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CHAPTER 1
INTRODUCTION
1.1. GENERAL INTRODUCTION
Invertebrates and more particularly arthropods are a major part of biodiversity
(Wilson, 1987). Although over a million species of arthropods have been described so
far (Stork, 1988), this number probably represents only a fraction of their total global
diversity. Among arthropods, spiders play a particularly important role in ecological
process. Spiders are known for their great species richness and diversity of predatory
behaviors (Coddington and Levi, 1991; Coddington et al., 1996), with an estimated
500,000 species worldwide (Grove and Stork, 2000).
Spiders belong to the Phylum: Arthropoda; Class: Arachnida and Order: Araneae.
They constitute the seventh largest Order in global diversity (Nyffeler and Benz, 1980;
Coddington and Levi, 1991) with approximately 44,032 species described worldwide
belonging to 3,905 genera and 112 families (Platnick, 2013).
Spiders play a significant role in the regulation of insect and other invertebrate
populations and thus balance the ecosystem. Spiders are the most diverse and
abundant invertebrate predators in terrestrial ecosystems (Specht and Dondale, 1960;
Tischler, 1967; Van Hook, 1971; Moulder and Reichle, 1972; Schaefer, 1974; Edwards
et al., 1976; Lyoussoufi et al., 1990; Wise, 1993), playing an important role in
ecosystem functioning throughout habitats (Van Hook, 1971). Their impact as generalist
predators on invertebrate herbivores (Clarke and Grant, 1968; Wise, 2006; Birkhofer et
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al., 2007) is an indicator of their key role in the trophic webs (Wise, 1993; Nyffeler,
2000). This ubiquity, diversity and ecological role of spiders make them a promising
focal group for invertebrate conservation and useful indicators of the effects of land
management on local biodiversity (Clausen, 1986; Churchill, 1997; Topping and Lövei,
1997; Marc et al., 1999; Riecken, 1999).
Spiders have been widely recommended as bio-indicators (Duchesne and McAlpine,
1993; Niemelӓ et al., 1993; Butterfield et al., 1995; Atlegrim et al., 1997; Beaudry et al.,
1997; Churchill, 1997; Bromham et al., 1999; Duchesne et al., 1999; Werner and Raffa,
2000; Heyborne et al., 2003). Spiders have also been used for pest control in agroecosystems (Nyffeler and Benz, 1987; Wyss et al., 1995; Marc and Canard, 1997).
Spiders respond to changing habitat conditions (Uetz, 1991; Ziesche and Roth, 2008)
either directly, or through changes in the physical characteristics of their environment
(Greenstone, 1984; Dennis et al., 2001; Dennis, 2003) and in the populations of their
prey (Marc et al., 1999; Schmitz, 2003). Hence, they are considered appropriate
organisms for the study of succession processes or medications to ecosystems caused
by human or natural disturbances (Maelfait and Hendrickx, 1998; Marc et al., 1999;
Buddle et al., 2000). Spiders have good potential for use as indicators of the availability
of exotoxic elements like lead and cadmium. The venom of some spiders is useful in
neuromuscular and cardiac pharmacology.
1.2. SPIDER ANATOMY
The body of spiders consists of two main parts: an anterior portion- the cephalothorax
or prosoma and a posterior part- the abdomen or opisthosoma (Figure 1.1). These two
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portions are connected by a narrow stalk, the pedicel. The prosoma mainly serves for
locomotion, food uptake, and for nervous integration and the opisthosoma is associated
with the process of digestion, circulation, respiration, excretion, reproduction and silk
production. The prosoma is covered by plates both dorsally (carapace) and ventrally
(sternum). The carapace and sternum helps in attaching the six pairs of appendages:
one pair of chelicerae (first pair of appendage); one pair of leg-like pedipalps (second
pair of appendage) and four pairs of walking legs. Chelicerae are used for capturing and
killing the prey, courtship and mating displays and defense. The mouth parts are located
at the base of the chelicerae and labium.
