UNUSUALLY LONG HYPTIOTES (ARANEAE, ULOBORIDAE) SEQUENCE FOR SMALL SUBUNIT (18S) RIBOSOMAL RNA SUPPORTS SECONDARY STRUCTURE MODEL UTILITY IN SPIDERS

2006·10 MB·English

by  J C Spagna

2006 (2007). The Journal of Arachnology 34:557-565
UNUSUALLY LONG HYPTIOTES (ARANEAE, ULOBORIDAE)
SEQUENCE FOR SMALL SUBUNIT RIBOSOMAL
(18S)
RNA SUPPORTS SECONDARY STRUCTURE
MODEL UTILITY IN SPIDERS
J. C. Spagna^ and R. G* Gillespie: Division of Insect Biology, Department of
Environmental Science, Policy and Management, 137 Mulford Hall, UC Berkeley,
Berkeley, California 94720, USA. E-mail: [email protected]
ABSTRACT. We report on the structure of the small-subunit ribosomal RNA (18S rRNA) sequence
from Hyptiotes gertschi (Araneae, Uloboridae), which is the largest 18S gene sequenced in any arachnid
to date. We compare this remarkable sequence to those from a range of other spiders and arachnids, and
develop base-pairing models of its insert regions to determine its overall secondary structure. The H.
gertschi sequenceof 1902 bases is 86 nucleotides longerthan anycomparable spidersequenceandcontains
5 inserts between 5 and 28 bases in length, all at regions characterized as among the most variable in
eukaryotic 18S genes. Inserts were also found in one of these variable regions in published sequences of
3 species of hard ticks (Acari, Ixodidae). Other arachnid taxa were remarkably uniform in 18S primary
sequence length, ranging from 1802 to 1816 nucleotides. Thermodynamic modeling of the H. gertschi
inserts suggests they are largely self-complementary, extending the stem portions of the variable regions.
Keywords: Phylogenetics, arachnids, Acari, gene inserts
The small subunit ribosomal RNA, or 18S at which different parts of the primary se-
rRNA, is one ofa small set ofcommonly used quence vary.
sequences for molecular phylogenetic recon- The genes coding for ribosomal RNAs con-
struction of arthropod relationships (reviewed tain regions that can accumulate and lose ba-
in Caterino et al. 2000). The gene coding for ses through insertion and deletion events (in-
the 18S rRNA contains sections that are high- dels) more easily than protein-coding genes,
ly conserved, and these provide informative which are constrained by the requirement that
characters for the assessment of relationships they maintain an open reading frame forprop-
between distantly related taxa, such as meta- er translation into a functional primary amino
zoan relationships (Giribet & Wheeler 2001; acid sequence. Indels can change the length of
Mallatt et al. 2004). The 18S rRNA has also the gene and make homology assessments of
been used in studies of divergence between individual base-pairs, and often long stretches
arachnid orders, as well as studies of diver- of base-pairs, difficult. The proper methodo-
gence between spider genera and species logical approach to this sequence-alignment
&
(Wheeler Hayashi 1998; Arnedo et al. problem is a source of controversy in phylo-
2004). In the context of arachnid molecular genetics, and opinions range from using static
phylogenetics, to understand both the poten- alignments, with regions that are difficult to
tial utility and drawbacks in the use of any align being either included or discarded (e.g.,
genetic marker, it is important to have knowl- Nardi et al. 2003); to using direct optimization
edge of the amount of variation in both the (Wheeler 1996), which avoids the arbitrary re-
primary sequence and secondary structures moval of data and possible evolutionary sig-
across taxa at different levels. This is because nal and avoids the problem of multiple-se-
the secondary structure can influence the rate quence alignment altogether. An additional
o^fCuIrlrleinnotisaddUrrebsasn:aD-eCphta,mpoafiEgnnt,om3o2lo0gyM,orUrnililverHsailtly, fineattuhreeporfodgirreacmt oPpOtiYmiz(aWthieoen,lears i1m9p9l9)emeisnttehde
505 S. Godwin Ave., Urbana, Illinois 61801. ability to treat inserts as multistate characters
E-mail: [email protected] with as many states as there are unique insert
557
558 THE JOURNAL OF ARACHNOLOGY
sequences, with a matrix of character-state hard ticks (Acari, Ixodidae) that demonstrate
&
transformation costs. See Giribet Wheeler the evolutionary conservation of the overall
(2001) for an example of implementation of structure of the arachnid 18s gene.
