CHAPTER I INTRODUCTION
Background
The elasmobranch fishes sharks and stingrays are the cartilaginous fishes and become one of the fishery resources in Indonesia. Almost all the elasmobranch fish is
important economically fish where the refine product and live specimen are heavily traded. Indonesia is also listed as one of the countries that most larger utilize the
elasmobranch resources in the world, with conjucture the catch was 105,000 tons in 2002 and 118,000 tons in 2003. After that years, the elasmobranch fish catch has decreased up
to 98 300 tons in 2006 Tull 2009. High market demand for elasmobranch fish product triggers the fisheries industry to maximally reach the production target without consider
the stocks sustainability in nature. One of the peculiarities elasmobranch fish is they have a low growth pattern and low reproduction. An Intensive catch activity without control has
caused the depletion of elasmobranch fish resource in Indonesia. Although Indonesia listed as a country with the highest production of elasmobranch fish, however not many
studies or publications conducted in Indonesia White et al. 2006.
Stingray is one of the cartilaginous fishes beside sharks and skates. In Indonesia, the skin and the meat of the stingrays are the favorite part. Himantura uarnak, H. undulata
and H. leoparda are stingrays belonged to genus Himantura under family Dasyatidae which is the most popular stingrays in Indonesian. These stingray are the major targets that
utilised as consumable product and other trading products as handbags, wallets, belts and other accessories Adrim 2008. Stingray fisheries conditions in Indonesian is not differ
from sharks that undergone over exploitation. According to the Fisheries Statistics 2010, from 6 recorded stingrays species, only flower ray and leopard whipray show an average
increasing of 1.46 in 2000-2010. Generally, stingray production decreased in 2005 to 2008, and slightly increased in 2009 and decreased again in 2010 by 16.49. In general,
only one stingrays species that has increased production is manta ray despite fluctuations in production still continued from 2005 to 2010. Whereas, production of another 3 stingray
decreased, even there is no record of production since 2000 to 2004. Regulation and supervision are still very low toward exploitation and trading of stingray products in
Indonesia. Very limited data collection, referring to the collections of the Museum of Zoology Bogoriense in Cibinong, and until now the new collection reaches 56 identified
species in Indonesian waters White et al., 2006, however this number only covers 11.2 from total stingray that exist in the world that predicted around 500 species Tricas
Taylor 1997. These data are needed for reference in conservation and monitoring planning efforts Anak 1997. Species from genus Himantura such as H. uarnak, H.
undulata and H. leoparda are species of stingray that still widely studied, because of frequent errors in identification. Conventional identification of the stingray is done
according to morphological characteristics including both reticulation patterns spotting pattern and disk form. Manjaji-Matsumoto Last 2008 have described new species H.
leoparda formerly misidentified as “H. undulata“. However, the description of H. leoparda relies on morphology exclusively, while molecular markers are new available to
assess species boundaries. Therefore a comprehensive research using molecular approaches is expected to answer such doubts.
One of molecular approaches that has been used to identify species and the origin of populations is DNA barcoding using genetic marker gene cytochrome c oxidase subunit
I COI Ward et al. 2005; Ward et al. 2008. Another marker in mitochondrial genome such as cytochrome b cyt b has also been shown to be able to answer the problems
associated with the geographical distribution patterns and the origin of the gene marker, known as phylogeography Knowlton et al. 1993; Knowlton et al. 2000; Emerson et al.
2011. Phylogeography can explain how populations spread and adapt to the local environment from the initial population and evolved until now. Genetic differences
between populations may be used in determining stock structure Randi Lucchini 1998.
Beside marker from the mitochondrial genome, using marker from the nuclear genome as intron 3 of calmodulin gene CAM, intron 1 from S7 ribosomal protein RP,
intron 6 of creatine kinase gene 6B CK6B, and intron 2 from actin gene Act2, is expected to clarify the species status and internal diversity of each species. That molecular
markers beside able to identify populations can also describe the genetic characteristics of the population Berrebi et al. 2006. Introns are noncoding sequences that criss-cross in
the genes and more varied due to selective pressures in intron regions that much lesser than in the exon region. Intron has the most varied of polymorphism length, so that widely
used as molecular markers because it has many desirable characteristics. For example, introns have specificity, codominant and neutral compared to the complementary DNA
cDNA Yang et al. 2007. To get the polymorphism properties and strengthens intron, PCR primers designed for the flanking exons, which are known as exon-primed intron-
crossing EPIC approach Palumbi Baker 1994; Bierne et al. 2000; Creer, 2007. This approach has several advantages: i by using primers from heterologous genes, cloning
and sequencing from target sequences can be avoided, ii amplification crossspecies will be easier than when primers designed in exon noncoding sequences because the sequence
is more conserved across species, iii for the same reason in the species, PCR artifacts such as null alleles are expected to be further reduced Palumbi Baker 1994; Palumbi
1995; Bierne et al. 2000.
