Phylogenetic Corroboration of Chiton Tuberculatus, Chiton Marmoratus, Chiton Viridis, Chiton Squamosus and Acanthopleura Granulata Using The 5' Dna Cytocrome Oxidase I

PHYLOGENETIC CORROBO RATION OF CHITON
TUBERCULATUS, CHITON MARMORATUS, CHITON VIRIDIS,

CHITON SQUAMOSUS AND ACANTHOPLEURA GRANULATA
USING THE 5 DNA CYTOCROME OXIDASE I
L U I S C O L N M E R C A D O , M AR I A N O C O L N C A R A B A L L O , N A T A L I E D E H O WI T T
Abbreviated Title: Phylogeny of chitons

Corresponding Author: Prof. Johanna Daz, Universidad de Puerto Rico en Ponce Tel: 787-844-8181
Email: johanna.diaz2@upr.edu
Figures and Tables: 34
Last Date of Revision: 15 December 2010
KEYWORDS
Barcoding
COI gene
Polyplacophora
Molecular phylogeny
DNA Parsimony Algorithm
Neighbor-Joining/UPGMA method
Nucleic acid sequence Maximum Likelihood method
Fitch-Margoliash method
N.De Howitt, M.Coln, L.Coln
2010 Phylogeny of chitons
ABSTRACT
Barcoding technique is applied to corroborate the relationship of five polyplacophora species, chiton
tuberculatus, chiton viridis, chiton squamosus, chiton marmortus, and acantopleura granulate. The
barcode sequence use was cytochrome c oxidase sub unit 1 (COI) found on the mitochondrial genome.
Organisms were donated by Dr. Cedar Garca. The aim of this study is to corroborate the stated taxonomy
of the polyplacophora species using phylogenetic analysis. Samples were between 5-15g and DNA was
extracted from this tissue using the DNeasy Tissue kit by Quiagen. Polymerase chain reactions (PCR)
were employed on the DNA to amplified de COI gene. PCR product was review by electrophoresis.
Bands were send to University of Puerto Rico in Rio Piedras for sequencing. The analysis of the sequence
was done with Bioedit. After analysing the sequence, four different un-rooted phylogenetic trees:
Parsimony, Neightbor-joining, Maximum likelyhood, and Fitch-margolialiash were used to corroborate
the taxonomy stated for the polyplacophora. This study shows chiton viridis as the least related from
the group of the Chitons. There is not clear view with the relation of marmoratus and
tuberculatus. They can be sister group as in the fig 1, tuberculatus can be divert first than
marmuratus like in fig 2 and 4, or the opposite in fig 3. But, the most important results is that
Chiton squamous and Acantopleura granula are sister groups.
INTRODUCTION
DNA barcoding is a novel system designed to provide rapid, accurate, and automatable
species identifications by using short, standardized gene regions as internal species tags. As a
consequence, it will make the Linnaean taxonomic system more accessible, with benefits to
ecologists, conservationists, and the diversity of agencies charged with the control of pests,
invasive species, and food safety (HEBERT, 2005). We used the technique of DNA barcoding
to identify the actual taxonomy of six chitons species on the Caribbean waters surrounding
Puerto Rico with collaboration of the marine ecologist, Cedar Garca. The six species of chitons
that were used as the following: Chitons tuberculatus, Chitons marmoratus, Chitons viridis,
Chitons squamosus, Acanthopleura granulata.
Chitons are been classified as mollusks. Mollusks are organisms that have similar body
structure;however, the basic body plan of mollusks has evolved in various ways in eight classes
of phylum (Campbell, Neil A.; Reece, Jane. B, 2007). One of those phyla is the Polyplacophora.
Polyplacophora are well known as chitons. Linneus was the first that describes the chitons and
realize various taxonomic studies of the species level; however, the taxonomic classification of
certain species in the group remained unsettled. In the beginning, traditional classifications of
chitons were performed using the valve characters (Van Belle 1983). More recent studies have
used egg hull morphology (smooth, cup-like, or spiny), gill placement and morphology (adanal
or abanal), radular morphology, perinotum morphology, shell spicular processes, and sperm
morphology in addition to the valve morphology (Buckland-Nicks 1995; Eernisse 1984;
Reynolds 1994; Sirenko 1993, 1997). Due the taxonomic studies, in a beginning by Linneaus and
then by others scientists, the Chitons are now a well identify organism. Chitons are divided in
families by their physical characteristics. Also, we can use different genes to classified Chitons
by families. Chitons are a group of morphologically conservative marine mollusks. Actually, 900
living species of chitons are distributed worldwide. Chitons are distinguish by their characteristic
appearances and shells; which consist of dorsal-ventrally body with bilateral symmetric, and
eight dorsal calcium carbonate plates or valves, a ventrally ciliated foot surrounded by the mantle
cavity and a dorsal perinotum with different kinds of protective and sensory organs that play an
important role in the reproductive cycle. The plates allows of taxonomy of the species. Each
plate or valve is composed of four layers, commonly named as the properiostracum layer, the
tegmentum layer, the articulamentum layer, and the myostracum layer. The first level or the
properiostracum is the thin level aragonite which is the thin and the hardest level of all. The
second level or tegmentum is the most relevance in taxonomy. The importance of the tegmentum
lies in the color and the shape. This layer contains sensory organs. In the case of chitons, these
organisms lack of primary true eyes. Instead, have secondary small eyes called aesthetes. These
latter are responsible for the photosensory, mechanosensory, and chemosensory functions. The
third level or the articulamentum is composing of aragonite. This level unites the plates with
each other. The fourth level or Myostracum it allows the plates to attach to the dorso-ventral
musculature (Schwabe and Wanninger 2006).
Another important chitons body structure is the radula. This buccal piece is responsible
for the uptake of nutrients necessary for the life span of the chitons. The radula is formed by a
membrane that contains 25 o 150 rows of teeth. Each of the teeth bears a large magnetite-capped
blade which is hardened and allows scraping in hard surfaces. (Schwabe, Enrico; Wanninger,
Andreas, 2006). Chiton creeps along slowly on a muscular foot. They have considerable power
of adhesion and can climb to rocks very powerfully. Ordinarily, adhesion is accomplished just by
means of the foot; however, when disturbed, the girdle clamps down on the hard substratum and
the inner margin is raised. This creates a vacuum that enables the chiton to grip the surface with
great tenacity. This is also the reason that chitons prefer smooth hard surfaces on which to live
for example rocks, shells of other molluscs, lobster traps sunken wood, anchors or other metal,
etc (Hickman 2005) (Ruppert et al, 2003). For this reason chitones are found in shores with rocks
and reefs with deeps up to 20 feet but there espices that live in the deep water. (Hickman 2005)
(Ruppert et al, 2003). Another important tool for the identification of chitons is their
reproductive cycle. Most polyplacophoran species have separate sexes and are free spawners
with external fertilization. As a result of the fertilization, a mature egg is produced. This egg has
many unique characteristics; among them we can mention the following: the hulls of mature eggs
show a characteristic ornamentation that is species specific, and are usually red or green in color
(Eernisse 1984). When the mature egg, reaches the necessary cleavage a trochophore larva is
formed. The next step in the cycle is the formation of the early juveniles. Early juveniles still
lack the eight valves, which can take many months to develop. Recents studies conclude that the
gene expression pattern analyses prove that the homeobox gene is involved in the
polyplacophoran plate formation (Jacobs, et al 2000; Moshel, et al 1998; Waninger and
Haszprunar 2001). In addition, one pair of protonephridia form the larval excretory system and
they persist in the juvenile stage. Based on all these characteristics Chitons are classified in two
major lineages. These lineages are the following: Lepidopleurida and Chotonida. Nevertheless,
these physical characteristics by their own are not sufficient to reveal certainly phylogenetic
relationships. The morphological characteristics of chitons contribute only to the genus level of
the taxonomic classification of chitons. For that reason, if we use the DNA Barcoding technique
to study different genes we can prove these phylogenetic relationships among the Chitons,
especially at the species level. The different species studied here are Chitons tuberculatus,
Chitons marmoaratus, Chitons vivridis, Chitons squamosus, Acanthopleura granulate, as seen
on TABLE 1.
Chiton tuberculatus- they are medium size to large. Adults reach 5 to 8 cm long. The belt is
covered with large, smooth and overlapping scales. The color of the integument is usually
olive green o dull green. The area center of the plates has deep grooves and curved
alternating with ribs. The jugum is smooth. The lateral area, that is well-defined, has four to
five cords or radial nodules ribs. Intermediate valves have one or more slits on each
side. Anal plate cephalic has several cracks. The breast is sutural denticulate. In the bottom
plates are blue green. The belt, with alternating bands, has scales light and dark green
color. It is very common under rocks, in the intertidal zone (Garca, 2003).
Chitons marmoratus- Medium size to large. Measure from 5 to 7 cm long. The belt is covered
with large scales, soft superimposed. The belt as alternating bands of gray scales and dark
green. The integument is staining varied, predominate in the reddish brown or purple, olive
green and shades of gray with betas "Marble" dark. The leaflets are smooth, soft, polished
and shiny. In the bottom plates are greenish blue. It is very common on the rocky shore in
onslaught of waves area (Garca, 2003).
Chitons viridis- Medium size to large. Is 5 to 7 cm long. The belt covered with scales large,
smooth and overlapping. The coat color is varied, speckled with brown spots and
green. Juveniles have bright red spots. The central area is fine channels in the form of
inverted "S", near the diagonal division and margins. This is the feature that most clearly
separates it from other species of the genus. There are about four cords with few pustules on
the lateral area. The rear edge of the plates is something sharp teeth by pustules. The interior
of the plates may be white Warmke and Abbott, 1961: 219), blue-green or light blue, usually
with a reddish brown mark near the mucro (Kaas, 1972: 115). Found on rocky coasts and
coral reefs from the intertidal zone (which is rare) to deeper areas (2 to 4 meters) where it can
be locally abundant (Garca, 2003)
Chiton squamosus-Medium size to large. Adults reach 5 to 7 cm long. The belt have
alternating bands of pale scales and grayish green. The coat is yellow coppery brown with
irregular markings. The sculpture is light. The central area of the plates doesnt have
ornamentation, except for cross scratches. Lateral areas with five to eight radial granular
cords. Have lines of black spots along the hind margins of the plates. In the bottom plates are
blue-green. Are common on the rocky shore on the rocks in the sweep of the waves.
Acanthopleura granulate- are large and oval. Measure from 5 to 7 cm long. The belt is thick
and muscular, densely covered with calcareous spines. The belt has alternating bands of
black and white. The integument with little sculpture is generally eroded, the surface is
brown with two light bands that run across all the plates. The plates are strong and thick. The
intermediate valves have several cracks. The articulamento is greenish blue with brown spots
in the center. The foot is light orange. The granulata is the only species of this genus known
in the Caribbean. They are common in the intertidal zone on the rocky coast, mainly on
sandstone rocks. It adheres strongly to the rocks.
DNA Barcoding is a new method to aid the improvement of the species level
identification and to the contribution of taxonomic and biodiversity research. DNA Barcoding is
based in the use of a standardized sequence to compare individuals of a species because genetic
variation between species exceeds that within species. (Herbert, Paul; Hickey, Donald; Singer,
Gregorie 2007). Species identification using barcoding is usually achieved by a short DNA
sequence, referred as the barcode, from a standard part of the genome (specific gene region)
from the specimen under investigation. The barcode sequence from each unknown specimen is
then compared with a library of reference barcodesequences derived from individuals of known
identity. A specimen is identified when its sequence closelymatches one in the barcode library
(Herbert, Paul; Hickey, Donald; Singer, Gregorie 2007).
Although, this method facilitates the work of the taxonomist, some scientists and
science experts do not accept this method as a valuable species identification tool. For that
reason, many scientists have been done different experiments using the DNA Barcoding to
prove the integrity, the effectiveness and the importance of this method. These studies have
been done using various groups organisms as animals, plants, macroalgae, fungi, protists,
bacteria and primates. In a same way, DNA Barcoding are been used in clinical trials to
improve the treatments for a certain disease.
Smith et al., (2005) used DNA Barcoding to study and obtain the biodiversity of ants in
Madagascar and it is now a routine element of a large scale biodiversity inventory on these
organisms. DNA barcoding has also been used to study groups such as Lepidoptera. By other
way, Hallwachs; Janzen; et al., (2003) used DNA Barcoding to understand the biodiversity of
the caterpillar fauna in northwest of Costa Rica. This project, which was started over 25 years
ago, was the base for the creation of a reference sequence library for more than 25,000
specimens for this type of organisms. In a same way, Clare and Lim (2007) used the DNA
Barcoding technique to identify different species of bats in the Guyana. The Guyanan bats were
able to classify 93% of their species correctly. Also, Kerr and Stoeckle (2007) used the DNA
Barcoding to classify correctly 95% of the North American breeding bird species. These
experiments mentions above do not make reference to the specific genes used in the DNA
Barcoding. However, the following experiments mention the specific genes used.
Pook and McEwing (2005) sustain that the inherent complexity of venom and its
variability needs that toxinocological and biochemical analysis includes accurate species
identification and specific geographic locality of the venom resource. For that reason, they
describe a novel method for venom identification that involves the extraction of mitochondrial
DNA from the source venom and its subsequent sequencing and comparison for species
determination. This experiment consist in the study of the cytochrome c oxidase subunit I gene
(COI) gene using the DNA Barcoding technique. In a same way, others experiment use animals
for strengthen the fact that DNA Barcoding is a useful technique for the identification of
species. Aliabadin and Nijman (2009) used the DNA Barcoding technique to identify primates.
The experiment is based in the used of various genes for the identification as: 16S ribosomal
RNA gene, cytochrome b (cob) and COI gene. However, among them, the most important gene
estipulate by the scientists was the COI gene. In this case, they used the COI gene because
itserves as a fast and accurate marker for the identification of primate species, and for the
discovery of new species across the tree of life.
DNA Barcoding can be used in the identification of different species of plants. Gao,
Song and Yao (2009) use the DNA Barcode internal transcribed spacer region 2 (ITS2) for the
identification of medical plants in the familiy Fabacae. Specifically, they want to test if the ITS2
region is an effective marker to authenticating the family Fabacae. The Fabacae is the second
largest family of medicinal plants and has many species that possess important medicinal
properties. For example, Glycyrrhiza uralensis, Glycyrrhiza inflata, and Glycyrrhiza glabra, are
generally used in traditional medicines and have inhibitory effects on HIV replication in vitro
and anti-Fas antibody induced hepatitis (Okamoto, 2000; Watanabe et al., 1996). The problem
consist that many plants have similar physical characteristics but have different toxins that
cause several problems. For example, Acacia rigidula has been shown to contain high levels of
toxic alkaloids (Clement et al., 1998), and many of species of the genus Crotalaria contain
pyrrolizidine alkaloids, which are toxic to mammals and birds (Williams and Molyneux,
1987).This experiment support the fact that the use of the DNA Barcoding permits an accurate
identification of species because the identification based only in morphological characteristics is