Figure 1.1: Diagrammatic representation of the general anatomy of spiders
Source: http://www.biodiversityexplorer.org/arachnids/spiders/images/anat1a.gif
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Eyes are usually eight in number but may vary in size, number and arrangement
pattern according to families and are often diagnostic of them. They are usually
arranged in two rows: anterior lateral eyes (ALE) and anterior median eyes (AME) in
anterior eye row and posterior lateral eyes (PLE) and posterior median eyes (PME) in
posterior eye row (Figure 1.2).
Figure 1.2: Diagrammatic representation of the arrangement of eyes in spiders
Source: http://bugguide.net/node/view/323910
The opisthosoma is generally soft and expansible and unsegmented except in the
Mesothelae (Platnick, 1995). On the dorsal side it has numerous patterns, stripes,
humps, etc. On the ventral side lie the reproductive system, respiratory system and the
silk producing glands (spinnerets). Towards the anterior end of the ventral side, is a pair
of book lungs and a single epigastric furrow. Both the male and female's reproductive
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organs are found beneath this furrow. Three pairs of spinnerets are situated usually at
the posterior end of the opisthosoma. Several spiders possess an additional spinning
organ, the cribellum which is a small plate located in front of the three pairs of
spinnerets. The region of the cribellum is often densely covered with many tiny spigots
through which it extrudes thin silk threads. These silk threads are combed out of the
cribellum by rhythmic movements of the calamistrum, a row of comb shaped hairs
situated on the metatarsi of the fourth legs.
In males the pedipalp is the copulatory organ, which is the modified form of the tibia,
metatarsus and tarsus and consists of a dorsal shield-like cymbium and a rounded
genital bulb. The genital bulb in the spiders with complex pedipalps consists of a well
sclerotized tegulum, within which are found an intromittent organ called the embolus,
the seminal duct and the seminal reservoir. A terminal apophysis is associated with the
embolus and a median apophysis is associated with the tegulum. The variously shaped
apophysis in adult males is heavily used for species identification. Female spiders
possess a pair of ovaries in the opisthosoma. The lumen of each ovary leads into an
oviduct, and the two oviducts unite to form a uterus or vagina. The uterus opens to the
outside in the epigastric furrow. In females, however, this furrow is normally sclerotized
forming an epigynal plate with a pair of pores, one on either side of the midline. The
males insert the pedipalps containing the semen into these pores in the epigynal plate
of the female during mating. Many spiders possess a complex structured sclerotized
plate just in front of the epigastric furrow. This plate, called the epigynum, extends over
the genital pore and bears the copulatory openings. The epigynal structures are
profoundly used to classify adult females to species level.
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1.3. DEVELOPMENT AND REPRODUCTION IN SPIDERS
Females lay eggs in an egg sac. Each egg sac contains anywhere from one to
thousands of eggs. Spider eggs normally take only a couple weeks to develop and
hatch. Once the spiderlings hatch, they disperse either by walking or by a process
called ballooning. Spiderlings undergo five or more molts before they reach their adult
size and while most spiders no longer molt after reaching maturity, some spiders
continue to molt for their entire lives.
Male spiders have no external sex organs. Instead they release their sperm into a
sperm web and hold it in their pedipalps until they can deposit it into the female’s
reproductive opening located on the ventral side of her abdomen. Female stores the
sperms in a spermathecae from which she can fertilize her eggs. Egg fertilization occurs
a few weeks later just before the eggs are laid into their egg sac.
1.4. SPIDER ECOLOGY
The interactions between spiders and their environment have been investigated
systematically only within the past few decades. Most spiders live in strictly defined
environments. The limitations are set by physical conditions such as temperature,
humidity, wind and light intensity (Schaefer, 1972; 1974), and also by biological factors
such as the type of vegetation, food supply, competitors and enemies (Tertzel, 1955).
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1.5. PHYLOGENETIC RESEARCH IN SPIDERS
Usually spider classification is based on morphometric characters and is dependent
on the structure of the spinnerets, eye arrangements, chelicerae, tarsal claws and the
labium. However, genital structures are used mainly for the separation of species and
are the only features that provide reliable identification. Subsequently, only adult
specimens may be accurately identified to species level.
