Table Of Content1991. The Journal ofArachnology 19:210-214
MITOCHONDRIAL DNA SEQUENCES CODING EOR A
PORTION OF THE RNA OF THE SMALL RIBOSOMAL
SUBUNITS OF TETRAGNATHA MANDIBULATA AND
TETRAGNATHA HAWAIENSIS {ARANEAE, TETRAGNATHIDAE)
Henrietta B. Croom: Department of Biology, The University of the South, Sewanee,
Tennessee 37375 USA
Rosemary G. Gillespie and Stephen R. Palumbi: Department ofZoology, The University
ofHawaii at Manoa, Honolulu, Hawaii 96822 USA
ABSTRACT. A region ofmitochondrial DNA coding for most ofthe third domain ofthe 12S rRNA ofthe
ribosomal small subunit has been sequenced from two spiders in the genus Tetragnatha (Araneae, Tetrag-
nathidae): a circumtropical species T. mandibulata and an endemic Hawaiian species T. hawaiensis. The
secondary structure ofthe spiderribosomal RNA shows strong similarity to that ofinsects. Acrossthis region,
the two Tetragnatha sequences are 22% different. The T. mandibulata sequence is 36% different from the
homologous segment in Drosophila yakiiba and 51% different from the same segment in Homo sapiens. The
spider sequences are sufficiently variable to be useful in studying genetic relationships among at least some of
the species in thisgenus.
A powerful approach to studying genetic re- tion make it often more instructivethan nuclear
DNA DNA
latedness of species involves sequence in comparing taxa (Wilson et al. 1985).
DNA
comparisons which can be used to estimate This evolves rapidly at the sequence level
branching order ofphylogenetic trees as well as in arthropods (DeSalle and Templeton 1988;
evolutionary distance between extant taxa (Fel- Palumbi and Benzie 1991) and has proven use-
senstein 1988). Recent application ofthe poly- ful for comparing recently-evolved taxa in Ha-
merase chain reaction and direct sequencinghas waii (DeSalle et al. 1987).
accelerated efforts to examine a wide range of In order to determine nucleotide sequence in
taxawithDNAcomparisons(Kocheretal. 1989; a particular mtDNA segment, the segment must
Martinetal. 1990). Todatetheuseofthismeth- first be amplified either by genetic cloning or by
od in studying spiders has not been reported. usingthe polymerasechain reaction (Mullis and
Here we describe a procedure we have used to Faloona 1987;Saikietal. 1988).Thelattermeth-
amplify and to determinethe sequence ofnucle- od depends upon knowledge ofoligonucleotide
otidesofa279-base-pairregionofmitochondrial sequences that flank the segment ofinterest and
DNA (mtDNA) from two species ofthe genus that serve as primers for enzymatic amplifica-
Tetragnatha (Araneae, Tetragnathidae). tion. Kocher et al. (1989) have described uni-
DNA
Sequencing hasthefollowingadvantages versal (highly-conserved) oligonucleotide se-
overothertechniques ofgeneticcomparison; (1) quences flankinga 300-base portion ofthe third
ithasgreaterresolvingpowerovera hierarchical domainofthe 12SribosomalRNAgenethatcan
range of intraspecific to intergeneric compari- be used to amplify mtDNA from animals as di-
sons, (2) sequences are easily compared with verse as humans and invertebrates. The conser-
known sequences from other species, and (3) vation of these primers makes them useful to
DNA
functional information on products encoded by investigators sequencingthe ofspecies for
DNA
allows strong inferences on the selective whichthereisnoprevioussequenceinformation
importance of mutations observed, allowing (Simon et al. 1990). We first used insect-specific
character weighting for sites that are not selec- primers for the 12S rRNA region, slightly mod-
tively neutral (Kocher et al. 1989). Mitochon- ifiedbyC.Simon(pers.comm.)fromtheoriginal
drial DNA was chosen for this work because its primers ofKocher et al. (1989), to amplify and
DNA
matriarchal inheritance and lack ofrecombina- sequence the of Tetragnatha mandibulata
210
5
CROOM ET Ah.-TETRAGNATHA MTDNA SEQUENCES 211
Walckenaer. We then designed a spider-specific location in the Drosophila yakuba mtDNA se-
primer based on this sequence to amplify and quenceofClary and Wolstenholme(1985), were
sequence the same region from a Hawaiian en- 12St-L (14503), a Tetragnatha-specific primer
demic species T.hawaiensis Simon. designed by H. Croom: 5’-GGTGGCATTT-
MATERIALS AND METHODS TATTTTATTAGAGG-3’and 12Sbi-H (14214),
an insect-specific primer designed by C. Simon:
Allsolutionsusedwereeithersterilizedorpre- 5’-AAGAGCGACGGGCGATGTGT-3’. One
paredusingsteriledeionized,distilledwater,and mL of the double-stranded product of the first
all glass- and plastic-ware were sterile with the amplification was cycled under the same con-
exceptionoftheCentricontubes(seebelow).The ditionsasaboveexceptthatonlyoneprimerwas
chelicerae and a front leg of each spider were added to the incubation mixture. This produced
placed in 70% ethanol as voucher specimens. a single-strand sequencing template, which was
Total (genomic) DNA was prepared by homog- processed in a Centricon 30 microconcentrator
enizing a single spider in a 1.5 mL Eppendorf (Amicon) to concentrate and purify the DNA
mM
tubein 200mLof25 TrisHCl (pH 7.5), 100 product. The template was then sequenced by
mM
EDTA, 2%SDS,and200ntg/mLProteinase thedideoxychainterminationmethodofSanger
K, followed by incubation for 1-2 hr in a water etal. (1977)asdescribedbyEngelkeetal.(1988),
bath at 65 °C. The homogenate was extracted using the primer that had not been used in the
DNA
first with phenol, previously-equilibrated with 1 second amplification. from three individ-
M
Tris HCl buffer (pH 7.5), then with 25:24:1 uals ofeach species was sequenced in both di-
phenol/chloroform/isoamylalcohol 1 to 4 times rections, and no intraspecific variation was ob-
(until all the protein-containing white interface served.
