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Swordfish (Xiphias gladius) is a migratory fish commercially exploited due to high export value. Presence of parasites in fish lead to economic losses in the export market and public health issues. This study was conducted to identify the parasite larvae inhabiting swordfish and to determine its phylogenetic origin using ribosomal subunit gene sequence. Parasite samples were extracted from swordfish muscles and five larvae belong to Molicola genus, confirmed by scolex morphology, were used for genomic DNA extraction. Polymerase chain reaction (PCR) was performed to amplify 18S and 28S ribosomal RNA (rRNA) subunit genes followed by Sanger sequencing. DNA sequences were edited by BioEdit software and assembled by CLC genomics version 8.0. Consensus sequences were aligned with NCBI blast to determine the species status. Isolated larval sequences were best aligned with genus Molicola followed by genus Gymnorhyncha. Out of the two published Molicola rRNA gene sequences, 99% identity was observed with Molicola sp. HP5 isolate from Indonesia. Due to lack of sequence data on other Molicola species (except M. thyristes) for comparison, our sequences were published as Molicola sp. Sri Lankan isolates. This is the first record of Molicola sp. in swordfish from Sri Lanka and the results will enhance the knowledge on the distribution of Molicola species while contributing to expand the genetic information on rRNA coding sequences.
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Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021
DOI : 10.20473/jafh.v10i1.20905
De Silva et al. (2021)
66
Molecular Identification of the Genus Molicola Larvae from
Swordfish (Xiphias gladius) Captured in Sri Lanka by Ribosomal
Subunit Gene Sequencing
D.P.N. De Silva1, 2*, J.L.C.S. Perera3, H.S.D. Fernando1, R.R.M.K.P. Ranatunga1 and
B.G.D.N.K. De Silva1
1Center for Biotechnology, Department of Zoology, Faculty of Applied Sciences, University of Sri
Jayewardenepura, Nugegoda, Sri Lanka
2Department of Animal Science, Faculty of Animal Science and Export Agriculture, Uva Wellassa
University, Badulla, Sri Lanka
3Laboratory of Veterinary Public Health, Graduate School of Agricultural & Life Sciences, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Abstract
Swordfish (Xiphias gladius) is a migratory fish
commercially exploited due to its high export value. The
presence of parasites in fish leads to economic losses in the
export market and public health issues. This study was
conducted to identify the parasite larvae inhabiting swordfish
and to determine its phylogenetic origin using ribosomal
subunit gene sequence. Parasite samples were extracted from
swordfish muscles and five larvae belong to Molicola genus,
confirmed by scolex morphology, were used for genomic DNA
extraction. Polymerase chain reaction (PCR) was performed to
amplify 18S and 28S ribosomal RNA (rRNA) subunit genes
followed by Sanger sequencing. DNA sequences were edited
by BioEdit software and assembled by CLC genomics version
8.0. Consensus sequences were aligned with NCBI blast to
determine the species status. Isolated larval sequences were
best aligned with genus Molicola followed by genus
Gymnorhyncha. Out of the two published Molicola rRNA gene
sequences, 99% identity was observed with Molicola sp. HP5
isolate from Indonesia. Due to the lack of sequence data on
other Molicola species (except M. thyristes) for comparison,
our sequences were published as Molicola sp. Sri Lankan
isolates. This is the first record of Molicola sp. in swordfish
from Sri Lanka and the results will enhance the knowledge on
the distribution of Molicola species while contributing to
expanding the genetic information on rRNA coding sequences.
INTRODUCTION
Capture fisheries provide 65% of the
animal protein requirement of the people
in Sri Lanka. According to the fisheries
statistics in 2016, its contribution to gross
domestic production (GDP) was around
1.8 % (marine 1.6 %, inland 0.2 %).
Among food fish Tuna species is the
highest catch while swordfish (Xiphias
gladius) is one of the seasonal fish which
has higher demand from importing
countries (Ministry of Fisheries & Aquatic
Resources Development, 2019). Swordfish
*Correspondence :
prasadi@uwu.ac.lk
Received : 2020-07-22
Accepted : 2020-09-05
Keywords :
Molicola, Swordfish,
Trypanorhyncha, rRNA
Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021
DOI : 10.20473/jafh.v10i1.20905
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67
is also called Broadbill, belongs to the
family Xiphiidae of order Perciformes.
