Molecular Phylogenetic Relationships of Exemplars of Four Spider Families from Ha ’ il Region , Northern Saudi Arabia and a Preliminary List of Spiders of Ha ’ il

Knowledge about spider fauna in Saudi Arabia is hugely inadequate compared to adjacent countries. The present work is a preliminary study of Hai’l’s spiders. Fourteen genera, in addition to five unidentified spider species, belonging to 16 families were recorded in Ha’il Region. This is the first study of spider’s fauna in Northern Saudi Arabia and may provide a basis for future studies. In addition molecular markers including Histone (H3) and 12S rDNA sequences were used to examine the phylogenetic relationships of exemplars of the most common four spider families. The constructed tree based on both maximum parsimony and maximum likelihood of the two studied sequences revealed that the studied representatives of family Agelenidae and family Sparassidae were grouped in one supported clade. However, the relationship of this clade with the other two studied families shows controversy in both H3 and 12S trees. Results for each of the studied genes, support their reliability for phylogenetic analysis of spiders. Ha’il, Spiders list, Molecular Phylogenetics, 12S rDNA, H3.

Hai'l Region is located in the center of the northern part of Saudi Arabia.Explorations for oil in the eastern and southern Arabia attracted people of professional interest to the oil fields and they contributed in gathering some biota.Ha'il area which does not have oil fields gained little attention.The Region comprises diverse ecosystems that provide interesting aspects for species diversity investigations.A comprehensive contribution to the Flora of Ha'il Region was found in literatures (Alshammari and Sharawy, 2010;El-Ghanim et al., 2010).On the other hand, the investigation of Ha'il's fauna is still infrequent and most studies focused on insects (Hölzel, 1982;Cranston and Judd, 1989;Albarrak, 2009) and reptiles (Dekinesh, 1991).Although arachnids represent a major component of desert's fauna, they have only recently attained little consideration in the region.In this respect, Sharawy and Alshammari (2009) recorded two highly poisonous scorpion species.Moreover both Al-Asmary et al. (2009) and Desouky and Alshammari (2011) reported eight scorpion species in the Region.However, to the best of our knowledge no studies have dealt with spider's fauna in Northern Saudi Arabia.
The present work is a preliminary study of Hai'l's spiders.The study's aim is to evaluate the diversity of spider species in this highly neglected region.In addition the phylogenetic relationships of exemplars of four recorded spider families are to be investigated using molecular markers including Histone (H3) and 12S rDNA sequences that represent nuclear and mitochondrial genes, respectively.

MATERIAL AND METHODS (a) Collection of spiders
Spiders of 16 families were collected from different habitats in the Ha'il Region during the periods of April-August 2010 and July-August 2011.Most of them were found under stones and sometimes on plants.All specimens were collected alive by hand.They were preserved in absolute ethanol and deposited in the Central Laboratory, Faculty of Science, Ha'il University.Adult specimens were identified to species.Juvenile and subadult specimens were, when possible, identified to genus level.Spider taxa were identified by the second author.

(b) DNA extraction
Four species representing the most common four families in the region were chosen for molecular phylogenetic studies.For this purpose, the whole organism was homogenized in 1 ml of DNA extraction buffer (50 mM Tris-HCl, pH 8.5, 10 mM EDTA, 100 mM NaCl and 2% SDS).The homogenate was centrifuged at 8,000 rpm for 5 minutes.The resulting supernatant was mixed with equal volume of phenol/chloroform to remove proteins.The mixture was centrifuged at 10,000 rpm for 10 minutes.DNA was precipitated from the aqueous layer with two volumes of absolute ethanol at -20  C for overnight.DNA was collected via centrifugation at 12,000 rpm for 10 minutes.After that, DNA was washed with 70% ethanol and recollected with centrifugation at 12,000 rpm for 10 minutes.The supernatant was decanted and the DNA pellet was air dried and then suspended in 50 µl of Tris-EDTA (TE) buffer (10 mM Tris-HCl, pH 8, 1mM EDTA) and kept at -20  C till use.The extracted DNA from all spiders was analyzed by running an aliquot of each DNA sample on 1% agarose gel electrophoresis containing 0.5 µg of ethidium bromide per ml of agarose.The applied voltage was 5-8 V/cm.After termination of electrophoresis process, DNA was visualized under UV to determine its quality.