difficult and inaccurate. Also, ensures a correct and safe application of the plants in the medical
treatment. In other hand, DNA Barcoding is commonly used to support epimiological studies.
Clark, Scicluna, and Tawari (2005) develop a simple method to subtyping the intestinal
protistan parasite Blastocytis using the DNA Barcoding technique. Blastocytis hominis is the
only stramenopile parasitic in humans. They used the small subunit ribosomal DNA (SSUrDNA) for subtyping the species. At the end of the experiment, they can identify that
the Blastocytis hominis is the species that cause diseases, using the DNA Barcoding technique.
In all these experiments we can see two things in common that the DNA Barcoding
technique is effective and the commonly used of the mitochondrial genome, specifically the
COI gene. Now is very important to mention that the COI gene is not suitable for studying all
groups of organisms. For example, rates of evolution of COI are too slow to allow this gene to
be useful in plant studies and other sequences have been proposed (Kress et al., 2005). In
relation with this information, we already saw that in the experiment realized with plants the
scientists used ITS 2, instead of the COI gene. In addition, anaerobic eukaryotes do not
normally have a mitochondrial genome and therefore lack the target gene altogether. For DNA
barcoding in such organisms an alternative target is required, a gene with the requisite
characteristics but that is present in the organism of interest.
However, the COI gene is commonly used due the following reasons: the mitochondrial
DNA presents in the cells permits multiple copies making PCR amplification of the target DNA
relatively easy, exists a large database of mitochondrial sequences against which novel
sequences can be compared, the gene is usually well conserved within species but sufficiently
variable that interspecific (inside the sequence) differences can be detected easily, and the gene
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segment has a size that can be easily amplified by PCR and then sequenced in a single reaction.
The resulting sequences can be used for both species identification and for phylogenetic
analysis. (Hebert et al. 2004; Hogg and Hebert 2004).
As we already saw, the COI gene permits an accurate identification of species and an
accurate phylogenetic analysis. For that reason, we want to study and prove the phylogenetic
relationships among the chitons (information already provided above). Specifically, we realize a
molecular analysis from various nuclear and mitochondrial protein coding genes (specially the
COI gene) of the chitons species, and then we should find and prove the phylogenetic
relationships among them. In addition, we can strengthen the fact that the mitochondrial genome
is most common used than the nuclear genome for molecular analysis to prove phylogenetic
relationships. This action would help us to sustain with real data our phylogenetic study.
Phylogenetic studies proved that the mitochondrial genome of animals is a better tool for
analysis than nuclear genome because the first one lacks of introns and have limited exposure to
recombination mechanism (Saccone et al. 1999). The continuously use of cytochrome c oxidase
subunit I gene (COI) is due because it presents two advantages. First, the primers for this gene
are very wide, enabling the recovery of its 59 end. These ends are representatives of most animal
phyla (Folmer et al.1994; Hewitt & Zhang 1997). Second, the COI gene has a greater range of
phylogenetic signal than any other mitochondrial gene.
This study presents the first molecular analysis using DNA Barcoding technique of
polyplacophoran relationships using the mitochondrial protein coding, COI. Since the gene is
found on the mitochondrial genome, we are using the foot as our tissue source. The gene is used
as the barcode to confirm the actual taxonomy of these organisms, chitons, with huge
morphological similarity that makes them a great study model in DNA Barcoding.
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MATERIALS AND METHODS

ORGAMISMS
Polyplacophoran species including: Chiton tuberculatus, Chiton scamosus, Chiton
marmaratus, Chiton viridis, and Acantopleura granulata, have been selected and collected for the
study to represent different families from the Lepidopleurida, Chitonida and Acanthochitonina
order. The specimens were collected from the nearshore environment of Western Coast of Puerto
Rico by Dr. Cedar Garca. The process used to collect the specimens include: identification of
the specimens, removal of the specimens from their habitat using different types of specialize
tools, and the appropriate information of the collection process. In the basic information was
included the date, locality, collector, and a good and brief description of the habitat as possible.
The material obtained was storage alive and fixed with 95% of Isopropanol and kept frozen in80C of temperature to maintain the integrity and the purity of the DNA.
METHODS
DNA EXTRACTION
To minimize the possibility of samples contamination we used the correct aseptic
techniques. To sequence the cytochrome c oxidase subunit 1 (COI), we extracted the DNA of
each tissue sample. We used the DNeasy Blood and Tissue Kits using mini Spin-Column
Protocol Qiagen. The principle of the DNeasy Blood and Tissue Kits using mini Spin-Column
Protocol is base on the Lysed of the tissue with the proteinase K. the Buffers provide optimal
DNA binding conditions . After the cell has being lysate is tranfere to mini sipin-column were
the DNA will bind the membrane and allowing the rests of the contaminants pass through. The
DNA will be extracted form the membrane will to elution with buffer. We identified each
microcentrifuge tube using the following code: A-1 to A11 (exclude A-4) and B-1 to B-11
12
(exclude B-4). The codes were based in two groups A and B. Cut the tissue in 5-15mg and add
the 180l of ATL buffer. The ATL buffer will help in the lysed of the cell. Next add 20l of the
proteinase K and vortex (Vortex Maxi Mix II (Model: M 37615 / Type: 37600 mixer) for 1 min.
After the vortex, adds 200l of ethanol at 96-100% and vortex for 1min. Then, incubate all the
samples in Fisher Isotemp 205 at 56C for 3 hours. We added the 4l of RNAasa to prevented
mix results. We transfer the product to a mini spin column of 2ml and centrifugation for 1 minute
at 6,000 x g 8,000 rpm used the Eppendorf Centrifuge 5415 R. We collected mini spin column
and discarded the flow and place the column in a new tube of 2ml. we added 500l of AW1
buffer and centrifugation for 1 minute 6,000 x g 8,000 rpm used the Eppendorf Centrifuge 5415
R. Again we collected the mini spin column and discarded the collection tube. We place the
mini spin column in a new column tube. We add the 500l of AW2 buffer and centrifugation for
3 mins. at 20,000 x g 14,000 rpm used Centrifuge 5810 R to dry the DNeasy membrane. Again
we collected the mini spin column and discarded the collection tube. We place the mini spincolumn in a 2ml collection tube and added 200l of water instead of the AE buffer and
centrifugation for 1mim. at 6,000 x g 8,000rpm used the Eppendorf Centrifuge 5415 R. We
collected the mini spin column and add 200l of AE buffer and centrifugation for 1minute at
6,000 x g 8,000 rpm used the Eppendorf Centrifuge 5415 R. After the centrifugation we keep
the collection tube with the AE buffer that traps ion to prevent any additional degradation of the
DNA. Any doubts see the Dneasy Blood and Tissue Kits using mini Spin-Column Protocol but
remember the modification we use are mark in bold. The speed was set with the right knob to
8,000 rpm and the time with the left knob to one minute and three minute respectably to each
step in the methodology. The temperature of all centrifugations was at 25oC. For further
reference check the Eppendorf Centrifuge 5415 R model instruction manual. For the
centrifugation of 14,000 rpm was done in the Eppendorf Centrifuge 5804 R model. To turn on
13
the centrifuge press the power key. To set the temperature press the tem key and use the
arrows to take to the 25oC. For the speed press the speed key and set to 14,000rpm and for the
time press the time key and using the arrow set to three minutes. For father references check the
Eppendorf Centrifuge 5804 R model instruction manual.
QUANTIFICATION OF THE CONCENTRATION OF THE DNA SAMPLES

