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Revised Hyrcanus group phylogeny based on ITS2 resolution
Ui Wook Hwang
a,b,c,d,*
, Ashraf Akintayo Akintola
a,d
a
Department of Biomedical Convergence Science and Technology, Kyungpook National University, Daegu 41566, Republic of Korea
b
Department of Biology Education, Teachers College, and Institute for Phylogenomics and Evolution, Kyungpook National University, Daegu 41566, Republic of Korea
c
Institute for Korean Herb-Bio Convergence Promotion, Kyungpook National University, Daegu 41566, Republic of Korea
d
Phylomics Inc., Daegu 4910
ARTICLE INFO
Keywords:
Mosquito
Anopheles
Hyrcanus
Species
ABSTRACT
This study presents a meticulous reassessment of the phylogenetic relationships within the Hyrcanus Group of
Anopheles (Anopheles) mosquitoes, a group of substantial medical and epidemiological importance. These vectors
are responsible for the transmission of various diseases, including malaria, posing a signicant public health
challenge in many regions. This research employs advanced molecular techniques, specically focusing on the
Internal Transcribed Spacer 2 (ITS2) region, to enhance taxonomic resolution within this complex group of
mosquitoes. The ITS2 region has proven to be a valuable molecular marker for differentiating closely related
species and rening our understanding of their evolutionary history. A total of 857 ITS2 sequences of Hyrcanus
group members were retrieved from the GenBank after using specic search keywords. 83 out of the initial
retrieved sequenced were then nally selected and utilized in the analysis based on sequence quality and length.
Sequences retrieved represent 22 out of the 25 members of the Hyrcanus group. This study claried the confusion
surrounding the taxonomic placement and synonymy of each member of the Hyrcanus group using the latest
available molecular ITS2 sequence data through Neighbor Joining (NJ), Maximum Likelihood (ML), and
Bayesian Inference (BI) phylogenetic tree analysis. The work also revised the taxonomic groupings based on
morphological characters observed within the group and evaluated the limitations within the classication based
on morphology and the challenges using molecular ITS2 data. A rened understanding of the Hyrcanus group
phylogeny provides a foundation for more precise and tailored approaches to combat malaria and other vector-
borne diseases in South Korea.
Introduction
Mosquitoes within the genus Anopheles (Diptera; Culicidae) are the
only mosquito taxon with the ability to transmit human malaria para-
sites. Yearly, more than 219 million cases of malaria are recorded
leading to about 400,000 deaths (World Health Organization, 2020).
They are also known to be vectors of larial worms (CDC, 2018) as well
as arboviruses of medical and veterinary importance (Yadav et al.,
2003a,b; Rezza et al., 2017; Brustolin et al., 2018). The taxonomical
classication and phylogenetic relationships within the genus Anopheles
(Diptera; Culicidae) mosquito therefore remain important for public
health initiatives.
To date, 485 species of Anopheles mosquitoes have been identied
(Harbach & Kitching, 2016), with 40 species recognized as medically
important due to their role in transmitting Plasmodium parasites (Sinka
et al., 2012). The Subfamily Anophelinae comprises of three genera:
Anopheles Meigen (472 species), Bironella Theobald (8 species), and
Chagasia Cruz (5 species). The genus Anopheles which includes the
vectors of malaria and larial parasites to humans has seven subgenera;
Anopheles (cosmopolitan, 185 species), Baimaia (Oriental, 1 species),
Cellia Theobald (Old World, 224 species), Kerteszia Theobald
(Neotropical, 12 species), Lophopodomyia Antunes (Neotropical, 6 spe-
cies), Nyssorhynchus Blanchard (Neotropical, 39 species) and Stethomyia
Theobald (Neotropical, 5 species) (Harbach & Kitching, 2016). These
taxonomic classications are based on morphological characteristics
such as the number and position of specialized setae borne on the gon-
ocoxites of the male genitalia (Christophers, 1915; Reid, 1968; Harbach
& Kitching 2005).