Despite the immense size of the Order, spiders have benefited from a relatively long
history of modern phylogenetic research. Ignoring overlaps in characters, these studies
involved 2,329 morphological characters. In contrast molecular data are available for
fewer than 50 taxa, and with a few exceptions were gathered in order to exemplify
Araneae in higher level studies on chelicerates or arthropods, or for intrageneric
studies. Due to the rapid developments in the field of molecular genetics, a variety of
different techniques have emerged to analyze genetic variation during the last few
decades (Whitkus et al., 1994; Karp et al., 1996; 1997a; b; Parker et al., 1998;
Schlötterer, 2004).
1.6. MOLECULAR MARKERS IN PHYLOGENETIC STUDIES
1.6.1. Enzyme markers
Genetic diversity can be evaluated with morphological characteristics, isozymes and
DNA markers (Gepts, 1993). Genetic analysis of data from enzymes with specific
substrates is often straight forward; extrapolations can be made from other eukaryotes
when the genetic bases of the polymorphism cannot be, or has not been formally
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demonstrated. Although isozyme polymorphisms have provided useful markers for
genetic and population studies, the number of marker loci are still limited; protocols are
system specific; considerable experience of individual systems are required for analysis
and interpretation; interpretation is confounded by developmental effects; and
polymorphism levels within populations are often limited. Isozyme analysis can be
influenced by environmental factors (Bailey, 1983) and tends to underestimate genetic
variability (Murphy et al., 1990).
1.6.2. DNA markers
DNA polymorphism is the variation in the sequence of genetic information and hence,
they provide basis of a powerful “marker-assisted selection”. The high frequency of DNA
polymorphism allows locating any desirable gene or a number of desirable genes at one
time. Alterations in non-transcribed regions, such as introns, regulatory sequences,
flanking sequences, pseudo genes and some types of satellite and repetitive sequences
can also be equally detected in nucleic acid analysis as those producing phenotypic
modifications. DNA provides many advantages that make it especially attractive in
studies of diversity and relationships. They include: 1) Freedom from environmental
effects, 2) A potentially unlimited number of independent markers are available (Judd et
al., 2002; Semagn et al., 2006; Kumar et al., 2009), 3) DNA characters can be more
easily scored as discrete states of alleles or DNA base pairs, and 4) Many molecular
markers are selectively neutral.
Various different techniques can be used to identify differences in nucleotide
sequences of DNA including but not limited to RFLP, AFLP and RAPD.
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1.6.2.1. Restriction Fragment Length Polymorphism (RFLP)
Restriction Fragment Length Polymorphism (RFLP) is the most widely used
hybridization-based molecular marker. RFLP markers were first used in 1975 to identify
DNA sequence polymorphisms for genetic mapping of a temperature-sensitive mutation
of adenovirus serotypes (Grodzicker et al., 1975). It was then used for human genome
mapping (Botstein et al., 1980), and later adopted for plant genomes (Helentjaris et al.,
1986; Weber and Helentjaris, 1989). The technique is based on restriction enzymes that
reveal a pattern difference between DNA fragment sizes in individual organisms.
Although two individuals of the same species have almost identical genomes, the
differences in DNA sequence may be due to single base-pair substitutions, additions,
deletions or gross chromosomal changes such as inversions or translocations. Hence,
RFLP result from specific differences in DNA sequences that alter the fragment sizes
obtained by digestion with a type II restriction enzyme.
RFLP mapping is often used to place cloned cDNAs encoding proteins of known and
unknown functions on genetic linkage maps. However, it is relatively slow, detection
system is dependent on genome size and polymorphisms, requires use of radioactivity
and large amount of material is required for DNA extraction. Hence, RFLP has been
increasingly substituted by other marker techniques, based on the polymerase chain
reaction (PCR) such as AFLP and RAPD (Lin et al., 1996).
1.6.2.2. Amplified Fragment Length Polymorphism (AFLP)
Amplified Fragment Length Polymorphism (AFLP) technique combines the power of
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RFLP with the flexibility of PCR-based technology by ligating primer-recognition
sequences to the restricted DNA (Lynch and Walsh, 1998). As described by Vos et al.,
(1995) the technique involves three steps: 1) DNA restriction and ligation of
oligonucleotide adapters, 2) selective amplification of sets of restriction fragments, and
3) gel analysis of the amplified fragments.
