APPENDIX : MOLECULAR SYSTEMATICS OF SHRIMPS USING …shodhganga.inflibnet.ac.in/bitstream/10603/11017/12/12... · 2015-12-04 · 98 CHAPTER – 3 APPENDIX : MOLECULAR SYSTEMATICS

98

CHAPTER – 3

APPENDIX : MOLECULAR SYSTEMATICS OF

SHRIMPS USING INTER-SIMPLE SEQUENCE

REPEAT (ISSR)

Introduction

The ISSR marker system is based on microsatellite sequences found

throughout eukaryotic genomes. Therefore, this technique involves the amplification

of DNA segments present between two identical microsatellites that are oriented in

opposite directions (Reddy et al., 2002). In other words, microsatellite repeats target

multiple genomic loci to amplify their inter-SSR sequences of different sizes.

Although the microsatellite repeat utilized as the primer, di-, tri-, tetra- or penta-

nucleotide repeats and particularly those with (AG), (GA), (CT), (TC), (AC), (CA)

repeats have been shown to reveal higher polymorphism than other di-, tri- or tetra-

nucleotides (Parsons et al., 1997). The primers can also be either unanchored or more

usually either 3'-anchored or 5'-anchored. Unanchored primers, consisting solely of

the microsatellite repeat as the primer, have a tendency to slip within the

microsatellite repeat unit during amplification leading to smears rather than clear

bands (Hess et al., 2000). Anchored primers, on the other hand, are extended at the 3'

or 5' end with 1 to 4 degenerate nucleotides which complement the flanking regions

surrounding the microsatellite, ensuring that annealing only occurs at the ends of a

microsatellite in the template DNA (Zietkiewicz et al., 1994). Not only does this

prevent internal priming and smear formation, the anchor also allows only a subset of

the microsatellite to serve as a priming site. The 5'-anchored primers produce

amplification products that include the entire microsatellite sequences, resulting in an

elevated number of bands and a higher degree of polymorphism. The 3'- anchored

99

primers also reveal high polymorphism but tend to give clearer banding patterns

compared to those anchored at 5'end. The variability within the genome revealed by

ISSRs is mainly attributed to the increased evolutionary rate of change of

microsatellites which is considerably higher than in most other types of DNA (Reddy

et al., 2002).

Overall, the ISSR markers are based on the presence of microsatellites

throughout the entire genome therefore describing the DNA characteristics of an

individual over several chromosomal loci. As a result, no two amplification patterns

will be identical, although those originating from the same species will be more

similar than those from more distantly related species. The characteristic of this multi-

locus marker system allows it to be useful in areas such as identifying individual-

specific differences, inter-species genomic fingerprinting, genetic diversity,

phylogenetic inferences, gene tagging, genome mapping and evolutionary biology.

(Reddy et al., 2002). For example, previous studies carried out on finger millet,

chrysanthemum, sorghum, maize, Pseudotsguga menzeisii and Cryptomeria japonica

have reported the successful use of this method in determining genetic diversity and

genomic origins of these species. In addition, studies suggest that this method can be

used to confirm the presence and evaluate the distribution of certain microsatellite

repeats within different genomes (Zietkiewicz et al., 1994).

There are a number of advantages associated with the ISSR multi-locus

technique. Firstly, ISSRs are universal in the sense that microsatellite repeats are

found in every eukaryotic genome studied to date. Secondly, unlike their RAPD

counterparts, ISSR have high reproducibility. This is most likely due to the longer

100

lengths of the primers which permit the use of higher annealing temperatures which in

turn, reduces nonspecific binding and results in higher stringency (Quan et al., 2001).

Furthermore, contrary to microsatellites, amplification does not require prior

knowledge of the DNA sequence. ISSRs are quick and easy to handle and reveal

multilocus, highly polymorphic patterns. Finally, because each band corresponds to a

DNA sequence delimited by two inverted microsatellites, the amplified products,

usually 200-2000bp long, are detectable by both agarose and polyacrylamide gel

electrophoresis (Reddy et al., 2002).

Reddy et al. (2002) used ISSR-PCR for fingerprinting of 13 diverse silkworm

strains and were successful in separating the diapauses strains and the nondiapause

strains. Nagaraja et al., (2004) using ISSR have assessed phylogenetic relationships in

nasuta subgroup of Drosophila. Kumar et al. (2001) have used ISSR-PCR to

differentiate the four disputed chilli samples (Caosicum annum) and proved the

reliability of the technique.