the latter method. METHODS
A very different approach, and one that has —
been promoted by many authors, is the use of Taxon sampling. We sampled exemplar
secondary structure models to aid in homol- sequences from all arachnid orders and major
ogy assessment and relative weighting (Dixon spider lineages for which at least one full se-
& Hillis 1993; Kjer 2004), Recently, it has quence of the entire 18S gene was available
been proposed that elements ofrRNA second- in GenBank (http://www.ncbi.nlm.nih.gov/
ary structure can provide a basis for choice Genbaek/). To sample unusually long arachnid
between multiple models to be used in a par- 18S sequences thoroughly, we extended our
titioned Bayesian analysis (Telford et al, search to include all “partial sequences” that
2005). For this technique, bases from a variety came within 90 bases (—5% of the gene
of taxa would be compared to a secondary length) ofthe primerregions, since this region
structure model for the gene for assignment to is highly conserved across arthropods and the
“stem'' and “loop” (and possibly transitional missing length could be estimated despite lack
or ambiguous) partitions. These partitions of primary sequence. In taxa where 18S gene
would be individually tested for the most ap- length was uniform and in the —1800 base
propriate likelihood models, then subsequent- pair (= bp) length typical of arachnids, one
ly analyzed using the partitioned, multiple- individual was sampled, while all complete or
model approach. nearly-complete sequences with large (> 3
The RNAs produced by the 18S genes form bp) inserts relative to theAphonopelma sp. se-
secondary structure by base-pairing with their quence were included in the sample, since a
own complementary stretches of sequence, complete secondary structure model is avail-
forming helical structures commonly referred able for this taxon (Hendriks et al. 1988).
to as stems, while the non-pairing regions Among spiders, lineages included represen-
form loop structures that connect multiple tatives from Mygalomorphae {Aphonopelma
stems, or form the terminal turn in the self- sp., Theraphosidae), Mesothelae {Liphistius
complementary stems. Because the overall bicoloripes, Liphistiidae), Orbicularia (H.
secondary structure is functionally important gertschi, Uloboridae), “derived orb-weavers”
for the ribosome, and because two comple- {sensu Coddington 1990), {Nesticus ceilulan-
mentary changes in primary sequence are re- us, Nesticidae) and the RTA (retrolateral tibia!
quired to maintain a stem structure, ithas been apophysis) clade {sensu Coddington et al.
proposed that the stems should be treated dif- 2004) {Coelotes terrestris, Amaurobiidae)
ferently in phylogenetic analysis than the less- (see Table 1), All spider sequences were from
constrained loops (Dixon & Hillis 1993). To Genbank except for the H. gertschi data, for
do so requires the ability to tell whether nu- which new sequence data were generated in
cleotides are in a loop or a stem structure, the current study. Specimens of H. gertschi
which can be modeled using computer algo- were collected by S. Lew at the Angelo Re-
rithms that develop secondary structures serve, Mendocino County (39°43'N,
based on comparative or thermodynamic in- 123°39'W), and from Del Norte County near
formation (reviewed in Gardner & Giegerich the Oregon border (4r59'N, 123°43'W), Cal-
2004). ifornia, USA, in May 2003. Voucher speci-
Here we present a comparison of 18S struc- mens are stored in the collection of the Essig
tures sampled from arachnid orders and spider Museum of Entomology, UC Berkeley, under
lineages to assess the utility of secondary the voucher codes EMEC50654 and
structure information currently available for EMEC50993, respective—ly.