Species complex is a group of closely related species, where boundaries among species are unclear and usually incomplete reproductive isolation. Example for species
complex are ring species, superspecies and cryptic species. This condition can be caused by the rapid speciation so that the separation mechanism was not fully developed. These
events can cause some paraphyletic species in species level and hybrids, that causing difficulties in phylogenetic analysis Mayr 1992, Irwin et al. 2001; Queiroz 2007.
Neotrygon kuhlii lineage in the Indo-West Pacific IWP is separated and closely related to the separation of the geographical area. Observations have been made against N. kuhlii and
found six different lineages. Geographical composition and mitochondrial lineages found in this species suggest that N. kuhlii may consist of several cryptic species Arlyza
Borsa 2010.
Research Problems
1. Three of Himantura uarnak species complex are stingray with high economic value and export commodity. Refine products and live specimens from the stingray are very
attractive to national and international markets, thus causing over exploitation that occurs in almost all of Indonesian waters in order to reach a maximum export
production. Therefore, the latest information on the population is needed to avoid scarcity and extinction Anak 1997.
2. Misidentification by the morphology of H. uarnak species complex H. uarnak, H. undulata and H. leoparda causes confusion. The ambiguity is due to the variation of
spotting pattern on the dorsal of three stingrays that tend to be similar, even often found three combinations of spotting pattern present in one individual. This variation
is thought to be the possibility cause of the species complex, and closely related to cryptic species at three species of Himantura.
3. Specifications geographical ranges. Not all members of H. uarnak species complex spread evenly in every area, but there are certain species- specific distribution.
Preliminary study results Table 1.1 showed the presence of the specific distribution. Table 1.1 Specific distribution of Himantura uarnak species complex in Indonesian
waters based on field indentification and genetic characterization
Species Locations
Total Indiv.
Banten Batang Yogyakarta
Bali Flores Selayar Makassar Kendari
H. uarnak -
- -
- 2
7 11
3 23
H. undulata -
1 -
- -
- -
- 1
H. leoparda 3
84 1
1 -
- -
- 89
Total species 1
2 1
1 1
1 1
1 113
As defined by Manjaji-Matsumoto Last 2008, but we show that this consists of two cyriptic species in this thesis
Research Objectives
The objectives of this study is to identify each species of Himantura uarnak species complex using molecular approaches. Specific objectives of this study are:
1. to identify each species of H. uarnak species complex through molecular approaches, 2. to estimate the evolutionary process of species divergence; gene flow and alelle specific
in H. uarnak species complex, and 3. to determine the relationship among species.
Hypothesis
1. There are genetic differences between Himantura undulata and H. leoparda in Indonesia.
2. There are alleles specific in H. uarnak species complex based on geographical distribution area.
3. There are gene flow in H. uarnak species complex based on molecular approach.
Benefits of Research
1. Molecular identification results can be help management of Himantura uarnak species complex more targeted.
2. The results are expected to provide a reference in determining the species of H. uarnak species complex that can be traded. Based on the latest status on the IUCN red list, this
species included in the vulnerable category, which means the species is vulnerable and threathened if the species sustainabikity not addressed.
Novelty
So far, research on H. uarnak species complex H. uarnak, H. undulata and H. leoparda has done through morphological characthers approach by researchers from
Australia and Malaysia. This is a first study that using molecular approaches to identify H. uarnak species complex in Indonesia. This approach produced 4 main groups with one of
the group was identified as different species than that have been released.
Framework
Himantura uarnak species complex was predicted as group of species due to variations in the spotting pattern on dorsal part of stingrays which tend to be similar.
Morphological identification has been done but still some question raise, therefore molecular approaches applied. This approach was done by using nuclear nDNA to
determine cryptic species in the H. uarnak species complex. Then, it was validated by
using the mitochondrial DNA mtDNA with genetic markers of COI for species identification. The results of species identification then verified using genetic markers cyt
b to obtain the identity of species in this species complex. The results of molecular approach were compared to the species identity by morphological approach through
spotting patterns. The results can be used to manage H. uarnak species complex in Indonesian. Figure 1.1.
Figure 1.1 Flowchart of research framework Himantura uarnak species complex
Molecular approach
Mitochondria genome Nuclear genome
Calmodulin
COI Cyt b
Actin
Species identification by molecular
Conservation and sustainable fisheries of Himantura uarnak
Morphological character spotting pattern
H. uarnak H. undulata
New species Cryptic species
Species identity
H. leoparda
CHAPTER II USE OF EPIC INTRONS AS NUCLEAR DNA MARKERS FOR THE
Kategori