(c) Amplification of a partial fragment of H3 gene
A partial fragment of the gene encoding for H3 was amplified via PCR using the primer set (H3aF and H3R, Table 1).The reaction mixture contained 5 µl of DNA, 25 µl of Dream Taq Master Mix (Fermentas Co.), 1 µl of each primer (10 pmol) and 19µl of nuclease free water.The PCR conditions of the thermocycler (pEQ lab.) were 95  C for 5 minutes for initial denaturation, 35 cycles, each cycle (94  C, 1 minute for denaturation, 50  C, 1 minute for annealing and 72  C, 1 minute for extension) and 72  C , 10 minutes for final extension.After termination of PCR run, 5 µl of the PCR product was checked on 2% agarose gel using electrophoresis.The expected PCR product was almost 350 bp in length.

(d) Amplification of a partial fragment of 12S rDNA
A partial fragment of 12S rDNA was amplified via PCR using the primer set (12S-ai and 12S-bi, Table 1).The reaction mixture contained 5 µl of DNA, 25 µl of Dream Taq Master Mix (Fermentas Co.), 1 µl of each primer (10 pmol), 1 µl of 25 mM MgCl 2 and 18 µl of nuclease free water.The PCR conditions of the thermocycler (pEQ lab) were 95  C for 5 minutes for initial denaturation, 35 cycles, each cycle (94  C, 1 minute for denaturation, 45  C, 1 minute for annealing and 72  C, 30 seconds for extension) and 72  C, 10 minutes for final extension.After termination of PCR run, 5 µl of the PCR product was checked on 2% agarose gel electrophoresis.The expected PCR product was almost 370 bp.

(e) Purification and Sequencing of PCR products
PCR products were purified to remove all components of PCR that would have an inhibitory effect on the sequencing reaction.Each PCR product was mixed with two volumes of absolute ethanol and kept at -20  C for an overnight.The amplified DNA was collected via centrifugation at 10,000 rpm for 10 minutes.After that, the PCR product was washed with 70% ethanol and recollected via centrifugation.It was then allowed to air dry and was suspended in 30 µl of nuclease free water.Approximately 30 g of the purified products were then used as templates for direct sequencing of both forward and reverse strands, using the same amplification primers in an automatic ABI 310 DNA Sequencer with Big Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer.All sequences generated in this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank.

(f) Determination of phylogenetic relationships
The programs PHYLIP package and MEGA version 5.0 were used for the determination of phylogenetic relationships.A Neighbor-Joining (NJ) tree based on uncorrected p-distances was constructed with MEGA version 5.0.Maximum Likelihood (ML) was performed in order to check consistency in the results using different algorithms based on different assumptions of molecular evolution.The ML analysis was performed using online PHYLIP package (www.atgcmontpellier.fr/phyml/)with model parameters fitted to the data by likelihood maximization.Maximum Parsimony (MP) analysis was performed in MEGA version.5.0.Branch swapping with 500 random stepwise additions of taxa were used.

RESULTS (a) Study area
The Ha'il Region of Saudi Arabia, located between 2535` & 2900` N longitudes and 3901` & 4445` E latitudes and covers an area of approximately

Gene
Primer name Sequence Reference  Olayan, 2003).Air temperature typically rises as high as 50  C in summer and falls to freezing point in winter, especially at higher altitudes.Rainfall is irregular reaching a few tens of centimeters and occurs mostly between November and March.
The region is recently subjected to climatic and topographic changes.The draught increases heavy sand storms, which are most frequent during spring and winter seasons.Therefore, the sand dunes from the Northern Nafud Sand Sea occasionally encroach upon cultivated lands and people settlements.Moreover, agriculture is significantly growing in this arid region.Agricultural development projects and reclamation of new desert land increasedd the area of cultivated land from 9,500 ha in 1972 to 139,300 ha in 2010 (Hereher et al., 2012).

Remarks
Family Agelenidae are called Funnel-web Spiders because they make funnelshaped webs, which they use to trap insects.They are also known as Grass Spiders because they construct their webs in tall grass, heavy ground cover and the branches of thick shrubs.Members of this family can be easily recognized by the elongated apical segment of the posterior lateral spinnerets and the paired colulus.Only three Agelenid species were reported in Yemen of which Benoitia lepida was new record (Santos and van Harden, 2007).As a result, the fauna of the large continental areas as the Arabian Peninsula are still in great need for revision.

