PHYLOGENETIC (MOLECULAR) SYSTEMATIC

Name : Mellya Rizki Pitriani

Student ID : B1B017031
Entourage : II
Group :2
Assistant : Abdi Rahman Nursyamsi

MICROBIAL SYSTEMATIC LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2019
 I. INTRODUCTION

Kinship is a picture of relations with one another, both of which now also
exist in the past during the development of phylogenetic history. Phylogenetic
kinship is determined based on the origin of the ancestors according to their
development or evolutionary process (Davis and Heywood, 1973). The phylogeny
comes from the Greek word Phylon which means tribe or clan and Genesis which
means origin. Phylogeny is the history of the evolution of living things which is
depicted in the form of a tree which has branches. The branching illustrates the
existence of divergences. molecular phylogeny describes relationships between
organisms based on their genetic makeup using DNA or protein sequences. The
existence of inequality from genetic sequences illustrates the results of divergence or
the evolution of the organism. Classical phylogenic only describes the morphological
character of an organism. Morphological analysis can be seen from DNA sequences,
RNA sequences and protein sequences (Patwardhan, 2014).
Things that must be considered in molecular phylogeny according to
Patwardhan (2014) include cladogram is a phylogenetic tree that describes the
evolution of organisms, hompolasy is the same sequence as a result of convergence
but does not reflect the direct evolution, internal Transcribed Spacers (ITS), i.e.
rRNA will be transcribed into a single transcript by this ITS, monophyletic, that is,
taxa in phylogenetic trees have a common ancestor and outgroup a taxa that is a
comparative character in making phylogenic trees. Molecular phylogenies are based
on information about the evolution of the organisms. Deducing phylogenies from
molecular information might be helped out through the point analysis of similarities
and differences in the studied sequences. The 16S rRNA gene sequence, a large
number of strains because of some important reasons, including to be common in
almost all bacteria, a useful evolutionary chronometer, and the size of 16S rRNA
gene is large enough to be used in bioinformatics. Universal primers, which are
usually chosen as complementary to the conserved regions, are used for comparative
taxonomy. GenBank, the largest databank of genetic sequence, has previously
deposited sequences, helping out to compare the sequence of an unknown strain
(Bekler et al., 2018).
16S rRNA molecules consist of variable regions and conserved regions, and
universal primers for amplification of 16S rRNA genes are usually selected from
conserved regions while variable regions are used for comparative taxonomies. The
initial step in phylogenetic classification is to isolate and purify chromosomal DNA
from each strain. The 16S RNA gene was further amplified by the PCR (polymerase
chain reaction) technique of each chromosomal DNA sample. The results of the
amplification are purified for sequencing (Prakash et al., 2007).
Phylogenetic analysis, outgroup groups are needed and cause character
polarization or characteristics, namely apomorphic and plesiomorphic characters.
Apomorphic characters are characters that change and are inherited and are found in
ingroups, whereas plesiomorphic characters are primitive characters found in
outgroups. Synapomorphic characters are inherited characters and are present in
monophyletic groups. Morphological characters have long been used in many
phylogenetic studies. With the rapid development of techniques in molecular biology
the use of DNA sequences in phylogenetic research has increased rapidly and has
been carried out at all taxonomic levels, such as families, genera, and species.
Molecular phylogenetics combines molecular biology techniques with statistics to
reconstruct phylogenetic relationships (Hillis et al., 1996).
Softwares used in this experiment were ClustalX, MEGA 7th version, and
Phydit. ClustalX do alignment of sequences based on their homology so they can be
compared. The sequence is obtained from the FASTA from NCBI website as an
input data. Total alignmet once obtains, then will be able to import to the Phydit
software (Suryawan, 2016). Phydit has function to align the identified 16S rRNA
sequence based on their secondary structure. Phydit also able to construct a
phylogenetic tree by treeing algorithm (Jeong et al., 2016). Aside from the
phylogenetic tree from Phydit, it can also construct the similarity and dissimilarity
matrix. Sequence alignment itself is a method of arranging two or more sequences
(DNA/RNA and amino acid) to be classified based on their similarity and homology
(Suyono, 2010). MEGA is mostly used for constructing the phylogenetic tree since it
has various of methods. The sequence data is got from ClustalX and should be
compatible first. There are two methods commonly used in MEGA for constructing
phylogenetic tree. First is Neighbor-Joining Tree, which used 1000 of bootstrap
replication and evolutionary range. If any data is missing, then use the complete
deletion method. Another one is Maximum Likelihood Tree, same like Neighbor-
Joining Tree, it uses 1000 of bootstrap replication and evolutionary range, but if any
data is missing then use the use all sites method (Triana, 2005).
 The purpose of this practicum is the students are know the ways and stages of
bacterial kinship analysis using molecular phylogenetic methods.
 I. MATERIALS AND METHODS

A. Material

The tools used in this laboratory activity are application on computer such as
MEGA, Phydit and ClustalX.
The materials used in this laboratory activity are data of sequence 16s rRNA
strain of Spingomonas sp. and Bifidobacterium adolescentis from NCBI.

