: Malacostraca: Phyllocardia,
Hoplocardia, Eucarida (Vol. 19). Clayton, Vic., CSIRO Publishing.
De Grave, S., & Fransen, C. (2011). Carideorum catalogus: The recent
species of the dendrobranchiate, stenopodidean, procarididean and
caridean shrimps (Crustacea: Decapoda). Leiden, the Netherlands:
NCB Naturalis Leiden.
HURZAID et al.
Dore, I., & Frimodt, C. (1987). An illustrated guide to shrimp of the
world. New York, NY: Van Nostrand Reinhold.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: A maximum likelihood approach. Journal of Molecular Evolution, 17(6),
368–376. https://doi.org/10.1007/BF017​34359
Fernandes, T. J., Silva, C. R., Costa, J., Oliveira, M. B. P., & Mafra,
I. (2017). High resolution melting analysis of a COI mini-barcode as a new approach for Penaeidae shrimp species discrimination. Food Control, 82, 8–17. https://doi.org/10.1016/j.foodc​
Flegel, T. (2007). The right to refuse revision in the genus Penaeus.
Aquaculture, 264(1–4), 2–8. https://doi.org/10.1016/j.aquac​
Flegel, T. (2008). Confirmation of the right to refuse revision in the
genus Penaeus. Aquaculture, 1(280), 1–4. https://doi.org/10.1016/j.
Folmer, O., Hoeh, W., Black, M., & Vrijenhoek, R. (1994). Conserved
primers for PCR amplification of mitochondrial DNA from different
invertebrate phyla. Molecular Marine Biology and Biotechnology,
3, 294–299.
Galal-Khallaf, A., Ardura, A., Borrell, Y. J., & Garcia-Vazquez, E. (2016).
PCR-based assessment of shellfish traceability and sustainability in
international Mediterranean seafood markets. Food Chemistry, 202,
302–308. https://doi.org/10.1016/j.foodc​hem.2016.01.131
Geller, J., Meyer, C., Parker, M., & Hawk, H. (2013). Redesign of
PCR primers for mitochondrial cytochrome c oxidase subunit
I for marine invertebrates and application in all-taxa biotic surveys. Molecular Ecology Resources, 13(5), 851–861. https://doi.
George, M. J. (1973). On the penaeid prawn Parapenaeopsis stylifera
and a new variety of the species from Cochin. Journal of the Marine
Biological Association of India, 15(1), 420–422.
Gusmão, J., Lazoski, C., Monteiro, F., & Solé-Cava, A. (2006). Cryptic
species and population structuring of the Atlantic and Pacific seabob shrimp species, Xiphopenaeus kroyeri and Xiphopenaeus riveti.
Marine Biology, 149(3), 491–502. https://doi.org/10.1007/s0022​
Gusmão, J., Lazoski, C., & Solé-Cava, A. (2000). A new species of
Penaeus (Crustacea: Penaeidae) revealed by allozyme and cytochrome oxidase I analyses. Marine Biology, 137(3), 435–446.
Hebert, P. D., Ratnasingham, S., & de Waard, J. R. (2003). Barcoding
animal life: cytochrome c oxidase subunit 1 divergences among
closely related species. Proceedings of the Royal Society of London
B: Biological Sciences, 270 (Suppl 1), S96–S99. https://doi.
Hebert, P. D., Stoeckle, M. Y., Zemlak, T. S., & Francis, C. M. (2004).
Identification of birds through DNA barcodes. PLoS Biology, 2(10),
e312. https://doi.org/10.1371/journ​al.pbio.0020312
Holthuis, L. B. (1980). FAO Species catalogue, Vol. 1. Shrimps and
prawns of the world. An annotated catalogue of species of interest to
fisheries. FAO Fisheries Synopsis, 125(1), 1- 271.
Hubert, N., Hanner, R., Holm, E., Mandrak, N. E., Taylor, E., Burridge,
M., … Bernatchez, L. (2008). Identifying Canadian freshwater
fishes through DNA barcodes. PLoS ONE, 3(6), e2490. https://doi.
Hung, K. W., Russell, B. C., & Chen, W. J. (2017). Molecular systematics of threadfin breams and relatives (Teleostei, Nemipteridae).
Zoologica Scripta, 46(5), 536–551. https://doi.org/10.1111/zsc.12237
HURZAID et al.
Johnson, K. P., & Clayton, D. H. (2000). Nuclear and mitochondrial
genes contain similar phylogenetic signal for pigeons and doves
(Aves: Columbiformes). Molecular Phylogenetics and Evolution,
14(1), 141–151. https://doi.org/10.1006/mpev.1999.0682
Kekkonen, M., & Hebert, P. D. (2014). DNA barcode-based delineation of putative species: Efficient start for taxonomic workflows. Molecular Ecology Resources, 14(4), 706–715. https://doi.