To calculate the total concentration of DNA in each sample we used the Eppendorf
BioPhotometer. First, we prepare the station work; in this case, we used the UV hook. Then, we
used the 400 l cuvettes for the quantification of the DNA concentration. Futhermore, we
withdraw the samples of the refrigerator, to get ready to use in this process. In other part, we set
the Eppendorf BioPhotometer. First, we need to prepare a blank sample. The blank sample
consists only of 400 l of distillated water. Now, we precede to pipet the samples in the cuvettes.
The total volume of the cuvette was 400 l. Therefore, we proceed to prepare the cuvettes with
the DNA samples of the organisms. The cuvettes consist of 395 l of distillated water and 5 l of
the sample for a total volume of 400 l. The samples tubes are labeled with a code (A-1 to B-11).
Then, we proceed to calculate the concentration of DNA in each sample using the Eppendorf
BioPhotometer. We already mention that the BioPhotometer was prepared and we corroborate
the correct functioning. Now, we need to insert the blank sample in the cuvette shaft of the
BioPhotometer and press the blank button. In the screen, you are going to see 0.000 A, that
means 0 absorbance. Then, you are going to withdraw the blank cuvette of the shaft cuvette. The
next thing you must to do is to set the dilution factor. Press the dilution button. After that, you
must register first the sample concentration, in this case 5 l, and press the enter button. Next,
you register the concentration of the distilled water, in this case 395 l, and press the enter button
again. Now, you must insert the cuvette A-1 in the cuvette shaft and press the sample button to
14
measure the concentration. Then, you will repeat this last step for every sample. Register your
results. (Appendix: Table # 2).
POLYMERASE CHAIN REACTION (PCR)
The polymerase chain reaction (PCR) is a commonly method for the amplification of the
sequences of DNA. In this case, the purified DNA obtained for the chitons was used as templates
for amplification of a portion of the cytochrome oxidase c subunit I (COI) using this technique.
Before begin the process, we followed the necessary antiseptic techniques. Primer analysis prior
to PCR included: optimal annealing temperature, expected product length, GC content and
melting temperature (Tm) among others (Diaz, 2007). Now, we used the Sigma-Genosys 1242
Reverse Primer (5TAAACTTCAGGGTGACCAAAAAATCA3) and the Sigma-Genosys
Forward Primer (5 GGTCAACAAATCATAAGATATTGG3). The solution primers have a
total volume of 50 l: 49 l of distilled water and 1 l of the primers. This dilution for the
forward and reverse primers was performed in different tubes. 2 l of each tube of the primer
solution was used in the PCR Beads mix and PCR High Fidelity. The Ready-to GoTM PCR Beads
from General Electric Inc. were used for PCR, as described manufacturer. The PCR Beads mix
reaction consist in: 19 l of distilled water, 2 l of the DNA sample, 2 l of reverse sequence,
and 2 l of the forward sequence, for a total volume of 25 l. The High Fidelity Super Mix from
Quiagen Corp.TM was used for PCR, as descried manufacturer. The PCR High Fidelity mix
reaction consists in: 21 l of High Fidelity mix, 2 l of reverse sequence, and 2 l of the forward
sequence, for a total volume of 25 l.
For both procedures, PCR was performing in the Eppendorf Thermal Cycler (Model
5331) using the following amplification parameters: 35 cycles of a denaturation at 95oC for 5
15
min., primer annealing at 45o- 49oC-for 30 sec. (depending on the primer combination used),
polymerase mediated extension at 72oc for 30 sec., and a final elongation step at 72oc for 1 min.
The products (samples) were run in a 1% agarose gel electrophoresis containing 56 l ethidium
bromide at 100 volts (V) for 1 hour and the desire bands purified by direct band elution during
electrophoresis. The TBE buffer 1X was used to realized the electrophoresis. Fragments of
interested were excised from agarose gel and centrifuged at 12,000 rpm in the Eppendorf
Centrifuge (Model 5415 R) for 8 minutes in Spin-X Tube Filters (0.22 m cellulose acetate).
DNA SEQUENCING
Sequencing took place at the University of Puerto Rico in Rio Piedras. They are to follow the
technique or method of Sanger. They do a Polymerase chain reaction with DNA templates followed by
primers and dideoxynucleosides (ddNTPs). A series of PCR reactions are done in other to use different
concentrations of each of the ddNTPs. This ddNTPs are incorporated randomly on the growing DNA
strand. When one of the ddNTPs are incorporated, in the strand, the synthesis stops because both
terminals of the ddNTPs contains hydrogen that cannot make a fosfo-diester bond. After the separated
reactions they are all join and run in electrophoresis is made to see the different bands and the color
fragments that are emitted by the ddNTPs. This creates a chromatogram that is the use to analyses data.
BIOEDIT
After receiving sequences from the UPR- Rio Piedras Campus, we analyze them in Bioedit. This program
is a mouse-driven, easy-to-use sequence alignment editor and sequence analysis program designed and
written by a graduate student who knows how frustrating and time consuming it can be to rely upon
word-processors and command-line programs for sequence manipulation. BioEdit is intended to supply a
single program that can handle most simple sequence and alignment editing and manipulation functions
that researchers are likely to do on a daily basis, as well as a few basic sequences analyses. For that, we
16
used this program to analyze the sequences that was send from the sequencing center. We establish a
consensus sequence for the five species, and then use the program to correlate them in un- rooted trees.
First tree was DNA Parsimony, compare the differences in base with lengthen. The longer the line,
the less related, with the rest of the subjects. Other, was the DNA maximum likelihood program
this one uses the variance and standard deviation having in mind that the population rests on the
premise that it behaves by the normal distribution. This program carries out the Fitch-Margoliash
and Least Squares methods, plus a variety of others of the same family, with the assumption that
all tip species are contemporaneous, and that there is an evolutionary clock (in effect, a
molecular clock). This means that branches of the tree cannot be of arbitrary length, but are
constrained so that the total length from the root of the tree to any species is the same. The
quantity minimized is the same weighted sum of squares described in the Distance Matrix
Methods documentation file. Last tree: Neighbor-Joining method constructs a tree by successive
clustering of lineages, setting branch lengths as the lineages join. The tree is not rearranged
thereafter. The tree does not assume an evolutionary clock, so that it is in effect an un-rooted
tree.
17
RESULTS
In order to obtain a sequence of the COI gene of six species of chitons to develop phylogenetic trees, we
used barcoding method to demonstrate the interrelationship of the chitons species. We decide to classify
the DNA sample of the six species of chitons in seven groups. First, we realized seven PCR reactions to
obtain the COI gene sequence.
PCR BEADS REACTIONS