According to Harbach (2013), the three largest subgenera of
Anopheles i.e. Anopheles, Cellia and Nyssorhynchus, are divided into
* Corresponding author at: Professor/Chief Director, Department of Biology, Teachers College & Institute for Phylogenomics and Evolution, Kyungpook National
University, Daegu 41655, South Korea.
E-mail address: uwhwang@knu.ac.kr (U.W. Hwang).
Contents lists available at ScienceDirect
Journal of Asia-Pacic Entomology
journal homepage: www.elsevier.com/locate/jape
https://doi.org/10.1016/j.aspen.2024.102346
Received 25 July 2024; Received in revised form 21 November 2024; Accepted 24 November 2024
Journal of Asia-Pacic Entomology 27 (2024) 102346
Available online 28 November 2024
1226-8615/© 2024 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology.
hierarchical systems of informal taxonomic categories. Subgenus
Anopheles is divided into two Sections based on the shape of the pupal
trumpet. The wide funnel-shaped trumpet having the longest axis
transverse to the stem of the Laticorn Section distinguishes the semi-
tubular trumpet having the longest axis vertical more or less in line
with the stem of the Angusticorn Section (Reid & Knight, 1961). Sections
of subgenera Anopheles are further divided into Series and with the
larger Series divided into species Groups. Some groups are also further
divided into Subgroups and species complexes. Harbach (2013) also
stated that most of the groupings at each level of classication are
presumed to represent natural groups of species, thus implying phylo-
genetic relationships.
Among members of the Anopheles subgenus is the Hyrcanus group.
The Hyrcanus group is a species complex consisting of 27 morphologi-
cally similar mosquitoes (Harbach, 2004). The high degree of morpho-
logical similarity has led to the misidentication of species or caused
synonymy among the group members. The unresolved taxonomical
classication among the group members has led to the continuous
revision of morphological keys to identify members of the group (Shin &
Hong 2001, Wilkerson et al., 2003, Li et al., 2005, Ma & Yang, 2005, Ma
& Xu, 2005, Hwang et al., 2004, 2006).
In South Korea, six species among the Hyrcanus group have been
reported so far −An. sinensis, An. lesteri, An. pullus, An. sineroides, An.
kleini, and An. belenrae (Li et al., 2005; Rueda, 2005; Hwang, 2007). An.
sinensis −the most widespread and most commonly found species in
South Korea is known as the primary vector species of vivax malaria
(Ree et al., 1967). It is also a vector of larial parasite Wuchereria ban-
crofti (Zhang et al., 1994) and arthropod roundworm −Romanomermis
jingdeensis (Yang et al., 1984) in China, as well as a roundworm Setaria
digitata in sheep and goats in Japan (Zhao, 1992; Anderson, 2000).
To address the problem of synonymy and misidentication in the
Hyrcanus group, molecular phylogeny has been utilized to resolve these
taxonomic uncertainties. The application of molecular markers has been
established as a useful tool for species differentiation, phylogenetic
inference, and population genetics studies (Ma & Xu, 2005; Mas-Coma &
Bargues, 2009; Ma et al., 2023). The second internal transcribed spacer
(ITS2) of ribosomal DNA (rDNA) is a useful marker for studying the
evolution of gene differences in mosquito populations (Walton et al.,
2007; Manni et al., 2015; Artigas et al., 2021). It is also used to distin-
guish closely related species in Anopheles species complexes or groups
(Collins & Paskewitz 1996; Ma & Xu, 2005). Interspecic ITS2 sequence
divergences have also been applied to infer phylogenetic relationships
(Wesson et al., 1992, Marinucci et al., 1999) and it has been utilized to
differentiate member species of the Hyrcanus group in China (Ma et al.,
1998, 2000a,b). Min et al. (2002), Park et al. (2003), Hwang et al.
(2004), and Wilkerson et al. (2003) have also used the marker to
distinguish members of the group such as An. Sinensis, An. pullus and
Table 1
Species name, GenBank accession numbers and the references of the 83
Hyrcanus group mosquitoes employed in this study.