101

Materials and methods

Sampling

Seven species belonging to four genera of the family Penaeidae, were

collected from in ArabainSea coast. All shrimps in the fish markets were captured in

or near Mangalore area. The species were identified by followingkeys proposed by

Liu and Zhong (1986) and Dall et al (1990). Selected specimens of all species were

reexamined and confirmed by CMFRI in Mangalore. Table 3.1 lists the name of

species studied and sampling details. Only adult specimens with carapace length

(distance from postorbital margin to the posterior edge in the middorsal line) longer

than 8 cm and body weight greater than 50 grams were studied. All specimens were

frozen until dissection.

Genomic DNA isolation

Total genomic DNA was isolated from adult male shrimps of penaeidae

species investigated. DNA was extracted according to standard procedure (Williams

et al., 1990). Briefly, the specimens were ground in liquid nitrogen using pestle and

mortar. Extraction buffer (100 mM Tris-Hcl (pH 8.0), 50 mM NaCl, 50 mM EDTA

and 1 % SDS) was added to the ground tissue and the resulted slurry was transferred

to a clean eppendorf tube, and Proteinase K was added to a final concentration of 100

µg/ml and contents were mixed by swirling and incubated in a water bath maintained

at 37 ºC over night with occasional swirling.

After incubation, equal volume of Tris saturated (equilibrated) phenol was

added to the tube, the contents were mixed by swirling for 5-10 minutes, and spun at

8000 rpm for 20 minutes. The clear aqueous phase was transferred to a new tube

102

using wide mouthened pipette tips and equal volume of saturated

phenol:chloroform:isoamyl alcohol (24:24:1) was added, mixed the contents and spun

at 8000 rpm for 10 minutes. The aqueous phase was transferred to a new tube

containing an equal volume of chloroform and spun at 8000 rpm for 10 minutes. After

centrifugation the aqueous phase containing DNA solution was transferred to a new

tube and precipitated with 1/10th

volume of 3 M Sodium acetate (pH 5.2) and 2

volumes of cold ethanol. The precipitated DNA was spooled out using pipette tips and

washed once with 70% ethanol, air dried and resuspended in TE buffer [(10 mM Tris,

1 mM EDTA (pH 8.0)]. RNase A (GeNei, Bangalore) was added (final concentration

of 100 µg/ml) to the TE buffer (pH 8.0) containing DNA and incubated at 37ºC for 2

hours. After RNase treated, the gDNA was extracted by repeating the phenol,

phenol:chloroform and chloroform extraction steps.

Quantification of genomic DNA

The quantity and purity of the extracted genomic DNA was checked

spectroscopically at 260/280 nm absorbance as well as on 0.8% agarose minigel

(Sambrook et al., 1989). The DNA quantity of each sample was estimated by

comparing band intensity with uncut λ (lambda) DNA (GeNei, Bangalore) control of

known concentrations. The DNA was diluted using TE buffer to a final concentration

of µg/ml for ISSR analysis.

ISSR-PCR Primers

Totally four [UBC (University of British Columbia) primers were used in the

present investigation (Table 3.1).

103

Amplification with ISSR primers

All DNA samples were amplified with each of the four primers (Table 3.1).

The ISSR amplification was carried out in accordance with the method described by

Hua et al., (2006) and Zietkiewicz et al. (1994), with some modifications. PCR

amplification parameters including MgCl2 concentration and annealing temperature

were standardized for each primer separately as shown in Table 3.1. The polymerase

chain reaction (PCR) amplifications were carried out in 20µl reaction mixture which

contained 10x PCR buffer, 2.5mM MgCl2, 0.2mM of each dNTP (dTTP, dATP, dCTP

and dGTP), 25x of primer, 1 unit of Taq polymerase and 20ng/µl of genomic DNA

template. Amplification was carried out in a DNA thermal cycler (Perkin Elmer, Cetus-

2400). The program was set to a hot start with an initial denaturation of 7 minutes at

94°C followed by 2 minutes at 85°C and then 45 cycles of 1 minute at 94°C, 1 minute

at 52°C and 2 minutes at 72°C, with a final 7 minute extension at 72°C.

