2004. The Journal of Arachnology 32:349±352 SHORT COMMUNICATION ITS2 rDNA VARIATION OF TWO BLACK WIDOW SPECIES, LATRODECTUS MACTANS AND LATRODECTUS HESPERUS (ARANEAE, THERIDIIDAE) Daiyuan Zhang1, William B. Cook and Norman V. Horner2: Department of Biology: Midwestern State University, Wichita Falls, Texas 76308-2099 USA ABSTRACT. The taxonomic status of two closely related species of Latrodectus, L. mactans and L. hesperus, has been debated for many years. Based on morphological characteristics and genitalia, some workers consider them as valid species and others as subspecies. This study was conducted to determine whether the internal transcribed spacers 2 (ITS2) of rDNA exhibit sequence differences between the two taxa that could delineate their taxonomic relationship. Individuals of L. mactans and L. hesperus from six populations were collected and identi®ed based on morphological characteristics. The ITS2 rDNA of nine individuals was sequenced and analyzed. Results indicate that the minimal differences present in the ITS2 sequences are taxonomically insigni®cant. Keywords: Theridiidae, Latrodectus, rDNA, internal transcribed spacer 2 The literature reveals con¯icting information tionships at many taxonomic levels. The level of concerning the taxonomic status of two black wid- divergence observed in the spacer regions is appro- ow species, Latrodectus mactans (Fabricius 1775) priate for detecting differences between speci®c in- and L. hesperus Chamberlin & Ivie 1935 (Araneae, dividuals, which provides a potentially useful mark- Theridiidae). Taxonomic work based on morpho- er with which to study the relationships of logical characteristics has produced controversial populations and closely related species (Cerbah & conclusions. The two spider taxa were designated Souza 1998; Hedin 1997; Vogler 1994). For ex- as subspecies by Levi (1959), and as separate spe- ample, Vogler (1994) separated tiger beetles (Cicin- cies by Kaston (1970, 1978). For this study, the dela dorsalis); Hedin (1997) separated cave spider spiders were identi®ed to species following the populations and species (Nesticus); Harris & Cran- characteristics outlined by Kaston. Specimens of dall (2000) separated closely related species and each species are on deposit in the Invertebrate Col- populations of freshwater cray®shes (Decapoda, lection at Midwestern State University, Wichita Cambaridae). Falls, Texas. This preliminary study of ITS2 rDNA variation The rRNA genes have long been recognized as was conducted using individuals from three popu- attractive markers for phylogenetic studies (Hillis lations for each species. Latrodectus mactans indi- & Dixon 1991). These genes are organized in clus- viduals were collected in Texas from Wichita ters of repeated units, each of which consists of (348009N, 988429W), Brown (318459N, 998009W) coding sequences, and several transcribed and non- and Kimble (308299N, 998469W) Counties. Latro- transcribed spacer regions (NTS). The transcription dectus hesperus individuals were collected from units include the 18S, 5.8S, and 28S genes as well Eddy County, New Mexico (328529N, 1048459W), as external and internal transcribed spacers (ETS Brewster (298339N, 1038479W) and Garza (338109N, and ITS). Coding regions and spacers differ greatly 1018209W) Counties of Texas. Genomic DNA was in their rate of evolution, and hence the rDNA clus- isolated from leg IV of each individual spider using ters have the potential to reveal phylogenetic rela- QIAGEN DNAeasy Tissue Kit (Qiagen, Inc., Va- lencia, CA). The DNA concentration was measured 1 Current Address: Dept. of Biological Science, using a UV spectrophotometer (Pharmacia, Ultra- University of North Texas, Denton, Texas 76203- spec III). 5220. The ITS2 region of rDNA was ampli®ed using 2 Corresponding author. 5.8S primer (59-GGGACGATGAAGAACGCAGC- 349 350 THE JOURNAL OF ARACHNOLOGY Figure 1.ÐAlignment of ITS2 regions from Latrodectus spp. and Enoplognatha ovata. Lat. 5 consensus sequence of eight Latrodectus mactans and Latrodectus hesperus individuals. Eno. 5 sequence of E. ovata (Araneae, Theridiidae). Underlined ITS2 sequences are ¯anked by 5.8S and 28S sequences. Asterisks indicate identical nucleotide positions. Alignment generated by CLUSTALW 1.4. 