They can be found in the Indian, Atlantic
and Pacific oceans as well as in the
Mediterranean, Marmara, Black, and Azov
seas (FAO, 2019). Due to over-fishing, this
species reduced by 28% over the past two
decades (IUCN, 2019). Swordfish migrate
to cold waters for feeding and returns to
warm waters during summer for
spawning. They eat small to relatively
large fishes and occasionally consume
crustaceans and squids (Williams and
Bunkley-Williams, 1996). These feeding
and breeding patterns increase their risk of
getting exposed to parasites. Most of the
marine helminths, especially
Trypanorhyncha species have multiple
hosts during their life cycle including
swordfish and crustaceans as intermediate
hosts (Palm and Caira, 2008).
The parasitism in fish is an
ecological coexistence between two
organisms in close contact. Some parasites
establish an equilibrium of host-parasitic
relationship being encysted or dormant
until it gets a favorable condition to
multiply (Schaperclaus, 1992). It is found
that there are 49 parasitic species infecting
swordfish. About 13 of them are host-
specific while six are super family-specific.
Swordfish harbors about 20 larval
tapeworm species particularly due to the
consumption of intermediate hosts
infested with parasitic larvae. There are 14
cestodes (tapeworms) recorded in
swordfish including Trypanorhynch
(Williams and Bunkley-Williams, 1996).
Parasites infesting swordfish
muscles include Pennella sp. (Copepod)
(Hogans et al., 1985; Castro-Pampillόn et
al., 2002), cestode Trypanorhyncha
plerocercoid larvae of Molicola
(Gymnorhyncus) horridus, and
Gymnorhynchus gigas, and flukes,
Maccalumtrema xiphiados. A zoonotic
parasite Anisakis nematode larvae were
also reported in swordfish muscle (Hogans
et al., 1983; Castro-Pampillόn et al., 2002;
Garcia et al., 2011).
In Sri Lanka, swordfish catch is
seasonal, and a large amount of flesh was
discarded due to parasitic infestation in
muscles. A whitish, thread-like parasite
found in muscles often mistook as the
zoonotic Anisakis species until a
preliminary study using morphological
evaluation of this parasite revealed as
plerocercoid larvae of the genus Molicola
belong to order Trypanorhyncha (De Silva
et al., 2017). But due to higher
morphological similarity within the genus
Molicola, this study was aimed at the
molecular identification methods to
identify up to the species level and to
construct its phylogenetic tree.
The main morphological
characteristics of order Trypanorhyncha
are, scolex (with two or four bothria) and
tentacular apparatus (consists of four
tentacles covered with hooks and attached
to four bulbs by tentacle sheaths) (Palm et
al., 2009). There are about 277 valid
Trypanorhyncha species identified so far
and new species are being added
continuously. Based on a more detailed
cladistic analysis, five Trypanorhyncha
superfamilies and 15 families were found.
The five super families are,
Tentacularioidea, Eutetrarhynchoidea,
Gymnorhynchoidea, Lacistorhynchoidea,
and Otobothrioidea (Palm, 1997).
Taxonomic reports of the genus
Molicola indicated the presence of
parasitic larvae in teleost muscles and
liver. Sunfish (Mola mola) were reportedly
infected by Molicola in France, the
Mediterranean region, Japan, New
Zealand, India, and Canada. Molicola was
also reported on muscles of Thyrsites sp.
from Holland (Knoff et al., 2004). Molicola
horridus possess elongated scolex, four
auriculate and elongated bothridia which
is curved and apically inclined with round-
edged, thick rims that are highly similar to
the morphological examination of the Sri
Lankan isolate of Molicola sp. reported
previously (Knoff et al., 2004; De Silva et
al., 2017). But there are other Molicola
species (Molicola uncinatus (also called
Molicola thyrsitae) (Johns et al., 2009) and
Molicola walteri) which share similar
morphological characteristics (Palm,
2004), indicating the necessity of
Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021
DOI : 10.20473/jafh.v10i1.20905
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68
molecular identification for the
determination of species.