Separation and Detection of PCR Products

PCR amplified products were separated by agarose gel electrophoresis. All

PCR products were loaded into 1.2% agarose gel in the 1x TBE buffer (89 mM Tris

base, 89 mM Boric acid and 2 mM EDTA),. Gels were pre-stained with 4µl of

ethidium bromide and run at 3.14V/cm for approximately 120 minutes.

Ten micro liter (10µl) of reaction was mixed with 10x loading buffer (20%

Ficoll Type 400, 0.1 M EDTA (pH 8.0) 0.25% bromophenol blue and loaded onto gel

and run 4-5 hours at 70 Volts. The gels were stained with ethidum bromide to a final

concentration of 0.5 µg/ml for one hour. The gel image was recorded using Bio-Rad

ChemiDoc system. Generuler 1 kb DNA ladder (GeNei, Bangalore) were used as a

molecular standard. The approximate molecular size of the amplification product was

calculated using computational program of Alphaview gel doc system.

104

ISSR analysis

ISSR assays of each population were performed at least twice. Only

reproducible amplified fragments were scored. For each sample, the presence or

absence of fragments was recorded as 1 or 0, respectively and treated as a discrete

character. The 0-1 matrix was calculated using Microsoft Excel program and the

dendrograms were constructed based on the 0-1 matrix using UPGMA and neighbor-

joining and UPGMA methods.

105

Results

The amplification condition was standardized with respect to primer

concentration, MgCl2 concentration, and Taq DNA polymerase, as these affect

reproducibility and consistency of ISSR assay (Kumar et al., 2001). The MgCl2

concentration for different primers is varied and optimized as shown in the table 2.

Since the ISSR assay is based on the primers, which are of repetitive in nature, they

produce more number of products and separation and resolution of products is very

important. For this, the percentage of agarose gel is important to obtain a good

resolution of banding profile. The 1.2% agarose gel for the USB primer amplified

products, and obtained a well-resolved profile.

To investigate the ISSR patterns, 4 ISSR primers were used. After initial

screening with gDNA of one of the species (P. indicus) a total 4 UBC primers were

selected to carryout ISSR assay on gDNA samples of all species under

investigation.The ISSR-PCR was performed twice with four primers (Table 3.1). As a

result, an excellent reproducible and informative profile was obtained. A large amount

of polymorphism was recognized both at interspecific as well as intraspecific levels.

The figures (3.1, , 3 and ) show ISSR profiles obtained by four primers. The

number and size of the amplified products varied depending on the species. As per the

data analysis using the formula of Nei (1978) the dissimilarity index (1-F) values

obtained for each pair wise comparison is varied and is shown in the Tables (3.3).

Two dendrograms were obtained based on UPGMA (Fig 3.5) and Neighbor-joining

(Fig. 3.6).

106

Figure 3.1 shows ISSR profile obtained by UBC 818 primer. The number and

size of the amplified products varied depending on the species. The size of the

amplified products ranged from 562-1892 bp. The smallest fragment belongs to

Trachypenaeus curvirostris and the biggest one belongs to Metapenaeus monoceros.

A considerable amount of polymorphism was detected with this primer. A total of 17

bands were scored. Of these bands, one was monomorphic band i.e., shared by all

species. This band is 865 bp in weight. Out of 17 bands, 16 (94%) bands were

polymorphic and shared by at least two species.

Figure 3.2 shows ISSR profile obtained by UBC 825 primer. The number and


amplified products ranged from 320-2000 bp. The smallest fragment belong to

Trachypenaeus curvirostris, Parapenaeopsis longipes and Metapenaeus monoceros

the biggest one belong to the Penaeus canaliculatus and Trachypenaeus curvirostris.

A considerable amount of polymorphism was detected with this primer. A total of 13

bands were scored. Of these bands, 13 (100%) were polymorphic and shared by at

least two species.

