Molecular Biology and Evolution 18:1512-1521 (2001)
© 2001 Society for Molecular Biology and Evolution
Phylogenetic Utility and Evidence for Multiple Copies of Elongation Factor-1
in the Spider Genus Habronattus (Araneae: Salticidae)
Department of Ecology and Evolutionary Biology, University of Arizona
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Abstract |
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In the continuing quest for informative genes for use in molecular systematics, the protein-coding gene Elongation factor–1
(EF-1) has rapidly become one of the most prevalent "single-copy" nuclear genes utilized, particularly in arthropods. This paper explores the molecular evolutionary dynamics and phylogenetic utility of EF-1 in the salticid spider genus Habronattus. As has been reported for other arthropod lineages, our studies indicate that multiple (two) copies of EF-1 exist in Habronattus. These copies differ in intron structure and thus in size, making it possible to easily separate PCR amplification products. We present data for an intronless EF-1 copy for three Habronattus species. The presence of nonsense mutations and generally elevated rates of amino acid change suggest that this copy is evolving under relaxed functional constraints in Habronattus. A larger taxon sample (50 species plus outgroups) is presented for an EF-1 copy that includes both intron and exon regions. Characteristics of both regions suggest that this is a functional, orthologous copy in the species sampled. Maximum-likelihood relative-rate comparisons show that exon third codon sites are evolving more than 100 times as fast as second codon sites in these sequences and that intron sites are evolving about twice as fast as exon third sites. In combination, the EF-1 data provide robust, species-level phylogenetic signal that is largely congruent with morphologically well supported areas of Habronattus phylogeny. The recovery of some novel clades, and the unexpected fragmentation of others, suggests areas requiring further phylogenetic attention. |
Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Recent progress in spider systematics has been most influenced by two advances: the development of new morphological character sets (made accessible through SEM) and the growth of cladistic thought (see Coddington and Levi 1991
; Griswold et al. 1999
). The development and general impact of molecular systematics has lagged somewhat behind, although this appears to be changing. This is particularly true at lower taxonomic levels, with many recent phylogeographic and species-level phylogenetic studies using molecular data (e.g., Gillespie, Croom, and Hasty 1997
; Zehethofer and Sturmbauer 1998
; Masta 2000
; Bond et al. 2001
; Hedin 2001
). To date, essentially all studies have relied on a single gene region (mitochondrial) and thus are potentially subject to the various pitfalls that such analyses present (see Maddison [1997
] and others). More, different types of molecular markers need to be developed for the same reasons that we should be interested in using both molecular and morphological evidence in a complementary manner. New character sets represent more evidence, independent evidence, and potentially heterogeneous evidence (i.e., with different rate dynamics, thus resolving different depths of phylogenetic divergence).
The protein-coding gene Elongation factor–1 (EF-1) is rapidly becoming one of the most prevalent "single-copy" nuclear genes utilized in arthropod molecular systematics. EF-1 is a key and conservative element in protein synthesis, thus facilitating universal use and phylogenetic data collection. To date, studies using EF-1 data have suggested a broad, but perhaps bimodal, distribution of phylogenetic utility. Utility has been impressive for Tertiary age divergences, with most information coming from silent substitutions at third codon positions (e.g., Cho et al. 1995
; Reed and Sperling 1999
). Using a combination of intron and exon sequence data, Danforth, Sauquet, and Packer (1999)
extend this utility to the species level in halictine bees. Third-position saturation, coupled with amino acid conservation, appears to limit utility at "intermediate" levels (e.g., Mesozoic divergences), but amino acids are again informative at deep levels (e.g., among arthropods; see Regier and Shultz 1997
; Shultz and Regier 2000
). Based on the generally encouraging results from the handful of published studies, Caterino, Cho, and Sperling (2000)
suggest that EF-1 should be a "focal gene" in insect molecular systematics, contingent on the prevalence and potential difficulties associated with multiple gene copies.
