ITS2 rDNA VARIATION OF TWO BLACK WIDOW SPECIES, LATRODECTUS MACTANS AND LATRODECTUS HESPERUS (ARANEAE, THERIDIIDAE)

2004·3.9 MB·English

by  Daiyuan Zhang

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 Zhang', William B. Cook and Norman V. Horner^: 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 ofL. mactans and L. Hesperus from six
populations were collected and identified based on morphological characteristics. The ITS2 rDNA ofnine
individuals was sequenced and analyzed. Results indicate that the minimal differences present in the ITS2
sequences are taxonomically insignificant.
Keywords^ Theridiidae, Latrodectus, rDNA, internal transcribed spacer 2
The literature reveals conflicting 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 specific in-
and L. Hesperus Chamberlin & Ivie 1935 (Araneae, dividuals, which provides apotentially usefulmark-
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 tigerbeetles (Cicin-
cies by Kaston (1970, 1978). For this study, the dela dorsalis)', Hedin (1997) separated cave spider
spiders were identified 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 crayfishes (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-
a&ttrDaicxtoivne 1m9a9r1k)e.rTshefsore pgheynleosgeanreetoircgasntiuzdeiedsin(Hcillulsi-s lvaitdiuoanlssfowreeraechcoslpleeccietse.d LiantroTdeexcatsusfmraocmtanWsicihnidtia-
ters of repeated units, each of which consists of (34°00'N, 98°42'W), Brown (3U45'N, 99°00'W)
coding sequences, and several transcribed and non- and Kimble (30°29'N, 99°46'W) 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 (32°52'N, 104°45'W),
as external and internal transcribed spacers (ETS
Brewster(29°33'N, 103°47'W) andGarza(33°10'N,
and ITS). Coding regions and spacers differgreatly 101°20'W) Counties of Texas. Genomic DNA was
in theirrate ofevolution, and hence therDNA clus-
isolated from leg IV ofeach individual spiderusing
ters have the potential to reveal phylogenetic rela- QIAGEN DNAeasy Tissue Kit (Qiagen, Inc., Va-
lencia, CA). The DNA concentration was measured
UV
' Current Address: Dept, of Biological Science, using a spectrophotometer (Pharmacia, Ultra-
University of North Texas, Denton, Texas 76203- spec III).
5220. The ITS2 region of rDNA was amplified using
^Corresponding author. 5.8S primer (5'-GGGACGATGAAGAACGCAGC-
349
—
350 THE JOURNAL OF ARACHNOLOGY
Lat CAGCTGCGAGACTTGGTGTGAATTGCAGGACACATTGAGCACTGATTTTTCGAACGCGCA
Eno CAGCTGCGAGACT-GGTGTGAATTGCAGGACACATTGAGCACTGATTTTTCGAACGCGCA
************* ****************•******•*•****•****•******•*********
Lat TTGCGCCCTCT-~~--TCCCCGGGGGCTCGCCTGTCTGAGGGTCGGATAAGACTTACCGA
Eno TTGCGGCCTCGGGTCCTGCCCGGGGCCTCGCCTGTCTGAGGGTCGGATAAGACTTGCAAA
***** **** * ******* ***************************** * *
Lat GGAGGGTTTCCCTCCCA-TTGGCTGCATCGGATGG---TGATCATCCGTCTGCCTAAGGT
Eno GGAAAGTTCGCTTTCCACTTGGCCGAACCGGATCGCTTTTGTGGTTCGCCGGCTTAAGGT
*** *** * * *** ***** * * ***** * * * ** * ** ******
Lat CTTCGTCC—GCGAATCCCCCTGGCCGAGAAGCGTCGCTCC--~TCGAATAGGTCCAGCATA
Eno TTACG— GATCCTCCCCGGCCGAGAAGCGTGGCTCCCACTTGACCAACGCTCGCGG-
* ** ** * *** ************* ***** * ** * * **
Lat GAGGGAGAACGACTGA-TCGTCTCCCCGCCGACTGGAGCTGGAGCTGATGACGCAGAGAG
Eno GCTGGAGAA-GACTGAGTAGTTTCTCCGCTGAAGCGAGCGACGGGAGCGAGCGCAGAGAG
* ****** ****** * ** ** **** ** **** * * *********
Lat TGTGCCGGGAAGAAGGGATGGTCGGATGGTCCGCTTG—GG—TGCTTT—CGTG—CTA—ATGCGTCC
—— —
Eno CTTGCCGAGGAGA— GATCCTCCAC-- GCGTGC
***** * *** *** *** * **** *
Lat CTCGTTCG—TATAACG—GCGTTGAACTACGTCCT—GAATTGAGGGTTCGCAGCGAAAGGTCAC
Eno C------ ---AC -TGGCACGGTAAACTCAA
* ** * *** * ** ***
Lat GACTGATATTCATATCTGTCGACCTCAGATCAGACGAGATGACCCGCTGAATTTAA
Eno AA—-- ATAT-TGTCGACCTCAGATCAGACGAGATGACCCGCTGAATTTAA
* **** ****************************************
—
Figure 1. Alignment ofITS2 regions fromLatrodectus spp. andEnoplognatha ovata. Lat. = consensus
sequence ofeightLatrodectus mactans andLatrodectus hesperus individuals. Eno. = sequenceofL. ovata
(Araneae, Theridiidae). Underlined ITS2 sequences are flanked by 5.8S and 28S sequences. Asterisks
indicate identical nucleotide positions. Alignment generated by CLUSTALW 1.4.
3') and 28S primer (5'-TCCTCCGCTTATTGA- CLUSTALW 1.4 (Higgins & Sharp 1988) andcon-
TATGC-3') (Hillis et al. 1996). Two nanograms of sensus sequences were obtained.
spider genomic DNA were used for each reaction First, the extent ofthe intergenic region between