In Sri Lanka, very few studies have
been conducted on marine capture fish
diseases. The presence of cestode and
nematode parasites from Sri Lanka was
reported as early as 1906 (Shipley and
Hornell, 1906). The earliest report of
Trypanorhyncha species from Sri Lanka,
named Halysiorhynchus microcephalus was
isolated from Himantura imbricate (Scaly
Whipray) from the Sri Lankan coast
(Southwell, 1929). A specimen at the
British Museum of Natural History, which
was received from Sri Lanka had been
identified as another Trypanorhynch
larva, Pseudo-gilquinia pillersi (Beveridge
et al., 2007). Since there are a number of
Trypanorhynch parasites inhabiting
various host species, it was a timely
necessity to identify the parasitic species
present in swordfish muscles.
This study focused on the
identification of Molicola plerocercoid
larvae up to the species level and to
develop a phylogenetic tree to determine
its origins. Ribosomal RNA genes of 18S
and 28S subunits were sequenced for
comparative analysis due to their
conserved nature during evolution. Based
on this study Molicola sp. Sri Lankan
isolates found in swordfish enhances the
rRNA gene database of Trypanorhyncha
parasites.
METHODOLOGY
Place and Time
Parasitic samples from the muscles
of swordfish (captured from the FAO
statistical zone 57 of the Indian Ocean),
were obtained from a fish processing
factory in Sri Lanka. Samples were
collected twice during 2015 and 2016,
directly from the fish processing factory
and transferred on ice to the Center for
Biotechnology, Department of Zoology,
University of Sri Jayewardenapura, Sri
Lanka, where all morphological analysis,
DNA extraction and polymerase chain
reaction (PCR) were performed. PCR
products were sent to Microgen, Korea for
the Sanger sequencing.
Research Material
The whitish, thread-like parasitic
samples obtained from swordfish muscle
were confirmed as Molicola plerocercoid
larvae by morphological analysis. To
examine the morphology of the parasite
using wet mounts, compound light
microscope (Olympus, CX22LED, U.S.A.)
and microscope slides were utilized and
the parasitic samples were fixed in 10%
formalin 1.5 ml. Five larvae were
confirmed as genus Molicola based on
their scolex characteristics and used for
genomic DNA extraction. For DNA
extraction, reagents mentioned in the
methods of Ballinger-Crabtree et al.
(1992) were used.
In order to amplify ribosomal
subunit genes, primers were designed for
18S rRNA (ssrRNA) and 28S rRNA
(lsrRNA) gene sequences as shown in
Table 1. Primer sequences and PCR
protocols for 18S rRNA and 28S rRNA
gene amplification were based on Palm et
al. (2009) with slight modifications.
PCR reagents such as magnesium
chloride (MgCl2), deoxyribose nucleotide
triphosphate (dNTP), buffer (5x), Taq
DNA polymerase (University of Colombo,
Sri Lanka) were used to prepare samples
for amplification. Thermal cycler (Master
cycler personal, Eppendorf, USA) was
used to run the PCR. Agarose gel
(Promega, USA) was used in gel
electrophoresis. ABI sequencer (U.S.A.)
was utilized to perform Sanger sequencing
at Microgen, Korea.
Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021
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69
Table 1. Primers used to amplify and sequencing of 18S rRNA and 28S rRNA gene
segments.