Figure 3.3 shows ISSR profile obtained by UBC 827primer. The number and



Metapenaeus dobsoni and the biggest one belongs to Parapenaeopsis longipes. A

considerable amount of polymorphism was detected with this primer. A total of 12


species. This band is 1500 bp in weight. Out of 11 bands, 11 (91.6%) bands were


107

Figure 3.4 shows ISSR profile obtained by UBC 866primer. The number and



Parapenaeopsis stylifera and the biggest one belongs to Penaeus canaliculatus. A

considerable amount of polymorphism was detected with this primer. A total of 16


species. This band is 1142 bp in weight. Out of 16 bands, 15 (94%) bands were


Table 3.3 shows the numbers of shared fragments between two species and

dissimilarity index (1-F) for a pair of species. As per the data analysis using the

formula of Nei and Li (1979) the dissimilarity index (1-F) values obtained for each

pair wise comparison is varied. This table exhibits the numbers of shared fragments

and dissimilarity index (1-F) between pair species based on analysis of ISSR

fragments for all four primers. The least 1-F value among all species is 0.258 between

Metapenaeus dobsoni and Metapenaeus monoceros, while the highest is 0.551

between Trachypenaeus curvirostris and M. monoceros.All of the pair species that

have been compared have some shared fragment but M. dobsoni and M. monoceros,

from same genera, have the lowest shared fragments which is four. Five is the most

common number for shared fragments between the pair species.

According to phylogenetic tree (Fig. 3.5), there are three clusters with

different members. One cluster consists of the members of Parapenaeopsis and

Penaeus namely Penaeus indicus and Penaeus canaliculatusParapenaeopsis stylifera

and Parapenaeopsis longipes. The second one includes only the members of

108

Trachypenaeus, namely, Trachypenaeus curvirostris. Parapenaeopsis longipes has

more affinity with the members of Peneaus rather than Parapenaeopsis stylifera. The

other cluster includes only the members of Metapenaeus, namely, Metapenaeus

monoceros, Metapenaeus dobsoni, that form a clade together.

With reference to dendrogram (Fig. 3.6), there are two clusters. The first one

includes members of Peneaus, Parapenaeopsis, Trachypenaeus and one species

ofMetapenaeus, namely, Penaeus indicus, Penaeus canaliculatus, Parapenaeopsis

stylifera, Parapenaeopsis longipes, Trachypenaeus curvirostrisand

Metapenaeusmonoceros. Among these Penaeus indicus and Penaeus canaliculatus

share a very close relationship together and Parapenaeopsis stylifera and

Parapenaeopsis longipes form a subclade. Trachypenaeus curvirostris has more

affinity with the members of Peneaus and Parapenaeopsis rather than Metapenaeus

Monoceros. The other cluster consists of one species, namely, Metapenaeus dobsoni.

It is revealed that Metapenaeus dobsoni has an independence lineage in this

phylogenetic tree.

109

Figure 3.1 : ISSR profile of genomic DNA from shrimp’s individuals amplified

using primer UBC 818. M is the gene ruler 1 kb DNA ladder

M

M. dob

son

i

M. m

on

oce

ros

P. lo

ngip

es

P. st

ylif

era

P. ca

nali

cula

tus

P. in

dic

us

T. cu

rvir

ost

ris

865 bp

2000 bp

500 bp

1500 bp

1000 bp

700 bp

M

M. dob

son

i

M. m

on

oce

ros

P. lo

ngip

es

P. st

ylif

era

P. ca

nali

cula

tus

P. in

dic

us

T. cu

rvir

ost

ris

110

Figure 3.2 : ISSR profile of genomic DNA from shrimp’s individuals amplified using

primer UBC 825. M is the gene ruler 1 kb DNA ladder

M

M. dob

son

i

M. m

on

oce

ros

P. lo

ngip

es

P. st

ylif

era

P. ca

nali

cula

tus

P. in

dic

us

T. c

urv

irost

ris

565 bp

1500 bp

500 bp

1000 bp

700 bp

111



M

M. dob

son

i

M. m

on

oce

ros

P. lo

ngip

es

P. st

ylif

era

P. ca

nali

cula

tus

P. in

dic

us

T. c

urv

irost

ris

1500 bp

2000 bp

500 bp

1500 bp

1000 bp

700 bp

112



M

M. dob

son

i

M. m

on

oce

ros

P. lo

ngip

es

P. st

ylif

era

P. ca

nali

cula

tus

P. in

dic

us

T. c

urv

irost

ris

1142 bp

500 bp

2000 bp

1500 bp

1000 bp

700 bp

113

Table – 3.1 : List of primers and nucleotide sequences

Sl.