Kimura, M. (1980). A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), 111–120. https://
Klinbunga, S., Penman, D., McAndrew, B., & Tassanakajon, A. (1999).
Mitochondrial DNA diversity in three populations of the giant tiger
shrimp Penaeus monodon. Marine Biotechnology, 1(2), 113–121.
Klinbunga, S., Siludjai, D., Wudthijinda, W., Tassanakajon, A.,
Jarayabhand, P., & Menasveta, P. (2001). Genetic heterogeneity of the giant tiger shrimp (Penaeus monodon) in Thailand revealed by RAPD and mitochondrial DNA RFLP analyses. Marine
Biotechnology, 3(5), 428–438. https://doi.org/10.1007/s1012​
Kubo, I. (1949). Studies on penaeids of Japanese and its adjacent waters.
Journal of the Tokyo College of Fisheries, 36, 1–467.
Lanfear, R., Calcott, B., Ho, S. Y., & Guindon, S. (2012). PartitionFinder:
Combined selection of partitioning schemes and substitution models
for phylogenetic analyses. Molecular Biology and Evolution, 29(6),
1695–1701. https://doi.org/10.1093/molbe​v/mss020
Lavery, S., Chan, T., Tam, Y., & Chu, K. (2004). Phylogenetic relationships and evolutionary history of the shrimp genus Penaeus sl derived
from mitochondrial DNA. Molecular Phylogenetics and Evolution,
31(1), 39–49. https://doi.org/10.1016/j.ympev.2003.07.015
Li, X., Xu, Y., & Kou, Q. (2014). Molecular phylogeny of Parapenaeopsis
Alcock, 1901 (Decapoda: Penaeidae) based on Chinese materials
and 16S rDNA and COI sequence. Journal of Ocean University of
China, 13(1), 104–114. https://doi.org/10.1007/s1180​2-014-2272-7
Lin, C. P., & Danforth, B. N. (2004). How do insect nuclear and mitochondrial gene substitution patterns differ? Insights from
Bayesian analyses of combined datasets. Molecular Phylogenetics
and Evolution, 30(3), 686–702. https://doi.org/10.1016/S1055​
Lo, P. C., Liu, S. H., Nor, S. A. M., & Chen, W. J. (2017). Molecular
exploration of hidden diversity in the Indo-West Pacific sciaenid
clade. PLoS ONE, 12(4), e0176623. https://doi.org/10.1371/journ​
Lovett, L. (1981). A guide to the shrimps, prawns, lobsters, and crabs of
Malaysia and Singapore. Occasional publication (vol. 2). Selangor,
Malaysia: Universiti Pertanian Malaysia.
Ma, K. Y., Chan, T. Y., & Chu, K. H. (2009). Phylogeny of penaeoid
shrimps (Decapoda: Penaeoidea) inferred from nuclear protein-coding genes. Molecular Phylogenetics and Evolution, 53(1), 45–55.
Ma, K. Y., Chan, T. Y., & Chu, K. H. (2011). Refuting the six-genus classification of Penaeus s.l. (Dendrobranchiata, Penaeidae): A combined
analysis of mitochondrial and nuclear genes. Zoologica Scripta,
40(5), 498–508. https://doi.org/10.1111/j.1463-6409.2011.00483.x
Maggioni, R., Rogers, A. D., Maclean, N., & D'Incao, F. (2001).
Molecular phylogeny of western Atlantic Farfantepenaeus and
Litopenaeus shrimp based on mitochondrial 16S partial sequences.
Molecular Phylogenetics and Evolution, 18(1), 66–73. https://doi.
Mantelatto, F. L., Carvalho, F. L., Simões, S. M., Negri, M., SouzaCarvalho, E. A., & Terossi, M. (2016). New primers for amplification of cytochrome c oxidase subunit I barcode region designed for
species of Decapoda (Crustacea). Nauplius, 24, e2016030.
McLaughlin, P. A., Lemaitre, R., Ferrari, F. D., Felder, D. L., &
Bauer, R. (2008). A reply to T.W. Flegel. Aquaculture, https://doi.
Miller, M. A., Pfeiffer, W., & Schwartz, T. (2010, November). Creating
the CIPRES Science Gateway for inference of large phylogenetic
trees. In 2010 gateway computing environments workshop (GCE)
(pp. 1–8). Piscataway, NJ: IEEE.
Moritz, C., & Cicero, C. (2004). DNA barcoding: Promise and pitfalls. PLoS Biology, 2(10), e354. https://doi.org/10.1371/journ​
Page, T. J., Humphreys, W. F., & Hughes, J. M. (2008). Shrimps down
under: Evolutionary relationships of subterranean crustaceans from
Western Australia (Decapoda: Atyidae: Stygiocaris). PLoS ONE,
3(2), e1618. https://doi.org/10.1371/journ​al.pone.0001618
Palumbi, S., & Benzie, J. (1991). Large mitochondrial DNA differences between morphologically similar penaeid shrimp. Molecular
Marine Biology and Biotechnology, 1(1), 27–34.
