new 1. general introduction -...

1. GENERAL INTRODUCTION

“DNA is not merely a double-helix molecule but a path towards infinity,

Where the meticulous mechanisms of genes lie undiscovered”

-I. Karenfil

Most segmented worms found in marine environments represent a major

evolutionary branch of annelids--Class Polychaeta (Russell and Denning, 2000).

Polychaetes are multi-segmented worms living in all environments in the world's

Oceans (Stabili et al., 2013). They are the most abundant and diverse group of

Phylum Annelida (segmented worms, with over 16,500 recognized species),

including more than 13,000 described species in more than 80 families (Fauchald

and Rouse, 1977; Read and Fauchald, 2013). Polychaetes differ from other

annelids in having a well differentiated head with specialized sense organs; paired

appendages (parapodia), on most segments; and no clitellum. As their name

implies, they have many setae, usually arranged in bundles on the parapodia.

Polychaetes (chaetopods) are the dominant macro faunal taxa in all marine

sediments from abyssal depths to shallow estuaries and rocky shores, and even

free swimming in open water (Khan and Murugesan, 2005), but are especially

abundant in the littoral zone. Most of them live a benthic life and are major

components of marine benthos.

1.1. Polychaetes

According to the American Heritage® Dictionary of the English Language,

pol·y·chete also spelled as pol·y·chaete is defined as “any form of the various

annelid worms of the class Polychaeta, including most marine worms such as the

lugworm, characterized by fleshy paired appendages tipped with bristles on each

body segment”. The word Polychaeta is obtained from Latin language; whereas

General Introduction

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the class name is derived from the Greek word polukhaits, with much hair (polu

means poly and khait stands for long hair).

1.1.2. Habit and habitat

Polychaete worms are present in virtually all marine habitats, including coastal

estuarine and rocky shore systems, continental shelf and deep sea benthos, and

some pelagic varieties are found in the water column (Glasby et al., 2000). These

worms cannot often be seen on the surface but sometimes they may create subtle

signs and traces of their presence. Only a few may be found exposed on bare rock

surfaces, but they are common cryptic animals within rock crevices on the

seashore and under boulders. Those that can survive the harsh environmental

conditions on top of rock usually live in colonies, protected inside tubes. Estuaries

are complex environments with varied physical and chemical conditions, causing

the existence of many localized micro-environments or niches (Cognetti and

Malatagliati, 2000). This range of environments promotes rapid speciation (Bilton

et al., 2002) as seen in many estuarine species, including polychaetes, where they

encompass a range of morphologically diverse types, matching the variety of

habitats.

Regarding feeding, the polychaetes are mostly raptorial feeders. They

include members of many families of surface dwelling, pelagic groups and

tubicolous groups. The prey consists of various small invertebrates, including

other polychaetes, which are usually captured by means of an eversible pharynx

(proboscis). A scavenger or omnivorous habit has evolved in many polychaetes.

Apart from this, few members are categorized under non- selective deposit feeders


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and selective feeders. The non- selective feeders consume sand or mud directly

when the mouth is applied against the substratum. In the selective feeders lack a

proboscis. Special head structures extend out over the substratum. Deposit

materials adhere to mucous secretions on the surface of the feeding structure

which is then conveyed to the mouth (Srikrishnadhas et al., 1998).

1.1.3. Distribution

Polychaetes includes large number of species with a wide geographic range

(Knowlton, 1993) – apparently cosmopolitan species, found on the coasts of more

than one continent and in more than one Ocean (Westheide and Schmidt, 2003).

Figure 1.1 shows the worldwide distribution of polychaetes based on BOLD

database, 2013. Polychaetes are also abundant in seagrass beds and mangrove

areas, where large concentrations of organic matter accumulate from shed leaves.

On intertidal reef flats, these soft-bodied worms are an important food source for

wading birds at low tide, and for fish and crustaceans at high tide.

Figure 1.1. Geographical distribution of the polychaetes according to BOLD

database

(http://www.boldsystems.org/index.php/Public_SearchTerms)

http://www.boldsystems.org/index.php/Public_SearchTerms


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The distribution of polychaetes is largely dependent on the type of substrate

present. For example:

the size and type of sediment for burrowers

the presence of suitable reef substrate for the borers and nestlers

hard substrates for the encrusting species to settle on

suitable algal substrate for species that live in seaweed

Additional factors such as exposure and water currents are important for filter-

feeding organisms. Species living in sediments need to have stable sediments, so

high energy beach environments are typically low in the number of species and

individuals.

