Paper in Molecular Systematics

Molecular Data Unable to Resolve Taxonomic Distinction

between Caranx ignobilis Freshwater and Marine Ecotypes
__________________________________________
In Partial Fulfillment of the Requirements for the Course:
BIO606/607M Advanced Systematics

Submitted to:
Ma. Carmen A. Lagman, PhD
Faculty

Submitted by:
Jovie E. Nicolas
MS Biology
Molecular Data Unable to Resolve Taxonomic
Distinction between Caranx ignobilis Freshwater and
Marine Ecotypes
Jovie E. Nicolas
MS Biology
De La Salle University

INTRODUCTION
 Caranx ignobilis, commonly known as Giant trevally of the Family Carangidae is one of the commercially-
important fishes of the Philippines. The annual production of this commodity was 188,722 kilograms valued at
P33,752,859.79 with more than half of the production was from capture fisheries while the remaining were from
aquaculture (Mutia, 2020). Moreover, C. ignobilis is fabled for its presence in the freshwater Taal Lake, Batangas
and has been a major fish commodity in the area.
In a study by Willette and Padin (2014), the freshwater ecotype of C. ignobilis presented morphological
distinction from marine counterparts. Despite the morphological similarities of marine and freshwater ecotypes,
there are discrete meristic and coloration differences between the marine and freshwater C. ignobilis. It is noticeable
that a pale red coloration is present at the lower jaw and the base of the anal fins and caudal peduncle of freshwater
specimens as compared to the yellowish coloration present in marine specimens. Also, the number of conical teeth
also differs between the marine and freshwater C. ignobilis. Freshwater specimens have four (4) canine teeth at the
lower jaw as compared to only two (2) canine teeth present in marine specimens. However, despite the
morphological differences between the marine and freshwater C. ignobilis, the taxonomic distinction between the
two remains unresolved.
Since Linnaeus, biologists have been using external morphological characters in identifying species, but
these methods can be difficult due to diverse developmental changes that lead to misidentification and a number of
synonyms (Teletchea, 2009). Moreover, these methods cannot discriminate organisms that are morphologically the
same but molecularly different (cryptic) (Mat Jafaar et al., 2012). In response to this, applications opened up a new
way of classifying and identifying organisms. During the past decade, there has been a rapid transition from
morphological to molecular characterization of species (Hebert & Gregory, 2005).
Molecular-based identification and phylogeny reconstruction offers a fast, robust identification of any
species in the planet using a standardized short DNA sequence from an agreed upon region of the genome (Costa &
Carvalho, 2007). It is used not only in identifying species but also in unmasking species that look alike. It enables a
more accurate view of biodiversity and can reveal dangerous organisms masquerading as harmless ones. These
technologies such as DNA barcoding also reduces ambiguity by providing a digital identifying feature, shapes,
colors, and more analog gradation of words (Stoeckle et al., 2004).
This paper intends to discriminate the Caranx ignobilis Freshwater ecotype of Taal Lake from its marine
counterpart using Cytochrome C oxidase subunit I (COI) gene. Results from this paper would back-up the study of
Willette and Padin (2013) on the possible specific distinction between the two ecotypes. Proper species
identification is crucial for the development of proper management and conservation strategies for the
commercially-important fish species of the country.

METHODS
DNA sequences Mining
Mitochondrial Cytochrome C Oxidase subunit I(COI) gene sequences of C. ignobilis from Taal Lake and
Pangasinan were mined from GenBank. For comparison, sequences of C. sexfasciatus, also a species belonging to
the same genus were mined from GenBank and were selected as outgroup.
Sequence Analysis
Sequence identity of the mined COI sequences were verified using BLASTn (table 1)
(https://blast.ncbi.nlm.nih.gov). Consensus sequences were assembled and aligned using Clustal W. Furthermore,
pairwise distances within C. ignobilis (Taal), between C. ignobilis (Taal) and C. ignobilis (Pangasinan), and between
C. ignobilis and C. sexfasciatus were calculated using Tamura-Nei Model (Tamura & Nei, 1993), and a maximum
likelihood tree was constructed with 500 pseudoreplicates using MEGA X (Kumar et al., 2018).