making homology judgments across arachnid Molecular methods. DNA was extracted
taxa, and for determining data partitions used from two legs from each of the H. gertschi
in model-based analyses. Included are new specimens using a Qiagen DNeasy Tissue
data from the species Hyptiotes gertschi Kit's standard protocol. The 18S gene was di-
&
Chamberlin Ivie 1935 (Araneae, Ulobori- rectly PCR-amplified in three parts using
dae), and remarkably similar sequences from primer pairs 1F-5R, 5F-9R, and 3F-7R (after
SPAGNA & GILLESPIE— 18S SECONDARY STRUCTURE 559
—
Table 1. Taxa examined, with total gene length and p-distance between primary sequence ofindivid-
uals and Aphonopelma sp. reference sequence. Length includes terminal primer regions (approximately
50 base pairs (= bp); 23 per primer + 4 bp downstream in the 3' direction from primer 9R). * indicates
partial sequences (see text), with lengths estimated by assuming uniform sequence length relative to
reference sequence at missing terminal regions.
Genbank Total Uncorrected
Accession length p-distance to
Order (lineage represented) Taxon sampled Number (bp) reference
Araneae (Orbicularia) Hyptiotes gertschi Chamber- DQO15708 1902 10.8%
lin & Me 1935 —
Araneae (Mygalomorphae) Aphonopelma sp. XI3457 1814
Araneae (Mesothelae) Liphistius bicoloripes Ono AF007104 1808 2.4%
1988
Araneae (derived orb-weavers) Nesticus cellulanus (Clerck AF005447 1816 7.8%
1757)
Araneae (RTA ciade) Coelotes terrestris (Wider AJ007986 1814 5.7%
1834)
Opiliones Odiellus troguloides (Lucas X81441 1810 6.2%
1847)
Scorpiones Androctonus australis (Lin- X77908 1812 6.7%
naeus 1758)
Pseudoscorpiones Roncus cf. pugnax (Navas AF05443 1808 11.0%
1918)
Acari (Ixodidae) Amblyomma glauerti Keir- AFl15372 1802 10.3%
ans, King & Sharrad,
1994
Solifugae Eusimonia wunderlichi Pie- U29492 1802 7.7%
per 1977
Amblypygi Paraphrynus sp. AF005445 1810 4.8%
Uropygi Mastigoproctus giganteus AF005446 1810 4.9%
(Lucas 1835)
Schizomida Stenochrusportoricensis AF005444 1809 5.5%
Chamberlin 1922
Ricinulei Pseudocelluspearsei (Cham- U91489 1813 5.1%
&
berlin Ivie 1938)
Palpigradi Eukoenenia sp. AF207648 1810 7.4%
Acari (Ixodidae) Sejus sp. AF287237 1880* 21.1%
Acari (Ixodidae) Lohmannia sp. AF287234 1887* 12.3%
Acari (Ixodidae) Alicorhagidia sp. AF022024 1872* 13.8%
Giribet et al. 1999), to amplify fragments of propriate buffers to stabilize the PCRreaction,
length —950 bp, —850 bp, and —1000 bp, which improved yield and quality of PCR
covering the first half, second half, and an products. Molecular extraction vouchers were
overlapping central portion of the gene re- stored at —80° C.
