AQUINAS SCHOOL

SAN JUAN CITY

AY 2020 – 2021

Morphological Phylogeny of Selected Sardinella Species

Daniel Ramos III, David Isaac, Jan Vince Sadang, Joaquin Manalang, Manuel Caswell and Ronin Villaroya
Aquinas School
*Corresponding Author: dann.ramm.iii@gmail.com

Abstract: The genus Sardinella comprises of herrings, sardines and other

similar organisms under the Clupeidae family. As part of Clupeidae, these
fishes have shared morphological features. These features are used in tracing
hypothesized lineage of species in a process called phylogenetic analysis.
The paper aims to use phylogenetic analysis in tracing the genetic lineage of
selected sardinella species, constructing a parsimonious phylogenetic tree
based on morphological features. The five species examined in this paper are
Sardinella albella, Sardinella fimbriata, Sardinella gibbosa, Sardinella
lemuru and Sardinella tawilis. Their scientific names are validated in an
online database, Fishbase. Established keys were used as references for the
morphology-based phylogenetic tree. The program PHYLogeny Inference
Package or PHYLIP is used to analyze the phylogeny of the species. The
program constructs a phylogenetic tree that outlines the shared history of the
selected species. The generated tree reveals two key features. First, an inner
clade is constructed with the species Sardinella fimbriata, Sardinella
gibbosa and Sardinella lemuru. Second, the aformentioned clade, S. albella
and S. tawilis form a polytomy structure. To further improve the study, more
discrete characters may be added or, instead, continuous traits may be
substituted. Alternatively, molecular phylogeny may augment the data by
reducing the ambiguity caused by the polytomy.

Key Words: phylogeny; sardinella; morphological phylogeny

Introduction
Sardinella genus
The genus Sardinella comprises of herrings, sardines and other similar organisms under the
Clupeidae family. As part of Clupeidae, these fishes have shared morphological features such as the
absence of scales in the head, mostly fusiform shape of the body and the presence of a single dorsal fin
around the midpoint of the body (fishbase n.d.).

Species under the Sardinella genus usually reside in the Indian, Atlantic and Pacific ocean
especially in warmer coastal areas. According to Fishbase, twenty-two species are recognized to be under
the Sardinella genus. The shape of their supramaxilla distinguishes the species to other clupeoid genera.
Sektiana, Andriyono and Kim (2017) highlighted four features that distinguish the species under
Sardinella from each other: gill raker, pelvic scute, scales and otholith.
 Because of these homologous traits, it is expected that they have shared history via evolution.
Phylogenetic analysis enables biologist to trace the an hypothesized lineage of species.

Phylogenetic analysis
Phylogenetic analysis can be either molecular or morphological. Molecular phylogenetics
involves comparing the nitrogenous bases of a shared genes among organisms. The similarity and
differences in the bases can lead to conclusions of their ancestry. Some applications such as Molecular
Evolutionary Genetic Application X (MEGA X) do the analysis automatically. Morphological
phylogenetics, on the other hand, compares morphological features such as the shape of supramaxilla or
the color of the dorsal fin.

The phylogenetic analysis generates a tree that summarizes the relationship of the selected
organisms or group of organisms called taxon (plural: taxa). Each node represent a common ancestor and
branches represent the differentiation of the ancestor to two different taxa. In Figure 1.1, Taxon B and
Taxon C have diverged from a common ancestor. When two taxa diverge from the same ancestor, they
are consider sister taxa. Taxon A, on the other hand, are not within the group of interest. Thus, it is an
outgroup to Taxon B and C (Brinkman & Leipe 2001).

Figure 1.1. A simple phylogenetic tree. Taxon B and C are sister taxa while Taxon A is
considered as the outgroup to Taxon A and B.

The phylogenetic tree also identifies clades. Clades are simply a set of taxa that include a
common ancestor and all of its descendants (Brinkman & Leipe 2001). For example, Figure 1.2 shows a
sample phylogenetic tree with multiple clades. In the sample, taxon A, B and C are part of a clade since
they are under the ancestor marked as red. Another clade would be taxon A, B, C and D which are
descendants of the ancestor marked as blue. Organisms in a clade is known to be in monophyly and, thus,
are called monophyletic group (Brinkman & Leipe 2001).