wasremoved),and finallywith 24:1 chloroformM/ HomologoussequencesofDNAfromdifferent
isoamyl alcohol. One-half volume of a 7.5 taxa were aligned to minimize deletions or ad-
solution ofammonium acetatewas addedto the ditions using software written by S. R. Palumbi
M
extractto achieve a final concentration of2.5 andC. Parrish. Pairwisepercentdifferenceswere
and mixed well before adding 2‘/2 volumes of calculatedbycountingonlysiteswherebothspe-
95% ethyl alcohol and mixing again. This solu- cies have nucleotides in our aligned sequences.
tion was incubated at room temperature for 1 One strand ofspider DNA was folded to show
min to allow for DNA precipitation. The DNA itssecondarystructureusingthefoldedsequenc-
was pelleted by centrifugation at 14,000 x g for esofinsectsasaguide(ClaryandWolstenholme
15 min at room temperature, washed with 500 1987; Simon et al. 1990).
mL of70% ethyl alcohol, dried under a vacuum,
and resuspended in 25 mLwater. Five ixL ofthis RESULTS AND DISCUSSION
preparation was electrophoresed on a 0.8% aga-
rose gel in Tris-borate buffer and stained with The DNA sequences from the two Tetragna-
ethidium bromide as described in Sambrook et tha species were compared with the 12S rRNA
al. (1989) to ensure that high molecular-weight genes from both Homo sapiens and Drosophila
DNA was present. The preparation is stable in- yakuba (Fig. 1). The two Tetragnatha sequences
definitely when stored at -20°C. are 22%different from each other, 36%different
One/iLofa 1:10dilutioninwaterofthisDNA from the homologous segment in Drosophila
and 2.5 units ofDNA polymerase from Themus (Clary and Wolstenholme 1985), and 51% dif-
aquaticus (Perkin-Elmer Cetus) were incubated ferent from the same segment in Homo (Ander-
mM
in 100 fxL ofbuffercontaining 67 Tris HCl son et al. 1981). In comparison, the Drosophila
(pH 8.8), 3 mM magnesium chloride, 16.7 mM andHomosegmentsdifferby45%. These values
ammonium sulfate, each of four deoxynucleo- are based solely on pairwise nucleotide differ-
side triphosphates at 200 mM and each oftwo ences, ignoring insertions and deletions, using
,
primers at 1 according to the protocol of the alignments in Fig. 1. It is difficult to align
DNA
Saiki et al. (1988). Thermal profile for45 cycles nonconserved regions of from distantly-
wasas follows: (1) DNAmeltingfor 1 min at 94 related groups, so other alignments may yield
°C, (2) annealing for 1 min at 50 °C, and (3) slightly different percentages. Likewise, the dif-
polymerizationfor2 min at 72°Ctoamplifythe ferenees here are uncorrected for multiple mu-
double-strandedsequence.Theprimersandtheir tations at the same site, which has the effect of
. .
212 THEJOURNALOFARACHNOLOGY
Tetragnatha mandibulata aacatgtttattaatcgacattacacgattatttt
Tetragnatha hawaiensis .. t .......a atc .......... c ..
.
Drosophila yakuba ... c ..... tg ....... t a c ..... ggacc
Homo sapiens g c ctg t . aa.c c . c acc .
. . . . . . . . . . . . . .
TACTTTTTTATAA ATTTTATATACCTCCGTCC--AGAATAAATTTTTAATA TTT
C AATA T G .... TT ..... AAAA ..........
