Table Of ContentA tamerican museum
Novitates
PUBLISHED BY THE AMERICAN MUSEUM OF NATURAL HISTORY
CENTRAL PARK WEST AT 79TH STREET, NEW YORK, NY 10024
Number 3652, 10 pp., 2 figures June 25, 2009
Mitochondrial Intergenic Spacer in Fairy Basslets
(Serranidae: Anthiinae) and the Simultaneous
Analysis of Nucleotide and Rearrangement Data
WM. LEO SMITH,1-2 KATHLEEN R. SMITH,2 AND WARD C. WHEELER3
ABSTRACT
We present the results of a study that implements a recently developed phylogenetic algorithm
that combines fixed-states nucleotide optimization with breakpoint analysis to identify and
examine the evolution of a mitochondrial intergenic spacer between the tRNAVal and 16S rRNA
loci in a clade of fairy basslets (Serranidae: Anthiinae). The results of the analysis indicate that this
spacer evolved once and that it may be increasing in size through evolutionary time. The resulting
molecular hypothesis corroborates much of the previous morphological phylogenetic work.
INTRODUCTION genetic elements and genomic rearrange¬
ment data. Therefore, we have used a recent¬
During recent investigations focusing on the ly developed genomic algorithm (Wheeler,
relationships of “mail-cheeked” fishes and sea 2007) to examine the evolution of this anthiine
basses (Smith and Wheeler, 2004, 2006; Smith intergenic spacer and the relationships within
and Craig, 2007), we discovered a mitochon¬ anthiine serranids. We then discuss how this
drial intergenic spacer in a clade of anthiine method can be expanded beyond this com¬
sea basses. After surveying the analytical paratively simple example to simultaneously
literature, we were concerned about the analyze genomic and sequence data in a
subjectivity of the methods that have been phylogenetic framework. During the last 20
previously used to identify and analyze novel years, evolutionary biologists have made
1 Department of Zoology, Field Museum of Natural History, 1400 South Lake Shore Drive, Chicago, IL 60605, USA
(lsmith@fieldmuseum. org).
2 Department of Ichthyology, American Museum of Natural History, Central Park West at 79th Street New York, NY
10024, USA ([email protected]).
3 Division of Invertebrate Zoology, American Museum of Natural History, Central Park West at 79th Street, New York,
NY 10024, USA ([email protected]).
Copyright © American Museum of Natural History 2009 ISSN 0003-0082
2 AMERICAN MUSEUM NOVITATES NO. 3652
tremendous advances by combining innova¬ sequenced mitogenomes passes 2000, there
tions in automated DNA sequencing and will be continued discovery of novel and
computational biology. This progress has convergent rearrangements; these mitoge-
provided the data necessary to test evolution¬ nomic rearrangements foreshadow the com¬
ary hypotheses using complete genomic se¬ plications that will be encountered as research¬
quences. Animal mitochondrial genomes (mi- ers begin to sequence the larger prokaryo¬
togenomes) represent the vast majority of tic genomes and eukaryotic plastid and
complete genomic data. As the number of nuclear genomes. The realization that these
sequenced mitogenomes increases, so does our rare mitogenomic rearrangements may arise
understanding of their organization and func¬ through convergent evolution (Mindell et al.,
tion. These studies confirm previous sugges¬ 1998; Curole and Kocher, 1999; Dowton et
tions that animal mitogenomes are largely al., 2003) or be complicated by recombination
conserved in terms of gene order and content (Piganeau et al., 2004) challenges the assump¬
(Boore and Brown, 1998; Miya et al., 2005), tions upon which previous analyses of these
but they also challenge previous assumptions genomic characters were based. Most previous
of mitochondrial genome evolution (e.g., the studies made a priori assumptions of locus
lack of recombination; Piganeau et al., 2004). homology based on sequence similarity, per¬
Although most sequenced animal mitogen¬ ceived secondary structure, or tRNA antico¬
omes (primarily vertebrate) share a particular don sequences (Dowton et al., 2003; Mabuchi
gene order, rearrangements and other trans¬ et al., 2004; Miya et al., 2005). None of these
formations have been described in a variety of methods use a historical, evolutionary frame¬
animal groups (e.g., amphibians, squamates, work for inferring or testing homology.