1.1.4. Types

Polychaetes are often divided into two groups based on their activity: sedentary

polychaetes and errant (free-moving) polychaetes. Sedentary polychaetes spend

much or all of their life span in tubes or permanent burrows (Figure 1.2. a and

2b). Many of them, especially those that live in tubes, have specialized structures

for feeding and respiration. Errant polychaetes (L. errare, to wander), include

free-swimming pelagic forms, active burrowers, crawlers, and tube worms that

only leave their tubes for feeding or breeding (Glasby et al., 2000; Rouse and

Pleijel, 2001).

Figure 1.2. (a) Sedentary polychaetes Figure 2. (b) Errant polychaetes

(Feather duster worms) (Lug worm and Fire worm) (http://www.usca.edu/biogeo/zelmer/sansal/polychaete/sedent)

http://www.usca.edu/biogeo/zelmer/sansal/polychaete/sedent


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Though not seen on the surface, they may create subtle signs and traces

of their presence. They include forms such as sand worms (Nereidae), tubicolous

worms (Ampharteidae), blood worms (Glyceridae), lug worms (Arenicolidae) and

feather cluster worms (Sabellidae).

1.1.5. Reproduction

Polychaetes have relatively great powers of regeneration. Tentacles, palps

and even heads ripped-off by predators are soon replaced. Asexual reproduction is

known in some polychaetes; it takes place by budding or division of the body into

two parts or number of fragments. Some polychaetes live most of the year as

sexually immature animals called atokes, but during the breeding season a portion

of the body becomes sexually mature and swollen with gametes. An example is

the palolo worm, which lives in burrows among coral reefs. During the swarming

period, the sexually mature portions, now called epitokes, break off and swim to

the surface. Just before sunrise, the sea is literally covered with them, and at

sunrise they burst, freeing eggs and sperm for fertilization. Anterior portions of

the worms regenerate new posterior sections. Swarming is of great adaptive value

because the synchronous maturation of all the epitokes ensures the maximum

number of fertilized eggs. However, this reproductive strategy is very hazardous;

many types of predators have a feast on the swarming worms. In the meantime,

the atoke remains safely in its burrow to produce another epitoke at the next cycle.

In some polychaetes, epitokes arise from atokes by asexual budding and become

complete worms.


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1.1.6. Shape, size and colours

Many of them are strikingly beautiful with varied colours, mostly red, pink

and green or sometimes in combination of colours. Some are iridescent, owing to

the presence of crossed layers of collagen fibers in the cuticle. Majority of them

are 5-10 cm long with the diameter ranging from 2 to 10 mm. Deep water forms

are no longer than 1 mm (Neotenotrocha (Dorvilleidae, Eunicida)) whereas one

species attains a length of 3 meters (Eunice, Eunicidae).

1.1.7. Importance

Polychaetes have a high reproductive potential and they can reach very high

densities in some areas. For example, Lerberg et al., (2000) recorded densities of

over 2400 individuals per m2, for Streblospio benedicti. At these densities,

polychaetes can contribute to over half of the total biomass in such areas.

Consequently, polychaetes play a large role in nutrient cycling (e.g. through

digestion), with many species consuming organic particles, through faecal

deposition, and when dead, nutrients are released back into the water column.

Nutrient cycling is also facilitated by the process of burrowing and tube building

in soft sediment this effectively aerates the mud to a depth that in most cases

would normally be anaerobic (Waldbusser and Marinelli, 2006). Aeration of the

surface sediments through burrowing also allows other sediment dwelling species

to subsist in the same area, when they may not otherwise, due to anoxic

conditions.

Polychaetes are often the dominant organism in the soft bottom of brackish

water habitats and, due to its abundance, digestibility and high energy content, it


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plays a fundamental role as a prey species in the estuarine food web (e.g. for

fishes and birds). Many soft-bodied polychaete species are also a source of food

for larger predators (Pallaoro et al., 2006). In some areas, polychaetes have been

found to be some fish species main food source (Laffaille et al., 2005), and have

also been found as part of the diet of cuttlefish (Alves et al., 2006). Polychaetes

affect benthic community structure, as found by Callaway (2006), where in high

and low densities of the species cause species richness.