spectively. The PCR protocol (modified from Gene size, purity and concentration were
Hedin & Maddison 2001) used 35 cycles of assessed by running out a portion of the PCR
30 s at 95° C melting temperature, followed product on a 1.5% TBE/agarose gel. PCR
by 30 s at an annealing temperature of 52° C, products were cleaned using Qiagen QiaQuick
followed by an extension step of 45 s at 72° PCR Purification Kit, and cycle sequenced in
C, with 3 s added to this extension for every both directions using dye terminators (after
cycle after the first. In addition, these cycles Sanger et. al 1977). Cycle sequencing prod-
were preceded by an initial melting at 95° C ucts were analyzed using an ABI 3730 capil-
for 2 min, with a 7 min final extension at 72° lary autosequencing machine. Individual se-
C. A MasterAmp PCR Optimization Kit (Ep- quences checked against their complementary
icentre Technologies) was used to choose ap- sequences, using Sequencher 3.1.1 (Gene
560 THE JOURNAL OF ARACHNOLOGY
Codes Coiporation). This program was also model using the same method, thee calculated
used to assemble contigs of all three overlap- the percentage of base-pairs and loop mem-
ping primer regions for each H. gertschi spec- bers in the algorithm output that correctly
imen, to create a single sequence foreach. Ad- matched the model.
ditional sequences (see Table 1 for accession RESULTS
numbers) were acquired from GenBank for
Comparisons of 18S sequence length and
comparison.
Alignment and Comparisons.-—Using primary sequence divergence can be seen in
Clustal X version 1.83 (Cheena et al. 2003), Tthaeblrean1g.eMoofst18a0r0a-ch1n8i1d01b8aSsespeaqiuresnc(einsclaurdeinign
a static alignment of multiple sequences was
primer regions), with the H. gertschi and tick
created using the default settings (gap inser-
sequences being the exceptions (greater by 85
tion cost 15, gap extension cost 6.66, transi- and 56-64 bases, respectively). Figure 1
tion cost 0.5 times traesversion cost). Treating
the Aphonopelma sp, sequence and model as shows a schematic of the Hendriks et. al
(1988) model ofspider 18S, with regions with
references, gaps in the aligned sequences were
H. gertschi and tick inserts marked with black
compared to the secondary structure model to
arrows and a hatched bar, respectively.
detect regions where insertions and deletions
Hyptiotes gertschi and tick insertions.”—
have occurred.
The two H. gertschi sequences are identical,
schMio,—deIlninargeasofwiitnhseirntserrteigoinosn>s 3inbpH.relgaetritv-e palsifairceatsieoqnuseonfcetshefDreolmNaonrtaeddCiotiuonntayl sseptecoifmaemn-.
to the Aphonopelma sp. reference sequence,
However, in contrast to the marked similarity
secondary structures were modeled using the
in length and structure across the arachnid ex-
web-interface version of the RNAfold pro-
emplars from GenBank, the H. gertschi se-
gram in the Vienna Package (http://www.tbi.
quences are 86 nucleotides longer than the
univie.ac.at/~ivo/RNA/; Hofacker2003). This
next-longest spider 18S (Nesticus cellulanus),
program uses a dynamic-programming algo-
rithm with a variety of parameters to estimate and we estimate it to be 21 nucleotides longer
than the next-longest arachnid 18S (Sejus sp.,
the secondary structure based on minimizing
the free energy of the possible stem-loop Acari, Ixodidae). This addi—tional sequence is
found in two large inserts a 28-base exten-
structures from the primary sequence. The pa-
rameters used include RNA base-pairing en- sion ofthe helical arm El0-1 (Figure—2a), and
27-base extension ofEl0-2 (Fig. 2b)' andthe
ergies plus a variety of experimentally deter-
mined adjustments to this cost, including rest is found as smaller inserts of 5, 6, and 13
energy estimates forloops ofvarious sizes and nucleotides in helices 6, 41, and 47 respec-
tively (Figs. 3a, b, and c). Three near-com-
locations, and for single- and double-stranded
plete tick sequences were also found to have
multi-base motifs known to affect local sta-
large inserts in the terminal arms ofbranch 10
bility (Mathews et al. 1999).
The expanded regions of the H. gertschi (see Table 1).