teleosts, hymenopterans [Macey et al., 1997; Investigators using these nonphylogenetic
Dowton et al., 2003; Miya et al., 2005]). These approaches for identifying homologous loci
transformations include dramatic reductions will be misled by convergent evolution. For
in genomic size in chaetognaths (Helfenbein et example, Rawlings et al. (2003) showed that
al., 2004) to increases in size ranging from tRNALeu had convergently altered its antico¬
small tandem repeats (Moritz et al., 1987) to don sequence at least seven times within
large-scale duplications (McKnight and Sha¬ metazoans. Therefore, it is clear that homol¬
ffer, 1997). The consequences of these dupli¬ ogy assessment should be explicitly tested—
cations range from multiple functional copies not assumed—in a comparative, evolutionary
of loci undergoing concerted evolution framework using all available evidence. When
(Kumazawa et al., 1996) to rearrangements these scenarios are not quantitatively tested,
(Macey et al., 1997; Dowton et al., 2003; Miya they are prone to investigator bias or subop-
et al., 2005) to intergenic spacers (McKnight timal explanations. This homology-assessment
and Shaffer, 1997; Bakke et al., 1999; Ma- problem will only be exacerbated as we move
buchi et al., 2004). Many authors have argued from the simple animal mitogenomes to the
that the assumed uniqueness and rarity of larger prokaryotic genomes and eukaryotic
these genome-level transformations make plastid and nuclear genomes. Sankoff and
them more reliable than nucleotide transfor¬ Blanchette (1998), recognizing the need for
mations for inferring deeper phylogenetic explicit methods to infer phylogenies using
relationships (Moritz et al., 1987; Curole and genomic rearrangement data, developed
Kocher, 1999). However, as additional mito¬ breakpoint analysis, which infers phylogene¬
genomes are published, it is clear that these tic relationships from locus order data. Un¬
transformations are more common than once fortunately, breakpoint analysis ignores se¬
believed (Curole and Kocher, 1999; San quence variation and requires that locus
Mauro et al., 2006), and, more importantly, homology be established prior to the analysis.
it is striking how frequently these rearrange¬ As mentioned above, this locus homology
ments occur in “hotspots” between transfer assessment is difficult for all but the simplest
RNAs, often evolving repeatedly within large cases. Recently, Wheeler (2007) described and
clades (Dowton et al., 2003; San Mauro et al., implemented an algorithm in POY (Wheeler et
2006). It seems clear that as the number of al., 2003) that combines a breakpoint algo-
2009 SMITH ET AL.: MITOCHONDRIAL SPACER IN FAIRY BASSLETS 3
rithm with a fixed-states nucleotide algorithm subfamily Anthiinae. The relationships of the
(Wheeler, 1999) to optimize chromosomal and sea basses (Serranidae and Epinephelidae)
nucleotide data simultaneously for inferring have received substantial attention (Johnson,
locus and nucleotide homology during phylo¬ 1983; Baldwin and Smith, 1998; Craig and
genetic analysis. This method does not assume Hastings, 2007; Smith and Craig, 2007), but
locus homology or nucleotide alignment prior the only explicit phylogenetic study, to date, of
to analysis; it requires only that the boundar¬ the Anthiinae is that of Baldwin (1990), who
ies of the loci be assigned (e.g., using start and examined the relationships among American
stop codons for protein-coding sequences). anthiines. Here, we report and characterize a
Using evidence from the sequence data, this vertebrate intergenic spacer discovered in H-
algorithm will infer homologous loci and strand transcription unit one (HI). Although
simultaneously analyze both the nucleotide comparatively rare, an HI rearrangement has
and genomic variation to recover the optimal been noted in some toadfishes (Miya et al.,
phylogenetic relationships and hypothesize 2005). The HI includes tRNAphe, tRNAVal,
both nucleotide and locus homology in an and both ribosomal RNAs (Fernandez-Silva
evolutionary framework. Furthermore, this et al., 2003). Within HI, this anthiine interge¬
method is an improvement over current nic spacer evolved between tRNAVal and the
similarity-based or function-based approaches 16S large ribosomal subunit (fig. 1). The
because it is automated, internally consistent, remaining 24 heavy-strand loci are transcribed
repeatable, explicit, does not presuppose a in H-strand transcription unit two (H2), along
mechanism for resulting rearrangements, al¬ with some transcriptional overlap with HI.