The polychaetes play an important role in the ecology both as consumers of

plankton and as food for many bottom feeding fin and shellfishes. They provide

key linkages between primary producers and higher trophic levels in the marine

food chains (Parulekar et al., 1980). The high level of adaptability allows these

worms to be easily cultured and is responsible for its characterization as a good

experimental animal (Smith, 1977). Additionally, as an omnivore, it feeds on a

variety of diets, ranging from bacteria to detritus.

The identification of polychaetes is essential for a number of reasons.

Polychaetes have long been an obvious choice as biological indicators, with the

presence or absence of species and with increase/decline of a species population

sensitive to polluting factors indicating environmental health. Polychaetes are

economically viable species in some countries. Nereid and Glycerid (or

bloodworm) species of polychaetes are used as baitworm in U.S.A. which

generates huge revenue annually. Some are grown aquaculturally (Olive, 1999),

but most are harvested from the wild. Identification species will monitor the levels

of wild populations and keep record of what species are currently present in these


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environments may help prevent declines in population biomass, like that which

occurred in Maine in the 1990‟s.

1.2. Can polychaetes be invasive?

Accurate identification of species increases biological security of

estuaries which appear to be particularly vulnerable to invasions (Wolff, 1973,

Bilton et al., 2002). Most of the introductions occur through ballast water

transport in ships or through accidental introduction with new aquaculture species.

Some species become invasive when introduced to nonnative ecosystems, and

adverse effects of such invasive species on the local environment may have

detrimental effects on aquacultural developments. For example, the introduction

and population establishment of the polychaete Marenzelleria wireni in the Dutch

Wadden Sea was most likely contributed to shipping activity, and may have

caused the decline in local bivalve populations and the resident population of the

polychaete, Nereis diversicolor (Essink and Dekker, 2002). Also, species of

Polydora and Boccardia (Read, 2001) burrow and cause blistering in the shells of

cultivated species such as the Pacific oyster (Crassostrea gigas), the green-lipped

mussel (Perna canaliculus), and the cockle (Austrovenus stutchburyii) in New

Zealand as well as in the United States (Bishop and Peterson, 2005).

1.3. Diagnostic features

Metamerism is the division of the body into similar parts which are

arranged in a linear fashion along the antero-posterior axis. This is the most

distinguishing characteristic feature of the phylum Annelida. The segmented part

is always limited to the trunk. In most polychaetes the additional segments are


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added throughout the life. The youngest segment occurs at the posterior end of the

series. A typical polychaete body has a “head,” or prostomium, which may or may

not be retractile and which often bears eyes, tentacles, and sensory palps.

Peristomium, the first segment surrounds the mouth and may bear setae, palps, or,

in predatory forms, chitinous jaws. Ciliary feeders may bear a crown of tentacles

that can be opened like a fan or withdrawn into the tube. Another most

distinguishing feature of polychaetes is the presence of parapodia, the paired

lateral appendages extending from the segments. A typical parapodium is a fleshy

projection extending from the body wall and is more or less laterally compressed

(Mettam, 1967). The parapodium is basically biramous, consisting of an upper

division, the notopodium, and a ventral division, the neuropodium. Each division

is supported internally by one or more chitinous rods, or acicula (Chamber and

Garwood, 1992). Parapodia are used in crawling, swimming, or for anchoring the

animal in its tube (Mettam, 1967). They usually serve as the chief respiratory

organs, although some polychaetes also have gills example: Arenicola the

burrowing lugworm, has paired gills on certain segments. Most of the worms, like

clam worms in the genus Nereis are predatory and equipped with jaws or teeth.

They have an eversible, muscular pharynx armed with teeth that can be thrust out

with surprising speed to capture prey (Fischer and Fischer, 1995). Identification of

some estuarine polychaete species may have been hampered by their

morphological similarity to their fully marine counterparts (Bilton et al., 2002).


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1.3.1. Problems/challenges in morphological identification

Most taxonomic identification methods rely heavily on morphological characters.