Models of the H. gertschi inserts based on
18S gene were modeled using the primary se- free-energy minimization show that the in-
quence of the insert region plus the four com-
serted sequences extend the helices in all of
plementary bases at each insertion site that
could be homologized with the reference se- the cases except structure 6, where the helical
quence and structure from Aphonopelma sp. psatnedmedi.s TshhoertReNnAedfoalnddptrhoegrtearmmianlaslo cloororpecetxl-y
Because the RNAfold program had difficulty replicated 90% (40 of 48) of the stem base-
aligning the complementary 4-base-pair ends
properly in some cases, we added a comple- pairs in the Hendriks et al. (1988) model in
the arms in which the inserts were found,
mmeinntuasrtyo 5a-nncuhcolreotthiedeendesxtoefnstihoenseatqueeancche atenrd- which is comparable with the 73% base-pair-
ing accuracy for this parameter set calculated
stand in for the rest of the structure, so that
by Mathews et al. (1999) using a variety of
each primary sequence input took the form:
other well-characterized genes.
5'-GGGGG- primary sequence -CCCCC-3k
We also tested the RNAfold program's ability DISCUSSION
to predict the structures in the variable arms The most remarkable results from the cur-
ofthe Hendriks et al. (1988) Aphonopelma sp. rent study were the number of sizable inser-
SPAGNA & GILLESPIE—//yPr/OrE5 18S SECONDARY STRUCTURE 561
—
Figure L General model for secondary structure of arachnid 18S ribosomal DNA. Small numbers
refer to general stem-loop numbering system after (Nelles et al. 1984). Those features preceded by an
“E” are specific to eukaryotic 18S sequences. Arrows and bold numbers refer to locations and size (in
bases relative to tarantula model) ofH. gertschi inserts, respectively. Hatched bar represents region with
large inserts found in 3 tick taxa.
tions in the H. gertschi sequences and, in par- able across a range of taxa (Van de Peer
ticular, the shared expansion of stem ElO 1996). The RNAfold models show that much
found in both H. gertschi and ticks. InH. gert- of the inserted H. gertschi sequence is self-
schi all insertions took place in known “var- complementary and these insertions function
iable regions” of the eukaryote 18S structure; to extend the stem regions, rather than in-
helices 6, El0-1 and -2, 41, and 47 belong to crease the loop size, except in Helix 6. This
regions that have been characterized as vari- extension and maintenance of complementary
A
562 THE JOURNAL OF ARACHNOLOGY
2A7phboansoepselmasp.Helix&loop6, 3Hy2pbtiaosteessgertschiHelix&loop6,
El0-2 U GGUU *••• GACU UGUCU° GC
u UGGU :UCUGlA\UAACU •• AG GCGGA.a gC
AGGC ACCA AGAUUAUU GGGGU U,U, A. A.CrU
U c U A AACC U ACI 1 CU
G
A
Aphonopelmafeatures 10, El0-1 CCUCUAA GGCGl\CCCGAUC
andEl0-2, bases 174-267 GGAGAUU UUGCGGGCUG
C U c U
A A sAtprhuoctnuorpee4l1,ma50spb.assteesm-ioop
CU GGGAGGAGGCCGU/GC\ AA UGGU CCUCUA \UAGGCGUCCGAUCU C
A CCCUCUUCCGGCAGU ACCA GGAGAUU:, ^UCCGCAGGCUGGG C
A A ucu' \U/ G
C c A
Hyptiotesgertschi Hyptiotesgertschistem-ioop
FeatureEl0-2 structure41 56
,
7
4
CAGGU-iAcGCGGCGGG U GGUC UCG UUGGCGCUCGG CGGCC
C CCAG AGC GGCCGUGGGCC GCUGG
GCCGGCGCCGCC A UU G A \l GC
A A Aphonopelmasp.stem-loopstructure47,
Hyptiotesgertschi 64bases
FeatureEl0-1
Figures 2-4.—Region ofarachnid 18S gene with 3GUCUUCG°AC.U.G.G.C.G.C..C.C.G.G.CGC.C.G.G.G.C.G..G.A.G.G.U M ^.
