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International Journal of Advanced Research and Development
ISSN: 2455-4030; Impact Factor: RJIF 5.24
Received: 13-12-2020; Accepted: 27-12-2020; Published: 25-01-2021
www.advancedjournal.com
Volume 6; Issue 1; 2021; Page No. 16-21
Genetic structure of Sinularia (Octocorallia: Alcyoniidae) from Sanya (China) using COI and 28S
markers
Yue Han1, Lu Hao1, Changqin Li2, Jingjing Tian2, Yaxing Liu2, Haolan Wen1, Zhihao Wang1, Weidong Li3*, Pei-Zheng
Wang4*
1 College of Fisheries and Life Sciences, Hainan Tropical Ocean University, Sanya, China
2 Administration of Hainan Sanya National Coral Reef Nature Reserve, Sanya, China
3 College of Ocean, Hainan University, Haikou, China
4 College of Ecology and Environment, Hainan Tropical Ocean University, Sanya, China
Abstract
In this present study we randomly collected 15 samples of Sinularia from Sanya coral reefs which there are belonging to 7
species including Sinularia grandilobata (1 specimen), Sinularia flexibilis (4 specimens), Sinularia querciformis (1 specimen),
Sinularia humilis (2 specimens), Sinularia ceramensis (2 specimens), Sinularia humesi (2 specimens), and Sinularia slieringsi
(3 specimens). Both COI and 28S markers used to study inter-specific genetic variation in Sinularia. Based on this, the
specific genetic variation analysis within the genus was analyzed. The results showed that the 28S sequence of Sinularia is
higher than the mitochondrial COI sequence.
Keywords: soft coral, genetic variation, phylogeny, species distribution
Introduction
Soft corals (Cnidaria: Octocorallia) are important structural
components of coral reef communities and contributors to
coral reef biomass. Most soft corals belong to the family
Alcyoniidae, which is abundant and ecologically important
members of coral (Mc Fadden et al., 2009; Benayahu et al.,
2018, Quattrini et al., 2019) [13, 6, 15]. Despite the abundance
and importance of Alcyoniidae, only few ecological studies
have been done (Fabricius, 1995, 1997, 1998; Fabricius &
Dommisse, 2000; Bastidas et al., 2004; Aratake et al.,
2012), partly due to the difficulties in correctly identifying
species in the field. Colony morphology is highly variable
(Benayahu, 1998), and species classification usually be
distinguished by sclerites found within the tissue
(Verseveldt, 1980; Benayahu et al., 2018) [19, 6]. Recently,
DNA molecular marker techniques have been providing
more evident to solve such problems (Lien et al., 2015).
Sinularia is a genus of Alcyoniidae with approximately 170-
190 described species (van Ofwegen 2002, McFadden et al.
2009) [13]. Sinularia live in extensive areas of habitats in
open water. They also show different size of body
(Fabricius and Alderslade 2001) [7]. Verseveldt (1980) [19]
classified Sinularia into five groups based on morphological
pattern of sclerites. McFadden et al., (2009) [13] used
molecular methods to divided Sinularia in six group, but
they were different with the Verseveldt’s classification.
Although The South China Sea has many coral reefs with
high biodiversity recorded in Taiwan (Benayahu et al.,
2018) [6] and Vietnam (Lien et al., 2015) but there is a lack
of information about taxonomy of Octocorallia specially
Sinularia in China coral reefs. Sanya, located in the south
coast of Hainan Province in China, including several coral
reefers. Therefore, in this study, 15 specimens of Sinularia
were collected randomly around Sanya. Objectives of the
research included: (1) to identify taxonomic status of
collected specimens. (2) to consider phylogenetic
relationship among specimens based on mitochondrial
fragment of COI and nuclear fragment of 28S. (3) to
characterize the difference of molecular variation between
mitochondrial COI and nuclear 28S.
Materials and Methods
Study area and sampling
A total of 15 specimens were collected from Sanya city
coral reefs (18°15′12″N-109°30′13″E, South
China Sea) (Fig. 1). Taxonomical status were identified
according to haplotype patterns of 28S sequence (McFadden
et al., 2009; Benayahu et al., 2018, Quattrini et al., 2019) [13,
6, 15].
Fig 1: Map of Sinularia sampling sites in Sanya. © Google Map, 2020.
International Journal of Advanced Research and Development www.advancedjournal.com
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DNA extraction, PCR amplification and sequencing
DNA was extracted from tissue using of corals using DNA
Isolation Kit. Two fragments of mitochondrial markers
(COI) and one nuclear marker (28S) were amplified. PCR
was carried out on a total volume of 20 μl containing 8 μl of
ddH2O, 10 μl Taq polymerase, 0.4 μl of DNA solution and
0.8 μl of each primer.
The partially combined fragments of mitochondrial CO)
were amplified using special soft colal primers (McFadden
et al., 2004). A fragment of the 28S nuclear ribosomal gene
was amplified using primers suggested by McFadden and
van Ofwegen (2013) [14].
Phylogenetic analyses
Phylogenetic trees were performed Based on the alignment
of COI and 28S using maximum likelihood (ML) in MEGA-
X (Kumar et al. 2018). To detect the genetic differentiation
of samples a median network was performed using the
median-joining algorithm in the Network program ver.
5.0.0.3 (Bandelt et al. 1999) [5].