and PCR amplification was performed in 100 piL, the 5.8S and 28S coding regions referredto as ITS2
using the following profile: 5 min at 94 °C, 40 cy- was determined by similarity with the published se-
cles (1 min at 94 °C, 2 min at 45 °C, 1.5 min at 72 quences of Lnoplognatha ovata (Araneae; Theridi-
°C), and 7 min at 72 °C. The products wereassessed idae) (Fig. 1) (Tan & Gillespie 1999). The length
by mini-gel electrophoresis using 5 |jlL aliquots. ofthe ITS2 region in different clones ofL. mactans
Successful amplifications generated a single 0.5 kb and L. hesperus is —360 bp. Three individuals
product. After purification, the PCR products were (LMWl, LMW2, LMW3) of the LMW population
digested with LcoK 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 L. coU DH5a cells. Sequences of For individual LMW3, four separate clones were
both strands weredeterminedby NorthwoodsDNA, sequenced to test variation within one individual.
Inc. (Center for Research and Innovation, 44526 For the otherpopulations, one individual from each
CNTY Rd 3, Becida, MN 56678). Results were re- was randomly selected for PCR amplification and
ceived as unprocessed nucleotide sequences andan- 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.—VARIATION IN LATRODECTUS 351
1 2 3 4
LMWl C T T T
LMW2 c T T C
LMJ T T A T
LMB T A T T
LHM T A T T
LMW3 T T A T
LHD T T T T
LHX T T A T
—
Figure 2. Alignment of Variable ITS2 sites
from eight Latrodectus individuals. Variable nucle-
otides occuratfourpositions withintheLatrodectus
consensus sequences in Figure 1:1 = 124; 2 = 153;
3 = 227; 4 = 422. LMW = L. mactans, Wichita —
County, Texas; LMJ = L. mactans. Llano County, Figure 3. Phylogenetic network of five haplo-
TLeHxaMs;=LML.Bhe=spLe.rumsa,ctEadndsy. CBoruonwtny,CoNuenwty,MeTxeixcaos;; tdiyvpiedsuaflrso.mOIpTeS2n ssehqaupeensc,esroepfreeisgehnttiLnagtrotdheecthuasplion--
L=HLD. h=esLp.erhuess,peGraursz,aBrCeowusntteyr,CToeuxnast.y, Texas; LHX tAynpaelsy,siisndiwcaastepteherfionrcmleuddedbyLaTtrCoSdec(tCulsemiennditvsideutalasl..
2001
).
the alignmentofsequences from individualLMW3. clones ofITS2 from a single mosquito,Aedessimp-
Three variable nucleotide positions were found in soni, while intraspecific variation inA. aegypti was
the alignment of three individuals from Wichita only 1.17%. Thedifferentiationeven within asingle
County population of L. mactans. Three variable individual may be caused by the existence ofpoly-
nucleotide positions were found in the alignmentof morphisms among repeat units of rDNA.
threeL. mactanspopulations. Only one variablenu- In a phylogenetic analysis ofITS2 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 five
eightindividuals from six populations variedatfour individuals, which included representatives ofboth
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 reflect 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 five haplo- liable means of distinguishing between L. mactans
LMW
types (Fig. 3). The three individuals repre- and L. hesperus populations.
sented three different haplotypes on two different Only those characters that are diagnostic for all
branches from aL. hesperus node. LMW3 and two individuals of an entire population can be used in
L. mactans individuals from outside Wichita Coun- the phylogenetic reconstruction ofpopulations (Vo-
ty represented a single haplotype. Latrodectus hes- gler 1994). Further exploration of the molecular
perus sequences collapsed into two haplotypes, one taxonomy ofthese taxa will require additional data,
of which supported the two L. mactans branches. including sequence comparisons of mtDNA, ITSl
Even though all the clones, individuals, popula- DNA, plus other nuclear genes.
tions and two taxa showed some level of sequence
LITERATURE CITED
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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
thatamong populations areeach 0.83%. TheL. hes- chromosomal evolution. Molecular Biology and
peruspopulations exhibitonly 0.27% variation.The Evolution 17:284-291.
variation between two taxa is 0.83% and does not Clement, M., D. Posada & K.A. Crandall. 2000.
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found in the ITS2 research of mosquitoes. Wesson nealogies. Molecular Ecology 9:1657-1659.
et al. (1992) reported 0.46% variation within 10 Harris, D.J. & K.A. Crandall. 2000. Intragenomic
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