No. Primer Nucleotide sequences 5 -3

1 UBC 818 CACACACACACACACAG

2 UBC 825 ACACACACACACACACT

3 UBC 827 ACACACACACACACACG

4 UBC 866 CTC CTC CTC CTC CTC CTC

114

Table – 3.2 : Details of the primers, Number of total bands, Molecular size range and

Number of Monomorphic and Polymorphic bands

No Primer Total bands Size range

(bp)

Monomorphic

bands

Polymorphic

bands

1 UBC 818 17 480-1900 1 16

2 UBC 825 13 200-1380 0 13

3 UBC 827 12 250-2000 1 11

4 UBC 866 16 265-1980 1 15

115

Table – 3.3 : Analysis of ISSR bands. Above the diagonal are the numbers of shared

fragments (F) between two species. Below the diagonal are the 1-F values for a pair of species

M. dobs M. mono Pa. long Pa. styl P. cana P. indi T. curv

M. dobs 4 6 7 7 6 5

M. mono 0.258 6 7 5 5 8

Pa. long 0.363 0.375 7 5 6 6

Pa. styl 0.411 0.424 0.388 5 5 5

P. cana 0.466 0.344 0.322 0.312 6 7

P. indi 0.387 0.333 0.375 0.303 0.413 5

T. curv 0.333 0.551 0.387 0.312 0.500 0.322

M. dobs:M. dobsoni, M. mono: M. monoceros, Pa. long: P. longipes, Pa. styl: P. stylifera, P. cana: P.

canaliculatus P. indi: P. indicus, T. curv: T. curvirostris

116

Figure 3.5 : Phylogenetic relationship among species of four Penaeid genera

based on ISSR assay. Neighbor-joining

117

Figure 3.6 : Phylogenetic relationship among species of four Penaeid genera

based on ISSR assay. UPGMA

118

Discussion

Different genes have different power in revealing genetic divergence among

given taxa because of the heterogeneity in the rate of evolution. Therefore selection of

appropriate gene(s) for the phylongeny construction is as critical as the method used

for data analysis in the phylogeny (Hillis and Moritz, 1996). Although the species

diversity is high, based on morphological characters and limited palaeontological

data, the Penaeidae forms a clade in the Decapoda (Burkenroad, 1963, 1983;

Glaessner, 1969). The present study clearly shows that the divergence among some of

the genera is low, while it may be up to 2-3 times higher among others. These results

indicate that although penaeids share considerable external features, the genetic

heterogeneity at the DNA level is high in the family. Although it is hard to test,

Palumbi and Benzie (1991) proposed an assumption that morphological changes in

penaeid shrimp may slow down, while DNA substitution accumulated normally.

There has been evidence in other animals that the molecular divergence and

morphological changes are independent, responding to different evolutionary

pressures and following different routes (Wilson et al., 1974, 1977).

The ISSR-PCR technique is shown to be a useful marker for biosystematics

and phylogenetic studies. The present ISSR profile is highly reproducible, consistent

and informative for all the primers used. The reducibility of ISSR lies in the principle

of ISSR-PCR, that is, the ISSR assay based on the use of primers, which are not

arbitrary, but designed a priori to anchor to anonymous simple sequence repeats

(SSR) loci (Wolfe et al., 1998) and are long and repetitive in nature. The primers

require a stringent annealing temperature, and due to low primer-template mismatch,

the ISSR-PCR yields highly reproducible patterns. The present assay has given an

excellent consistent and reproducible profile.

119

The ISSR assay has yielded the multiple products with differential molecular

weight that ranges from 250-3200 bp. The present study has proven the efficiency of

ISSR-PCR in generating high level of polymorphisim in shrimp. A total 65 distinct

products (bands) amplified by four primers were recorded. Out of 58 fragments,

94.8% (55) were polymorphic and 3 bands were monomorphic (Table 3.2). So, the

resultant of ISSR assay with 58 bands from 4 primers showed both quantitative and

qualitative profile of species under study.

A total of 58 distinct products amplified by 4 primers were recorded and

94.8% polymorphic bands reveal the ubiquitous and hypervariable nature of ISSR

markers and it suggests that the applicability of ISSR-PCR in genome fingerprinting

at the interspecies level (Zietkiewicz et al., 1994).The observations show that, not

surprisingly, the performance of ISSR primers varies across taxa, which reflects

different relative frequencies of microsatellite motifs in different species. The number

of amplified products generated by a primer vary from 24-29. For example, the primer

UBC 866has amplified 29 scorable products, while the minimum are the 24 products

amplified by the primer UBC 825. This suggests that the hypervariable nature and

type of simple sequence repeats in the genomes of different species under study.