lows evaluation of competing hypotheses, and Given the smaller size and 20-fold greater
is grounded in a phylogenetic framework. transcription rate of HI (Fernandez-Silva et
Herein, we use this new method to examine al., 2003), this first described HI intergenic
the evolution of this novel seabass intergenic spacer may affect its posttranscriptional pro¬
spacer. Typically, vertebrate mitogenomes cessing.
have two large intergenic spacers: the origin
of light-strand replication, which has been lost MATERIALS AND METHODS
in some snakes, crocodiles, and birds (Ma-
ACQUISITION OF NUCLEOTIDE
cey et al., 1997), and the control region
SEQUENCES AND TAXON SAMPLING
(Fernandez-Silva et al., 2003). Additional
intergenic spacers are rare in vertebrate Fish tissues were preserved in 80% ethanol
mitogenomes, but have been described be¬ or were used fresh prior to extraction of DNA.
tween tRNAThr and tRNAPro in a variety of Genomic DNA was extracted from mus¬
vertebrate clades, e.g., Xenopus (Roe et al., cle using a DNeasy Tissue Extraction Kit
1985), Struthio (Harlid et al., 1997), ambysto- (Qiagen) following the manufacturer’s proto¬
matid salamanders (McKnight and Shaffer, col. Polymerase chain reaction (PCR) was
1997), and gadiform fishes (Bakke et al., used to amplify three overlapping frag¬
1999). McKnight and Shaffer (1997) showed ments crossing three rDNA fragments (12S,
that the salamander intergenic spacer had the tRNAVal, and 16S) and the intergenic spacer,
highest substitution rate among sequenced when present (fig. 1). Multiple overlapping
regions of the salamander mitogenome (in¬ primer pairs were used to ensure that we were
cluding the control region). Furthermore, amplifying the intergenic spacer region in the
these authors noted that the persistence of mitochondrial genome instead of amplifying a
this intergenic spacer contradicted Rand’s nuclear pseudogene. Double-stranded ampli¬
(1993, 2001) hypotheses that mitogenomic size fications were performed in a 25 pL volume
is minimized over time. Additional mitoge¬ containing one Ready-To-Go PCR bead
nomic modifications and rearrangements have (Amersham Bio sciences), 1.25 pL of each
also been described and discussed by Mueller primer (10 pM) and 2-5 pL of DNA. To
and Boore (2005) for other salamander taxa. amplify and sequence the tRNAVal, intergenic
The intergenic spacer described in this study is spacer (when present), and 5' end of 16S
restricted to a subset of sea basses in the fragment, the primers 12SL13-L 5'-TTA-
4 AMERICAN MUSEUM NOVITATES NO. 3652
A
Plectranthias
16S Large Ribosomal Subunit V 12S Small Ribosomal Subunit dip <
-12L13-Tltusl
_16L2A-165H10
16Sar-16Sbr
B
Nemanthias
16S Large Ribosomal Subunit i... V 12S Small Ribosomal Subunit IBS
12L13-TitusI
16L2A-16SH10
16Sar-16Sbr
Fig. 1. Illustration of partial HI in anthiine sea basses that (A) lack or (B) have evolved the intergenic
spacer (IGS = intergenic spacer, V = tRNAVal) with overlapping primer pairs used to amplify
DNA mapped.