Remarks
The jumping spider (Family Salticidae) contains 575 described genera and 5,423 described species, (Platnick, 2012) making it the largest family of spiders with about 13% of all species.Jumping spiders have some of the best vision among invertebrates and use it in courtship, hunting, and navigation.Though they normally move quietly and fairly slowly, most species are capable of very agile jumps, notably when hunting, but sometimes in response to sudden threats.Jumping spiders are easily distinguished from other spiders by their four big eyes on the face and four smaller eyes on top of the head.
Family Salticidae are notably studied in Saudi Arabia and adjacent countries of the Arabian Peninsula.In Yemen, Family Salticidae was extensively studied by Wesolowaska and van Harten (1994;2002;2007).Such studies increased the number of Salticid species in Yemen to 81.However, only 30 Salticid species containing one new genus and ten new species were described in Saudi Arabia (Prószyński, 1989;1993).

Remarks
The mature Black Widow Spider of the genus Latrodectus has a characteristic glossy black colour and sometimes an hourglass configuration on its ventral surface.Despite its medical importance, the taxonomic situation is still unsatisfactory (Knoflach and van Harten 2002).Currently, 31 species of Latrodectus are known worldwide (Platnick, 2012), two of them (L.cinctus Blackwall, 1865 and L. reinvulvatus Dahl, 1902) were reported in the southern part of Saudi Arabia by Knoflach and van Harten (2002) for the first time.
Latrodectus tredecimguttatus, commonly known as the Mediterranean Black Widow or Steppe Spider, is commonly found throughout the Mediterranean region.In Arabian Peninsula, this species was early recorded in Socotra (Pocock, 1903).According to Platnick (2012) the distribution of the species extends from the Mediterranean across Central Asia to China.

Molecular studies
A representative from the common four families was used for molecular studies.This include Stegodyphus lineatus (Family: Eresidae), Artema atlanta (Family: Pholcidae), Benoitia lepida (Family: Agelenidae) and Eusparassus walckenaeri (Family: Sparassidae).Comparison of some major morphological characteristics of these families is shown in Table (2).347bp sequences were obtained from H3 gene and 308bp from 12S rDNA of the studied spider species.All sequenced gene segments were deposited in NCBI GenBank.To the best of our knowledge, 12S rDNA and H3 sequences were found to be new records for three from the studied four species (Table 3).Data about these sequences and their GenBank Accession Numbers (submitted) are shown in Table (3).For the H3 gene fragment, the tree revealed two maximally supported clade in ML analysis (Fig. 15), one containing the families Pholcidae, Sparassidae and Agelenidae which represented by species, A. atlanta, B. lepida.and E. walckenaeri.96 % bootstrap value in ML but only 59% in MP analysis was recorded for this clade.The clade of Benoitia lepida and Eusparassus walckenaeri had a very strong support in both ML and MP analyses (100%).The 12S rDNA phylogenetic tree revealed only one maximally supported clade (Fig. 16) which was between the family Sparassidae, represented by E. walckenaeri and Agelenidae represented by B. lepida with 91% bootstrap value in ML but with low bootstrap support in MP analysis.In the combined mitochondrial and nuclear gene fragments (682 bp), the tree revealed two maximally supported clade in ML analysis.(Fig. 17  In order to clarify where the studied spiders are placed within their representative families, the phylogenetic tree was constructed for each family separately using gene sequences already published whenever these data are available.The only available sequences on data base are 12S rDNA and H3 sequences for considerable numbers of representatives of family Pholcidae and family Eresinidae respectively. ML Phylogenetic tree of pholcid species based on partial 12S rDNA sequences retrieved from data base is shown in Fig. 18.Maximum likelihood under the GTR+G+I model of evolution revealed that A. Atlanta from Saudi Arabia forms a maximally supported sister clade with the same species from with 97% bootstrap value.The most relative species to A. atlanta are Trichcyclus sp. and Ninetis subtillissima.On the other hand, ML Phylogenetic tree of Arsenid species based on partial H3 sequences retrieved from data base (Fig. , 19) revealed that S. lineatus from the present study forms maximally supported sister clade with the same species from Netherland with 87% bootstrap value in ML analysis.Moreover, the tree showed that S. lineatus appears as sister to all other studied arsenids.