AFLP is robust and reliable because stringent reaction conditions are used for primer
annealing with the reliability of RFLP (Botstein et al., 1980; Tanksley et al., 1989)
combined with the power of the PCR (Mullis and Faloona, 1987; Saiki et al., 1988;
Erlich et al., 1991). Their high genomic abundance and generally random distribution
throughout the genome make AFLP a widely valued technology for gene mapping
studies (Vos et al., 1995). Nevertheless, it requires good quality DNA, requires
denaturing or sequencing polyacrylamide gel and radiolabelling.
1.6.2.3. Random Amplified Polymorphic DNA (RAPD)
Williams et al., (1990) described Random Amplified Polymorphic DNA (RAPD) as an
efficient and useful technique that allows the detection of sequence variation at multiple
sites in the DNA. A selected oligonucleotide primer is hybridized prior to priming of DNA
replication using PCR amplification. RAPD-PCR amplification is based on the use of 10base pair primers with arbitrary sequences comprising 50–70 % of Guanine + Cytosine
contents and lacking self-complementary ends (Welsh and McClelland, 1990; Williams
et al., 1990). The use of a low annealing temperature in the cycle increases the
probability of such short primers binding to sites in the genome by allowing mismatching
between primers and priming sites in addition to specific priming. A single primer is
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used which must anneal in two opposite places on the DNA template and region
between the primers will be amplified, that provides the priming sites within the
amplifiable distances (Tingey et al., 1993). The amplification products are then
visualized with ethidium bromide after gel electrophoretic separation without the use of
radioactive isotopes. The presence or absence of PCR fragments is assumed to
represent mutations in the primer-binding sites of genomic DNA (Wolfe and Liston,
1998). Polymorphism is scored by the presence or the absence of amplification
products and the similarity among samples is then computed from the banding patterns
to produce a similarity matrix for constructing a clustering pattern.
RAPD markers offer significant advantages. Using a set of universal primers, they
can be generated without preliminary investigation apart from the isolation of a small
amount of DNA. Furthermore, some RAPD markers can be used to generate other
types of markers such as Sequence Characterized Amplified Regions (SCARs).
RAPD have been used in a wide range of applications such as gene mapping
(Williams et al., 1993), population genetics (Aitken et al., 1994) and molecular
systematics (Fujimori and Okuda, 1994) in a range of eukaryotes, including humans
(Potsch et al., 1992), plants (Virk et al., 1995) and fungi (Gosselin et al., 1996). RAPD
analyses are efficient, economical and tend to produce genetic markers suited to the
assessment of population, race and species-specific genetic variation. The primers pick
up polymorphisms which appear to be randomly distributed throughout the genome
(Williams et al., 1990). Such polymorphisms can function as genetic markers and can
be used to construct genetic maps (Williams et al., 1991).
However, RAPD markers are dominant markers and heterozygous loci are not easily
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distinguished from homozygotes; this has to be accounted for in the design of mapping
experiments (Rafalski et al., 1991). Another problem concerns homology of co-migrating
bands; only the primer sequence at each fragment end is known in RAPD analysis.
There is no sequence information for the rest of the fragments and therefore homology
is assumed for bands that migrate equal distances. Genetically speaking, this means
that co-migrating fragments may not be allelic (Newbury and Ford-Llyod, 1993).
However, taxonomic studies indicate that RAPD data are consistent with other forms of
molecular data (Demeke et al., 1992; van Huesdan and Bachmann, 1992) which
suggest that phylogenetic trees are consistent whether generated from RAPD, VNTR or
Isozyme data. This consistency among various molecular data, including RAPD
suggests that co-migrating RAPD electromorphs must be homologous.
1.7. CONSTRUCTING A PHYLOGENETIC TREE WITH MOLECULAR MARKERS
1.7.1 Phylogenetic trees
A phylogenetic tree is a graph composed of branches and nodes used to represent
the historical or evolutionary relationships among groups of organisms, often at the
species level. It consists of nodes, which represent taxonomic units (species,
populations and individuals; both extant and their presumed ancestors) and branches
that define the relationship between taxonomic units in terms of descent and ancestry.