We used the PCR Beads from GE Health Care to amplificate the COI gene of the six species of chitons
of the groups 1, 2 and 3. After the PCR, we realized the electrophoresis method to obtain the band with
the DNA sequence. In the first PCR reaction we used the 1A and 1B groups. The lanes 2 to 6 of the
electrophoresis gel of the PCR products of the group 1A1 to 1A6 show the presence of the DNA.
However, in the lane 6, we observed two lanes. In the lane #7, we can observe that the control was
negative (Figure #1). In the electrophoresis gel, the lanes 2 to 6 of the PCR products of the group 1A7 to
1A11 show the presence of DNA. In the lane #7, the control was negative (Figure #2).
In the second PCR reaction, we decided to use the group 2A and 2B. In the gel electrophoresis, the lanes
2 to 6 of the PCR products of the group 1B1 to 1B6 show the presence of DNA. However, in the lane 6,
we observed two lanes. In the lane #7, the control was negative (Figure #3). The lanes 2 to 6 of the
electrophoresis gel of the PCR products of the group 1B7 to 1B11 show the presence of the DNA. In the
lane #7, we can observe that the control was negative (Figure #4). In the next electrophoresis gel, the
lanes 2, 4, and 6 of the PCR products of the group 2A1 to 2A3 show the presence of DNA (Figure #5).
The lanes 2, 4, and 6 of the electrophoresis gel of the PCR products of the group 2A5 to 2A7 show the
presence of the DNA (Figure# 6). In the next electrophoresis gel, the lanes 2, 4, and 6 of the PCR
products of the group 2A8 to 2A10 show the presence of DNA (Figure #7). The lanes 2 and 6 of the
electrophoresis gel of the PCR products of the group 2A11 to 2B1 show the presence of the DNA (Figure
18
# 8). The control in the lane 4 was negative. In the next electrophoresis gel, the lanes 2, 4, and 6 of the
PCR products of the group 2B2 to 2B5 show the presence of DNA (Figure # 9). The lanes 2, 4, and 6 of
the electrophoresis gel of the PCR products of the group 2B6 to 2B8 show the presence of the DNA
(Figure # 10). In the next electrophoresis gel, the lanes 2, 4, and 6 of the PCR products of the group 2B9
to 2B11 show the presence of DNA (Figure # 11).
In the third PCR reaction, we used the group 3A and 3B. In this case we only run the electrophoresis of
the group 3A6, 3B1, and 3B6. The lanes 2, 4, and 6 of the electrophoresis gel of the PCR products of the
group 3B1, 3A6 and 3B6 show the presence of the DNA (Figure # 12).
HIGH FIDELITY PCR REACTIONS