GAAGAGGCA AGTCGT AACATGGTA - 3' DNA sequences from the complementary
and Titusl-H 5'-GGTGGCTGCTTTTAGG- heavy and light strands. Sequences were edited
CC-3' (Titus, 1992; Feller and Hedges, 1998) in Sequencher and Bioedit (Hall, 1999). All
were used. To amplify the middle section of novel sequences were deposited in GenBank
the 16S fragment, the primers 16SL2A-L 5'-C- under accession numbers FJ548764-FJ548784.
C A A ACG AGCCT AGT GAT AGCT GGTT - 3' The remaining three sequences (Myripristis
and 16SH10-H 5' -TGATTACGCTACCTTT- berndti [NC 003189], Caranx melampygus
GCACGGT-3' (Hedges, 1994) were used. To [NC 004406], and Holanthias chrysostictus
amplify the final section of the 16S fragment, [AY141436]) were taken from GenBank and
the primers 16S ar-L 5'-CGCCTGTTTATC- were derived from studies by Miya et al. (2005)
AAAAACAT-3' and 16S br-H 5'-CCGG- and Chen et al. (2003). Mitochondrial DNA
TCTGAACTCAGATCACGT-3' (Kocher et sequences were analyzed for 18 serranid and
al., 1989; Palumbi, 1996) were used. Ampli¬ epinephelid species and three outgroups
fications for all fragments were carried out in 36 (Myripristis, Caranx, and Acanthistius). We
cycles using the following temperature profile: focused our taxon sampling on representatives
initial denaturation for 360 sec. at 94° C, of the smaller fairy basslets (e.g., Luzonichthys,
denaturation for 60 sec. at 94° C, annealing Nemanthias, Pseudanthias, Tosana) in the
for 60 sec. at 48° C, and extension for 75 sec. at subfamily Anthiinae because these genera were
72° C, with an additional terminal extension at initially found to posses the intergenic spacer in
72° C for 360 sec. The double-stranded ampli¬ HI. We also included representatives of four
fication products were desalted and concen¬ additional anthiine genera (Hemanthias,
trated using AMPure (Agencourt® Bioscience Holanthias, Plectranthias, and Pronotogra-
Corporation) following the manufacturer’s mmus), three serranine genera (Diplectrum,
protocol. Both strands of the purified PCR Paralabrax, and Zalanthias), two epinephelid
fragments were used as templates and directly genera (Epinephelus and Grammistes), and the
cycle-sequenced using the original amplifica¬ former anthiine, now percoid, genus Acan¬
tion primers and Prism Dye Terminator thistius to examine the phylogenetic distribu¬
Reaction Kit vl.l (ABI). The sequencing tion of the intergenic spacer. Furthermore, we
reactions were cleaned and desalted using included four specimens of the species
CleanSEQ (Agencourt® Bioscience Corpora¬ Pseudanthias tuka to assess haplotype diversity
tion) following the manufacturer’s protocol. for the intergenic spacer region. A11 four of
The nucleotides were sequenced on an ABI these specimens were procured at one time
3730X1 automated DNA sequencer. Contigs from the aquarium trade, so their collection
were built in Sequencher (GeneCodes) using localies are unknown.
2009 SMITH ET AL.: MITOCHONDRIAL SPACER IN FAIRY BASSLETS 5
ANALYSIS to the POY analysis, we manipulated and
compared sequence data using ClustalX
For the phylogenetic analysis, 17,285
(Thompson et al., 1997) using default values,
aligned base pairs (based on the implied
and we examined possible secondary struc¬
alignment; Wheeler, 2003) were analyzed.
tures of the intergenic spacer transcript using
This large number of base pairs resulted from
the program MFOLD (Zuker et al., 1999)
our inclusion of the complete mitogenomes for
using default values.