B. Methods

The method used in this laboratory activity are:

a. Data collection
1. 16S rRNA sequence data from 10 isolates of Sphingomonas sp. and one
Bifidobacterium adolescentis (outgroup) isolate downloaded from the NCBI
website.
2. Each one is copied to the Words or Notepad program. The data is in the form of a
text file and saved as a file.
b. 16S rRNA sequence preparation
1. The sequence data above is then opened with PFE (Programmer File Editor) and
the sequences of each isolate are given a code name on the front of the sequence
(eg ‘SG1, SG2 and so on).
2. Furthermore, in front of the sequence code is marked with fasta format (>), this
can be read by the program to align, namely CLUSTALX.
3. Then the file is saved and given a code name (save as), this file is already in the
form of a fasta file format that is ready to be loaded into CLUSTALX for
alignment.
c. Alignment of 16S rDNA sequences (ClustalX)
1. Sequence data of each strain (How it works b) is loaded into the CLUSTALX
program to align. Alignment aims to arrange sequences so that each other is
placed in accordance with the position of homology between sequences. That is,
homologous regions must be placed in the same position (conserved region with
conserved region, variable region with variable region). With the alignment
between 16S rRNA gene sequences from each strain can be compared. All
sequences dialigned are arranged in one file by the ClustalX program as output
files in several format choices. So that the alignment file can be read by the
 MEGA and PHYLIP programs used to construct phylogeny trees based on these
sequences, then this file is created in phylip format (file name.phy). This can be
done by selecting the output file format when aligning in CLUSTALX.
2. The output file is stored in the CLUSTALX directory (Eg C: \ ClustalX \ SG.phy),
so that after alignment, the results of the alignment can be found in the form of a
file named as an example, in the ClustalX directory.
3. This file is then used to construct the phylogeny tree with the MEGA or PHYLIP
program.
d. Phylogeny Tree Construction
• MEGA 7 Program
All 16S rDNA sequences from the alignment were used to construct the
phylogeny tree with the Mega 7 program.
1. The Mega 7 program is opened, select Alighment, then Alighment Explorer, then
select Retrieve.
2. Next take the file with the format ". Aln" results from Clustalx. Click the file, then
select Export Alighment, select Mega format, and save. Close, then type 16S then
"OK", then "no" and select close.
3. Next comes the open data in mega command? Select "yes", then minimize.
4. Select phylogeny, Construct phylogeny, select Neighbor Joining, then Test
phylogeny, Bootstrap with 1000 replication. Then choose compute.
 III. RESULT AND DISCUSSION
A. Result

Sphingomonas sp. JSS-28

SG6
100 0.00

82
0.01 Sphingomonas sp. JSS-27
SG7
0.00
0.00
70
0.02
Sphingomonas
SG1 hengshuinensis
0.00
90 Sphingomonas phyllosphaerae
SG4
0.01 0.02
Sphingomonas
SG3 zeicaulis
0.02
100
0.04
100 0.01
Sphingomonas
SG9 sp. SB5
0.04
SG5
Sphingomonas sp. D16
65 0.00
0.01
0.06
Sphingomonas sp. ZL5
SG8
100 0.00
0.03
SG2
Sphingomonas sp. BRW2
0.00