We used the High Fidelity Super Mix from Invitrogen to amplificate the COI gene of the six species of
chitons of the groups 4, 5, 6 and 7. During the process of PCR while using the Eppendorf Master Cycler ,
the PCR tubes containing the groups 4, 5, and 6 were open inside of the machine. However, we decided to
realize the electrophoresis method to obtain the band with the DNA sequence.
The fourth PCR reaction was performed using the groups 4A and the 4B. The lanes 2, 4, and 6 of the
electrophoresis gel of the PCR HF products of the group 4A7 to 4A9 do not show the presence of the
DNA because we cannot observe the bands (Figure #13). In the next electrophoresis gel, only the lane 6
of the PCR HF products of the group 4A7 to 4B1 shows the presence of DNA. However, the lanes 2 and
4 of the PCR HF products of the group 4A7 to 4B1 do not show the presence of DNA because we cannot
observe the bands (Figure #14). The lanes 2 and 6 of the electrophoresis gel of the PCR HF products of
the group 4B2 to 4B3 show the presence of the DNA. The control in the lane 4 was negative (Figure #15).
In the next electrophoresis gel, the lanes 2, 4, and 6 of the PCR HF products of the group 4B5 to 4B7 do
not show the presence of DNA because we cannot observe the bands (Figure #16). The lanes 2, 4, and 6
of the electrophoresis of the PCR HF products of the group 4B8 to 4B10 show the presence of the DNA.
19
In the next electrophoresis gel, the lane 2 contains the PCR HF product of the group 4B11 and the lane 3
contains the control. In this case, the group 4B11 does not show the presence of DNA because we cannot
observe the band. Also, the control in the lane 3 was negative (Figure #17).
The groups 5A and 5B were used to realize the fifth PCR reaction. The PCR HF products of group 5A1 in
the lane 2 and the group 5A2 in the lane 2 and 4 of the electrophoresis gel do not show the presence of the
DNA. However, the PCR HF product of the group 5A3 in the lane 6 shows presence of the DNA (Figure
#18). In the next electrophoresis gel, the PCR HF products of the group 5A5 in the lane 2 and group 5A6
in the lane 6 do not show presence of the DNA. Also, the control in the lane 4 was negative (Figure # 19).
The PCR HF products of the groups 5A8 to 5B4, including control # 4 do not have photos, that
demonstrate the presence or the absent of DNA. Is very important to mention that we realized the
electrophoresis gel for each sample of the above groups.
The sixth PCR reaction was performed using the groups 6A and the 6B. The PCR HF products obtained
were used to realize the electrophoresis gels. In this case, again we do not take photos of the groups to
demonstrate the presence of absent of DNA in each sample.
The groups 7A and 7B were used to perform the seven and last PCR reaction. In the next electrophoresis,
the lane 1 contains the control # 1. The band of the control do not appears in the electrophoresis gel. The
PCR HF products of the group 7A1 in lane 4 and 7A2 in the lane 6 show presence of DNA (Figure #20).
The PCR HF products of group 7A3 in the lane 2, and 7A5 in the lane 4 of the electrophoresis gel show
the presence of the DNA. However, the PCR HF product of the group 7A6 in the lane 6 do not shows
presence of the DNA (Figure #21). The next electrophoresis gel containing the PCR HF product of the
group 7A7 in the lane 3 shows presence of DNA. However, the PCR HF product of the group 7A8 does
not show presence of DNA. In the lane 7, the band of the control do not appears in the electrophoresis gel
(Figure #22). In the next electrophoresis gel, the PCR HF products of the group 7A9 in the lane 2 and
7A10 in the lane 4 do not show the presence of DNA. The PCR HF product of the group 7A11 in the lane
6 shows the presence of DNA (Figure #23). The electrophoresis gel contains the PCR HF products of the
20
group 7B1, 7B2, and 7B3 in the lanes 2, 4, and 6 respectively. In this case we do not take photos of the
electrophoresis gel that have the samples. In the next electrophoresis gel, the PCR HF products of the
groups 7B5, 7B6, and 7B7 in the lane 2, 4, and 6 respectively show the presence of the DNA. In the case
of the group 7A7 in the lane 6, the electrophoresis gel shows a double band (Figure #24). The next
electrophoresis gel containing the PCR HF products of the group 7B8 in the lane 2, 7B9 in the lane 4, and
7B10 in the lane 6 do not shows presence of DNA (Figure #25). The last electrophoresis gel contains the
PCR HF products of the group 7B11 in the lane 2 and the control 3 in the lane 4 (Figure # 26).
During the process, we can saw the continuity of the electrophoresis gels of the groups the absence of
DNA. Between the most common reasons to explain this fact we can mention that the samples of DNA
were stored in TBE Buffer. TBE Buffer (Tris Boric Acid and EDTA) contains EDTA that is a chelator
agent responsible of the removing of magnesium ions. The magnesium ions function as co factors for
nucleases, enzymes that catalyze the break of the fosfodiester bonds of the DNA molecule. We can
predict that the EDTA agent in the TBE does not work efficiently in preserving intact the DNA samples.
In other words, the EDTA failed in removing the magnesium ions in the preservation solution. For that
reason, the nucleases catalyze the breaking of the bonds in the DNA. Another reason to explain the fact of
the absent of DNA in the electrophoresis gels is that the Tris HCl component in the TBE buffer does not
work appropriately. The Tris HCl component is the responsible to maintain the pH of the solution. If the
pH solution decrease (quantity of hydrogen increase), this fact can cause the degradation of the DNA
molecule. Also, the pH affects the velocity of migration of the molecules. If the DNA molecule was
degraded or the velocity of migration was affected, the absent of bands in the electrophoresis is common.
Also, if the dilution of the buffer was realized wrong, it can cause interference with the PCR, and the
absence of DNA concentration. The ineffectively of buffer could be cause by external contamination with
the environment, by expiration of the product or by inappropriate the storage condition.
Also, during the process we identified the presence of double bands in the groups 6 and 7. The presence
of double bands can occur by contamination of the sample. If the method of PCR was not performed
21
using the appropriate aseptic precautions that could contaminate the sample. Also, some quimicals can
defragmented the DNA molecules. The defragmentation appears as a double band in the electrophoresis
gel.
QUANTIFICATION OF DNA
To corroborate the presence of DNA in the PCR Beads Reactions products and PCR High Fidelity
Reactions products of the six species of chitons, we realize a quantification of DNA in each product of
PCR. Before realize the quantification of each sample, we prepare a blank solution to calibrate the
machine. The results demonstrate that in some samples the concentration of DNA was not cero (Table
#4). The others samples that do not appear in the table demonstrate the absence of DNA. This fact support
the absent of bands in the electrophoresis gels of the PCR products.
In many samples the DNA concentration was cero. However, this fact do not means absence of DNA in
the sample. For that reason, we decided to send for sequencing some samples that show absence of DNA.
Also, we sent samples that show presence of DNA that include samples of the groups 6 and 7 which show
double bands.
Turbid measuring solutions show increased absorbance for all wavelengths. To corroborate the purity of
each sample we used the parameters A320 and A260. The parameter A320 tells us the how pure is each
sample. With pure samples, the absorbance at 320 nm should be approximately zero. If the parameter
A260 show measured solutions with absorbance lower to apparent 0.002 to 0.003, the sample should not
be used. This is because with such low absorbance, disturbances such as small particles, microbubbles or
turbidity have a great deal of influence upon the measuring result and often lead to unreliable results
(Table # 4).
22
SEQUENCE EDIT
In the DNA parsimony algorithm we acquired the tree (fig 1 of the work) in which the out groups
is as expected is the Cyrtopodion. The viridis follows as the frist to divert form the chiton
mitochondrial genetic line. And the spices granulata and squamosus are sisters grupos and the
marmoratus and tuberculatos are also sister grupos
In the Neighbor-Joining/UPGMA method we acquired the tree (fig 2 of the work) in which the
out groups is as expected is the Cyrtopodion. The viridis follows as the frist to divert form the
chiton mitochondrial line genetic line. This tree suggests that tubercula diverts from the group.
Marmorata is sister grupo with the sister group of Squamous and granulata.
In the Nucleic acid sequence Maximum Likelihood method we acquired the tree (fig 3 of the
work) in which the out groups is as expected is the Cyrtopodion. Marmorata is the first to divert
form the chiton mitochondrial line genetic line. Tuberculato divertes from the grupo third and
virides forms a sister grupo with the sister grupo of the squamous and granulata
In the Fitch-Margoliash method with contemporary tips we acquired the tree (fig 4 of the work)
in which the out groups is as expected is the Cyrtopodion. This tres is a monoclade in which all
grupos have sister groups with the one or ones before him with the exemption of the sister