Primer type
Primer ID
Primer sequence
ssrDNA (18S rRNA gene)
Forward primer
ssrDNA F1
5`-GCGAATGGCTCATTAAATCAG-3`
Reverse primer
ssrDNA R1
5`-CTTGTTACGACTTTTACTTCC-3`
Internal primers
300F2
5`-AGGGTTCGATTCCGGAG-3`
600R
5`-ACCGCGGCKGCTGGCACC-3`
1270F
5`-ACTTAAAGGAATTGACGG-3`
930F
5`-GCATGGAATAATGGAATAGG-3`
1200F
5`-CAGGTCTGTGATGCCC-3`
lsrDNA (28S rRNA gene)
Forward primer
lsrDNA F1
5`-ACCCGCTGAATTTAAGCATAT-3`
Reverse primer
LsrDNA R1
5`-GCTATCCTGAGGGAAACTTCG-3`
Internal primers
300F
5`-CAAGTACCGTGAGGGAAAGTTG-3`
ECD2
5`-CTTGGTCCGTGTTTCAAGACGGG-3`
400R
5`-GCAGCTTGACTACACCCG-3`
1090F
5`-TGAAACACGGACCAAGG-3`
Research Design
Parasitic samples from swordfish
muscles were obtained by adhering to the
complete randomization method. A total
of 20 parasitic samples was obtained for
morphological diagnosis and from them,
five confirmed Molicola samples (by scolex
morphology) were selected for DNA
extraction followed by a molecular
examination. Each parasitic sample was
tested for both 18S rRNA and 28S rRNA
genes and sequences were compared for
determination of their individual
variations and phylogenetic distance.
Work Procedures
Genomic DNA from five parasites
was separated into two sets for the
detection of two regions of the ribosomal
RNA gene, 18S rRNA and 28S rRNA
respectively. PCR reaction was performed
using the reagents and amounts as below:
Total volume of the PCR product was 25
µl. Genomic DNA 2 ng were used from
each sample as the template. Double
distilled water was added as the negative
control. PCR master mix was prepared by
adding 1 µl each from forward and reverse
primers, 6 µl of magnesium chloride
(MgCl2), 1.25 µl deoxyribose nucleotide
triphosphate (dNTP), buffer (5x) 5 µl, Taq
DNA polymerase (University of Colombo,
Sri Lanka) 0.2 µl into 8.55 µl of double
distilled water to make it 25µl of total
volume.
The thermocycler was run according
to the following conditions: To amplify
18S rRNA gene, initial denaturation at
94°C for 2 minutes followed by 40 cycles
of 94°C, 30 seconds, 54°C, 30 seconds and
72°C, 2 minutes. For 28S rRNA gene
amplification, 95°C, 5 minutes initial
denaturation followed by 40 cycles of
95°C, 30 seconds, 55°C, 30 seconds and
72°C, 2 minutes. Both PCRs were finally
extended at 72°C for 7 minutes before it
was kept at 4°C until electrophoresis. Final
PCR products were observed by one
percent (1%) Agarose gel (Promega, USA)
electrophoresis.
All amplicons of 18S rRNA and 28S
rRNA genes were sequenced by the Sanger
method (ABI sequencer at Microgen,
Korea) and sequences were analyzed to
confirm the species and constructed the
phylogenetic tree.
Data Analysis
Poor quality bases on both ends of
the sequences were trimmed by using Bio
Edit software version 7.2.5 (Hall, 1999).
Multiple alignments was performed using
Clustal W and assembled by de novo
assembly of contigs by CLC Bio genomics
work bench version 8.0 (www.clcbio.
com). Consensus sequences were
compared with the reference sequences
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deposited in National Center for
Biotechnology Information (NCBI) by
basic local alignment tool (BLAST).
Phylogenetic analysis and the taxonomic
tree were performed by using Molecular
Evolutionary Genetics Analysis (MEGA)
version 6.0 (Tamura et al., 2013)
neighbour-joining method (Saitou and
Nei, 1987).
RESULTS AND DISCUSSION
Morphological identification of the
parasitic larvae found on swordfish muscle
was reported previously by our research
group as Molicola plerocercoid larvae
belong to the Order Trypanorhyncha (De
Silva et al., 2017).
The amplicon for 18S rRNA and 28S
rRNA gene, obtained by PCR using region-
specific universal primers for trematodes
showed electrophoresis band size over
1500 bp which formed the consensus
sequences of 18S rRNA and 28S rRNA
genes of about 2030 bp and 1500 bp
respectively. Based on the sequence
analysis of 18S rRNA and 28S rRNA gene,
all five isolates from Sri Lanka were
aligned to Molicola sp. HP5 (Indonesia)
with 99% and 100% identity, followed by
Molicola thyristes isolate Moli (Australia)
and Gymnorhynchus isuri. All three species
belonged to the family Gymnorhynchidae
(Table 2).