Studies on morphological structures to assign taxonomic identifications can be

tedious and can lead to misidentification in cryptic species, and the morphology of

most of the species can be described in detail in the adult stages only. Moreover,

histological anomalies may also affect morphological identification.

In the area of taxonomy, it is well known that there is a need for finding

new and fast methods for identification of species, to aid in the discovery of new

species and for accurate biological diversity assessments. Presently, very few

taxonomic specialists are there and the species are becoming extinct more rapidly

than can be catalogued. The inability to correctly identify species hinders

ecological research, including the areas of comparative ecology and biological

diversity analysis (Hebert et al., 2003).

With many species complexes all over the world, is particular the

Nereididae family of polychaetes are considered to be one of the most cryptic.

Morphological identifications of invertebrates usually focuses on a specific

characters, such as coloration patterns, the structure of wings in wasps (Yu and

Kokko, 1992), legs, head and mouth-part arrangement, and genetalia as in spiders

(Jocque, 2002). Differentiation in these structures can be ambiguous and it can be

hard to distinguish between species. Many cryptic species delineation depends on

very specific complex structural components whose identification requires close

and time-consuming viewing of structure under the microscope.


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In the Neredidae, structures such as parapodial and chaetal organization

(Bakken, 2002; Sato and Nakashima, 2003), and the number and arrangement of

paragnaths on the eversible pharynx (Fiege and Damme, 2002; Breton et al., 2004;

Bakken and Wilson, 2005) have been used for identification. Some of the

structures such as the parapodia of many polychaetes used in identification are

very small and fragile. Identification of polychaetes involves observation of these

structures on a slide under a microscope, and any damage to these structures either

during collection or during observation will hinder identification. The limitation of

morphological identification and a lack of expertise of taxonomy makes

polychaete classification even a greater challenge.

1.4. Molecular identification – Need of the hour!

Due to anthropogenic and environmental stress the marine biodiversity is

at great risk. Each day habitats are changed due to human activity, and each day

organisms are disappearing forever. However, a required step prior to protection is

biodiversity assessment, usually conducted at the species level of biodiversity.

Therefore, species identification has a paramount importance (Radulovici et al.,

2009). Marine habitats are no exception to this. At the same time, the number of

taxonomists who are to study the remaining biodiversity is dwindling (Iseley,

1972; Gaston and May, 1992; Daly, 1995; Buyck, 1999; Lammers, 1999;

McAllister, 2000; Hopkins and Freckleton, 2002).

Scientists took the opportunity provided by the development of

molecular methods to clarify many ambiguities in traditional taxonomy. With

more than 72,500,000 hits on Google search engine (October 2013), the concept


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of molecular identification is becoming a commonplace to access genetic

techniques for the detection of potentially cryptic species complexes within

recognized morphological species (Hateley et al., 1992; Abbiati and Maltagliati,

1996; Rohner et al., 1997; Sato and Masuda, 1997; Manchenko and Radashevsky,

1998; Maltagliati et al., 2000; Scaps et al., 2000; Maltagliati et al., 2001; Sato and

Nakashima, 2003).

The rapid development of methods to sequence and analyze DNA has

revolutionized the study of genetic variation in organisms. Various genes have

been explored, for recognizing delineating species boundaries, quantifying

diversity and clarifying distributions in understudied groups (Westheide and

Schmidt, 2003; Worheide et al., 2005; Witt et al., 2006).

1.5. Molecular markers

Several markers have been used in genetic diversity. They are broadly divided into

three classes based on the method of their detection: hybridization-based; PCR-

based; and sequencing-based (Collard et al., 2005; Gupta et al., 1999).