grupos of granulata and squamosus. Virides si the frist to divert from the group followed by
tuberculata net marmorata and finally the sister grupo of granulata and squamosus.
23
DISCUSSION
In this investigation we used the DNA Barcoding technique and the mitochondrial genome,
specifically the COI gene sequence to evaluated relatedness at species level in Chiton
marmoratus, Chiton tuberculatus, Chiton squamosus and Acanthopleura granulata.
Now is very important to mention that the COI gene is not suitable for studying all groups of
organisms. For example, rates of evolution of COI are too slow to allow this gene to be useful in
plant studies and other sequences have been proposed (Kress et al., 2005). In addition, anaerobic
eukaryotes do not normally have a mitochondrial genome and therefore lack the target gene
altogether. For DNA barcoding in such organisms an alternative target is required, a gene with
the requisite characteristics but that is present in the organism of interest.
The COI gene permits us to develop an accurate identification of species and an accurate
phylogenetic analysis. Using a molecular analysis from the mitochondrial protein coding genes
(specially the COI gene) of the chitons species, we prove the phylogenetic relationships among
them. Phylogenetic studies proved that the mitochondrial genome of animals is a better tool for
analysis than nuclear genome because the first one lacks of introns and have limited exposure to
recombination mechanism (Saccone et al. 1999). The continuously use of cytochrome c oxidase
subunit I gene (COI) is due because it presents two advantages. First, the primers for this gene
are very wide, enabling the recovery of its 59 end. These ends are representatives of most animal
phyla. Second, the COI gene has a greater range of phylogenetic signal than any other
mitochondrial gene (Folmer et al. 1994; Hewitt & Zhang 1997).
In order to prove the relatedness of this species we realized severe techniques. First of all, we
realized the collection of the tissue sample of the each species of Chitons (Table # 1 and Table #
2).
24
After sequencing of bands from our electrophoresis, the sequence was analyze indenpendently,
and we use Bioedit to work on filogenetic trees to corroborate relationship between the species.
Cyrtopodion was choose as the outgroup for the un-rooted trees. In the tree 1,2, and 4 presents
chiton viridis as the first it devert from the group of the Chtiones in study only in fig 3
marmuratus is the frist to divert. There is not clear view with the relation of marmoratus and
tuberculatus. The can be sister group as in the figure # 1, tuberculatus can be divert first than
marmuratus like in figure # 2 and figure #4, or the opposite in figure # 3. But the most important
results are that Chiton squamous and Acantoplura granulata are sister grups in all the trees
(figure # 1- figure #4). What this result means is that there must be a taxomy revision that takes
in consideration that Acantoplura granulata can be places in the Chiton gruops like Chiton
squamosus, tuberculatus, viridis and marmoratus.
After doing this research with just one sequence, its kind of logic, that there should be more
studies with this organism using other barcodes, like the ribosomal 16S rRNA in the
mitochondrial. With our sequence we had different facts that differ from the ones establish. And
more than taxonomically, future studies most be done on how does they metabolize the
magnetite in their teeth, and the force of adhesions that they use to stick to the rocks. This could
be significant for biology matters.
25
CONCLUSION
The DNA Barcoding technique is an effective technique to corroborate the interrelationships
between species. We decide to use the mitochondrial genome, specifically the COI gene to
realize the interrelationships between five species of Chitons. In addition, we can strengthen the
fact that the mitochondrial genome is most common used than the nuclear genome for molecular
analysis to prove phylogenetic relationships. This action helps us to sustain with real data our
phylogenetic study.
The phylogenetic studies shows chiton viridis as the least related from the group of the Chitons.
There is not clear view with the relation of marmoratus and tuberculatus. The can be sister group
as in the fig 1, tuberculatus can be divert first than marmoratus like in figure # 2 and figure #4,
or the opposite in figure # 3. But, the most important results is that Chiton squamosus and
Acantopleura granulata are sister groups
26
FIGURES AND TABLES
27
Figure # 1
PCR products of PCR Beads Reaction from DNA samples of Chiton tuberculatus,
Acantopleura granulate, and Chiton squamosus using 1 kb and 123 ladders.
Twenty L of DNA samples were introduced in the gel to realize an electrophoresis gel of 1% of
agarose. Also, two L of the 1 kb ladder and three L of the 123 ladder were used.
Lane 1 shows the 1 kb ladder
Lane 2 shows the presence of DNA of the organism Chiton tuberculatus (1A1)
Lane 5 shows the presence of DNA of the organism Acantopleura granulata (1A5)
Lane 6 shows the presence of DNA of the organism Chiton squamosus (1A6)
Lane 7 shows the Control # 1
Lane 8 shows the 123 ladder
28
Figure # 2
PCR products of PCR Beads Reaction from DNA samples of Chiton marmoratus, and
Acantopleura granulata using 1 kb and 123 ladders.
Twenty L of DNA samples were introduced in the gel to realize an electrophoresis gel of 1% of
agarose. Also, two L of the 1 kb ladder and three L of the 123 ladder were used.
Lane 3 shows the presence of DNA of the organism Chiton marmoratus (1A8)
Lane 7 shows the Control #2
29
Figure # 3
PCR products of PCR Beads Reaction from DNA samples of Chiton tuberculatus,
Acantopleura granulate, and Chiton squamosus using 1 kb and 123 ladders.
Twenty L of DNA samples were introduced in the gel to realize an electrophoresis gel of 1%
of agarose. Also, two L of the 1 kb ladder and three L of the 123 ladder were used.
Lane 2 shows the presence of DNA of the organism Chiton tuberculatus (1B1)
Lane 5 shows the presence of DNA of the organism Acantopleura granulata (1B5)
Lane 6 shows the presence of DNA of the organism Chiton squamosus (1B6)
30
Figure # 4
Lane 3 shows the presence of DNA of the organism Chiton marmoratus (1B8)
31
Figure # 5
PCR products of PCR Beads Reaction from DNA samples of Chiton tuberculatus using 1 kb
and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
32
Figure # 6
PCR products of PCR Beads Reaction from DNA samples of Chiton squamosus and
Acanopleura granulata using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
33
Figure # 7
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
34
Figure # 8
Chiton tuberculatus using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
35
Figure # 9
PCR products of PCR Beads Reaction from DNA samples of Chiton tuberculatus and
Acantopleura granulate using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
36
Figure # 10
PCR products of PCR Beads Reaction from DNA samples of Chiton marmoratus, Chiton
squamosus and Acantopleura granulata using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
37
Figure # 11
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
38
Figure # 12
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton squamosus and
Chiton tuberculatus using 1 kb and 123 ladders.
Lane 4 was empty
Lane 5 was empty
Lane 7 was empty
39
Figure # 13
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton marmoratus,
and Acantopleura granulata using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
40
Figure # 14
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton tuberculatus
and Chiton marmoratus using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
41
Figure # 15
PCR products of High Fidelity Reaction from DNA samples of Chiton tuberculatus using 1
kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
42
Figure # 16
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
43
Figure # 17
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton marmoratus
using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 6 was empty
Lane 7 was empty
44
Figure # 18
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
45
Figure # 19
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
46
Figure # 20
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
47
Figure # 21
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton squamosus,
Chiton tuberculatus and Acantopleura granulata using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
48
Figure # 22
Lane 2 was empty
Lane 4 was empty
Lane 6 was empty
Lane 7 shows the presence of Control #2
49
Figure # 23
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton marmoratus, and
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
50
Figure # 24
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
51
Figure # 25
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton marmoratus,
and Acantopleura granulata using 1 kb and 123 ladders.
Lane 3 was empty
Lane 5 was empty
Lane 7 was empty
52
Figure # 26
PCR products of PCR High Fidelity Reaction from DNA samples of Chiton marmoratus
Lane 3 was empty
Lane 5 was empty
Lane 6 was empty
Lane 7 was empty
53
Figure #27
IDNA parsimony algorithm, version 3.6a2.1
One most parsimonious tree found:
+---------------viridis
|
|
+---------granulata
|
+----------4
|
|
+---------squamosus
1-----------2
|
|
+---------------marmorata
|
+-------3
|
+------------------tuberculat
|
+--------------------Consensus
requires a total of 1855.000