Species GenBank ID Author
Anopheles sinensis AF543861 Gao et al., 2004
AY375465 Wilkerson et al., 2003
AY576907 Gao et al., 2004
AY130469 Min et al., 2002
AY339278 Ree et al., 2005
AY130470 Min et al., 2002
Anopheles belenrae AY375466 Wilkerson et al., 2003
AF187728 Yajun and Mu, 2005
EU789794 Park et al., 2008
GU384707 Joshi et al., 2010
LC634741 Sawabe et al., 2021
Anopheles engarensis AB159604 Sawabe et al., 2003
LC634749 Sawabe et al., 2021
LC634750 Sawabe et al., 2021
Anopheles kleini EU789793 Ma et al., 2008
FJ875072 Ma et al., 2008
AY753740 Li et al., 2005
FJ875074 Ma et al., 2008
KC797431 Choochote et al., 2014
KC797434 Ree et al., 1967
Anopheles sineroides AJ620895 Sawabe et al., 2003
AB159605 Sawabe et al., 2003
EU789795
OP902521
Park et al., 2008; Hong et al.,
2022
Anopheles kweiyangensis AF261150 Ma & Xu, 2005
Anopheles kunmingensis AY170920 Ma & Xu, 2005
Anopheles liangshanensis AY170922 Ma & Xu, 2005
KU682194 Fang et al., 2007
KU682195 Fang et al., 2007
AF146750 Ma & Xu, 2005
Anopheles crawfordi AB839803 Hempolchom et al., 2013
AB779159 Saeung et al., 2012
AB839801 Hempolchom et al., 2013
AB779135 Saeung et al., 2012
Anopheles paraliae AB839838 Hempolchom et al., 2013
AB733023 Taai et al., 2012
AB839839 Hempolchom et al., 2013
AB826117 Hempolchom et al., 2013
AB733027 Taai et al., 2012
Anopheles lesteri AJ620902 Ma & Xu, 2005
AJ620899 Ma & Xu, 2005
AJ620900 Ma & Xu, 2005
AB733021 Taai et al., 2012
AB733020 Taai et al., 2012
AF384172 Ma & Xu, 2005
Anopheles
anthropophagus
AJ620897 Hwang, 2006
AF543860 Gao et al., 2004
AY375467 Wilkerson et al., 2003
AY803793 Fen and Bao, 2005
AY803792 Fen and Bao, 2005
Anopheles pseudopictus AM773810 Poncon et al., 2008
GU478907 Oshaghi et al., 2010
GU478906 Oshaghi et al., 2010
Anopheles hyrcanus AM773809 Poncon et al., 2008
AY515177 Djadid & Zakeri, 2003
AY533855 Djadid & Zakeri, 2003
DQ243835 Djadid & Gholizadeh, 2005
AM773808 Poncon et al., 2008
DQ243836 Djadid & Gholizadeh, 2005
AF261149 Ma & Xu, 2005
Anopheles junlianensis AY170192 Ma & Xu, 2005
Anopheles
yatsushiroensis
AY576908 Gao et al., 2004
AY186792 Ma & Xu, 2005
Anopheles pullus AY339273 Hwang et al., 2004
AY170923 Ma & Xu, 2005
AY339272 Hwang et al., 2004
EU789792 Park et al., 2008
AY375471 Wilkerson et al., 2003
AY444345 Hwang et al., 2004
AY170924 Ma & Xu, 2005
Anopheles peditaeniatus AF543862 Gao et al., 2004
Table 1 (continued )
Species GenBank ID Author
AB715036 Saeung et al., 2012
AY129958 Ma & Xu, 2005
AB715034 Saeung et al., 2012
AB715039 Saeung et al., 2012
Anopheles argyropus AB826053 Thongsahuan et al., 2013
AB826055 Thongsahuan et al., 2013
AB826056 Thongsahuan et al., 2013
Anopheles nigerrimus AB778780 Songsawatkiat et al., 2013
AB778779 Songsawatkiat et al., 2013
Anopheles nitidus AB777799 Songsawatkiat, et al., 2014
AB777802 Songsawatkiat, et al., 2014
AB777800 Songsawatkiat, et al., 2014
Anopheles pursati AB826070 Thongsahuan et al., 2014
AB826071 Thongsahuan et al., 2014
AB826072 Thongsahuan et al., 2014
Anopheles argyropus AB826053 Thongsahuan et al., 2014
AB826055 Thongsahuan et al., 2014
AB826056 Thongsahuan et al., 2014
U.W. Hwang and A.A. Akintola
Journal of Asia-Pacic Entomology 27 (2024) 102346
2
An. lesteri in South Korea, the Philippines, China, and Thailand. Other
than its use as a standard complementary effective barcode of accurate
identication to COI, more ITS2 sequences of Hyrcanus group members
are readily available in the GenBank database for members of the group,
therefore presenting the opportunity to carry out more robust analyses
within the group.