Hybridization based methods detect differences in affinity to a specific template,

such as restriction fragment length polymorphisms (RFLPs) and oligonucleotide

fingerprinting for single nucleotide polymorphisms (SNPs) detection (Gupta et al.,

1999). PCR-based methods use a single primer or a pair of primers in an

amplification reaction which results in the production of several discrete DNA

products. Examples of PCR based methods are random amplified polymorphic

DNA (RAPDs) simple sequence repeats (SSRs), inter simple sequences repeats

(ISSR), sequence-tagged sites and amplified fragment length polymorphisms


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(AFLPs) (Gupta et al., 1999). DNA sequencing is a straightforward approach for

identifying variations at a given locus, but until recently it was too expensive,

laborious and inaccurate for routine use (Joshi et al., 1999). Recent advances in

sequencing technology allow large-scale use for many fragments and many

individuals (Meyer et al., 1999; Schlotterer and Harr, 2002), and are improved

with respect to cost and accuracy (Marziali and Akeson, 2001; Maturana et al.,

2011). Examples of sequencing-based methods are amplification of mitochondrial

genome, mitochondrial control region (D-loop), cytochrome oxidase subunit B

(cytB) genes, nucleotide sequence of DNA barcoding region (CO1), internal

transcribed spacer regions (ITS), nuclear small ribosomal subunit 18S (SSU), the

nuclear large ribosomal subunit 28S (LSU), the mitochondrial version of the large

ribosomal subunit 16S, mitochondrial small ribosomal subunit 12S, Elongation

factor-1α is nuclear gene involved in part of the cell‟s protein synthesis machinery

EF-1α, the nuclear H3 (Histone subunit 3) and U2 snRNA genes.

1.5.1. Non PCR based methods

1.5.1.1. Restriction Fragment Length Polymorphism (RFLP)

Restriction fragment length polymorphism (RFLP) is a polymorphism in an

individual defined by restriction fragment sizes of distinctive lengths produced by

a specific restriction endonuclease. The rapid rate of evolution, the maternal mode

of inheritance and the relatively small size of mtDNA make the RFLP (Restriction

Fragment Length Polymorphism) analysis of this molecule one of the methods of

choice for many population studies (Ferguson et al., 1995). Several factors such

as, time consumption, laborious process, low sensitivity to detect the


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polymorphism, high cost and most importantly need of sequence information for

probing, restricts the usage of RFLP in genetic variation studies in polychaetes.

1.5.1.2. Single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) are polymorphisms due to

single nucleotide substitutions (transitions/transversions) or single nucleotide

insertions/deletions. These variants can be detected employing microchip arrays

and fluorescence technology. SNPs are the most popular markers because they are

abundant in the genome, highly reproducible, amenable to automation, relatively

easy to score, and relatively cheap per analysis. However their development costs

are high. Major applications of SNPs are genomic studies and diagnostic markers

for diseases.

1.5.2. PCR-based methods

1.5.2.1. Random amplified polymorphic DNA (RAPD)

RAPD is a random amplification of anonymous loci by PCR. This

method is simple, rapid and cheap; it has high polymorphism, only a small amount

of DNA is required. There is no need for molecular hybridization and most

importantly, no prior knowledge of the genetic make-up of the organism in

question (Hadrys et al., 1992). RAPD markers allow creation of genomic markers

from species of which little is known about target sequences to be amplified.

Disadvantages of this include difficulty in reproducing results, subjective

determination of whether a given band is present or not, and difficulty in analysis

due to the large number of products. This is because RAPDs are not sensitive to


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any but large-scale length mutations. Therefore, variation might be underestimated

(Brown and Epifanio, 2003).

1.5.2.2. Microsatellites

Microsatellites or SSRs are tandem repeats of 1-6 nucleotides. They are

highly polymorphic and ubiquitous DNA markers based on length polymorphisms

in DNA repeats that can be easily scored using PCR technology. Due to their high

level of potential polymorphism, locus-specificity, multi-allelic and codominant

nature, relative abundance and reproducibility, SSRs have become valuable

genetic makers for linkage mapping, comparative mapping, QTL mapping,

association mapping, and diversity analysis (Nickerson et al., 1990; Powell et al.,

1996; Jones et al., 1997; Schlotterer, 2004; Varshney et al., 2005). For the first

time Weber and May (1989) introduced the use of SSRs.

1.5.2.3. Amplified fragment length polymorphism (AFLP)

The AFLP technology was first described by Vos et al. (1995). AFLP is

a PCR-based method, which involves restriction enzyme digestion of the genomic

DNA. High reproducibility, rapid generation, a high frequency of polymorphisms

and no requirement of a priori sequence information make AFLP an attractive

technique for developing polymorphic markers and for constructing a linkage map

from a segregating population (Schlotterer, 2004; Mohan et al., 1997; Meudt and

Clarke, 2007). AFLPs are a valuable tool for constructing a linkage map for a non-

model organism (Meudt and Clarke, 2007).