Table 2. Species comparison using NCBI blast of 18S and 28S rRNA gene sequences of Sri
Lankan isolates with species belong to superfamily Gymnorhynchoidea.
Species name
Family
28S rRNA
Identity
NCBI
accession
Identity
NCBI
accession
Molicola sp. HP5
Gymnorhynchidae
99%
FJ572913
100%
FJ572949
Molicola thyristes
Gymnorhynchidae
99%
DQ642908
98%
DQ642746
Gymnorhynchus isuri
Gymnorhynchidae
98%
DQ642909
97%
DQ642747
Pintneriella
musculicola
Rhopalothylacidae
98%
FJ572912
96%
FJ572948
Chimaerarhynchus
rougetae
Gymnorhynchidae
95%
DQ642906
93%
DQ642744
Vittirhynchus squali
Gilquiniidae
95%
DQ642905
93%
DQ642743
Sagittirhynchus
aculeatus
Gilquiniidae
95%
DQ642907
93%
DQ642745
Gilquinea robertsoni
Gilquiniidae
95%
FJ572910
93%
FJ572944
Aporhynchus
norvegicus
Aporhynchidae
95%
FJ572911
93%
FJ572947
This clearly confirmed that the
plerocercoid larvae isolated from
swordfish from Sri Lanka (Indian ocean)
belonged to the genus Molicola, family
Gymnorhynchidae. There were point
mutations and insertions in 18S and 28S
rRNA gene sequences possibly due to the
individual variations. The evolutionary
distance between the sequences of five
isolates was not significantly different (p
> 0.05). Therefore, all the parasitic larvae
isolated from swordfish in this study
belong to the same species.
Though the analysis of 18S rRNA
and 28S rRNA gene sequences were
performed in five individual parasitic
samples, there were less than ten
nucleotide variations found (more than
95% identity) indicating that they all
belong to the same species. The significant
identity of Sri Lankan isolates with
Molicola sp. Hp5 from Indonesia obtained
from Taractes rubescens, suggests that
swordfish captured near the Sri Lankan
coast might have migrated via Indonesian
coasts. Furthermore, both T. rubescens and
swordfish are pelagic fish that shows
similar feeding patterns (Scott and Tibbo,
1968; mez-Morales et al., 2008),
justifying the possible entry of Molicola
larvae through their food chain. The other
species of close identity with Sri Lankan
Journal of Aquaculture and Fish Health Vol. 10(1) - February 2021
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71
isolates was Molicola thyristes isolated
from Australia in Thyrsites atun. It is also
an intermediate host of Molicola sp. which
feeds on pelagic crustaceans (Nakamura
and Parin, 1993).
Phylogenetic analysis of 18S rRNA
and 28S rRNA sequences of Molicola Sri
Lankan isolates were compared with 20
Trypanorhyncha species belonged to
different families. Based on the
phylogenetic analysis, the highest
significance with Molicola sp. HP5 isolate
from Indonesia was observed and Sri
Lankan isolates shared the common
ancestor with members from the
Gymnorhynchidae family (Figure 1). The
comparison of both phylogenetic trees
showed similarity in 18S rRNA and 28S
rRNA genes. Accordingly, it confirms the
morphologically recognized genus of the
parasite. Nucleotide sequences of 18S and
28S ribosomal subunit gene of Molicola sp.
Currently, in the NCBI database only two
Molicola species 18S and 28S rRNA gene
sequences were available for comparison.
Therefore, our results were submitted as
Molicola sp. SL 01 to 05. The country of
origin, Sri Lanka was stand by ‘SL’ and one
to five indicates the individual
plerocercoid larval isolates. The Sri
Lankan isolates identified from this study
were deposited at NCBI GenBank with the
accession numbers for 18S rRNA partial
gene sequences starting from KX712332 to
KX 712336 and for 28S rRNA partial gene
sequences from KX712337 to KX712341.