The nodes at the tips of the tree are constructed from observable features such as
protein sequences or morphological features and are called Operational Taxonomic
Units (OTUs) (Page and Holmes, 1998; Prevost and Wilkinson, 1999; Brocchieri, 2001).
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A node can be external, internal, or at the root of a tree. Internal nodes represent a
common ancestor of two other OTUs that can represent many types of comparative
taxa. Any branch that divides splits into two daughter branches. The splitting of lineages
usually assumes that it is a binary process that results in the information of two species
from a single ancestral species. This occurrence may not always happen, or a lack of
suitable data may make it impossible to resolve the order in which species descended
from a single common ancestor. The branching pattern of a tree, defined by the
relationships among the taxa in terms of ancestry, is called topology (Morrison, 1996;
Prevost and Wilkinson, 1999; Brinkman and Leipe, 2001).
Phylogenetic trees may be unscaled or scaled. In an unscaled tree or cladogram only
the branching order of nodes is shown and the branch lengths are not proportional to
the information represented at an external node (Morrison, 1996). This may be
especially useful if the tree has numerous of OTUs. Scaled trees called phylograms
display both branching order and distance information. Distance is the number of amino
acid or nucleic acid substitutions that have taken place along a branch. A phylogram
has the helpful feature of conveying a clear visual idea of the relatedness of different
characters within the tree (Morrison, 1996; Page and Holmes, 1998). A tree is said to be
additive if the distance between any two OTUs is equal to the sum of the lengths of all
the branches connecting them (Hall, 1942; Morrison, 1996).
1.7.2. Methods of constructing phylogenetic trees
The principal methods of making trees can be classified into two types: distancebased and character-based methods. Distance-based methods begin the construction
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of a tree by calculating the distances for all pairs of taxa and build a tree by considering
the relationship among these distance values. The main distance-based methods are
the Unweighted Pair Group Method with Arithmetic mean (UPGMA) and Neighbor
Joining (NJ). In contrast, character-based methods analyze candidate trees based on
relationships inferred directly from sequence alignments. These approaches are distinct
from distance-based methods, since they do not involve an intermediary summary of
the sequence data in the form of a distance matrix or resemblance matrix. There are
two main character-based methods: Maximum Parsimony (MP) and Maximum
Likelihood (ML) (Nei and Kumar, 2000; Brocchieri, 2001).
1.7.3. Making trees using UPGMA distance-based method
The UPGMA is a simple tree-making algorithm that works by clustering the sequence
data, DNA profile, morphology data and other evidence based on a distance matrix. The
program first searches the pair of OTUs with the smallest distance between them. The
branching pattern between them is defined as half of that distance resulting in placing a
node at the midpoint. Then the two OTUs are grouped together into a cluster and the
matrix is re-written with distances from the cluster to each of the remaining OTUs. As a
result, the cluster serves as a substitute for two OTUs and the entire number in the
matrix is now reduced by one. This process is repeated on the new matrix and is
continued until the new matrix consists of a single entry OTU. Then, that set of matrices
is used to construct the tree by starting at the root and moving out to the first two nodes
represented by the last two clusters (Hall 1942; Sneath and Sokal, 1973; Morrison,
1996; Page and Holmes, 1998; Nei and Kumar, 2000).
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1.8. AIMS OF THE STUDY
The application of molecular methods to spiders which have been classified on the
basis of morphology has the potential to accelerate the accumulation of genetic
information as compared to traditional methods. The goal of this research was
therefore, to take the current physical identifications and compare the types of spiders
at the genomic level to detect an evolutionary relationship. Accordingly, the study was
carried out on the following lines:
1. To identify and characterize the spiders collected for the present study
2. To optimize the DNA extraction protocol from appropriate tissues
3. To design appropriate primers suitable for RAPD amplification of the spiders
under study
4. To characterize the genomic DNA of the collected spiders through RAPD-PCR
5. To analyze the genetic variations present among the spiders for the assessment
of phylogenetic relationships
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