SG10
0.08 Sphingomonas sp. W2.09-2
BA1
0.15 Bifidobacterium adolescentis

Figure 3.1 Result of Phylogeny tree use MEGA application

From this dendogram, the closest and furthest kimship among these strains
has been proofen as clear as possible. Because this compares of all strains not only
two. This dendogram or phylogenetic tree shows the closest kinship occur between
Sphingomonas sp. JSS-28 and Sphingomonas sp. JSS-27. They even have an
evolution range of 0.00 with the possibility of being ranked as the closest in 1000 of
replication is 100. While the furthest one occur in Sphingomonas sp. JSS-27 and
Sphingomonas hengshuinensis with evolutionary range is more or less larger than
0.01 and the possibility of being in the same rank of 1000 bootstrap replication is 79.
Actually, the furthest kinship occur between Sphingomonas sp. JSS-28 and
Bifidobacterium adolescentis since Bifidobacterium adolescentis is an outgroup. This
is in accordance with Salanki et al. (1994), that stated evolutionary tree plays a role
on classifying organisms based on their similarities and dissimilarities in order to get
them all neat.
Table 3.1 Dissimilarity Matrix of Phylogenetic Analysis
SG6 SG3 SG9 SG4 SG7 SG1 SG5 SG8 SG2 SG1 BA1
0
SG6 ---
SG3 52/1 ---
422
SG9 125/ 102/ ---
1408 1429
SG4 55/1 71/1 132/ ---
440 443 1429
SG7 0/14 52/1 125/ 55/1 ---
41 422 1408 440
SG1 48/1 76/1 127/ 75/1 48/1 ---
441 443 1429 480 441
SG5 113/ 105/ 18/1 123/ 113/ 121/ ---
1436 1423 410 1437 1436 1437
SG8 89/1 95/1 104/ 94/1 89/1 101/ 101/ ---
435 424 1408 436 435 1436 1436
SG2 85/1 92/1 109/ 99/1 85/1 101/ 106/ 7/14 ---
435 424 1408 436 435 1436 1436 37
SG1 195/ 196/ 203/ 194/ 195/ 203/ 208/ 211/ 210/ ---
0 1439 1442 1428 1478 1439 1482 1436 1435 1435
BA1 329/ 331/ 341/ 327/ 329/ 340/ 338/ 339/ 339/ 335/ ---
1436 1440 1425 1475 1436 1484 1433 1433 1433 1479

Based on the dissimilarity matrix of nucleotide above, it can be seen that the
furthest relation occur among all the strains tested occur in SG9 (Sphingomonas sp.
SB5) and BA1 (Bifidobacterium adolescentis). The proof is they have the nucleotide
sequence difference as many as 341 of all the 1425 nucleotides. Bifidobacterium
adolescentis is actually an outgrup that function to compare the edge both of these
two genera kinds. De Peer et al. (1996), stated that the more different nucleotide
sequence between organisms compared, the furthest kinship they’re in. Some of
cases even might said as placed into different genera.
 Table 3.2 Simmilarity Matrix of Phylogenetic Analysis
SG6 SG3 SG9 SG4 SG7 SG1 SG5 SG8 SG2 SG10 BA1
SG6 ---
SG3 96.34 ---
SG9 91.12 92.86 ---
SG4 96.18 95.08 90.76 ---
SG7 100.00 96.34 91.12 96.18 ---
SG1 96.67 94.73 91.11 94.93 96.67 ---
SG5 92.13 92.62 98.72 91.44 92.13 91.58 ---
SG8 93.80 93.33 92.61 93.45 93.80 92.97 92.97 ---
SG2 94.08 93.54 92.26 93.11 94.08 92.97 92.62 99.51 ---
SG10 86.45 86.41 85.78 86.87 86.45 86.30 85.52 85.30 85.37 ---
BA1 77.09 77.01 76.07 77.83 77.09 77.09 76.41 76.34 76.34 77.35 ---

The closest kinship based on phylogenetic (molecular) systematics on

Sphingomonas strains occur between SG6 (Sphingomonas sp. JSS-28) and SG7
(Sphingomonas sp. JSS-27). The proof is the nucleotide among those two is 100 of
percentage, which means all the same. Moriguchi et al. (2001), stated that the
relation between organisms tested may be seen from their nucleotide sequence
similarities. The more they have similarity, the closer of kinship they get.
 IV. CONCLUSION AND SUGGESTION
A. Conclusion
Based on the results and observations it can be concluded that :
1. The steps used in molecular phylogenetic analysis are data collection from NCBI,
16S rRNA sequence preparation using PFE, 16S rRNA sequence alignment using
ClustalX, phylogeny tree construction using MEGA 7 and the last step is the
construction of the matrix similarity using PHYDIT.
2. Results of molecular analysis with isolates Sphingomonas sp. various strains
obtained data strain SG7 and SG6 had the highest similarity value of 100%, and
the smallest similarity index was seen in strains of SG2 and SG9 with similarity
values of 65%.
B. Suggestion
Suggestions that can be given for this practicum event is that it is better to
carefully pay attention to the steps when making the practicum and note it so it is
easier to remember.
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