between
and
length
-------------1
viridis
0.267228
1
2
0.198496
2
4
0.188547
4
granulata 0.156546
4
squamosus 0.170180
2
3
0.138535
3
marmorata 0.267608
3
tuberculat 0.320493
1
Consensus 0.351190
(viridis:0.26723,((granulata:0.15655,squamosus:0.17018):0.18855,
(marmorata:0.26761,tuberculat:0.32049):0.13853):0.19850,Consensus:0.35119);
54
Figure # 28
6 Populations
Neighbor-Joining/UPGMA method version 3.6a2.1
Neighbor-joining method
Negative branch lengths allowed
+--------------------viridis
!
! +---------------------tuberculata
! !
2---3
+---------squamosus
! ! +-----------1
! +-4
+-------granulata
! !
! +-----------------marmorata
!
+-----------------------------------------------------Consensus
Figure # 29
remember: this is an unrooted tree!
Between
And
Length
-------------2
viridis
1.04566
2
3
0.21202
3
tuberculata
1.10733
3
4
0.08377
4
1
0.61702
1
squamosus
0.49274
1
granulata
0.40236
4
marmorata
0.91338
2
Consensus
2.67534
(viridis:1.04566,(tuberculata:1.10733,((squamosus:0.49274,
granulata:0.40236):0.61702,marmorata:0.91338):0.08377):0.21202,Consensus:2.67534);
6
Consensus 0.0000 3.9976 4.2435 3.8295 3.9321 3.7210
tuberculat 3.9976 0.0000 2.1884 2.1444 2.2431 2.3621
squamosus 4.2435 2.1884 0.0000 1.8702 0.8951 2.5939
marmorata 3.8295 2.1444 1.8702 0.0000 2.0857 2.2300
granulata 3.9321 2.2431 0.8951 2.0857 0.0000 2.2736
viridis 3.7210 2.3621 2.5939 2.2300 2.2736 0.0000
55
Figure # 30
Nucleic acid sequence Maximum Likelihood method, version 3.6a2.1
Empirical Base Frequencies:

A
0.29047
C
0.19123
G
0.19747
T(U) 0.32083
Transition/transversion ratio = 2.000000
+marmorata
|
| +---------------------------------------------------viridis
| +--4
| | | +squamosus
2--1 +--3
| | +granulata
| |
| +--tuberculat
|
+-----------------------------------------------------------Consensus
remember: this is an unrooted tree!

Ln Likelihood = -5136.59212
Between
And
--------2
2
2
1
4
4
3
3
1
Length
Approx. Confidence Limits
------------ ---------- ------
Consensus
marmorata
1
4
viridis
3
squamosus
granulata
tuberculat
55.58907 ( zero, infinity) **

0.56036 ( zero, 1.51086)
0.65149 ( zero, 1.60199)
1.77452 ( 0.76328, 2.77469) **
48.68463 ( zero, infinity) **
0.03795 ( zero, 1.03813)
0.19607 ( 0.08909, 0.27886) **
0.43426 ( 0.34086, 0.52768) **
3.16109 ( 1.27708, 5.04509) **
* = significantly positive, P < 0.05

** = significantly positive, P < 0.01
(marmorata:0.56036,((viridis:48.68463,(squamosus:0.19607,
granulata:0.43426):0.03795):1.77452,tuberculat:3.16109):0.65149,
Consensus:55.58907);
56
Figure # 31
6 Populations
Fitch-Margoliash method with contemporary tips, version 3.6a2.1
__ __
2
\ \ (Obs - Exp)
Sum of squares = /_ /_ -----------2
i j
Obs
negative branch lengths not allowed
+-------------granulata
+---------------4
+---3
+-------------squamosus
! !
+--2 +-----------------------------marmorata
! !
+-----------------------5 +--------------------------------tuberculat
!
!
--1
+-----------------------------------viridis
!
+----------------------------------------------------------Consensus
Sum of squares =
0.059
Average percent standard deviation = 4.58338