two taxa (Myripristis and Caranx). The
remaining terminals sequenced in this study
RESULTS
included the sequenced regions of the
tRNAVal, intergenic spacer (when present),
The POY analysis results in 15 equally most
and 16S, encompassing 2180 aligned base
parsimonious trees (strict consensus shown in
pairs (fig. 1). These sequences were initially
fig. 2). Each of the resulting optimal hypoth¬
analyzed simultaneously under the optimality
eses has a cost of 24,036 weighted steps, which
criterion of parsimony with nucleotide trans¬
includes the traditional nucleotide transfor¬
formations (e.g., transversions, transitions,
mation costs as well as POY’s rearrangements
indels) equally weighted with a cost of one, a
costs. Based on this analysis, we hypothesize a
breakpoint cost (the cost for separating
single origin of the intergenic spacer in the
adjacent loci) of 100, a locus gap cost (the
ancestor of the anthiine genera Pseudanthias,
constant fraction of the cost of locus origin-
Luzonichthys, Tosana, and Nemanthias. Be¬
loss) of 200, and a locus-size gap cost (the
cause the analysis includes two complete
variable fraction of the cost of locus origin-
mitogenomes, it explicitly tests whether the
loss based on the size of the locus) of one. This
intergenic spacer evolved de novo (e.g.,
analysis was repeated with a variety of other
duplication, insertion) or is a rearrangement
plausible weighting schemes with weights from
from elsewhere in the mitogenome. The
one to 500 for the various breakpoint param¬
analysis indicates that the intergenic spacer is
eters. The limited changes in results that we
a novel element because it was not aligned
recovered will be discussed below, but all
(i.e., inferred to be homologous) with any
inferences must be based on the explicit costs.
mitogenomic element in the analyses that
This analysis was conducted in POY
included the entire ~2100 base-pair region
(Wheeler et al., 2003, 2006) and run on the
that was sequenced. Subsequent POY and
American Museum of Natural History paral¬
lel computing cluster. The analysis began with ClustalX analyses that compared the interge¬
500 random addition sequences that were nic spacer sequences individually to both
improved with TBR branch swapping and included mitogenomes consistently aligned it
150 parsimony ratchet replicates (Nixon, with tRNAVal despite the inclusion of all
1999). The results of these analyses were mitochondrial transfer RNAs for Myripristis
submitted to a final round of tree fusing and Caranx. This suggests that the intergenic
(Goloboff, 1999a) and TBR branch swapping spacer is a duplication of this adjacent tRNA.
using fixed-states character optimization Using the resulting phylogenetic framework,
(Wheeler, 1999) and breakpoint analysis we describe the general features of and discuss
(Sankoff and Blanchette, 1998). To estimate the evolution of this novel mitochondrial
the robustness of the clades recovered in the element as well as compare our phylogenetic
phylogenetic hypotheses, Bremer supports results to those of Baldwin (1990). The
(Bremer, 1994) were calculated using tRNAVal and 16S fragments that flank the
TreeRot (Sorenson, 1999) in conjunction with intergenic spacer, when present, align well
PAUP* 4.0bl0 (Swofford, 2002), and jack¬ with the homologous regions in the taxa that
knife resampling analyses were performed in lack the intergenic spacer and were identified
NONA (Goloboff, 1999b; 1,000 replications, as homologs in the POY analysis. The
heuristic searches, 10 random additions per intergenic spacers of all 10 terminals have
replication via the WinClada interface [Nixon, unique sequences with high sequence variation
2002]) using the implied alignment, which does (uncorrected “p” distances): 0.025-0.618
not include the breakpoint costs. In addition (mean: 0.409 ± a: 0.149) between species.