unusually large inserts. 2. Basic structureforhelices :CAG A.G. GGCCGUGGGCCG GGCCCGCCUCUA c
10, El0-1 and El0-2 in arachnids. Primary se- U A^A Aa UA a MU C c
quence and model after Hendriks et al. (1988); 3. U C
Expanded El0-2 stem-loop structure in H. gertschi', Hyptiotesgertschi.Stem-loopstructure
4. Expanded El0-1 stem loop in H. gertschi. Boxes — 47,77bases
represent areas of homology where expanded arms Figures 5-7. Smaller inserts and secondary
attach to conserved, adjacent parts ofthe structure. structure model comparisons. 5. Structure 6 with 5
base pair insertion in H. gertschi; 6. Structure 41
with 6 inserted bases in H. gertschi; 7. Structure47
with 13 inserted bases in H. gertschi.
stems has remarkable parallels with the find-
ings of Hancock & Vogler (2000), who re-
vealed a similar pattern of complementary not a pseudogene or an experimental artifact.
stem expansion in the evolution of hypervari- One would expect to see decay ofpseudogene
able regions of 18S in tiger beetles. sequence relative to the selectively-con-
A variety of factors suggest the H. gertschi strained functional gene (Giribet & Wheeler
sequence represents the functional 18S rRNA 2001), and this decay (in the form of random
gene rather than a pseudogene copy or exper- mutations) should be equally distributed
imental artifact. The presence of insertions in throughout the sequence rather than restricted
areas amplified by three different primerpairs, to known variable regions. Though some
the replication of the sequence in individuals metazoans, such as helminths, are known to
from two populations, the maintenance (and, carry two functional copies of the 18S gene
in some cases, increase in length) ofthe base- with differing sequences (Carranza et al.
pairing in stem structures, and the non-ran- 1996), we found only single bands in our aga-
dom, self-complementary sequence found in rose gel visualizations of all three PCR prod-
both large and small inserts, make it likely ucts, and no second allele or double-peak pat-
that we have sequenced a functional gene and terns were seen in the raw sequence data,
SPAGNA & GILLESPIE—//FPr/Or^^ 18S SECONDARY STRUCTURE 563
despite repeating the amplification and se- While the RNA folding algorithm used may
quencing processes, suggesting the existence not predict intra-molecular folding dynamics
of a single, uniform 18s sequence in H. gert- of rRNA accurately, it provides a repeatable
schi. method for producing a plausible structure for
How exceptional is the H. gertschi 18S sequence data which lacks obvious homologs
rRNA? It seems to be an anomaly for spiders, from related taxa for comparison. The algo-
which, based on those sampled in the current rithm also gives a reasonable basis forjudging
study, generally vary little even between the whether an insert extends a helix, a loop, or
most distantly related lineages. Along with the both, particularly with short sequences such as
tick data, these anomalous sequences show those modeled in this study. With changes af-
that the presence of large inserts in arachnids, fecting sequence length concentrated in the
though rare, is consistent with a gen—eral eu- terminal ends of known stem-loop structures,
karyotic 18S evolutionary tendency overall the H. gertschi and tick 18S genes appear to
structure is maintained, while rare inserts ap- be the exceptions that prove the rule; bases
pear in known variable areas. It is worth not- come and go, albeit very rarely, but the helical
ing that only the H, gertschi gene contains backbone and location ofstem and loop struc-
RNA
expansions of several variable regions while tures of the small subunit ribosomal
the tick inserts are restricted to the ElO region. have remained conserved throughout arachnid
With only two populations of H. gertschi evolution.