Several studies, such as those completed Nkogaoka et al., (2002) and by

Matais et al (2000), have compared ISSR and RAPD markers with varying results

depending on the species studied. In these studies, the two systems generated similar

levels of polymorphism. These results suggest that the two techniques are comparable

and effective in the expression of polymorphism between species, which has also

been observed (Nkongolo et al., 2002). RAPD and ISSR target different non-coding

120

regions of the genome, but these results suggest that it is possible that a fraction of

these RAPD regions could be contained within the ISSR regions of the genome, and

this could be the cause for some of the close results achieved for levels of

polymorphism between the two techniques.

Some studies show similar results when comparing RAPD and ISSR markers.

Matais et al., (2000) obtained Percent Polymorphic Band values of 66% and 60% for

RAPD and ISSR markers, respectively, in Phaseolus vulgaris. But, in Citrus, Fang

and Roose (1997) showed that PCR of RAPD has the most variation, while many

other reports indicate that ISSR marker screening produces more variation than

RAPD primers (Quan et al., 2001). These discrepancies could be caused by the fact

that ISSR and RAPD primers target different regions of the genome.

The complexity of patterns as well as the level of polymorphism detected per

single PCR experiment exceeds those of RAPDs. Thus, the ISSR assay in contrast to

RAPD is more advantageous with a large number of fragments and high level of

polymorphism between taxa. Since, the ISSR primers are designed to specific SSR

(microsatellite) locus, these primers are longer than RAPD primers, and have becom

appropriate markers in genomic fingerprinting (Reddy et al., 2002).

Zietkiewicz et al., (1994) have reported species specific patterns obtained

from a variety of eukaryotic taxa. The results suggest that inter- SSR-PCR can be

used to identify the presence of repeated elements targeted by the different primers

and to evaluate their distribution within different genomes (Zietkiewicz et al., 1994).

121

Neighbor-joiningphylogenetic tree revealed the relative positions of the species of

Penaeidae. The species of penaeusand Parapenaeopsis form a cluster which is

separated from Trachypenaeus and Metapenaeus those showing two separate clusters

where Trachypenaeus curvirostris occupies an intermediate position between two

lineages. Among the four species of the upper cluster penaeus indicus and penaeus

canaliculatus are very close in their evolutionary relationship compared to

Parapenaeopsis longipes andParapenaeopsis stylifera which are divergent to some

extent from these two species. This relationship between these four species is congruent

with studies of (John et al., 1998). The relationship between penaeus indicus and

penaeus canaliculatus is also congruent with studies (Palumbi and Benzie, 1991) and

allozymes and RAPD of the present study. Parapenaeopsis longipes has more affinity

with Penaeus rather than Parapenaeopsis stylifera and this species has low affinity with

Penaeus rather than Parapenaeopsis longipes and this is well supported by studies of

(Li, 2003).Among the species of other cluster Metapenaeus monoceros and

Metapenaeus dobsoni are more closely related and form a subclade.

According to UPGMA pylogenetic tree, there are two clusters. One cluster

consists of the species, namely, the members of Penaeus and Parapenaeopsis,

namely, Parapenaeopsis stylifera, penaeus indicus, penaeus canaliculatus and

Parapenaeopsis longipes and of these Parapenaeopsis stylifera and Parapenaeopsis

longipes share very close relationship and form a subclade as much as , penaeus

indicus andpenaeus canaliculatus. In this cluster Trachypenaeus curvirostris and

Metapenaeus monoceros have independent lineage e that Trachypenaeus curvirostris

has close relationship with the members of Penaeus and Parapenaeopsis than

Metapenaeus monoceros. Metapenaeus dobsoni occupiesa direct descent froms

ancestor and form a cluster with only one species.

122

It is surprising that the two Metapenaeus species were grouped with different

genera, which was apparently different from NJ tree. The members of Metapenaeus,

which was morphologically very similar and formed a strictly monophyletic group in

NJ tree, failed to form a monophyletic clade, so did Trachypenaeus.