Shared traits in a monophyletic group are described as a synapomorphic trait. Once a trait
changes in a lineage, the trait is known to be apomorphic. Autapomorphic traits are apomorphic traits
unique to a taxon.
 Figure 1.2. The red box encloses a clade consisting the taxon A, B and C. The blue box
consists of the clade with Taxons A, B, C and D. Taxon E is seen as the outgroup.

PHYLogeny Inference Package or PHYLIP

The package PHYLogeny Inference Package or PHYLIP has a set of diverse options for both
molecular and morphological methods among others. The documentation of the program package details
four general methods based on input data—molecular sequence, distance matrix, continous characters and
discrete characters (Felsenstein 2005).

The molecular sequence methods involve comparing actual DNA sequences. The distance matrix
methods involve inputing an array of pairwise distances based on molecular sequence data, gene
frequency genetic distances, amounts of DNA hybridization, or immunological distances. Continuous
character methods involve comparing continuous character among selected taxa. On the other hand,
discrete character methods involve comparing discrete characters. Each of these category contains more
specific programs with varying algorithm used for analysis. Such algorithms include maximum-likelihood
and parsimony.

The programs generate a file output and a tree output. The package also contains a program that
reads the tree output and constructs a more readable format.

Objective
The paper aims to use phylogenetic analysis in tracing the genetic lineage of selected sardinella
species. Specifically, it aims to construct a parsimonious phylogenetic tree based on the discrete
characters recorded among the selected speecies.

Methodology
Trait selection
The five species examined in this paper are Sardinella albella, Sardinella fimbriata, Sardinella
gibbosa, Sardinella lemuru and Sardinella tawilis. The scientific names are validated in fishbase.org
(n.d).

Morphological keys analyzed are based on Peter Whitehead’s species catalogue (1985). Some
absent or outdated morphological traits are overridden from more recent sources (Willete, Santos &
Aragon 2011). Species of the sardinella genus may be identfied with the presence or absence of the
following traits:

 Fimbriated scales
 Overlapping striae
 Black caudal fin tips
 Black dorsal fin
 Black spot dorsal fin origin
  Dark spot at the hind of the gill cover
 Black spot at the margin of operculum

Table 2.1 shows the representative species and their morphological features.

Table 2.1. Morphological characters of the representative Sardinella species.

black spot at black spot at

fimbriated overlapping tips of caudal dorsal fin dark at hind
margin of dorsal fin
scales striae fin black blackish of gill cover
operculum origin
Sardinella
Yes No No No No Yes Yes
albella
Sardinella
Yes No Yes Yes Yes Yes Yes
gibbosa
Sardinella
Yes No No Yes Yes Yes No
lemuru
Sardinella
Yes No Yes Yes No No Yes
fimbriata
Sardinella
Yes Yes Yes Yes No Yes Yes
tawilis

Phylogenetic analysis using Phylip

The established characters by the aforementioned study is used as references for the morphology-
based phylogenetic tree. PHYLogeny Inference Package or PHYLIP is used to analyze the morphological
phylogeny of the selected taxa.

The discrete character method, specifically parsimony, is used since the gathered data is
dichotomous (existing in two states). The data in Table 2.1 is converted to an acceptable input format for
the program (Figure 2.1).

Figure 2.1. Input file for the discrete character parsimonious tree program. Every 1
indicates the presence of a trait and every 0 indicates otherwise. The first number in the
first row is the number of selected taxa and the second number indicates the number of
characters used.

The output tree is then drawn by the Drawgram program in the package. Then, the features of the
tree such are discussed.
 Results and Discussion

Overview of generated phylogenetic tree

Figure 3.1 shows the phylogenetic tree generated by Phyllip using the morphological characters in Table
2.1. The tree reveals two notable features. First, an inner clade consisting of Sardinella gibbosa,
Sardinella fimbriata and Sardinella lemuru is formed. Another interesting feature is the polytomy
exhibited by the aforementioned clade, Sardinella albella and Sardinella tawilis. Such features is
discussed in the future sections.