Palumbi, S., Martin, A., Romano, S., McMillan, W. O., Stice, L., &
Grabowski, G. (1991). The simple fool's guide to PCR. Ver. 2.0.
Honolulu, Hawaii: University of Hawaii.
Pathan, D. I., & Jalihal, D. R. (1998). On the taxonomic status of the
Indian kiddi prawn Parapenaeopsis stylifera (H. Milne Edwards,
1837). Indian Journal of Marine Science, 27, 367–372.
Pérez Farfante, I., & Kensley, B. (1997). Penaeoid and sergestoid
shrimps and prawns of the world. Keys and diagnoses for the families and genera. Paris, France: Editions du Museum national d'Histoire naturelle.
Puillandre, N., Lambert, A., Brouillet, S., & Achaz, G. (2012).
ABGD, Automatic Barcode Gap Discovery for primary species
delimitation. Molecular Ecology, 21(8), 1864–1877. https://doi.
Quan, J., Zhuang, Z., Deng, J., Dai, J., & Zhang, Y.-P. (2004).
Phylogenetic relationships of 12 Penaeoidea shrimp species deduced
from mitochondrial DNA sequences. Biochemical Genetics, 42(9–
10), 331–345. https://doi.org/10.1023/B:BIGI.00000​
Racek, A. A., & Dall, W. (1965). Littoral Penaeinae (Crustacea,
Decapoda) from northern Australia, New Guinea, and adjacent
waters. Verhandelingen der Koninklijke Nederlandse Akademie van
Wetenschappen, afdeeling Natuurkunde (2), 56(3), 1–119.
Rajkumar, G., Bhavan, P. S., Udayasuriyan, R., & Vadivalagan, C.
(2015). Molecular identification of shrimp species, Penaeus
semisulcatus, Metapenaeus dobsoni, Metapenaeus brevicornis,
Fenneropenaeus indicus, Parapenaeopsis stylifera and Solenocera
crassicornis inhabiting in the Coromandel coast (Tamil Nadu, India)
using MT-COI gene. International Journal of Fisheries and Aquatic
Studies, 2, 96–106.
Rambaut, A. (2002). Se-Al sequence alignment editor, version 2.0 a11.
Edinburgh, UK: Institute of Evolutionary Biology.
Rambaut, A. (2012). FigTree v1.4.2. http://tree.bio.ed.ac.uk/softw​are/
figtr​ee/. Accessed March 10, 2019.
Rambaut, A., Suchard, M. A., Xie, D., & Drummond, A. J. (2014).
Tracer v1.6. Retrieved from http://beast.bio.ed.ac.uk/Tracer
Ravindranath, K. (1989). Taxonomic status of the Coromandel shrimp
Parapenaeopsis stylifera coromandelica Alcock (Decapoda,
Penaeidea). Crustaceana, 257–262. https://doi.org/10.1163/15685​
Robalino, J., Wilkins, B., Bracken-Grissom, H. D., Chan, T. Y., &
O'Leary, M. A. (2016). The origin of large-bodied shrimp that
dominate modern global aquaculture. PLoS ONE, 11(7), e0158840.
Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling,
A., Höhna, S., … Huelsenbeck, J. P. (2012). MrBayes 3.2: Efficient
Bayesian phylogenetic inference and model choice across a large
model space. Systematic Biology, 61(3), 539–542.
Sakai, K., & Shinomiya, S. (2011). Preliminary report on eight new genera formerly attributed to Parapenaeopsis Alcock, 1901, sensu lato
(Decapoda, Penaeidae). Crustaceana, 84(4), 491–504. https://doi.
Schram, F. R. (1977). Paleozoogeography of late paleozoic and Triassic
Malacostraca. Systematic Biology, 26(4), 367–379. https://doi.
Schram, F. R. (1982). The fossil record and evolution of Crustacea.
Biology of Crustacea, 1, 93–147.
Schweitzer, C. E., Feldmann, R. M., Garassino, A., Karasawa, H., &
Schweigert, G. (2010). Systematic list of fossil Decapod Crustacean
Species. Crustaceana Monographs, vol. 10. Leiden, the Netherlands:
Sharawy, Z., Abbas, E., Desouky, M., & Kato, M. (2016). Descriptive
analysis and molecular identification of the green tiger shrimp
Penaeus semisulcatus (De Haan, 1844) in Suez Gulf, Red Sea.
International Journal of Fisheries and Aquatic Studies, 4(5),
Sodsuk, S. (1996). Genetic differentiation and population structure of
Penaeus monodon in Thailand. Technical Paper No. 12 (pp. 19).