Hwang (2007) had earlier conrmed the monophyly of the Hyrcanus
group as well as the reexamination of unidentied and misidentied
ITS2 sequences. The research showed that An. yatsushiroensis is a syno-
nym for An. anthropophagus just as An. pullus is a synonym for An. lesteri.
Similar taxonomic discrepancies can also be seen in the case of An.
kunmingensis and An. liangshanensis as well as in An. hyrcanus and An.
pseudopictus (Hwang, 2007). Hwang (2007) also reported the cases of
unidentied members within the Hyrcanus group previously reported
by earlier works of (Li et al., 2005). Similar taxonomic resolutions have
led to renaming two previously unidentied samples as An. belenrae and
An. kleini by Rueda (2005).
Building on the work of Hwang (2007), this study incorporates
additional and recent sequence data to conduct a detailed comparative
analysis of ITS2 sequences from 22 Hyrcanus group species retrieved
from GenBank. This study therefore seeks to enhance the taxonomic
placement and resolve genetic relationships within the Hyrcanus group
using molecular phylogenetic analysis and genetic sequence
comparison.
Fig. 1. Adapted ITS2 sequence alignment showing the interspecic variations among the Hyrcanus group members. The red box highlights the area of focus that
clearly distinguishes the species.
U.W. Hwang and A.A. Akintola
Journal of Asia-Pacic Entomology 27 (2024) 102346
3
Materials and methods
ITS2 sequence retrieval and sequence alignment
To retrieve the ITS2 sequences, the keywords “ITS2/INTERNAL
TRANSCRIBED SPACER 2 +Anopheles +species name of members of
the Hyrcanus group” were searched on the NCBI database. 857 ITS2
nucleotide sequences from 22 Hyrcanus group members available in the
GenBank as at 14th of December 2023 were therefore retrieved using
this procedure. Information of the ITS2 sequences is contained in
Table 1. The retrieved ITS2 sequences for each species were then aligned
individually using the ClustalX2 multiple alignment program (Larkin
et al., 2007) to examine the intraspecic variations among the ITS2
sequences of the Hyrcanus group members. Selections of the represen-
tative sequences of each species were then made based on species
availability (some species only have two representatives), sequence
quality and length. These were then subsequently used for comparative
and phylogenetic analysis. 83 sequences, representing the 22 Hyrcanus
group members were nally selected and available in Table 1 alongside
their author information. Likewise, the genetic distances among the
examined Hyrcanus group members were calculated using the Kimura-
2-parameter method. Finally, for the phylogenetic analysis, represen-
tatives of the 22 species were aligned using the Clustal X2 (Larkin et al.,
2007) and BioEdit (Hall, 1999). The resultant alignment was rened by
eye with care. The two extruding ends caused by the differences in the
sequenced lengths were trimmed to construct a rened alignment sub-
jected to subsequent phylogenetic analyses.
