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2003

An Analysis of the Secondary Structure of the Mitochondrial Large Subunit rRNA Gene (16S) in and Its Implications for Phylogenetic Reconstruction

Stacey DeWitt Smith University of Nebraska - Lincoln, [email protected]

Jason E. Bond East Carolina University

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Smith, Stacey DeWitt and Bond, Jason E., "An Analysis of the Secondary Structure of the Mitochondrial Large Subunit rRNA Gene (16S) in Spiders and Its Implications for Phylogenetic Reconstruction" (2003). Faculty Publications in the Biological Sciences. 111. https://digitalcommons.unl.edu/bioscifacpub/111

This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in the Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 2003. The Journal of Arachnology 31:44±54

AN ANALYSIS OF THE SECONDARY STRUCTURE OF THE MITOCHONDRIAL LARGE SUBUNIT rRNA GENE (16S) IN SPIDERS AND ITS IMPLICATIONS FOR PHYLOGENETIC RECONSTRUCTION

Stacey D. Smith1: Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 USA Jason E. Bond: East Carolina University, Department of Biology, Howell Science Complex, Greenville, North Carolina 27858 USA

ABSTRACT. We investigated the pattern of molecular variation with respect to secondary structure in the 16S ribosomal RNA gene and its phylogenetic implications for with a focus on spiders. Based on a model by Gutell et al. (1996), secondary structures were proposed for the 3Ј half of 16S in the mygalomorph atomarius. Models were also constructed for a hypervariable length of the 16S in three other arachnids, which revealed a trend of stem and loop reduction in more advanced arachnids. Using a simple statistical approach to compare functional regions, we found that internal and external loops are more variable than stems or connection regions. Down-weighting or excluding regions which code for the more variable loops improved tree topologies by restoring the monophyly of the Aptostichus, a group supported by combined 16S, COI, and morphological data in other analyses. This study demonstrated the utility of considering secondary structure for DNA sequence alignment and phy- logenetic reconstruction in spiders. Keywords: Secondary structure, 16S rRNA gene, Aptostichus, phylogenetic utility

The most important aspect of any molecular The 16S rRNA gene, which encodes the phylogenetic study is, unequivocally, gene mitochondrial large ribosomal subunit (mt choice. Choosing a gene that strikes the ap- LSU) in , has been employed exten- propriate balance between molecular conser- sively to explore phylogenetic relationships in vation and variability is essential to the suc- at most phylogenetic levels [e.g., cess of phylogenetic reconstruction. Without ordinal (e.g., Flook & Rowell 1995), familial question the functional role of a gene and its level (e.g., Black & Piesman 1994) and the rate of evolution are tightly coupled although genus level and below (e.g., DeSalle et al. within genes, this relationship is variable be- 1992; Bond et al. 2001)]. The wide range in cause regions of a single gene can evolve at utility of 16S at various taxonomic levels sug- different rates. This tight relationship between gests that the differential rates of molecular rate of nucleotide substitution and gene com- evolution within 16S, due to varying func- ponent ``form and function'' results in a single tional constraints, greatly affect its phyloge- gene being useful at different, sometimes netic utility. Thus, understanding the function- quite disparate phylogenetic levels. Such mul- al roles of different portions of the gene tilevel phylogenetic utility, strongly tied to through secondary structure modeling should gene function (i.e., the functional secondary lead to a more robust use of 16S in phyloge- structure) is particularly true for the mito- netic studies. chondrial 16S rRNA gene in arthropods The main focus of this paper is the second- (Flook & Rowell 1995), the gene of focus for ary structure of 16S in spiders and other this paper. arachnids. One of the ®rst models of 16S sec- 1 Current address: Stacey D. Smith, Department of ondary structure in arthropods was created by Botany, 430 Lincoln Dr., University of Wisconsin, Clary & Wolstenholme (1985) using the Dro- Madison, WI 53706-1481. E-mail: sdsmith4@ sophila yakuba sequence. Subsequent models wisc.edu by Gutell and colleagues (Gutell & Fox 1988;

44 SMITH & BONDÐSPIDER 16S SECONDARY STRUCTURE 45

Table 1.ÐSpider taxa sampled.