DISCUSSION
Knowledge about the spider fauna in Saudi Arabia is hugely inadequate compared to adjacent countries.For example, More than 80 Salticid species were recorded in Yemen (Wesolowaska and van Harten 2007) compared with only 30 species from Saudi Arabia (Prószyński, 1989;1993) which is considerably larger country.This fact underlines the necessity for further studies on the spider's fauna in this highly neglected country.In the present study, 14 genera, in addition to five unidentified spider species, belonging to 16 families were recorded in Ha'il Region.This is the first study of spider's fauna in Northern Saudi Arabia and may provide a basis for future studies.
Biogeographically, the Arabian Peninsula is classified as belonging to Palaearctic region.However, spiders of African origin were recorded from regions south of Baha till Yemen (Knoflach and van Harten, 2002;El-Hennawy, 2011).Therefore, Grashoff and van Harten (2007) conclude that there is close biogeographical relationship between the Arabian Peninsula and Africa.Many people think of Arabian Peninsula as a part of Asia.From the viewpoint of geological history, this is erroneous; because the geological history of this area leads to the conclusion that the Arabian Peninsula was a part of Africa (Thompson, 2000).
In recent times, after the discovery of advanced methods utilizing DNA it has become of great significance to use these modern techniques in association with classical taxonomy which relies on measurements and description of morphological characters.Recently, DNA techniques, specifically sequencing, have been introduced into systematic and phylogeny of spiders (e.g.Bruvo-Madaric et al., 2005;Bond et al., 2012;Griswold et al., 2012).In this way, sequence comparisons provide clues to clarify the view of relationships among species.In the present study, exemplars of four different spider families, which are the most common in the Region, were examined for gene composition.According to Flook et al., (1999) combination of data sets with varying rates of evolution (e.g., mitochondrial and nuclear genes), can efficiently accommodate for variation in results of individual analyses.Therefore, nucleotide sequence data were generated from one mitochondrial gene (12S rDNA) and one nuclear (H3) gene for which we assumed they might be informative.This assumption was based on the fact that these genes were previously used with success in a range of phylogenetic analyses in spiders (e.g., Fang et al., 2000;Maddison and Hedin, 2003;Arnedo et al., 2004).
The constructed tree based on both Maximum Parsimony and Maximum Likelihood revealed that the studied representatives of family Agelenidae and family Sparassidae were grouped in one supported clade.However, the relationship of this clade with the other two studied families appears controversy in both H3 and 12S trees.Results for each of the studied genes support their reliability for phylogenitic analysis of spiders.In this respect, sequence data from a portion of the mitochondrial 12S rDNA have yielded large data sets for phylogenetic analysis of spiders than the nuclear gene (e.g.Fang et al., 2000;Vink et al., 2002;Bruvo-Madaric 2005).Zehethofer and Sturmbauer (1998) found that 12S rRNA was especially suitable for resolving relationships higher than the species level.Generally, studying the mitochondrial DNA is very important to build the phylogenetic tree of living organisms, as it is the most useful molecule to infer the phylogeography.The mtDNA size is smaller compared to genomic DNA, and its sequence evolution rate is generally high; this high rate is the product of both high mutation rate and a high mutation fixation rate.The high mutation rate results in part from the mtDNA's lack of protective histones, inefficient DNA repair systems and continuous exposure to mutagenic effects of the oxygen radicals.The high mutation fixation rate is due to the efficient intracellular sorting of mutant molecules in the female germ line and the rapid genetic drift of mtDNAs in the general population (Wallace, 1994;Hartl, 1998).In spite of these facts, the current results revealed that the H3 nuclear gene sequence is also highly reliable for phylogenetic analysis of spiders which may be due to the high conservative nature of molecular gene.
In an effort to gain a better understanding of intergeneric and subfamilial relationships of some of the studied families, we used nucleotide sequence data previously published to construct phylogenetic tree for representatives of families Pholcidae and Arsenida separately.Regarding the studied representatives of these two families, it was found that A. Atlanta was derived species relative to the other pholcids.On the other hand, S. lineatus was found to basal clades which represent sister clades to all other arsenids with high support.However, the current molecular study is a preliminary step in phylogenetic analysis of spiders' fauna in Saudi Arabia and may represent valuable basis for further studies including a greater taxon sampling.Our knowledge about gene sequences of spiders is so limited that molecular data about many families are completely lacking in database.Further gene sequencing of other representatives of the studied families will hopefully allow spider Systematists to get a stable and more detailed idea about phylogenetic relationships both within and among spider families.
km 2 .It is named after the large Wade Ha'il (formerly called Al-Adair Valley) in the Shamir Mountain region.According toChapman (1978) the area belongs to the Arabian shield and the great An-Nafud (Nafud Al-Kabir), which is connected by Dahma, to the Rub Al-Khali to the South of Saudi Arabia.Ha'il Region is characterized by its variation in topography and geomorphology.It is characterized by several landscape units, such as isolated mountains, escarpments, wadis (valleys) and sand-seas.The Ha'il valley extends to the North-East through a narrower corridor linking the Capital City with the Shammar Mountain.The Shammar Mountains form a major feature of Ha'il Region and consist of two great Granitoid mountain ranges namely Aja and Salma.The entire region is about 1000 m.The principal part of Ha'il is An-Nafud Desert.It forms the northern part of the Ha'il Region, covering about 64,000 km 2 and is composed of reddish sand and lies at an elevation of 900 m above sea level.Topographic map of Ha'il Region as obtained from the satellite SRTM DEM (2000) is shown in Fig.1.According toHereher et al. (2012) the region is topographically classified into four classes: plains, sand dunes, mountains and cultivated lands occupying 47.77%, 35.13%, 15.88% and 1.17% respectively of the total area.Ha'il Region is generally arid to extremely arid zone (Al-Turki and Al-