Figure 3.1. The morphology-based phylogenetic tree of the selected Sardinella species
shows two striking features—the inner clade formation and the polytomy structure.

Sardinella gibbosa clade

The monophyly of S. gibbosa, S. fimbriata and S. lemuru may be attributed to the synapomorphic
trait of black dorsal fins since it is the only trait they have in common (see Figure 3.1). However, black
dorsal fins are also evident in S. tawilis which indicates homoplasy.

The S. fimbriata diverges from S. gibbosa with the autapomorphic traits, black pigment at hind of
gill cover and black spot at posterior margin of operculum. On the other hand, the species S. lemuru
diverges from S. gibbosa with the autapomorphic traits, black caudal fin tips and black spot in dorsal fin
origin. Again, these autapomorphic traits are also evident in taxa outside the clade which implies
homoplasy. The synapomorphic and autapomorphic traits are summarized in Figure 3.2.
 Figure 3.2. Adoption of synapomorphic and autapomorphic traits. The monophyletic
group contains the black dorsal fins as a synapomorphic trait. S. fimbriata and S. lemuru
diverged with the autapomorphic traits shown.

Polytomy structure
Polytomy may arise from two origins—insufficient data (known as soft polytomy) and rapid
speciation (known as hard polytomy). Soft polytomy indicates that the genetic lineage cannot be inferred
since it lacks necessary data. Thus, such polytomies should be resolved. Hard polytomy occurs when .
There are no evidence that the S. tawilis and S. albella are a result of simultaneous cladogenesis; thus, the
polytomy may be due to insufficient data.

Figure 3.3 shows the possible ways on how to resolve the polytomy. First, two clades may be
made to remove the polytomy (Figure 3.3b). In this hypothesized lineage, both S. tawilis and S. albella
become more distant to the S. gibbosa clade. Another solution would be merging the S. tawilis to the S.
gibbosa clade (Figure 3.3c). This solution makes S. tawilis closer to the rest of the species while making
the S. albella the outgroup of the tree. Conversely, S. albella can be merged into the clade instead of S.
tawilis (Figure 3.3d). In this hypothesis, S. albella is less distant to the rest of the species while S. tawilis
becomes the outgroup.

Figure 3.3. Multiple ways on resolving the polytomy. Boxed taxa are part of a clade while
unboxed taxons may be seen as outgroups.

Among the three solutions, Figure 3.3b is the most likely since studies reveal that S. tawilis is
closer to S. albella than other species. Samonte et al. (2000) suggest that S. albella is the closest marine
relative of S. tawilis. The study analyzes the molecular phylogeny of S. tawilis to other sardinella species
(S. fimbriata, S. longiceps and S. albella) using mitochondrial DNA. They found that the control sequence
in the mtDNA of the S. tawilis and S. albella differs by only 1.3% - 3.5%.
 Their findings are strengthened by another study (Samonte et al 2009) where the researchers
focused on morphometric variables instead of molecular variables. Consistent to the previous findings,
principal component analysis shows that features of S. tawilis and S. albella are indistinguishable to each
other. These findings imply that the two species are more closely related to each other than with other
species, making the second solution the more likely one.

Limitations of the results

Like any phylogenetic analysis, any results shown is simply a hypothesis. As more evidence
supports the hypothesis, the hypothesis are more likely to be true. With this, the results of the study may
be seen merely as a working hypothesis, needing further proof as the presence of polytomy makes the
results slightly inconclusive. More discrete characters may be added or, instead, continuous traits may be
substituted. Research simulating the difference between discrete character and continuous character
phylogeny shows using continuous traits improves accuracy (Parins-Fukuchi 2017). Additionally,
differentiation in the Sardinella species mainly involves morphometric and metristic characters such as
standard length, number of scutes and number of gillrakers (Whitehead 1985; Strauss & Bond 1990).

Alternatively, molecular phylogeny may augment the data by reducing the ambiguity caused by
the polytomy.
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