Higher level Reference/GenBank classi®cation Species Origin accession # Heptathela nishihirai Haupt 1979 Japan Huber et al. 1993 Clubionidae pallidula (Clerck 1757) Austria Huber et al. 1993 Ctenidae Cupiennius coccineus F. Pickard-Cam- Costa Rica Huber et al. 1993 bridge 1901 Ctenidae Cupiennius getazi Simon 1891 Costa Rica Huber et al. 1993 Ctenidae Cupiennius salei (Keyserling 1877) Mexico Huber et al. 1993 Ctenidae Phoneutria boliviensis (F.O. Pickard- Costa Rica Huber et al. 1993 Cambridge 1897) Lycosidae Pardosa agrestis (Westring 1861) Austria Huber et al. 1993 Pisauridae Dolomedes ®mbriatus (Clerck 1757) Austria Huber et al. 1993 Pisauridae Pisaura mirabilis (Clerck 1757) Austria Huber et al. 1993 Antrodiaetus unicolor (Hentz 1841) Virginia AY241258 Cyrtaucheniidae Aptostichus atomarius Simon 1891 California AY241254 Cyrtaucheniidae Chamberlin 1917 (3 California AF307969, populations sampled, Bond et al. AF307964, 2001) AF307960 Cyrtaucheniidae Aptostichus sp. California AY241255 Cyrtaucheniidae Entychides arizonicus Gertsch & Wal- Arizona AY241257 lace 1936 Cyrtaucheniidae Promyrmekiaphila gertschi Schenkel California AY241256 1950