Fig. 1 :
Fig.1: Location and topographic map of Ha'il Region as obtained from the satellite SRTM DEM (2000), 1 km spatial resolution.(Courtesy of Mohamed E. Hereher).(b) Species account All collected spider specimens, Order Araneae, (Figs. 2-14) are listed below with their identification, localities, and collecting dates.

Fig. 15 :
Fig.15: Phylogenetic tree for the partial H3 sequences of the studied spider species obtained with PHYML.Numbers by the nodes indicate: ML bootstrap values (>50%) are given above the nodes, and Maximum Parsimony (MP) are given below the nodes.H3 sequence from Alopecosa kochi.(Family: Lycosidae) (GenBank Accession Number, DQ628635.1)was used as an outgroup.

Fig. 16 :
Fig.16: Phylogenetic tree for the partial 12S sequences of the studied spider species obtained with PHYML.ML bootstrap values (>50%) are given above the nodes, and Maximum Parsimony (MP) are given below the nodes.12S sequence from Alopecosa kochi.(Family: Lycosidae) (GenBank Accession Number, DQ 019755.1)was used as an outgroup.
), one containing the families Pholcidae, Agelenidae and Sparassidae which represented by species, A. atlanta, B. lepida and E. walckenaeri respectively, with 97% bootstrap value in ML and only 60% in MP analysis.The clade of B. lepida and E. walckenaeri had a very strong support in both ML and MP analyses.

Fig. 17 :
Fig. 17: Phylogenetic tree for the partial H3 sequences of the studied spider species obtained with PHYML.ML bootstrap values (>50%) are given above the nodes, and Maximum Parsimony (MP) are given below the nodes.Alopecosa kochi.(Family: Lycosidae) was used as an outgroup.

Fig. 18 :
Fig. 18: ML Phylogenetic tree of pholcid species based on partial 12S rDNA sequences retrieved from data base (with their GenBank Accession Numbers) showing the place of Artema Atlanta within the Family: Pholcidae.ML bootstrap values (>50%) are given below the nodes,.12S rDNA sequence from Alopecosa kochi.(Family: Lycosidae) (GenBank Accession Number, DQ 019755) was used as an outgroup.

Fig. 19 :
Fig.19: ML Phylogenetic tree of Arsenid spider species based on partial H3 sequences retrieved from data base (with their GenBank Accession Numbers) showing the place of Stegodyphus lineatus within the Family: Arsenidae.ML bootstrap values (>50%) are given below the nodes.H3 sequence from Alopecosa kochi.(Family: Lycosidae) (GenBank Accession Number, DQ628635) was used as an outgroup.

Table 1 :
Primers used for PCR.

Table 2 :
Jocqué and Dippenaar- Schoeman (2006)l characteristics of the four studied families, taking in consideration that they are all araneomorph spiders.This table is based onJocqué and Dippenaar- Schoeman (2006).

Table 3 :
Data for gene sequences of the studied spider species