National Aquaculture Genetics Research Institute, Department of
Fisheries, Ministry of Agriculture and Cooperatives.
Song, H., Buhay, J. E., Whiting, M. F., & Crandall, K. A. (2008). Many
species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified.
Proceedings of the National Academy of Sciences, 105(36), 13486–
13491. https://doi.org/10.1073/pnas.08030​76105
Stamatakis, A. (2014). RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9),
1312–1313. https://doi.org/10.1093/bioin​forma​tics/btu033
Swofford, D. L. (2003). PAUP*. Phylogenetic Analysis Using Parsimony
(*and Other Methods). Version 4.0b10. Sunderland, MA: Sinauer
Associates, Inc.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013).
MEGA6: Molecular evolutionary genetics analysis version 6.0.
Molecular Biology and Evolution, 30(12), 2725–2729. https://doi.
Tassanakajon, A., Klinbunga, S., Paunglarp, N., Rimphanitchayakit,
V., Udomkit, A., Jitrapakdee, S., … Lursinsap, C. (2006). Penaeus
monodon gene discovery project: The generation of an EST collection and establishment of a database. Gene, 384, 104–112. https://
Tavares, C., & Gusmao, J. (2016). Description of a new Penaeidae
(Decapoda: Dendrobranchiata) species. Farfantepenaeus isabelae
sp. nov. Zootaxa, 4171(3), 505–516. https://doi.org/10.11646​/zoota​
View publication stats
HURZAID et al.
Timm, L., Browder, J. A., Simon, S., Jackson, T. L., Zink, I. C., &
Bracken-Grissom, H. D. (2019). A tree money grows on: The first
inclusive molecular phylogeny of the economically important pink
shrimp (Decapoda: Farfantepenaeus) reveals cryptic diversity.
Invertebrate Systematics, 33(2), 488–500. https://doi.org/10.1071/
Tong, J. G., Chan, T. Y., & Chu, K. H. (2000). A preliminary phylogenetic analysis of Metapenaeopsis (Decapoda: Penaeidae) based
on mitochondrial DNA sequences of selected species from the
Indo-West Pacific. Journal of Crustacean Biology, 20(3), 541–549.
Tsang, L., Ma, K., Ahyong, S., Chan, T.-Y., & Chu, K. (2008). Phylogeny
of Decapoda using two nuclear protein-coding genes: Origin and
evolution of the Reptantia. Molecular Phylogenetics and Evolution,
48(1), 359–368. https://doi.org/10.1016/j.ympev.2008.04.009
Tsoi, K. H., Ma, K. Y., Wu, T., Fennessy, S. T., Chu, K. H., & Chan,
T. Y. (2014). Verification of the cryptic species Penaeus pulchricaudatus in the commercially important kuruma shrimp P. japonicus (Decapoda: Penaeidae) using molecular taxonomy. Invertebrate
Systematics, 28(5), 476–490. https://doi.org/10.1071/IS14001
Vazquez-Bader, A. R., Carrero, J. C., Garcia-Varela, M., Grcia, A.,
& Laclette, J. P. (2004). Molecular phylogeny of superfamily
Penaeoidea Rafinesque-Schmaltz, 1815, based on mitochondrial
16S partial sequence analysis. Journal of Shellfish Research, 23(3),
Voloch, C. M., Freire, P. R., & Russo, C. A. (2005). Molecular phylogeny of penaeid shrimps inferred from two mitochondrial markers.
Genetics and Molecular Research, 4(4), 668–674.
Ward, R. D., Zemlak, T. S., Innes, B. H., Last, P. R., & Hebert, P. D.
(2005). DNA barcoding Australia's fish species. Philosophical
Transactions of the Royal Society B: Biological Sciences, 360(1462),
1847–1857. https://doi.org/10.1098/rstb.2005.1716
Yang, C. H., Sha, Z., Chan, T. Y., & Liu, R. (2015). Molecular phylogeny of the deep-sea penaeid shrimp genus Parapenaeus (Crustacea:
Decapoda: Dendrobranchiata). Zoologica Scripta, 44(3), 312–323.
Yu, H. P., & Chan, T. Y. (1986). The illustrated Penaeoid prawns of
Taiwan. Taipei, Taiwan: Southern Materials Center.
Zhang, J., Kapli, P., Pavlidis, P., & Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29(22), 2869–2876. https://doi.org/10.1093/
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Hurzaid A, Chan T-Y, Mohd
Nor SA, Muchlisin ZA, Chen W-J. Molecular
phylogeny and diversity of penaeid shrimps
(Crustacea: Decapoda) from South-East Asian waters.
Zool Scr. 2020;00:1–18. https://doi.org/10.1111/