Gutell et al. 1993) were based on extensive used in this analysis, the classi®catory place- surveys of sequences as well as studies of po- ment of these taxa, and the source of their sitional covariance; these are thus considered sequences. Decisions regarding taxon choice to be more accurate and have been used in within the Mygalomorphae re¯ect an attempt recent studies modeling secondary structure in to examine taxa across a number of phyloge- arthropods (e.g. Buckley et al. 2000). Existing netic levels within this clade. secondary structure models of 16S in arach- The major objectives of this study are to: nids are limited to a tick (Black & Piesman 1) construct a secondary structure model for 1994), which used the Clary & Wolstenholme mygalomorph spiders using the preferred Gu- (1985) model and two spider species of the tell model, 2) examine trends in secondary infraorder Araneomorphae (Huber et al. 1993; structure evolution in spiders and other arach- Masta 2000), which used the Gutell model nids, 3) analyze the pattern of molecular var- (Gutell & Fox 1988). To obtain a more com- iation with respect to structure in mygalo- plete picture of 16S secondary struc- morphs and araneomorphs, and 4) assess the ture, this study will examine the secondary effects that differential weighting of molecular structure of the other ``primitive'' infraorder characters based on secondary structure has of spiders, the Mygalomorphae (Coddington on phylogeny reconstruction in spiders. This & Levi 1991), and, to a lesser extent, primi- is the ®rst study of arachnids to examine the tive liphistiid spiders, Acari (ticks) and Scor- rates of variation in 16S rRNA relative to sec- piones using the Gutell model. Spider taxa ondary structure in a rigorous statistical man- from disparate groups were examined as part ner and to analyze secondary structure trends of a concerted effort to sample across all of across the class. It is also the ®rst to consider the major clades. Table 1 summarizes the taxa the implications of secondary structure in se- 46 THE JOURNAL OF ARACHNOLOGY quence alignment and phylogenetic recon- that the location of stems and loops would be struction in arachnids. very similar because the LSU rRNA second- ary structure has been found to be widely con- METHODS served (Buckley et al. 2000; Masta 2000). The DNA extractions.ÐTotal genomic DNA A. atomarius sequence was chosen because it was extracted from leg tissue using a Pure- had the highest similarity to Drosophila, fa- gene DNA extraction kit, which comprises a cilitating comparison. lysis step in which ground tissue is incubated To examine evolution of secondary struc- in Tris-EDTA buffer with SDS and Proteinase ture in arachnids, models were also created for K for three hours, a protein precipitation step a hypervariable portion of the molecule (Fig. using potassium acetate, followed by DNA 1) in distantly related arachnid taxa: the tick precipitation in isopropanol, and a 70% etha- Ornithodoros moubata (Murray 1877) (Black nol wash. DNA was resuspended in Tris- & Piesman 1994), the scorpion Vaejovis car- EDTA buffer and diluted 1:100 for subsequent olianus (Beauvois 1805) and the mesothele reactions. Heptathela nishihirai Haupt 1979 (Huber et Mitochondrial gene PCR and sequenc- al. 1993) (Fig. 2). All structures were drawn ing.ÐThe polymerase chain reaction (PCR) using Canvas᭧ graphics software (Deneba was used to amplify the 16S rRNA gene with Systems Inc.). the 16S universal primers 16sar-5Ј (5Ј- Analysis of variability with respect to CGCCTGTTTATCAAAAACAT-3Ј) and secondary structure.ÐEach nucleotide po- 16sbr-3Ј (5Ј-CCGGTCTGAACTCAGAT- sition was assigned a single letter designating CACGT-3Ј) (Hillis et al. 1996). The primers its structural function (S ϭ stem, I ϭ internal 16sar-5Ј and 16sbr-3Ј correspond to Drosoph- loop, L ϭ external loop, C ϭ connecting re- ila melanogaster mitochondrial genome posi- gion) and was coded as variable (1) or in- tions 13398 and 12887 respectively. Standard variable (0). In this study, stems refer to a se- PCR reactions were carried out in 50 ␮l vol- ries of bonded nucleotides. Internal loops are umes and run for 35 cycles, each consisting those unbonded nucleotides, which occur of a 30 sec denaturation at 95 ЊC, 30 sec an- within a stem; external loops occur at the end nealing at 50ЊC and 45 sec (ϩ3 sec/cycle) ex- of stems. Connecting regions link stems. tension at 72ЊC, with an initial denaturation Variation was analyzed separately for the step of 95ЊC for 2.5 min and a ®nal extension two spider infraorders, Mygalomorphae and step of 72ЊC for 10 min. Ampli®cation prod- Araneomorphae. For each structural category, ucts were electrophoresed on a 0.8% agarose variability was calculated by dividing the gel, excised from the gel and puri®ed using number of variable positions by the total num- Qiagen QIAquick gel extraction columns. Pu- ber of positions. Statistical analyses were per- ri®ed products were sequenced from both di- formed using SAS (SAS Statistical Systems). rections with an ABI PRISM 377 and 310 au- To compare the pattern of variation across tomated sequencers using the ABI PRISM structural regions to random variation within Dye Terminator Cycle Sequencing Ready Re- the molecule, the percent variability of ran- action Kit with Amp1iTaq DNA Polymerase, dom blocks within the sequence was com- FS. pared to the percent of actual variability with- Secondary Structure.ÐMygalomorph 16S in structural units (S, I, L, C). A simple sequences generated for six taxa were crudely random number generator program written for aligned (alignment of stem regions was im- Mathematica (Wolfram 1996) produced nucle- proved by eye once the secondary structure otide blocks in lengths of 5 or 13 consecutive was obtained) with sequences from eight ar- numbers between 1 and 503 base pairs. The aneomorph taxa (Table 1) and Drosophila me- block size corresponds to the maximum and lanogaster using ClustalX (Higgins et al. minimum average sizes of the structural re- 1996). The 16S rRNA secondary structure gions, and the random numbers corresponded was predicted for the mygalomorph Aptosti- to positions along the molecule. The mean chus atomarius Simon 1891 (Fig. 1) by com- variability in the random blocks provided the parison with sequences and models from Dro- expected values for a Chi-Squared Test to de- sophila yakuba (Gutell et al. 1993) and an termine if the pattern of variability was dis- araneomorph (Huber et al. 1993). We assumed tinct from the random model. SMITH & BONDÐSPIDER 16S SECONDARY STRUCTURE 47