Phylogenetic analyses
Three different tree-making algorithms were employed in the present
phylogenetic analyses: the neighbor joining (NJ), maximum likelihood
(ML) and bayesian inference (BI) methods. The NJ tree was constructed
with the MEGAX (Kumar et al., 2020) software with the bootstrap values
set 1000. The GTR +G +I (general time reversible model +among site
rate variation +invariable sites) model was selected as best-t substi-
tution model by the Model Test conducted using the IQ-Tree software
package (https://www.iqtree.org) for the ML tree. The ML tree was
constructed on the IQ-TREE webserver (https://iqtree.cibiv.univie.ac.
at), and each sequence dataset was set to 1000 maximum iterations
with 1000 replicates. The BI tree was reconstructed using MrBayes 3.22
(Ronquist et al., 2012) under two parallel runs for 10 million iterations
with a sampling frequency of 1,000 iterations. After determining that
the Markov chain Monte Carlo (MCMC) generations reached a stationary
level, the initial 20 % of the generations were removed as burn-in. The
Bayesian Posterior Probability (BPP) is also displayed at each node.
Results
Intra- and Interspecic variation of the ITS2 sequences
A total of 857 ITS2 sequences of Hyrcanus group members were
retrieved from the GenBank. Sequences that were previously denoted as
synonyms were also retrieved to make the right inference of their syn-
onymy from the phylogenetic tree. Apart from An. kunmingensis, An.
Fig. 2. ITS2 sequence alignment showing the lineage (subgroups) among the Hyrcanus group members.
U.W. Hwang and A.A. Akintola
Journal of Asia-Pacic Entomology 27 (2024) 102346
4
junlianensis and An. kweiyangensis with one ITS2 sequence each, at least
three ITS2 sequences were retrieved for all the Hyrcanus group members
to infer relationship within and among the species. All sequences of the
same species were aligned individually, and a selection of the best se-
quences were made based on the sequence quality, variability and
length. Based on initial assessment, the ITS2 sequences of synonymous
group members were also compared. 83 ITS2 sequences out of the initial
selections were nally selected and used in the analysis based on
sequence quality and length (Fig. 1).
From the comparative analysis, seven lineages or subgroups (Fig. 2)
of the Hyrcanus group members were made such as the Sinensis lineage
(An. sinensis, An. kleini, An. belenrae and An. engarensis), Sineroides
lineage (An. kweiyangensis, An. kunmingensis, An. sineroides, and An.
liangshanensis), Lesteri lineage (An. paraliae, An. anthropophagus, and An.
lesteri), Hyrcanus lineage (An. hyrcanus, An. pseudopictus, An. yatsushir-
oensis, An. pullus, and An. junlianensis), Argyropus lineage (An. niger-
rimus, An. nitidus, An. pursati, and An. argyropus) as well as An.
peditaeniatus and An. crawfordi that are also denoted as Peditaeniatus
and Crawfordi lineages respectively. The intraspecic variations can be
seen in Table 2.
Phylogeny
For species with more than one similar sequence, at least one or two
sequences were randomly selected to perform comparative and phylo-
genetic analysis. Only 22 Hyrcanus group members have their ITS2 se-
quences available in GenBank. These sequences were then aligned and
rened. As shown in Fig. 2, the seven lineages described earlier bear
sequence similarity among each other as evident in their high node
condence values in the unrooted phylogenetic tree based on the NJ,
ML, and BI algorithms (Figs. 3–5). The phylogeny also shows the simi-
larities and variations that exist among the Hyrcanus group members.
Interestingly, there are variations among members of the Lesteri lineage
due to a few nucleotide differences. The genetic distance between them
can however distinguish them into type A, B and C.
Discussion
The Hyrcanus group of mosquitoes is of signicant medical impor-
tance, yet their phylogenetic relationships remain unresolved. The
taxonomic classications based on morphological characteristics pre-
sent challenges due to the high degree of similarity and variation within
the group (Harbach, 2004; Harbach & Kitching, 2005). The latest clas-
sication by Harbach & Kitching (2016), which is available online at htt
ps://mosquito-taxonomic-inventory.info/, identies 25 species within
the Hyrcanus group. However, morphological characteristics alone are
insufcient for ascertaining their phylogeny.