Here we report on the molecular evolutionary dynamics and phylogenetic utility of EF-1 in the spider genus Habronattus. Habronattus is one of the most species-rich spider genera in the Americas, including over 90 described species distributed from Canada south to northern South America (Griswold 1987
). The genus is remarkable in other respects, with interesting patterns of sex chromosomal evolution (Maddison 1982
), fine-scale morphological and behavioral divergence (Maddison and McMahon 2000)
, and a diversity of communication modalities (e.g., visual, stridulatory, etc.; Peckham and Peckham 1889
; Maddison and Stratton 1988
). Morphology-based phylogenetic analyses have been conducted by Griswold (1987)
and are summarized in figure 1
.
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iw-2]Two apparent phylogenetic patterns are important to the focus of this paper. The first pattern is the "bundling" of species into species groups. The character support for some of these groups is so strong (e.g., the americanus, dorotheae, and agilis groups) that we would be surprised if molecular analyses failed to recover coincident monophyletic lineages. Alternatively, there are species groups lacking robust character support (e.g., the decorus and pretiosus groups), where independent analyses are needed to confirm or perhaps refute hypothesized groupings. Furthermore, relationships among groups, particularly near the base of the genus, are currently poorly understood (fig. 1 ). A second interesting pattern, essentially opposite that of the "bundling" pattern, is the presence of distinctive Habronattus species lacking clear phylogenetic relatives (e.g., H. tarascanus, H. pugillis, H. hallani; fig. 1 ). It would be interesting to know if such species are components of "cryptic" species groups (i.e., strongly supported molecular clades that lack obvious morphological synapomorphies), which would thus comprise sets of very distinctive taxa. Of course, it might be the case that such phylogenetic "stragglers" are real, which would have equally interesting implications for speciation/extinction dynamics in Habronattus.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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We gathered sequences from 50 Habronattus species, representing a large percentage of the phylogenetic stragglers, and multiple species for all but two of Griswold's (1987)
nine recognized species groups (fig. 1
and table 1
). Within species groups we targeted taxa hypothesized to be phylogenetically distant (i.e., species from opposite sides of a proposed root node). Although within-species geographic variation is prevalent in Habronattus, we sampled only a single population per species for this study. Sequences from two species of Pellenes and a single species of Hawaiian "Sandalodes" were included as outgroups, following molecular phylogenetic analyses of salticid relationships that strongly support a ((Pellenes, Habronattus) "Sandalodes") grouping (unpublished data).
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Genomic DNA was extracted from either fresh or ETOH-preserved leg tissue using the CTAB protocol of Shahjahan et al. (1995)
. Otherwise-intact spiders are preserved as alcohol voucher specimens at -80°C in the personal collection of W.P.M. Initial EF-1 sequences were generated using universal degenerate primers (Cho et al. 1995
) available in the UBC Insect Nuclear DNA Oligonucleotide Set. From these preliminary sequences, spider-devoted primers were designed by identifying conserved regions shared across Habronattus and available spider EF-1 exon sequences (Regier and Shultz 1997
). For most genomic templates, these primers (M46-1SPID [5'-GAG GAA ATC AAG AAG GAA G-3'] and rc1007-1 [5'-GAC CTG AGC AAA GAA TTC TTG AGT AC-3']) amplify a single fragment approximately 700 bp in length. A small percentage of templates produced multiple bands; these products were size-separated for further analysis. PCR cycle parameters were as follows: 2 min 30 s initial 95°C denaturation, followed by 35 cycles of 30 s at 95°C, 30 s at 52°C, and 45 s at 72°C + 3 s per cycle, followed by a final 72°C extension for 10 min. All PCR reactions included TaqStart Antibody (Clontech Laboratories) at dilutions suggested by the manufacturer.
Double-stranded PCR products were polyacrylamide gel-purified (Sambrook, Fritsch, and Maniatis 1989
) and directly sequenced using ABI dye chemistry on an ABI 377 machine. Both strands were determined for most templates using PCR primers as sequencing primers. T-rich regions at the 3' end of intron I (see below and fig. 2
) caused premature stops in some sequencing reads; sequences for these templates were thus determined using single reads from opposite sides of the intron. Because we were uncertain of the exact number of repeated T's in some of these sequences, we excluded a 6-bp region at the 3' end of intron I from all phylogenetic analyses. Sites including two peaks of equivalent intensity were interpreted as heterozygosity and entered into the phylogenetic matrix using IUBMB ambiguity codes. Of approximately 38,000 total aligned sites, 53 sites were scored as ambiguous (31 exon sites, 22 intron sites).