Figure 1.ÐProposed secondary structure model for the 3Ј half of 16S of the mygalomorph species Aptostichus atomarius based on the Gutell et al. model (1993). Highly conserved regions indicated by bold-lettered nucleotides. Dashes represent Watson-Crick bonds; circles are U-G bonds. The hypervariable area examined in the study and modeled in Figure 3 is corresponds to nucleotide positions 237±359. 48 THE JOURNAL OF ARACHNOLOGY

Figure 2.ÐProposed secondary structures of a hypervariable region of 16S based on Gutell et al. model (1993) for 4 Arachnid taxa. Solid lines indicate tertiary interaction with strong comparative data and dashed lines indicate those with less support (Gutell 1996). (a) Aptostichus atomarius (Mygalomorphae) (b) Ryuthela nishihirai (Mesothelae) (c) tick Ornithodoros moubata (d) scorpion Vaejovis carolianus.

Phylogenetic analysis.ÐPhylogenetic erwise, gaps were treated as missing charac- analyses were performed using PAUP* ver- ters. sion 4.0b2a (Swofford 1999) run on a Power Heuristic searches were performed using Macintosh 6500/275. The phylogenetic signal random addition stepwise (1000 replicates) of in the data set was evaluated using the g1 sta- taxa followed by TBR (tree bisection-recon- tistic (Hillis & Huelsenbeck 1992) based on nection) branch swapping. Branches with a 100,000 random trees generated in PAUP*. maximum length of zero were collapsed. Mea- All characters were treated as reversible, un- sure of branch support is based on decay (Bre- ordered, and all characters were initially mer 1988; Donoghue et al. 1992) and boot- weighted equally. Unambiguous gaps were strap analyses (Felsenstein 1985). Decay scored as binary characters. These binary indices were computed using the computer scorings were retained and the individual nu- program Autodecay (Eriksson & Wikstrom cleotide positions from which they were 1996). Bootstrap values are based on 500 rep- scored were excluded from the analysis. Oth- licates using strict parsimony in PAUP*. SMITH & BONDÐSPIDER 16S SECONDARY STRUCTURE 49

RESULTS Table 2.ÐSummary of genetic variability with- in each structural category. Secondary structure model.ÐA 503 base pair segment of the 3Ј half of 16S was ob- Mygalomorphs Araneomorphs tained for 6 mygalomorph taxa. Figure 1 shows the proposed secondary structure for Number Per- Number Per- the mygalomorph Aptostichus atomarius of cent of cent posi- vari- posi- vari- based on the Gutell model. While the myga- Structure tions able tions able lomorph model shares substantial physical similarity with the araneomorph and Drosoph- Stems 65 32% 54 24% ila models, the underlying RNA sequences Inner loops 62 45% 62 66% Loops 103 51% 99 46% vary greatly except in some highly conserved Connecting 217 38% 235 32% regions (Fig. 1). The location of these con- served regions serve as anchor points for es- tablishing the molecule's overall secondary structure. The structural similarity among the models has been maintained through compen- are most reduced in Aptostichus although the satory changes and by slight shifting of struc- ``AUU'' sequence in the loop is conserved in tural regions to accommodate nucleotide sub- all arachnids sampled. The importance of this stitutions. sequence is supported by Buckley et al. Structural evolution in the hypervariable (2000), who found it to be conserved through- region.ÐAlignment of 16S sequences from out insect taxa. Finally, the length of connec- the various arachnid taxa sampled revealed a tor 7 is fairly conserved, ranging from 14 nu- highly variable length of DNA; models of this ``hypervariable'' region showed marked vari- cleotides in Heptathela to 20 in the tick. ation in the size of loops and the length of Pattern of variation with respect to struc- stems (Fig. 2). Interestingly, bases identi®ed ture.ÐTable 2 summarizes the pattern of var- as highly conserved (Buckley et al. 2000) are iation within the 16S molecule for mygalo- found adjacent to and even within this region. morphs and araneomorphs. A Wilcoxon Rank Evolution in the hypervariable segment of Sum Test shows that the variability of each 16S (Fig. 2) reveals an overall trend towards structural class (S, I, L, C) is statistically dif- reduction in more advanced arachnids. We ferent for both mygalomorphs (P Ͻ 0.05) and will describe this trend moving clockwise araneomorphs (P Ͻ 0.0001). through the region (Fig. 2). The models dem- The mean observed variability of the four onstrate extreme variability in loop 1, which structural classes differed from both the small varies in length from 9 nucleotides in Aptos- (expected (e) ϭ 0.29, X2 ϭ 40.34, P Ͻ 0.005) tichus to 30 in Ornithodoros (tick). The stem and large (e ϭ 0.33, X2 ϭ 26.93, P Ͻ 0.005) supporting this loop is variable as well, rang- random blocks for the araneomorphs. How- ing from 3 base pairs in Aptostichus and Hep- ever, the mean variability values for mygalo- tathela to 11 base pairs in the tick. The con- morphs were not statistically distinct from the necting region 2 is short in spiders as it is in random model (small blocks: e ϭ 0.42, X2 ϭ humans and Drosophila but is longer in Vae- 4.49, P Ͼ 0.10; large blocks: e ϭ 0.43, X2 ϭ jovis (scorpion) and the tick than in spiders. 4.68, P Ͼ 0.10). Stem 3, which contains the sequence of four Phylogeny reconstruction.ÐWe consider G's common to all organisms, is slightly the phylogenetic signal in this data set to be shorter in the spiders than in the tick or the signi®cant (g1 ϭϪ0.55, P Ͻ 0.01). A strict scorpion. Area 4 consists of approximately the parsimony analysis with all positions weight- same number of nucleotides in the arachnids ed equally resulted in two equally parsimo- sampled; the morphological differences result nious trees (Figs. 3a & b), 719 steps in length from different amounts of base pairing. Re- (CI ϭ 0.61, RI ϭ 0.66). Subsequent analyses gion 5 is maintained by two sets of bonds, with loop and inner loop positions ®rst down- which uphold the structural integrity of the weighted to 0.20 and then excluded each re- stem but permit suf®cient freedom for tertiary sulted in one most parsimonious tree with interactions. The stem and loop of region 6 slightly improved CI and RI values (Fig. 4). 50 THE JOURNAL OF ARACHNOLOGY