C. .CAACACA.C.TT..GG.CT.AA.TTCC..GGC.C.......... GG.T ...A.. TATTTCC . . C. .A. . TCTCC. . AGA. AG. GAAG.GTCAATA.CAAA
. . . . . . . .
TATTCAAAATAATATTATAATAATTTA GGTAAAGGTGTAGACTTTAAATTAGT-TT
AT C GA A A T AG A A
T.-.T.T..A.......A...... TATCA.A.C. . . . . . . C.T...A.. .. TT..A.. . .. A. A
A G A GCGC GTACCC CGT AAG CGTTA C C CA G GG G. C. AAG
. . . . . . . . . . . . . . . .
AAATGTGTTACATTAAAAATTATTT AAGAATTATTTTTTATAA CAATATATGA
......A ... AA ........... G ... AAC AA C T AGT T A
T .... G ......A.... -T ... ACGGAT.AAAA. . G. A.A. . .A . T . T. . .
G.C. TTCT CCCCAGAAAACTACGA GCCC . . . .. G.. AAC.T.T.. . G. GG.TC . .
. . . . . . . . . . . . .
AAGAGGATTTATAAGCTACTTTTTAATTAAAATTTTAACTTGAATTAAAAA--TAAATGCG
.. A.............. TAA T A ... T ...... TA
G T GGT TA .AA . . . TAA. G T AA... T ....... TT GCTC ....AT.AT
. G . T GC . . TA . AC . A.-AG. .G. .G.. G.. GC.T.G. .-C. GG.GCCC G GCGC
. . . . . . . . . . . .
Figure 1.—Comparison ofMitochondrial 12S Ribosomal DNA from Tetragnatha mandibulata, Tetragnatha
hawaiensis.Homosapiens,andDrosophilayakuba.Dotsrepresentpositionsthatareidenticaltothetopsequence,
anddashesrepresentgapsin the sequencesrequired to maximizealignment. The sequence from theDrosophila
isthatofClaryandWolstenholme(1985)betweentheirbasepairs 14236and 14502.TheHomoDNAsequence
is that ofAnderson et al. (1981) between their bases 1201 and 1475.
making the more distantly-related taxa appear tional ribosomal gene. The third domain ofthe
deceptively similar. small rRNA encoded by nuclear DNA is both
Whenusingthepolymerasechainreactionwith larger than, and has a structure distinct from,
genomic DNA, one must always consider the that represented in Fig. 2 (Woese et al. 1983;
possibility that nontarget nuclear or mitochon- Dams et al. 1988).
drialDNAhasbeenamplified. Usingthemethod We have sequenced most ofthe homologous
of Palumbi and Wilson (1990), we separated regionfrom 19otherspiders:Aphonopelmachal-
mtDNA from nuclear DNA of25 specimens of codes Chamberlin (Araneae, Theraphosidae),
T. mandibulata on a cesium chloride gradient Doryonychus raptor Simon (Araneae, Tetrag-
before amplifying and sequencing the mtDNA nathidae), and 17 endemic Hawaiian Tetrag-
fraction. The sequence obtainedwas identical to nathataxa.InthecaseoftheA.chalcodes,cesium
that in Fig. 1. In order to verify that the spider chloride gradient purified mtDNA was used for
sequencescodeforthethirddomainof12SrRNA, the amplification instead ofgenomic DNA. We
a single strand was folded to generate the sec- foundeachofthesesequencestobemoresimilar
ondarystructurestabilizedbyhydrogen-bonding to the spidersequences in Fig. 1 than tothose of
between complementary bases. In all ofthe taxa any otherknown taxa (Croom and Palumbi, un-
studied to date (Dams et al. 1988; Simon et al. published). Such similarity suggests that neither
1990), the foldedstructure ofthisdomain forms ofthe sequences reported in this paper is from
helical paired stems and unpaired loops. The contaminating DNA.
structure obtained from T. mandibulata (Fig. 2) Interestingly, 83% ofall bases in the two Te-
isessentiallythesameasthoseoftheotherknown tragnatha sequences are either A or T. In the
taxa. Conservation of secondary structure, de- strand shown in Fig. 2, the frequencies for bases
spitethe largeoverallsequencedifferencesamong are: 39% A, 43% T, 8% C, 10% G. The percent
taxa (Fig. 1), suggestswe have sequenceda func- AT across this region is 79% forDrosophila and
8
CROOM ET AL.-TETRAGNATHA MTDNA SEQUENCES 213
ACKNOWLEDGEMENTS
C C
WethankBaileyKessing,ChrisParrish,Chris-
G-C
C-G tine Simon, and Rob deSalle for their help and
encouragement during this study. Lei-Anna
Willmanprovidedexcellenttechnicalassistance.
ThisresearchwassupportedbyNSFgrantBSR-
8604969 and by the Faculty Research and Fac-
ulty Development Funds of The University of
the South.
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