RESULTS
Data Mining and BLASTn Analysis
A total of eight (8) COI sequences were mined from GenBank. Four (4) of which are sequences of C.
ignobilis collected from the Freshwater Taal Lake, Batangas. Another two (2) sequences of C. ignobilis were from
specimens collected from Pangasinan. In addition, two (2) sequences of C. sexfasciatus from Pangasinan were also
mined from GenBank for comparison and were assigned as outgroup (table 1). After data mining, sequences were
evaluated for percent identity using BLASTn. All sequences were assigned to their corresponding species with
100% identity (table 1).

Table 1. Sequences mined from GenBank used in this paper and BLASTn analysis results
Pairwise Distances and Maximum Likelihood Tree
After alignment, the nucleotide composition and pattern of nucleotide substitution were analyzed. The
average nucleotide composition for all taxa were T/U=30.2%, A=24.2%, C=27.0% and G=18.5%. The estimated
transition/transversion bias ratio was 10.904, with higher rates of transitional substitution for C↔T (62.79) than
A↔G (28.28%).
Sequence distance between congeners ranged from 0.200% to 10.368%. The intraspecific distances within
C. ignobilis from Taal Lake were from 0.200% to 0.500% with an average of 0.341%. Furthermore, the average
genetic distance between C. ignobilis and C. sexfasciatus were 9.839%, indicative of their specific distinction from
each other. However. The conspecific distance between the freshwater and marine ecotypes of C. ignobilis were
ranging from 0.170% to 1.027% with an average of 0.635%, which is much lower than the average interspecific
distance. The mean genetic distance between the two ecotypes of C. ignobilis is consistent with the mean
intraspecific distance within the C. ignobilis from Taal Lake.

Table 2. Summary of percent genetic distances within and between species

In the phylogenetic reconstruction of COI gene using maximum likelihood, C. ignobilis from Taal lake and
Pangasinan formed individual subclades. However, these subclades were statistically insignificant with the absence
of bootstrap support. The absence of bootstrap support in these subclades are consistent with the low genetic
distance between the two ecotypes.

16
C. ignobilis Taal 2 | HQ654678.1

39
C. ignobilis Taal 3 | HQ654677.1

67
C. ignobilis Taal 1 | HQ654679.1

C. ignobilis Taal 4 | HQ654676.1

C. ignobilis Pangasinan 1 | KF714905.1

98
C. ignobilis Pangasinan 2 | KC970457.1

C. sexfasciatus Pangasinan 1 | KF714908.1

Figure 1. Caranx COI phylogeny. Maximum likelihood tree illustrating the reconstructed phylogeny of Marine and
Freshwater C. ignobilis based on 500 psuedoreplicates.C. sexfasciatus Pangasinan 2 | KF714907.1
100

Phylogenetic reconstruction of the two ecotypes of C. ignobilis using extracted sequences from GenBank
revealed weak divergence between the two ecotypes having very low genetic distance. Also, the maximum
likelihood tree showed weak divergence between the two ecotypes with the absence of bootstrap support, which
0.010

supposed to prove the significance of each subclade. The results of this paper are consistent with the study of
Aquilino and colleagues in 2011, surveying the Ichthyofauna of Taal Lake using COI barcodes. However, this result
does not support the reports of morphological distinctions between the two ecotypes as discussed in the study of
Willette and Padin in 2014.
 The contradictions between morphological and molecular evidences warrants further studies to delineate
the C. ignobilis population of Taal lake from marine populations. Jacks, which includes C. ignobilis are important
fishery resource of the country. Proper identification of these species is important as framework for the construction
of conservation and management strategies for these economically important fisheries of the country.

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