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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 1 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 2 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 3 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 4 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. 5 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). 6 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 7 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. 8 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 9 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 10 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 11 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). 12 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 13 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). 14 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 15