Figures 3 A, B.ÐTwo equally parsimonous trees based on 16S sequences in which all positions are weighted equally, both 719 steps in length (CI ϭ 0.61, RI ϭ 0.66). ``C.'' stands for the genus Cupiennius and ``A.'' for the genus Aptostichus. Locations for A. simus populations are Leo Carillo State Beach, Los Angeles County, CA (lcn), Sycamore Cove Beach, Ventura County CA (sc) and Zuma Beach County Park, Los Angeles County, CA (zb).

DISCUSSION ogy by identifying conserved regions are most Secondary structure.ÐWith the exception likely to re¯ect true homology and thus cor- of a few hypervariable regions of the mole- rect gene phylogeny, a consideration that may cule, the mygalomorph secondary structure not be re¯ected in alignment approaches that bears close resemblance to those structures invoke overall similarity (e.g., Higgins et al. proposed for prokaryotes (Gutell 1996; Larsen 1996) or concurrent optimizations of align- 1992), insects (Buckley et al. 2000), and ar- ment and phylogenetic inference (e.g., Wheel- aneomorphs (Huber et al. 1993). This supports er & Gladstein 1994). the ®ndings of Wheeler & Honeycutt (1988), Trends in secondary structure evolution which demonstrated that Darwinian selection in the hypervariable region.ÐExamination must be operating strongly on these genes to of the hypervariable region (Fig. 2) suggests maintain the functional aspects of secondary a general trend toward the reduction of stems structure. and loops in Araneae. The Aptostichus and The alignment of DNA sequence is com- Heptathela models exhibit a large deletion in putationally dif®cult (Swofford et al. 1996: the stem and loop of region 1 (Fig. 2), similar Slowinski 1998). Comparison of the mygalo- to that observed by Masta (2000) in Habron- morph model to other secondary attus oregonensis (Peckham & Peckham structures facilitated the identi®cation of con- 1888). The size of region 1 in the scorpion served areas, which were integral to improv- and the tick (39 and 53 nucleotides, respec- ing the alignment (Buckley et al 2000). Ri- tively) is an intermediate between Drosophila bosomal RNA sequences add another level of (55 nucleotides) and Aptostichus (15 nucleo- complexity to the problem of sequence align- tides). This suggests a pattern of evolution to- ment because nucleotide site position align- wards reduction of region 1, a possible syna- ment is determined by secondary structure po- pomorphy of spiders. However, based on sition homology. Sequence alignments that Wheeler & Hayashi's (1998) chelicerate phy- account for true molecular structural homol- logeny, additional 16S sequence data are re- SMITH & BONDÐSPIDER 16S SECONDARY STRUCTURE 51 quired for at least palpigrades, amblypygids, rRNA secondary structure for phylogenetic and schizomids before this can be stated un- reconstruction. Differentially weighting seg- equivocally. The main exceptions to this re- ments of the 16S rRNA based on their struc- ductionary trend are those areas involved in tural properties prevents the overemphasis of tertiary interactions, such as region 5 and con- homoplasic nucleotide positions. This was necting region 7 (Fig. 2). The size of these most evident for the mygalomorph taxa. areas varies little from Drosophila to humans Down-weighting the variable loops and inner to arachnids. loops improved the phylogenetic tree, which The extreme variability of region 1 in spi- recovered Aptostichus as a monophyletic ders may be due to its position in the tertiary group (47), a grouping supported by com- structure of the fully-formed ribosome. When bined analyses of morphological and molec- addressing why a certain segment of a ribo- ular data (Bond 