sampled in the current study, we cannot es- Because the expanded sequence has only
tablish the phylogenetic distribution of the been found in a single spider species, it is im-
18S rDNA extension, whether it occurs in oth- possible to argue that this is evidence of any
er Hyptiotes species, other uloborid genera, or trend in 18S evolution in spiders. However,
their sister group, the Deinopidae. Such ex- the increase in size of the 18S gene seen here
tensions have not been described from other differs from the documented trend (relative to
Orbicularia, the sister-group of the deinopoid other metazoans) toward reduction in size in
(Uloboridae + Deinopidae) clade, but may be the hypervariable region of the mitochondrial
more widespread within the deinopoids. The 16S gene (Smith & Bond 2003) and in the
large number of inserts found in H. gertschi arms of some tRNA genes (Masta & Boore
in this study suggests that there might be var- 2004) of spiders. It should be noted that the
iation in number and size of inserts found in cited trends are in mitochondrial genes,
different taxa, since it seems unlikely that all whereas 18S is part of the nuclear genome,
these inserts appeared simultaneously. It and the two genomes may be subject to dif-
would be interesting to map any such varia- ferent selective pressures or constraints.
tion onto the existing phytogeny ofDeinopoid To understand the broader pattern of 18S
genera (Coddington 1986) to determine the size change, it is useful to look at the evolu-
pattern ofevolution ofthe 18S gene in greater tion of the 18S gene in other metazoan taxa.
detail. Alternatively, if variation seems to be Giribet & Wheeler (2001) showed that the
limited to a subset of Hyptiotes or a small general pattern of 18S evolution across a
number of uloborid genera, the inserts them- much broader sampling of taxa, including
selves could be evaluated as phylogenetic hexapods, chelicerates, myriapods, and crus-
characters within these groups. taceans, is one of conserved length in the
Once the taxonomic range and amount of 1800 bp range, with occasional increases in
variation of these inserts has been character- size. Deletion events appear to be exceedingly
ized, hypotheses about causal biological, rare. The data presented here are in keeping
physiological or ecological factors allowing with those findings, although the total length
such inserts to occur can be tested rigorously. for the H. gertschi 18S gene is greater than
Comparative studies could also be performed that of any of the 49 chelicerate taxa reported
with the wide range of metazoan taxa which by Giribet & Wheeler (2001) or of any full
show similar inserts, such as myriapods, pro- arachnid 18S sequence currently found in the
turans, and helminth worms, to find general Genbank database. Complete or nearly-com-
correlations between 18S size and phenotypes plete sequences are required for secondary
&
(Carranza et al. 1996; Giribet Wheeler structure modeling since there is long-range
2001). complementary base-pairing in 18S secondary
564 THE JOURNAL OF ARACHNOLOGY
structure (Telford et al. 2005). Unfortunately gertschi specimens, to K. Rudolph for labo-
for the pursuit ofwhole-gene comparisons and ratory assistance, and to S. Crews and S.
structural modeling, many arachnid studies Longhorn for comments on the text. This
have used only half of the 18S gene, (e.g., work was partially supported by grants from
Wheeler & Hayashi 1998; Arnedo et al. the NSF, the Walker Fund for Systematics and
2004), and there is a sizable and important the Vincent Roth Fund for Spider Systematics.
spider lineage, the haplogynae, for which no LITERATURE CITED
comApsleftoer 1t8heS osveeqruaelnlceutiilsitayvaoilfab1l8e.S data for Arnedo M.A., J. Coddington., I. Agnarsson & R.G.
arachnid phylogeny, this data set shows that Gillespie. 2004. From a comb to a tree: phylo-
genetic relationships of the comb-footed spiders
the Hendriks et al. (1988) model is sufficient (Araneae, Theridiidae) inferred from nuclearand
for locating structural changes in the 18S gene mitochondrial genes. Molecular Phylogenetics
for all known arachnid genotypes, though the and Evolution 31:225-245.
value ofmodeling based solely on primary se- Carranza, S., G. Giribet, C. Ribera, N. Baguna &
quence using thermodynamic predictions on a M. Riutort. 1996. Evidence thattwo types of 18S
single taxon is limited. In the majority ofcas- rDNA coexist in the genome of Dugesia
es, alignment is trivial with a mean of less {Schmidtea) meditarranea (Platyhelminthes, Tur-
than 1% sequence length divergence between bellaria, Tricladida). Molecular Biology and
most taxa. This similarity in sequence-length Evolution 13:824-832.
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