6 AMERICAN MUSEUM NOVITATES NO. 3652
This variation is approximately three times a single intergenic spacer sequence of observed
that found in the flanking tRNAVal and 16S length. In our diversity of analyses, the only
fragments for the same species: 0.010-0.181 topological changes that we found occurred
(0.140 ± 0.041). All four Pseudanthias tuka when the breakpoint costs approached 1 (i.e.,
haplotypes have distinct intergenic spacer evidentially equivalent to a single substitution
sequences (two to nine variable sites) but or indel). These results suggest that the
identical tRNAVal and partial 16S sequences. intergenic spacer evolved independently (i.e.,
Additionally, the intergenic spacer sequences it was not homologous) in all species (except
are highly length variable, ranging from 77 to Pseudanthias tuka and Nemanthias carberryi)
259 bps (174 ± 62) between species (fig. 2). due to the extreme sequence variability in the
Despite the high variability, there is a region intergenic spacer sequences found among
of —25 base pairs at the 3' end of the species. These scenarios, as well as others,
intergenic spacer sequences that is highly should be examined further as additional
conserved among these 10 terminals. anthiine taxa are sequenced for the intergenic
spacer region. Our discovery of this large,
DISCUSSION highly variable, yet persistent, intergenic
spacer has implications for future studies
As Wheeler (1995) described for nucleotide examining the systematics and population
sequences, the results of this and all other genetics of sea basses, as well as for the
numerical phylogenetic methods are affected evolutionary dynamics of the mitogenome.
by explicit assumptions (e.g., costs of transi¬ Our phylogenetic hypothesis recovered a
tions, transversions, indels, breakpoint cost, monophyletic Anthiinae (sensu Smith and
locus gap cost, locus-size gap cost) and Craig, 2007) and corroborates Baldwin’s
implicit assumptions. Different costs for these (1990) and Smith and Craig’s (2007) hypoth¬
various parameters may result in different esis that Plectranthias (sensu Eschmeyer, 1998)
hypotheses of relationships and evolutionary is not monophyletic and Baldwin’s (1990)
scenarios, as is seen with different weighting hypothesis that among included taxa,
schemes in parsimony analyses or different Hemanthias, Holanthias, and Pronotogra-
evolutionary models in model-based phyloge¬ mmus form a monophyletic group. Clearly,
netic approaches (i.e., MrBayes or maximum additional work on anthiine relationships is
likelihood). Our resulting phylogeny was still desperately needed, but our study clearly
recovered in almost all breakpoint cost re¬ traces and documents the evolution and
gimes that were examined. The specific break¬ persistence of this intergenic spacer in a
point costs that we present for the calculations diverse clade of anthiine serranids (presum¬
were chosen because they are roughly equiv¬ ably 65+ species in Luzonichthys, Nemanthias,
alent to the range of sizes for the intergenic Pseudanthias, and Tosana [Eschmeyer, 1998]).
spacer sequences. They were chosen so that The persistence of this large intergenic spacer
the breakpoint costs would be equivalent to is as surprising as its increase in size through
the number of independent substitutions and evolutionary time (fig. 2). Among the taxa
insertions required to explain the evolution of sequenced in this study, the most basal taxon
Fig. 2. The strict consensus cladogram of the 15 most parsimonious trees. Anthiine serranids have black
branches. The two-dimensional secondary structure of the transcript predicted by MFOLD for the
intergenic spacer is adjacent to the taxon name, the free-energy calculation resulting from the secondary
structure prediction of the intergenic spacer is listed above the taxon name, and the number of base pairs in
the intergenic spacer are listed below the taxon name. Bremer supports are listed above the branches and
jackknife values >50% are listed below the nodes (* = 100%). Nodes that lack Bremer support are recovered
in the analysis that include breakpoints costs, but they are not recovered in the support value analyses based
exclusively on the nucleotide transformations inferred from the implied alignment. Varied branch lengths do
not represent amounts of change; they are varied solely to provide sufficient space for illustrating predicted
secondary structures.