1999; Bond & Opell 2002). somal subunit is more variable than others, This ability to ®ne tune a data set, omitting or Simon et al. (1994) proposed that it is impor- differentially weighting hypervariable regions, tant to consider the position of the segment in may allow 16S sequence data to be applied the molecule. Peripheral secondary structure more broadly. At the population level, highly elements tend to be more variable than those variable loops would provide phylogenetic on the interior and more frequently undergo signal while those same loops could be dis- modi®cation or elimination. The hypervaria- regarded for comparison of intermediate or bililty of this portion of 16S (Fig. 2) suggests very disparate taxa in favor of more con- that it is probably located on the periphery of served stems or connecting regions. the molecule. Because this study utilizes only the 3Ј half Pattern of variation with respect to struc- of the 16S, application of our differential ture.ÐWithin the Mygalomorphae and Ara- weighting scheme may require adjustment neomorphae, both the stem (S) and connecting when considering the entire molecule. Simon (C) regions exhibited lower variability than (1991) pointed out that smaller short range the loops (I, L), although this difference was stems (terminal stems) vary substantially most distinct in the araneomorphs (Table 2). more than long range stems (supporting Because stems must base pair to maintain stems) and can evolve as rapidly as some their integrity, changes that disrupt base pair- loops. Simon (1991) suggested that, if differ- ing along the stem will be less likely to occur. ential weighting were employed, these two Connecting regions are probably more con- types of stems should be distinguished and the served because they have a vital function in rapidly evolving shorter stems should be maintaining the spatial arrangement of the down-weighted. In the segment of 16S used molecule. Loops are less constrained as in this study, there were 16 short range stems changes in their sequence will not interrupt and only one strand of two long range stems. base pairing and at most will threaten tertiary Therefore, the mean variability for stems interactions. Additionally, changes in length could be considered representative of short- of the loops will have a small effect on the range stems. Perhaps, if the system of differ- overall form of the molecule. This disparity in ential weighting used in this study were em- variability between stems and loops is differ- ployed on a longer sequence of LSU rRNA, ent from what has been observed for other the two stem types would have to be distin- ribosomal RNA molecules. Wheeler & Ho- guished. neycutt (1988) found stems to be more vari- Summary.ÐThe majority of sequence able than loops for 5.8S rRNA, the small nu- based phylogenetic studies rely solely on clear subunit (SSU). They suggest that this DNA sequence alignment algorithms and was due to the compensatory nature of nucle- weighting schemes based on nucleotide com- otide base changes in the stems. Mutations in positions. Not surprisingly, this study dem- one base pair would result in a selection pres- onstrates that greater understanding of the sure for changes in the other, causing stem processes underlying nucleotide sequence nucleotide pairs to evolve in concert. evolution will increase the likelihood of re- Effects of differential weighting of struc- covering the correct phylogenetic relation- tural regions.ÐThe results of this study dem- ships. We show that at very ``deep'' phylo- onstrate the importance of considering 16S genetic levels gross secondary structure, the 52 THE JOURNAL OF ARACHNOLOGY