This is the first study on rRNA genes of
Molicola sp. infested in the muscles of
swordfish captured from the Sri Lankan
coast to the best of our knowledge. Further
studies on other Molicola species are
needed to develop a better sequence
comparison and speciation of the genus
Molicola.
A
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Figure 1. Evolutionary relationships of Molicola Sri Lankan isolates with other species
belong to different taxonomic families of order Trypanorhyncha, obtained by
Neighbor-joining method using (A) 18S rRNA and (B) 28S rRNA gene sequences.
Scientific names of the species and their NCBI accession numbers were written
at respective nodes and the branch lengths indicate the evolutionary distance
drawn to a scale bar of 0.01 for 18S and 0.02 for 28S rRNA gene. E. harfordi
belong to the order Diphyllidea was used as an outgroup.
It is mandatory to perform fish
quality checks before exporting to other
countries and the difficulty arose when
both Trypanorhyncha and Anisakis species
look alike grossly in color and consistency
(Muscolino et al., 2012). Although
Molicola is not a zoonotic parasite as
Anisakis studies on mice have shown
anaphylaxis due to Molicola horridus
allergens (Gomez-Morales et al., 2008).
However, the chance of allergic reactions
to occur in consumers by using frozen
swordfish slices is very low, but the
parasitized flesh reduces the consumer
preference (Muscolino et al. 2012). Hence
this method can be used for confirmatory
and differential diagnosis of
Trypanorhyncha parasites from other fish
parasites.
CONCLUSION
The whitish parasite isolated from
swordfish muscles belonged to the genus
Molicola, order Trypanorhyncha.
Molecular phylogenetic analysis and
sequence comparison of 18S and 28S
ribosomal subunit genes revealed a higher
similarity to Molocola sp. HP5 from
Indonesia. This is the first record of
molecular analysis of ribosomal subunit
genes in Molicola species isolated from
swordfish captured near the Sri Lankan
coast.
ACKNOWLEDGMENT
The authors would like to
acknowledge the staff members of the
Center for Biotechnology, Department of
Zoology, University of Sri
Jayewardenapura, Sri Lanka.
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... The scarcity of genetic data is well reflected in public databases, so currently only published sequences of M. uncinatus (Olson et al., 2010;Palomba et al., 2021) and Molicola sp. (De Silva et al., 2021;Palm et al., 2009) are available in GenBank database. In this study, the molecular characterization of the 18S-ITS1-5.8S-ITS2-28S ...
... Our genetic results confirmed the identity of this cestode as belonging to the genus Molicola. The general configuration of our phylogenetic tree was consistent with previous analyses described by De Silva et al. (2021), Palomba et al. (2021 and Santoro et al. (2020) and grouped the obtained sequence with those of Molicola sp. infecting the swordfish from Sri Lanka and the Keeltail pomfret from Indonesia, which indicates the wide geographic and host range of this species. ...
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Muscle gross lesions, associated to parasites, were routinely found during self-inspection in a Spanish fish plant processing Atlantic swordfish. To determine the taxonomic status of these parasites, molecular analysis was performed based on 18S, ITS1, 5.8S, ITS2 and 28S rDNA sequences, obtaining a consensus sequences of 4581 bp for the cestode and 4200 bp for the trematode. Taxonomic affiliation was determined by phylogenetic analysis of combined SSU + LSU rDNA regions using maximum likelihood models. Molecular characterization allows us to identify the trematode Maccallumtrema xiphiados and the cestode Molicola sp. infecting the musculature of the Atlantic swordfish. Both parasites are responsible of significant economic loss to fish industry due to commercial rejection of parasitized products.
... Phylogeny, as well as the genetic distance matrix in Table 2, also confirmed the high similarity of M. uncinatus and G. gigas, while G. isuri represented a separate more basal clade. Although our analysis was performed with a single genetic marker, our results were in agreement with studies including multiple markers (i.e., [7,20,21,23]), supporting this approach as an invaluable method to discriminate among Gymnorhynchidae members. Indeed, it has been suggested that some morphological characters might not have taxonomic value for the genus diagnosis in this family [7]. ...
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