2009 SMITH ET AL.: MITOCHONDRIAL SPACER IN FAIRY BASSLETS 7
Myripristis berndti
Caranx melampygus
40 Paralabrax maculatofasciata
* *- Diplectrum formosum
goi Epinephelus adscenscionis
sj *1 Epinephelus itajara
61Acanthistius ocellatus
11 Grammistes sexlineatus
Zalanthias kelloggi
AMERICAN MUSEUM NOVITATES NO. 3652
with an intergenic spacer, Pseudanthias lori, has longer be discounted.” Therefore, we have
the smallest spacer (77 bp), roughly the size of a analyzed our data using POY’s new algorithm
tRNA. At each successive node, there is an (Wheeler, 2007) that combines Sankoff and
increase in the size of the intergenic spacer, Blanchette’s (1998) chromosomal rearrange¬
culminating in more highly derived taxa with ment (breakpoint analysis) and Wheeler’s
spacers as large as 259 bp (fig. 2). This trend of (1999) fixed-states nucleotide optimizations to
insertions within the intergenic spacer region is examine simultaneously the identity and evolu¬
surprising considering current opinions about tion of this novel genomic locus as well as
mitochondrial DNA evolution that argue for examine relationships among a subset of sea
mitogenome size reduction through evolution¬ basses. This method for simultaneously com¬
ary time (Rand, 1993, 2001). Rand (1993, 2001) bining chromosomal order and nucleotide data
has argued that the lack of introns and to assess locus homology (i.e., genomic annota¬
intergenic spacers in animal mitochondrial tion) and nucleotide alignment is preferred over
DNA is due to strong purifying selection for other methods because it is automated, inter¬
small mitogenomic size (and thus rapid replica¬ nally consistent, repeatable, explicit, does not
tion time). Given this argument, one would presuppose a mechanism for resulting rear¬
expect large, evolutionarily stable intergenic rangements, allows evaluation of competing
spacers to serve a function. None of the sea hypotheses, and is grounded in a phylogenetic
bass intergenic spacer sequences possess an open framework. Our results suggest a single evolu¬
reading frame of realistic size, suggesting that tion of an intergenic spacer in the highly
they are not protein coding. As a further transcribed H-strand transcription unit one,
exploration of its function, we examined the presumably through duplication and subsequent
predicted secondary structures of their RNA evolution of tRNAVal. Finally, contrary to cur¬
transcripts using a free energy minimization rent mitochondrial genomic selection theory,
model in MFOLD. Although the intergenic this intergenic spacer is not only persistent, but
spacer sequences often form complex two- seems to be increasing in size as it evolves.
dimensional stem and loop structures (fig. 2),
these structures differ between species and ACKNOWLEDGMENTS
haplotypes. Furthermore, they are not particu¬
larly stable, with free energies ranging from We would like to thank J. Smith (Los
-4.0 to -20.3 kcal/mol, making it unlikely that Alamos National Laboratory) and J. Fai-
they form functional secondary structures in vovich, T. Grant, K. Pickett, J. Sparks, M.
organello. Additionally, we compared this Stiassny, and K. Tang (all at or formerly at
tRNAVal-16S region with more than 600 the American Museum of Natural History
acanthomorph species from more than 250 [AMNH]) for discussing aspects of this project
families, and none of these taxa had a similar with us. We are grateful to H. Endo (Kochi
sequence between the tRNAVal and 16S. Finally, University), the Gahan Family, J. Leis and M.
we are unable to find any sequences similar to McGrouther (Australian Museum), Reef and
the intergenic spacer sequences by performing Fin (Stamford, CT), and H. Walker (Scripps
BLAST searches in GenBank. Therefore, it Institution of Oceanography) for providing
seems likely that the only functional role the specimens used in this study. This project was
intergenic spacer may play is in posttranscrip- supported by funding from the AMNH Lerner-
tional processing. We speculate that this con¬ Gray Program for Marine Research, the
served 25-bp region is important for accurate NASA-Ames Fundamental Space Biology
cleavage at the 5' end of the adjacent 16S rRNA, Program, the Field Museum of Natural
a function that would have to have been retained History, and the National Science Foundation
from the ancestral tRNAVal following the (DEB-0405246 and DEB-0732642).
hypothesized duplication to ensure proper
posttranscriptional processing of the 16S
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