Figure 4.ÐTree produced when loop and inner loop positions of 16S are down-weighted to 0.20 (520.8 steps, CI ϭ 0.63, RI ϭ 0.69) and when loops and inner loops were excluded (471 steps, CI ϭ 0.63, RI ϭ 0.70). Bootstrap values and decay indices refer to the tree with loops downweighted.

morphology of the molecule, may very well tical approach employed may be useful for contain phylogenetic information useful in re- studies of secondary structure in other groups. constructing arachnid relationships. At more intermediate phylogenetic levels (inter-famil- ACKNOWLEDGMENTS ial) we show that by down-weighting loop nu- This study formed the 1999 undergraduate cleotides phylogenetic signal may be im- honor's thesis project for the ®rst author, who proved. Although the conclusions drawn here wishes to express her gratitude to several may be limited to arachnid studies, the statis- members of the Virginia Tech Biology de- SMITH & BONDÐSPIDER 16S SECONDARY STRUCTURE 53 partment who served on the thesis committee: the reconstruction of the phylogeny of an insect A. Schultheis, A. L. Buikema, A. C. Hen- order (Orthoptera). Molecular Phylogenetics and dricks and B. D. Opell. This project was Evolution 8:177±192. supported by NSF grant DEB 9700814 to J. Gutell, R.R. 1996. Comparative sequence analysis Bond and B. Opell. and the structure of 16S and 23S rRNA. Pp. 111± 128. In Ribosomal RNA: Structure, Evolution, LITERATURE CITED Processing, and Function in Protein Biosynthe- Black, W.C. & J. Piesman. 1994. Phylogeny of sis. (R. A. Zimmermann & A. E. Dahlberg, eds.). hard- and soft-tick taxa (Acari: Ixodida) based CRC Press, New York. on mitochondrial 16S rRNA sequences. Proceed- Gutell, R.R. & G.E. Fox. 1988. A compilation of ings of the National Academy of Sciences, USA large subunit RNA sequences presented in struc- 91:10034±10038. tural format. Nucleic Acids Research 16:R175± Bond, J.E., & B.D. Opell. 2002. Phylogeny and tax- R313. onomy of the genera of south-western North Gutell, R.R., M.N. Schnare & M.W. Gray. 1993. A American Euctenizinae trapdoor spiders and their compilation of large subunit (23S and 23S-like) relatives (Araneae: Mygalomorphae, Cyrtauch- ribosomal RNA structures. Nucleic Acids Re- eniidae). Zoological Journal of the Linnean So- search 21:3055±3074. ciety 136:487±534. Higgins, D.G., A.J. Bleasby & R. Fuchs. 1996. Bond, J.E., M.C. Hedin, M.A. Ramirez, & B.D. Clustal V: Improved software for multiple se- Opell. 2001. Deep molecular divergence in the quence alignment. Computer Applied Bioscience absence of morphological and ecological change 8:189±191. in the Californian coastal dune endemic trapdoor Hillis, D.M. & J.P. Huelsenbeck. 1992. Signal, spider Aptostichus simus. Molecular Ecology 10: noise and reliability in molecular phylogenetic 899±910. analyses. Journal of Heredity 83:189±195. Bond, J.E. 1999. Systematics and Evolution of the Hillis, D.M., B.K. Mable, A. Larson, S.K. Davis & Trapdoor Spider Genus Aptostichus Simon (Ar- E.A. Zimmer. 1996. Nucleic Acids IV: Sequenc- aneae: ). Ph.D. Dissertation, Depart- ing and cloning. Pp. 321±385. In Molecular Sys- ment of Biology, Virginia Polytechnic Institute tematics. (D.M. Hillis, C. Moritz & B.K. Mable, and State University. eds.). Sinauer, Sunderland. Bremer, K. 1988. The limits of amino acid sequence Huber, K.C., T.S. Haider, M.W. Miller, B.A Huber, data in angiosperm phylogenetic reconstruction. R.J. Schweyen & F.G. Barth. 