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1.5.3. Sequencing-based methods

Various conserved genes have been explored, some of which are suitable

enough for assessment of high - level relationships (e.g. 18S and 28S rDNA),

while others evolve at higher rates, revealing differences and unique similarities

among closely related taxa and within species. The most popular of the latter

category are the mitochondrial COI, CytB and 16S genes, and the internal

transcriber spacers ITS, of the nuclear genome.

1.5.3.1 Nuclear gene

28S also known as the nuclear large ribosomal subunit (LSU), is

physically linked to the 18S in the tandem repeat and is typically ~2800–3000

nucleotides in length. Several studies (Brown et al., 1999; Colgan et al., 2001;

Rousset et al., 2003; 2004) have focused on partial sequences of this gene

surrounding divergent domains. In some of these cases, when comparing across

life, this gene contains regions both more variable and more conservative than the

18S, and thus, it should be applicable over a broader range of evolutionary history.

18S is the nuclear ribosomal gene and commonly referred to as the

nuclear small ribosomal subunit (SSU). This gene is part of a tandem repeated

element in the nuclear genome. There are hundreds of copies of this repeat in the

genome that are typically homogenized by concerted evolution. 18S data have

been used to address intra-species relationships mainly for historical reasons.

Additionally, conserved regions throughout animals has allowed for the

development of universal primers for amplification via polymerase chain reaction

(PCR), and variation in nucleotide sequence in different gene regions facilitates


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obtaining information at several different phylogenetic levels. 18S has both pros

and cons (Hillis and Dixon, 1991; Abouheif et al., 1998; Halanych, 2004). Issues

with variation of nucleotide substitution rates across lineages are well known and,

to some degree, can be factored into analyses.

Internal transcribed spacers: Ribosomal DNA (rDNA) is widely used

as phylogenetic marker for taxonomic studies and phylogenetic inferences. rDNA

is composed of three subunits (18S, 5.8S, and 28S) and two internal transcribed

spacers (ITS1 and ITS2), each with a different evolution rate (Williams and

Barclay, 1988; Hillis and Dixon, 1991; Eickbush and Eickbush, 2007; Poczai and

Hyvonen, 2010) (Fig.1.3). The length and sequences of ITS regions of rDNA

repeats are believed to be fast evolving and therefore may vary. Universal PCR

primers designed from highly conserved regions flanking the ITS and its

relatively small size (600-700 bp) enable easy amplification of ITS region due to

high copy number (up to-30000 per cell, Dubouzet and Shinoda, 1999) of rDNA

repeats. For phylogenetic purposes, each region can be considered separately, and

the choice of a given region depends on the taxonomic level targeted by the

study. As such, regions that evolve quickly are used for phylogenetic inferences

of closely related species or genera. This makes the ITS region an interesting

subject for evolutionary/ phylogenetic investigations (Baldwin et al., 1995;

Hershkovitz et al., 1996; 1999) as well as biogeographic investigations (Suh et

al., 1993). ITS regions have been used for phylogenetic analyses at the species to

generic level. The sequence data of the ITS region has also been evaluated as


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potential DNA barcodes for Fungi and Plants (Schoch et al., 2012). However,

meager information is available on its applicability to identify invertebrates.

Figure 1.3. Schematic view of ribosomal DNA showing ITS region and the

primers used for amplification (Primers for routine sequencing are shown in

orange colour)

1.5.3.2. Mitochondrial gene

Mitochondrial genome in most animals has been is ~15 000 bp and holds

phylogenetic information that can be examined as gene rearrangement data, amino

acid data, or nucleotide data (Fig. 1.4). This is due to its dynamics in evolutionary

rates of different genes and among different position (Brown,1985; Kondo et al.,,

1993). The nearly compete genomes are the result of difficulties with amplifying

the control region (also called the D-loop or unknown region) of mtDNA genomes

(Boore and Brown, 2000; Jennings and Halanych, 2005). mtDNA genomes show

a remarkable degree of conservation in gene order suggesting that analysis of

concatenated coding and ribosomal genes may be more promising.


