A Study of the Cyrtophora Species (Araneae: Araneidae)
Total Page：16
File Type：pdf, Size：1020Kb
IJMBR 1 (2013) 21-34 Combined analyses of genetic and morphological data indicate more than one species of Cyrtophora (Araneae: Araneidae) in South Africa P. Z. N. Franzini1*, A. S. Dippenaar-Schoeman2,3, K. Yessoufou4 and F. H. Van der Bank1 1Department of Zoology, African Centre for DNA Barcoding, University of Johannesburg, P. O. Box 524, Auckland Park 2006, South Africa. 2ARC-Plant Protection Research Institute, Private Bag X134, Queenswood, Pretoria, 0121, South Africa. 3Department of Zoology and Entomology, University of Pretoria, Pretoria, 0001, South Africa. 4Department of Botany and Plant Biotechnology, African Centre for DNA Barcoding, University of Johannesburg, P. O. Box 524, Auckland Park 2006, South Africa. Article History ABSTRACT Received 25 February, 2013 Forty-three (43) Cyrtophora species are known throughout the world whereas Received in revised form 02 only Cyrtophora citricola is suspected to occur in South Africa. Earlier April, 2013 Accepted 29 April, 2013 investigations of South African representatives of the genus revealed a wide range of morphological features including colour and abdominal shapes, thus Key words: raising the need for a thorough investigation of species diversity of the genus. Arachnida, Using fresh and museum specimens sampled from 2006 to 2011, we combined Cryptic species, DNA data - the mitochondrial cytochrome c oxidase subunit 1 (CO1) and nuclear Cyrtophora citricola, histone H3 genes - with morphological features of the genitalia to investigate the DNA barcoding. question. Our genetic analysis differentiates between three to 10 potential species in our dataset based on H3 and CO1, respectively. Based on morphological characteristics, we found diverse abdominal colour variations but only two genitalia shapes were distinguishable in the collections; also suggesting that at least two species do occur in South Africa. Interestingly, specimens exhibiting these two distinguishable genitalia shapes also differ in both genetics and morphological colour variation. These specimens correspond Full Length Research Article to C. citricola and a potentially new species from the Malelane region. ©2013 BluePen Journals Ltd. All rights reserved INTRODUCTION The correct identification of many spider species is still specimens (Huber, 2004). Genital characteristics, on problematic due to complex variation in morphology which most taxonomists rely on for the description of (Barrett and Hebert, 2005). Sexual dimorphism occurs in spiders, can however be occasionally misleading, due to the family Araneidae, making it difficult to match the polymorphism in some species (Huber and González, males with females. Identification of immature spiders is 2001; Jocqué, 2002). Given all these constraints, even more problematic due to underdeveloped identifying spiders to species level can be a challenging morphological characters that are useful for species task. discrimination. For example, most identification keys are A potential tool to overcome impediments in mainly based on the morphology of genitalia of adult morphological species identification is provided by DNA barcoding (Hebert et al., 2003a; b; Barrett and Hebert, 2005; Nagoshi et al., 2011; Van der Bank et al., 2012). DNA barcoding approach is increasingly acknowledged *Corresponding author. E-mail: [email protected]. as a tool to identify closely related species (Edwards et Int. J. Mod. Biol. Res. 22 al., 2008; Sun et al., 2012; Van der Bank et al., 2012). features of the abdomen and genitalia. Over the past 10 years, the barcoding approach became a widely used method for accelerating species identification, where traditional taxonomy failed, for MATERIALS AND METHODS example, when only small fragments of organisms devoid of morphological features are available (Hebert et al., Sampling 2003a; b). It has been acknowledged not only as a tool for species identification, but also important in We undertook a five-year long data collection from 2006 discovering new species (Hebert et al., 2004; Hajibabaei to 2011. Our data collection included 34 specimens of the et al., 2005b). It also assists in controlling biological genus Cyrtophora which are currently housed at the materials at ports of entry (Yassin et al., 2008; National Collection of Arachnida (NCA), Plant Protection Newmaster and Ragupathy, 2009), conservation planning Research Institute (PPRI) of the Agricultural Research (Bickford et al., 2007; Forest et al. 2007), and Council (ARC) in Pretoria, South Africa. Additional surveillance of disease vectors (Nadin-Davis et al., 2012). sampling of specimens was undertaken from sites across Although recent analyses revealed the need for refining South Africa to cover as much as possible the known the approach (Taylor and Harris, 2012), DNA barcoding geographical ranges of the genus (Figure 1). Three remains a complementary or alternative tool to traditional specimens from Zimbabwe, one from Botswana and one taxonomic approaches (Derocles et al., 2012; Sun et al., from Madagascar were also included in