From To
---- -4
3
4
2
3
5
2
1
5
1
Length
Height
-----------
granulata
0.44755
4
0.53557
squamosus
0.44755
3
0.11212
marmorata
0.98312
2
0.07942
tuberculat
1.09524
5
0.79007
viridis
1.17466
Consensus
1.96473
1.96473
1.51718
1.96473
0.98161
1.96473
0.86949
1.96473
0.79007
1.96473
1.96473
(((((granulata:0.44755,squamosus:0.44755):0.53557,marmorata:0.98312):0.11212,
tuberculat:1.09524):0.07942,viridis:1.17466):0.79007,Consensus:1.96473);
57
Table # 1: Taxon Collection

Identification Number
Species
Chiton tuberculatus
Chiton tuberculatus
Chiton tuberculatus
Chiton tuberculatus
Acontopleura granulata
Chiton squamosus
Chiton marmoratus
10
Chiton marmoratus
11
Chiton marmoratus
58
Table # 2: Tissue Sample (mg)

Tissue sample of each individual
Quantity of tissue extracted (mg)
A-7.4
1
B-5.2
A-11.8
2
B-12.2
A-10.7
3
B-11.6
NONE
4
NONE
A-12.6
5
B-12.5
A-10.7
6
B-13.1
A-13.4
7
B-14.3
A-11.5
8
B-14.5
A-14.6
9
B-12.5
A-12.0
10
B-11.3
A-7.8
11
B-8.7
59
Table # 3: Primers Sequence

Name
Length
Tm
% GC
Sequence (5-3)
COFW
25
61.1
32.00%
5-GGTCAACAAATCATAAAGATATTGG-3
CORV
26
66.9
34.62%
5-TAAACTTCAGGGTGACCAAAAAATCA-3
Table # 4: Cycling Parameters for the PCR

Cycling Parameters
Initial Denaturation
Denaturation during cycling
Annealing
Elongation
Total number of cycles
Final elongation
Value used in the PCR

95C for 5 min
35 cycles at 95C for 30 s
45 to 49C for 30 s
72C for 1 min
25-35
72C for 1 min
60
Sample
B3b6
B3a6
B2a11
B2a10
B2a9
B2a8
B2a7
B2a6
B2a5
B2a3
B2a2
B2a1
B2b1
B2b2
B2b3
B2b5
B2b6
B2b7
B2b8
B2b9
B2b10
B2b11
Hf7a3
Hf7a5
Table # 5: Samples sent to sequence

SampleConcentration
Base Pair
(ng/l)
Concentration
2
15
1
7.50
0
0
1
7.50
2
15
1
7.50
0
0
0
0
1
7.50
1
7.50
0
0
0
0
0
0
1
7.50
1
7.50
1
7.50
1
7.50
1
7.50
1
7.50
1
7.50
1
7.50
1
7.50
0
0
0
0
Lecture of Purity
A 320
0.029
0.015
0.000
0.009
0.013
0.002
0.000
0.000
0.000
0.003
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.004
0.000
0.001
0.003
0.000
0.000
61
PCR Reaction
(Beads or High
Fidelity)
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
PCR Beads
Sample
1A1
1A2
1A3
1A5
1A6
1A7
1A8
1A9
1A10
1A11
1B1
1B2
1B3
1B5
1B6
1B7
1B8
1B9
1B10
1B11
2A1
2A2
2A3
2A5
2A6
2A7
2A8
2A9
2A10
2A11
3A6
3B6
2B1
2B2
2B3
2B5
2B6
2B7
2B8
2B9
2B10
2B11
Table # 4: Quantification of DNA

Sample
Purity
Viable
Concentration
Lecture (Yes or No)
(ng/l)
A 320
6
0.000
Yes
46
0.002
Yes
12
0.000
Yes
0
-0.001
No
70
0.013
Yes*
3
0.000
Yes*
0
0.000
Yes
3
0.000
Yes
12
0.001
Yes
0
0.000
No
15
0.000
Yes
0
0.000
Yes
31
0.004
Yes
41
0.001
Yes
24
0.000
Yes
24
0.001
Yes
49
0.004
Yes
42
0.001
Yes
77
0.001
Yes
59
0.003
Yes
0
0.000
Yes
0
0.000
Yes
1
0.003
Yes
1
0.000
Yes
0
0.000
Yes
0
0.000
Yes
1
0.002
Yes
2
0.000
Yes
1
0.009
Yes
0
0.000
Yes
1
0.015
Yes*
2
0.029
No*
0
0.000
Yes
1
0.000
Yes
1
0.000
Yes
1
0.001
Yes
1
0.004
Yes
1
0.000
Yes
1
0.003
Yes
1
0.001
Yes
Lecture at
A 260
Viable
(Yes or No)
0.002
0.017
0.004
-0.001
0.036
0.001
0.000
0.001
0.003
0.000
0.005
0.000
0.014
0.015
0.008
0.009
0.020
0.015
0.027
0.023
0.000
0.004
0.000
0.010
0.009
0.003
0.012
0.040
0.016
0.005
0.025
0.036
0.000
0.012
0.010
0.013
0.015
0.011
0.014
0.014
Yes
Yes
No
No
Yes
No
No
No
No
No
No
No
No
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
Yes
No
Yes
Yes
No
No
No
No
Yes
No
No
No
62

PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
PCR HF
5A1
5A2
5A3
5B2
5B5
6A1
6A2
6A3
6B1
6B2
6B3
7A1
7A2
7A3
7A5
7A7
7A9
7B1
7B2
7B3
7B5
7B6
7B7
7B9
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.007
0.000
0.000
0.000
0.000
0.000
0.000
0.003
0.004
0.000

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
0.010
0.002
0.008
0.003
0.000
-0.000
0.000
0.000
0.000
0.018
0.000
0.001
0.001
0.000
0.007
0.000
0.007
0.004
0.009
0.001
0.000
0.015
0.011
0.022
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
Yes
No
Yes
63
ACKNOWLEDGEMENTS
Dr. Cedar Garca
University of Puerto Rico in Ponce
Prof. Johanna Daz
J2 Laboratory Investigation
REFERENCES

Phylogenetic Corroboration of Chiton Tuberculatus, Chiton Marmoratus, Chiton Viridis, Chiton Squamosus and Acanthopleura Granulata Using The 5' Dna Cytocrome Oxidase I

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Phylogenetic Corroboration of Chiton Tuberculatus, Chiton Marmoratus, Chiton Viridis, Chiton Squamosus and Acanthopleura Granulata Using The 5' Dna Cytocrome Oxidase I

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PHYLOGENETIC CORROBO RATION OF CHITON

TUBERCULATUS, CHITON MARMORATUS, CHITON VIRIDIS,

Abbreviated Title: Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

identification of species because the identification based only in morphological characteristics is

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

MATERIALS AND METHODS

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

QUANTIFICATION OF THE CONCENTRATION OF THE DNA SAMPLES

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

POLYMERASE CHAIN REACTION (PCR)

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

PCR BEADS REACTIONS

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

HIGH FIDELITY PCR REACTIONS

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

N.De Howitt, M.Coln, L.Coln

2010 Phylogeny of chitons

FIGURES AND TABLES