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Figure 1.4. Genes encoded by mitochondrial genome

The mitochondrial version of the large ribosomal subunit 16 S is a short

450–500 nucleotide fragment of this gene. This region is typically useful for

intraspecific and intrageneric level relationships (Dahlgren et al., 2001; Halanych

et al., 2001; Jolly et al., 2006; Schulze, 2006) and has limited utility at higher

levels (Struck et al., 2006). However, the utility of a larger region of, or the

complete, 16S gene is unknown. With the increase of known mitochondrial

genomes available for annelids, it should be possible to explore the utility of the

16S and design novel primers that span a longer region.

1.5.4. DNA Barcoding

DNA barcoding is a DNA-based species identification method in which

molecular biology and bioinformatics are combined. PCR and sequencing

techniques coupled with IT technology have provided a new method of

classification, termed DNA taxonomy. Hebert et al. (2003) had suggested a

section of the mitochondrial DNA gene cytochrome-c oxidase subunit I (COI).

Once sequenced, this gene fragment could be used as a „barcode‟ to distinguish


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between species. COI is the best candidate for this taxonomic tool, as it has a high

degree of conservation and insertions and deletions are rare (Moritz and Cicero,

2004). It also has many rapidly evolving nucleotide sites, which will allow for

differentiation between even recently evolved species (Nylander et al., 1999).

Compared to the nuclear genome, the mitochondrial genome lacks introns, has had

restricted exposure to recombination, and has a haploid mode of inheritance

(Saccone et al., 1999). Hebert et al. (2003) demonstrated that the presence of high

level of diversity between species sequences allowed for the successful

assignment of 98% of samples of cryptic lepidopteran species. However, DNA

barcoding and taxonomy is still controversial (Moritz and Cicero, 2004; Will and

Rubinoff, 2004). mtDNA sequences divergences have also been successfully used

to distinguish between species of North American birds (Hebert et al., 2004b),

spiders (Hebert and Barrett, 2005), cryptic species of butterflies (Hebert et al.,

2004a), mosquitoes (Besansky et al., 2003), leeches (Siddall and Budinoff, 2005),

springtails (Stevens and Hogg, 2003; Hogg and Hebert, 2004), beetles (Monaghan

et al., 2005), oligochaetes (Nylander et al., 1999), naidid worms (Bely and Wray

2004), extinct moas (Lambert et al., 2005), and various other species of

vertebrates and invertebrates (Saccone et al., 1999; Hebert et al., 2003).


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1.5.5. Need of Barcoding Polychaetes

To date, local and regional scale DNA barcoding studies on polychaetes

are scarce. As of October 2013, there are only 9168 polychaete DNA barcodes

deposited in

BOLD (The Barcode of Life Data Systems, www.barcodinglife.org)

(Ratnasingham and Hebert, 2007), among which only 1614 are effective. To

satisfy the demand for quick and accurate polychaete classification during

ecological and biodiversity surveys using DNA barcoding and other molecular

identification methods, it is essential to develop a comprehensive library of DNA

sequences (Ekrem et al., 2007).

The ability to use COI to identify species will enable the identification of

cryptic and polymorphic (where a single species may exhibit a range of different

morphologies) taxa of polychaetes, and also identify and associate individuals of

life stages other than adult to their correct species (Schander and Willassen, 2005).

Moreover, several other genes that have been used in deeper level annelid analysis

include the mitochondrial 12S (or mitochondrial small ribosomal subunit) and

CytB (cytochrome oxidase subunit B) genes, and the nuclear H3 (Histone subunit

3) and U2 snRNA genes. The utility of the mitochondrial genes for such issues is

not well known, whereas these nuclear genes are of limited use because they are

too conserved or too short (Brown et al., 1999). Clearly, additional markers need

to be developed and application of 18S and ITS genes needs to be explored for its

effective utilization in polychaete identification.


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These tools can be particularly useful in marine organisms where there

is a poor understanding of species boundaries and broad-scale distributions. This

lack of understanding is driven by the assumption that there are few barriers to

gene flow and thus many ubiquitous species (Palumbi, 1994; Radulovici et al.,

2010), coupled with the reliance on morphological differences for species

recognition (Knowlton, 1993).

new 1. general introduction -...

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