Although ISSR have some noises to the phylogenetic reconstruction in the

present study, it was found to be a good candidate for phylogeny at species and genus

level in other decapod crustaceans (Tam et al., 1996; Chu et al., 2002). Nevertheless,

at the amino acid level, this gene has some phylogenetic information and resolves a

similar clade structure as that based on the ISSR, although the position of

Metapenaeus dobsoni clade is different and the tree has much less detailed internal

node structure because of the low level of divergence, as suggested by Machado et al.,

(1996) is under high functional constraints in shrimp. Similar results have been

previously reported in two penaeid genera, Penaeus and Metapenaeus (Palumbi and

Benzie, 1991; Machado, et al., 1996). The present study provides further information

on the evolution of ISSR in the Penaeidae and support a previous assumption

proposed by Boore et al., (1995) and Tautz et al., (1995).

Metapenaeus is found to be a very compact genus in the present study.

Members of this genus are morphologically similar (Dall et al., 1990). Both allozyme

(Tam and Chu, 1993) and RAPD data suggest that Metapenaeus monoceros and

Metapenaeus dobsoniare close together.. However, phylogenetic position of the genus

was not clearly resolved based on current morphological characters. The present study

showed that it was quite distant between two species to which it was designated by

Burkenroad (1983) and Kubo (1949). Although Metapenaeuswas in different group

123

with Trachypenaeus clade in NJ tree failed to resolved Metapenaeus and

Trachypenaeus with confidence. In maximum parsimony analysis, if Trachypenaeus

and Metapenaeus were constrained in order to be grouped with other members of the

first cluster of UPGMA tree considerably more steps are required. This strongly

suggests that Metapenaeus and Trachypenaeus are genetically less similar to the other

members of the the first cluster of UPGMA tree. Therefore, the first cluster of

UPGMA tree is then defined as less homogeneous based on ISSR, although the two

genera Parapenaeopsis and Penaeus are closely related, and apparently a result of

recent radiation. Burkenroad (1983) stated that compared with other two tribes

(peneini and Parapeneini) of his system, Trachypeneini is less compact. Furthermore,

analysis of divergence based ISSR suggesting a possibly closer relationship of

Trachypenaeusto Parapenaeus and Penaeus group than to Metapenaeus group. This

is somewhat different from current taxonomic classification using morphological

characters. Then these data suggest probable convergence for Trachypenaeus and

Metapenaeus in the morphological characters currently applied to taxonomy.

The distribution patterns of genera provide some information to the evolution

of Penaeidae (Dall et al., 1990, Dall, 1991). Many penaeid genera have a wide

distribution; some extending throughout most of the Indo-West Pacific, while others

appears to be restricted to a relatively small area. Taking into account the artifacts

arising from the biased collection, there appears to be substantial difference in

distribution pattern. Both between and within genera. Based on geographic

distribution pattern. It is believed that several genera of Penaeidae, such as

Metapenaeopsis, Parapenaeopsisand Trachypenaeopsis, are recent (Dall et al., 1990).

Probably there are some more recent genera but there are no data to estimate their

124

divergence and separation. Dall et al. (1990) estimated the divergence of the species

within and between two genera. Penaeus and Metapenaeus, based on allozyme data of

Mulley and Latter (1980). The divergence between congeneric species ranged from

0.9-1.8 million years, and the divergence of the two genera was estimated as 4.7

million years. The present data clearly demonstrated that many of the genera in

Penaeidae diverged from each other more recently compared to their divergence from

Penaeus indicating that some genera in Penaeidae are exceptionally closely related

and the results of recent radiation.

Based on limited fossil record (Glaessner, 1969) and functional morphological

comparison (Burkenroad, 1934; Bauer, 1990), the evolutionary polarity of Penaeidae

has been believed to be from Penaeus group to Trachypenaeus group. This

evolutionary direction was supported by the present data. However, the present data

show that Parapenaeopsis, rather than Penaeus, exhibits the highest level of

divergence from the other genera studied. Parapenaeopsis and Penaeus are grouped

together and consistently form the deepest branch of all trees. As fossil records are

incomplete, no fossil evidence is available to test the present observation. Other

molecular markers such as mDNA of Parapenaeopsis and Penaeus should be

examined to confirm the phylogenetic status of Parapenaeopsis.

Documents

APPENDIX : MOLECULAR SYSTEMATICS OF SHRIMPS USING …shodhganga.inflibnet.ac.in/bitstream/10603/11017/12/12... · 2015-12-04 · 98 CHAPTER – 3 APPENDIX : MOLECULAR SYSTEMATICS