39) and 28S primer (59-TCCTCCGCTTATTGA- CLUSTAL W 1.4 (Higgins & Sharp 1988) and con- TATGC-39) (Hillis et al. 1996). Two nanograms of sensus sequences were obtained. spider genomic DNA were used for each reaction First, the extent of the intergenic region between and PCR ampli®cation was performed in 100 mL, the 5.8S and 28S coding regions referred to as ITS2 using the following pro®le: 5 min at 94 8C, 40 cy- was determined by similarity with the published se- cles (1 min at 94 8C, 2 min at 45 8C, 1.5 min at 72 quences of Enoplognatha ovata (Araneae; Theridi- 8C), and 7 min at 72 8C. The products were assessed idae) (Fig. 1) (Tan & Gillespie 1999). The length by mini-gel electrophoresis using 5 mL aliquots. of the ITS2 region in different clones of L. mactans Successful ampli®cations generated a single 0.5 kb and L. hesperus is ;360 bp. Three individuals product. After puri®cation, the PCR products were (LMW1, LMW2, LMW3) of the LMW population digested with EcoR I and Hind III and ligated into (L. mactans from Wichita County, Texas) were se- pUC19 vector. The recombinant plasmids were lected to analyze the variation in one population. transformed into E. coli DH5a cells. Sequences of For individual LMW3, four separate clones were both strands were determined by Northwoods DNA, sequenced to test variation within one individual. Inc. (Center for Research and Innovation, 44526 For the other populations, one individual from each CNTY Rd 3, Becida, MN 56678). Results were re- was randomly selected for PCR ampli®cation and ceived as unprocessed nucleotide sequences and an- one or more independent clones covering the ITS2 alyzed manually. Some visual adjustments were were sequenced from each of these individuals. made. The individual sequences were aligned using Five variable nucleotide positions were found in ZHANG ET AL.ÐVARIATION IN LATRODECTUS 351 Figure 2.ÐAlignment of Variable ITS2 sites from eight Latrodectus individuals. Variable nucle- otides occur at four positions within the Latrodectus consensus sequences in Figure 1: 1 5 124; 2 5 153; 3 5 227; 4 5 422. LMW 5 L. mactans, Wichita Figure 3.ÐPhylogenetic network of ®ve haplo- County, Texas; LMJ 5 L. mactans, Llano County, types from ITS2 sequences of eight Latrodectus in- Texas; LMB 5 L. mactans, Brown County, Texas; dividuals. Open shapes, representing the haplo- LHM 5 L. hesperus, Eddy County, New Mexico; types, indicate the included Latrodectus individuals. LHD 5 L. hesperus, Brewster County, Texas; LHX Analysis was performed by TCS (Clements et al. 5 L. hesperus, Garza County, Texas. 2001). the alignment of sequences from individual LMW3. clones of ITS2 from a single mosquito, Aedes simp- Three variable nucleotide positions were found in soni, while intraspeci®c variation in A. aegypti was the alignment of three individuals from Wichita only 1.17%. The differentiation even within a single County population of L. mactans. Three variable individual may be caused by the existence of poly- nucleotide positions were found in the alignment of morphisms among repeat units of rDNA. three L. mactans populations. Only one variable nu- In a phylogenetic analysis of ITS2 sequence var- cleotide position was found among three popula- iations, L. mactans individuals from Wichita Coun- tions of L. hesperus. The consensus sequences of ty represented as many haplotypes as the other ®ve eight individuals from six populations varied at four individuals, which included representatives of both nucleotide positions (Fig. 2). L. hesperus and L. mactans. Furthermore, the three The phylogenetic analysis of the aligned se- LMW haplotypes represented two distinct branches quences was performed as described by Templeton from LHM. Assuming that the Wichita County L. et al. (1992) using the TCS software package mactans individuals re¯ect a typical level of re- (Clement et al. 2000). ITS2 sequences from eight gional variation, ITS2 sequences do not offer a re- Latrodectus individuals collapsed into ®ve haplo- liable means of distinguishing between L. mactans types (Fig. 3). The three LMW individuals repre- and L. hesperus populations. sented three different haplotypes on two different Only those characters that are diagnostic for all branches from a L. hesperus node. LMW3 and two individuals of an entire population can be used in L. mactans individuals from outside Wichita Coun- the phylogenetic reconstruction of populations (Vo- ty represented a single haplotype. Latrodectus hes- gler 1994). Further exploration of the molecular perus sequences collapsed into two haplotypes, one taxonomy of these taxa will require additional data, of which supported the two L. mactans branches. including sequence comparisons of mtDNA, ITS1 Even though all the clones, individuals, popula- DNA, plus other nuclear genes. tions and two taxa showed some level of sequence variation in the ITS2 region of rDNA, the separa- LITERATURE CITED tion between species was not well supported on Cerbah, M. & T. Souza. 1998. Molecular phylogeny these grounds. For L. mactans, variation within an of the genus Hypochaeris using internal tran- individual is 1.4%, variation among individuals and scribed spacers of nuclear rDNA: Inference for that among populations are each 0.83%. The L. hes- chromosomal evolution. Molecular Biology and perus