the DNA analysis 2012) especially when the preferred method of traditional to allow comparison with the South African specimens. morphology is helpless (Packer et al., 2009). Newly sampled specimens were identified by the In this study, we focus on the genus Cyrtophora Simon, collectors to genus level using a field guide (Dippenaar- 1864 in South Africa. Members of the genus are Schoeman and Van den Berg, 2010) and identification commonly referred to as tent-web spiders due to the was confirmed by the second author. Information on unique shape of their orb-web (Levi, 1997; Dippenaar- localities, GPS coordinates, altitude, habitats, and Schoeman and Van den Berg, 2010). The genus photographs for all specimens (excluding PZNF L1 and comprises 43 species, as well as 9 sub-species, that are L2) is available on the international Barcode of Life wide-spread across the world (Platnick, 2012). Five Database (iBOLD). DNA sequences of the barcode species are found in Africa namely: C. caudata region CO1 are also available on the BOLD system Bösenberg and Lenz, 1895; C. citricola Forsskål, 1775; (www.boldsystems.org) and the sample and process ID C. gemmosa Thorell, 1899; C. larinioides Simon, 1895 are shown in Table 1. In total, 86 specimens were and C. petersi Karsch, 1878 (Platnick, 2012). The included in this study, consisting of 59 females, 8 males diversity of the southern African species is little explored and 19 juveniles. and only the cosmopolitan species C. citricola has so far been reported to occur for instance, in South Africa (Foord et al., 2002; Dippenaar-Schoeman et al., 2005; DNA extraction, amplification and sequencing Dippenaar-Schoeman, 2006). The need for revisions of the genus Cyrtophora has been felt worldwide, leading to Tissues (mainly legs) were sampled and placed in a the multiple initiatives observed in several countries. For microplate with a drop of 100% ethanol. Specimens were example, Yaginuma (1968) revised the Cyrtophora of preserved and handled following the guidelines set in Japan, Tikader (1982) focused on the fauna of India, Levi Hajibabaei et al. (2005a). The microplate was sent to the (1997) revised the species of the Americas and Levy Canadian Centre for DNA Barcoding (CCDB) where the (1998) undertook similar initiatives in Israel. In South CO1 gene was sequenced using standard CCDB Africa, although the genus has been suspected to be protocol (www.dnabarcoding.ca). more diverse than currently thought, no formal proof has DNA extraction, amplification and sequencing of the H3 been provided as yet. gene (and the CO1 gene of PZNF L1 and 2) were done The first world-wide revision of the genus was at the African Centre for DNA Barcoding (ACDB; undertaken by Corey (1993), who studied 19 species and www.acdb.co.za), University of Johannesburg in South noted some important morphological variations in a Africa. QIAprep Spin Miniprep Kit (Qiagen, Switzerland) number of cases. Cyrtophora citricola, the only species protocols and equipment were used for these purposes. currently known in South Africa is one of the species in The polymerase chain reaction (PCR) of the nuclear H3 which colour variation has been reported (Corey, 1993; gene was done using the primer pair H3aF (5’ Edwards, 2006), thus raising the need for in-depth ATGGCTCGTACCAAGCAGACVGC 3’) and H3aR (5’ investigations. The aim of this study was to determine the ATATCCTTRGGCATRATRGTGAC 3’). Each PCR species diversity of the genus Cyrtophora in South Africa. reaction contained 22 µl PCR Master Mix (Thermo To reach this objective, we combined DNA barcode data Scientific, Japan), 1 µl forward primer, 1 µl reverse (CO1) with the DNA nuclear region H3 and morphological primer, 0.5 µl BSA and 0.5 µl DMSO. The volume of DNA Franzini et al. 23 Figure 1. Sites where Cyrtophora specimens were sampled in South Africa. template added was calculated according to the Genetic distances between specimens’ DNA concentration of DNA after extraction. The PCR thermal sequences regime started with one cycle at 94°C for 3 min, a further 30 cycles of 94°C for 1 min, 48°C for 1 min and 72°C for Sequences were edited and aligned using the MUltiple 3 min and a final cycle of 72°C for 7 min. PCR products Sequence Comparison by Log Expectation (MUSCLE) were cleaned according to the QIAquick (Qiagen, program (Edgar, 2004). Genetic distances (mean, Switzerland) protocol. minimum, maximum and pairwise distances between Cycle-sequencing was done using 1.5 µl sequencing specimens) were calculated for CO1, H3 and CO1 + H3 buffer, 0.3 µl primer, 0.5 µl DMSO, 0.5µl Big Dye (combined). The standard model of evolution used in Terminator (Applied Biosystems, SA) and 35ng DNA DNA barcoding studies, that is, the Kimura 2-parameter template. The cycle sequencing thermal regime consisted (K2P) model was used for the calculation of genetic of 26 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 distances (Kimura, 1980). The genetic variation within min. The cycle sequencing products were cleaned and DNA regions was also assessed using the Tajima’s K DNA sequencing was done on an ABI 3130x1 genetic index of divergence (Tajima, 1983).