Results and Discussion
With regard to our results, collected samples from Sanya
belonging to 7 species including Sinularia grandilobata (1
specimen), Sinularia flexibilis (4 specimens), Sinularia
querciformis (1 specimen), Sinularia humilis (2 specimens),
Sinularia ceramensis (2 specimens), Sinularia humesi (2
specimens) and Sinularia slieringsi (3 specimens). Figure 2
and 3 show the photos of studied Sinilaria and morphology
of sclerate for each species
Fig 2: Photos of Sinularia soft coral in Sanya
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Phylogenetic tree based on mitochondrial COI marker
divided Sinularia samples in three major clads that S.
grandilobata located as a basal clad (Fig. 4). In the other
hand, phylogenetic tree using nuclear 28S marker divided
studied samples in two major clads while S. grandilobata
located as a sister clad for S. flexibilis, S. querciformis, S.
humilis (Fig. 5).
Fig 4: Phylogenetic tree of Sinularia using COI sequences based on the ML approach. The number behind major nodes denotes bootstrap
confidential values.
Fig 5: Phylogenetic tree of Sinularia using 28S sequences based on the ML approach. The number behind major nodes denotes bootstrap
confidential values.
Figure 6 showed the COI haplotype distribution network of
the Sinularia among different species. The 15 COI
sequences of Sinilaria have represented 14 distinct
haplotypes, that only two specimens of S. flexibilis
represented same haplotype (H2) (Fig. 6). Figure 7
displayed the 28S haplotype distribution network of the
Sinularia. Fifteen sequences have showed 14 separated
haplotypes. Same as COI haplotype network, two specimens
of S. flexibilis have same haplotype (H4). Haplotypes
information has been summarized in Table 1. According to
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result of COI sequences, S. grandilobata showed significant
differentiation in genetic structure than other Sinularia species and located as basal clad in phylogenetic tree while
this species has low variation in 28S gene.
Fig 6: The relationship of COI haplotypes distribution among species.
Fig 7: The relationship of 28S haplotypes distribution among species.
Table 1: Haplotypes information for COI and 28S sequences in
Sinularia
Haplotype
COI
28S
Species
Individual
No.
Species
Individual
No.
H1
S. querciformis
1
S. humilis
1
H2
S. flexibilis
2
S. humilis
1
H3
S. flexibilis
1
S. flexibilis
1
H4
S. flexibilis
1
S. flexibilis
2
H5
S. grandilobata
1
S. flexibilis
1
H6
S. humesi
1
S. grandilobata
1
H7
S. humilis
1
S. querciformis
1
H8
S. humilis
1
S. ceramensis
1
H9
S. ceramensis
1
S. ceramensis
1
H10
S. ceramensis
1
S. slieringsi
1
H11
S. slieringsi
1
S. humesi
1
H12
S. humesi
1
S. slieringsi
1
H13
S. slieringsi
1
S. humesi
1
H14
S. slieringsi
1
S. slieringsi
1
In animals, generally mitochondrial markers show 5 to 10
times faster alteration than nuclear genes (Allio et al., 2017)
[1]. This trait has caused mitochondrial markers identified as
an important evolutionary markers to distinguish species
and study the genetic variation of animals (Avise, 1994) [3],
also COI has showed important results to identify animal
species (Smith et al., 2005; Hajibabaei et al., 2006; Tavares
and Baker 2008; Baker et al., 2009) [16, 9, 17, 4]. Benayahu et
al. (2012) documented that COI mitochondrial marker
cannot identify species of Klyxum and Cladiella, but nuclear
28S could distinguish species of these genera. Same as our
results, Lien et al. (2015) also confirmed that different
molecular markers show different result for phylogenetic
tree. Recently in Sinularia, sequences of MutS and 28S
sequences can use to identified species of soft coral
(Benayahu et al., 2018; Quattrini et al., 2019) [6, 15].
Based on network distribution, total numbers of mutation
have been reported 54 and 89 mutations for COI and 28S,
respectively. This result documented that 28S nuclear
marker has high level of genetic differentiation than COI
mitochondrial gene.
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21
First comprehensive investigation on soft corals taxonomy
has been done by Benayahu et al. (2012) from the Penghu
Archipelago (Taiwan). They could identify 13 species of
Sinularia based on morphology of sclerites or corals with
identified species from Penghu Archipelago are completely
different with species from Sanya. Lien et al. (2015) studied
on soft coral of Nha Trang bay (Vietnam) from
South China Sea. They identified 12 species of genus
Sinularia using MutS marker which only one species (S.
humilis) are identical as our identified species from Sanya.
In a recent study, Benayahu et al. (2018) [6] have reported 27
species of Sinularia from that six species are same with
species from Sanya (S. ceramensis, S. flexibilis, S. humesi,
S. humilis, S. querciformis, S. slieringsi). Totally it can be
conclude that distribution of genus Sinularia is completely
dissimilar in different localities in South China Sea. It can
be attributed to different ecological condition in South
China Sea which can effect species distribution.
In conclusion, Sinularia, is a common genus of Alcyoniidae
in the Indo-Pacific coral reef (van Ofwegen 2002,
McFadden et al. 2009) [13]. Our results showed that in Sanya
among 15 randomly collected samples, S. flexibilis has high
species density than other species of this genus.
Additionally, mitochondrial (COI) and nuclear (28S)
markers displayed different position of species on
phylogenetic trees which it can be referred to disconnected
evolutionary history for nuclear and mitochondrion
genomes.
Acknowledgements
This project was funded by 2019 Undergraduate Innovation
and Entrepreneurship Program (201811100006) and
cooperative agreement provided by Hainan Province
Science and Technology Department Key Research and
Development Programme (ZDYF2019154).
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