1993. DNA se- Evolution 42:795±803. quence data indicates the polyphyly of the family Buckley, T.R., C. Simon, P.K. Flook, & B. Misof. Ctenidae (Araneae). Journal of Arachnology 21: 2000. Secondary structure and conserved motifs 194±201. of the frequently sequenced domains IV and V Larsen, N. 1992. Higher order interactions in 23S of the insect mitochondrial large subunit rRNA rRNA. Proceedings of the National Academy of gene. Insect Molecular Biology 9:565±580. Sciences, USA 89:5044±5048. Clary, D.O. & D.R. Wolstenholme. 1985. The ri- Masta, S.E. 2000. Mitochrondrial sequence evolu- bosomal RNA genes of Drosophila mitochon- tion in spiders: Intraspeci®c variation in tRNAs drial DNA. Nucleic Acids Research 13:4029± lacking the T␺C arm. Molecular Biology and 4045. Evolution 17:1091±1100. Coddington, J.A. & H.W. Levi. 1991. Systematics Simon, C. 1991. Molecular systematics at the spe- and evolution of spiders (Araneae). Annual Re- cies boundary: exploiting conserved and variable view of Ecology and Systematics 22:565±592. regions of the mitochondrial genome via direct DeSalle, R., J. Gatesy, W. Wheeler, & D. Grimaldi. sequencing from enzymatically ampli®ed DNA. 1992. DNA Sequences from a fossil termite in Pp. 33±71. In Molecular Techniques in Taxono- Oligo-Miocene amber and their phylogenetic im- my. (G.M. Hewitt, A.W.B. Johnston & J.P.W. plications. Science 257:1933±1936. Young, eds.). NATO Advanced Studies Institute, Donoghue, M.J., R.G. Olmstead, J.F. Smith, & J.D. H57, Springer, Berlin. Palmer. 1992. Phylogenetic relationships of Simon, C., F. Fratl, A. Beckenbach, B. Crespi, H. Dipscales based on rbcL sequences. Annals of Liu & P. Flook. 1994. Evolution, weighting, and the Missouri Botanical Garden 79:333±345. phylogenetic utility of mitochondrial gene se- Eriksson, T. & N. Wikstrom. 1996. AutoDecay. quences and a compilation of conserved Poly- Stockholm University, Stockholm. merase Chain Reaction primers. Annals of the Felsenstein, J.H. 1985. Con®dence limits on phy- Entomological Society of America 87:651±686. logenies with a molecular clock. Systematic Zo- Slowinski, J.B. 1998. The number of multiple ology 34:152±161. alignments. Molecular Phylogenetics and Evo- Flook, P.K. & C.H.F. Rowell. 1995. The effective- lution 10:264±266. ness of mitochondrial rRNA gene sequences for Swofford, D.L. 1999. PAUP*: Phylogenetic Anal- 54 THE JOURNAL OF ARACHNOLOGY

ysis Using Parsimony (and other methods). Sin- eny of the extant chelicerate orders. Cladistics auer, Sunderland. 14:173±192. Swofford, D.L., G.J. Olsen, P.J. Waddell & D.M. Wheeler, W.C. & R.L. Honeycutt. 1988. Paired se- Hillis. 1996. Phylogenetic inference in molecular quence difference in ribosomal RNAs: evolution- systematics. Pp. 407±514. In Molecular System- ary and phylogenetic implications. Molecular Bi- atics. (D.M. Hillis, C. Moritz & B.K. Mable, ology and Evolution 5:90±96. eds.). Sinauer, Sunderland. Wolfram, S. 1996. Mathematica. Cambridge Uni- Wheeler, W.C. & D. Gladstein. 1994. MALIGN: A versity Press, Cambridge. multiple sequence alignment program. Journal of Heredity 85:417. Manuscript received 13 November 2000, revised 26 Wheeler, W.C. & C.Y. Hayashi. 1998. The phylog- February 2002.