Previous studies that have utilized molecular phylogeny as a tool to
understand the variations within the group have encountered several
challenges. Hwang (2007) examined the limitations in the study of Ma &
Xu (2005) that utilized the ITS2 sequences for the phylogenetic infer-
ence within the group. These include inappropriate outgroup selected
and rened alignment of the 23 ITS2 sequences of the Hyrcanus group
members. 3 of these ITS2 sequences which were previously unidentied
were later concluded to be identical to An. belenrae. Likewise, An. lesteri
was found to have three types. This work also resolved some synonymy
issues among group members.
Furthermore, to achieve a robust phylogenetic tree that addresses the
challenges within the group requires more comprehensive analysis. The
Harbach & Kitching (2016) latest classication framework of the group
presents conicts with the sequences available online for members of the
species. However, as pointed out by Harbach (2013), formal (e.g family
Culicidae, genus Anopheles and species gambiae) and informal (e.g the
Pyretophorus Series, Hyrcanus Group or Gambiae Complex) taxonomic
entities are conceptual constructs invented by taxonomists to create
some order in the diversity of species. Thus, the Hyrcanus Group, which
Fig. 3. Adapted unrooted phylogenetic tree using the 22 Hyrcanus group members reconstructed based on the neighbor joining (NJ) method. The tree was con-
structed using ITS2 sequences with the MEGA X software under the K2P model with percentage bootstrap values (1000 replicates) shown on the branches.
U.W. Hwang and A.A. Akintola
Journal of Asia-Pacic Entomology 27 (2024) 102346
5
are human-conceived taxonomic concepts, cannot be observed as en-
tities or visualized under a microscope.
In this study, the Neighbor-Joining (NJ), Maximum Likelihood (ML),
and Bayesian Inference (BI) phylogenetic trees were reconstructed,
providing further insight into the classication patterns among group
members. Relying solely on morphological characteristics fails to cap-
ture the extensive variations within the group. This research identies
seven major subgroups (lineages) based on the aligned ITS2 sequences of
Hyrcanus group members, underscoring the need to address synonymy
issues within the group.
It is important to note that the identication and naming of the se-
quences submitted to GenBank lie solely with the authors. Due to the
dearth of sequence information for pairwise comparison between se-
quences, authors may inadvertently misname the sequences when sub-
mitting to GenBank to receive accession numbers, creating confusion for
phylogenetic and population genetics studies that rely on such second-
ary data. The gaps observed in alignment analysis are partly because
these sequences are not protein-coding genes; thus, the gaps represent
polymorphic sites for comparison within the group.
This study highlights the necessity of integrating molecular data with
morphological characteristics to achieve a more accurate and compre-
hensive understanding of the Hyrcanus group’s phylogeny. Future
research should focus on acquiring more sequence data and rening
alignment techniques to resolve the taxonomic ambiguities and improve
the reliability of phylogenetic inferences.
Conclusion
It is necessary to address the unresolved taxonomic classications
within the Hyrcanus group members due to the importance of some of
their members as vectors of infectious diseases. Traditional eld tax-
onomy has been utilized as the gold standard for the classication of
mosquitoes. However, recent molecular (ITS2) data made available in
the GenBank allows for exploring the taxonomic resolution of the group
further. The latest morphological data establishes 25 members of the
Hyrcanus group while only 22 species are available based on ITS2 mo-
lecular sequences. Both conclusions are not without their limitations.
Thus, this work attempts to shed more light on the issue to enable re-
searchers in the eld to decipher the right approach to the work. More
work should be done to reconcile the differences between these two
methods of classication to establish the right taxonomic framework for
the Hyrcanus group members.
CRediT authorship contribution statement
Ui Wook Hwang: Writing – review & editing, Validation, Supervi-
sion, Methodology, Investigation, Data curation, Conceptualization.
Ashraf Akintayo Akintola: .
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Fig. 4. Adapted unrooted phylogenetic tree using the 22 Hyrcanus group members reconstructed based on the maximum likelihood (ML) method. The tree was
constructed using ITS2 sequences with the IQtree online site with GTR +G +I model.
U.W. Hwang and A.A. Akintola
Journal of Asia-Pacic Entomology 27 (2024) 102346
6
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