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Parsimony constituted the primary phylogenetic estimation procedure, carried out using the program PAUP*, version 4.0b2a–b4a (Swofford 1999
), in combination with MacClade, version 4 (Maddison and Maddison 2000)
. Searches were heuristic with TBR branch rearrangement, using stepwise taxon addition from 500 random replicates, saving no more than 500 most-parsimonious (MP) trees per replicate. Relative clade support was assessed using nonparametric bootstrap analyses (Felsenstein 1985
). Bootstrap analyses comprised 200 full heuristic search replicates, each including five random-addition sequence searches with TBR branch swapping, saving no more than 500 MP trees per search. Phylogenetic congruence between exon versus intron sites was assessed using the incongruence length difference (ILD) test (Farris et al. 1995
; as implemented in PAUP*), considering only variable sites (Cunningham 1997
). Each of 100 replicates included a heuristic search strategy as per bootstrap analyses. In all analyses considered above, multistate taxa (i.e., taxa including heterozygous sites) were scored as polymorphic, and gaps in intron sequences were treated as "fifth states."
Treating gaps in this manner is a compromise. In inspecting the EF-1 intron matrices (see figs. 2 and 3
), it is clear that some indel events are phylogenetically informative (e.g., consistent with unambiguous exon signal). If we were to either exclude indel sites or treat gaps as missing, we would be excluding available phylogenetic information. Alternatively, some strings of adjacent gaps are almost certainly nonindependent but are counted as independent in a "gaps as fifth" analysis. The result is that the number of independent character changes (and bootstrap support, etc.) is potentially overestimated. We pay particular attention to this possibility in interpreting the phylogenetic results presented below.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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A small fraction of PCR experiments resulted in the amplification of two strong bands, one band corresponding to a
700-bp product present in all taxa, and a smaller band at 490 bp. Analysis of a sample of genomic templates for which both bands were gel-purified and sequenced indicated that the larger product included one complete and two partial exons, separated by two introns (fig. 4 ). The smaller fragment corresponded to an exon-only product (referred to below as the "intronless copy"). Alignment of Habronattus exons from both copies revealed high amino acid similarity to other arthropods, including other spiders (fig. 4
). As compared with the copy with introns, the intronless copy appeared to be evolving under relaxed functional constraints, as evidenced by single-nucleotide deletions (ultimately resulting in stop codons) and relatively high rates of amino acid evolution (figs. 4 and 5 ). This variability contrasted strongly with extreme amino acid conservation in sequences that contained introns (see below).
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The remainder of the results and analyses presented consider only those EF-1
sequences that contain introns. Such sequences were gathered for 50 Habronattus species plus 3 outgroup taxa (see table 1
for GenBank accession numbers). Available evidence suggests that these sequences correspond to an orthologous, apparently functional gene copy across the taxa surveyed. All sequences included exon data coding for 159 amino acids. There were no amino acid substitutions observed across the Habronattus-plus-Pellenes matrix, and there were only three physiochemically similar changes distinguishing the Sandalodes sequence (Thr<>Gly, Thr<>Ser, Leu<>Ileu; fig. 4
). Exon nucleotide variation was less conserved, with 78, 6, and 1 variable sites at third, first, and second codon positions, respectively. The two introns that intervened in the exon sequences both appeared functional, possessing 5' (GTAWGT) and 3' (WAG) splice site signal sequences (fig. 4 ) consistent with proposed Metazoan consensus signal sequences (Senapathy, Shapiro, and Harris 1990
; Mount et al. 1992
). Although raw intron sequences varied in length (table 2
), both sets of sequences were readily aligned manually (see figs. 2 and 3 ). A large fraction of the aligned intron sites were variable (193) and parsimony-informative (147). Both exon third positions and introns were AT-rich, and there was no significant variation in base composition across taxa for either intron or exon data (PAUP* chi-square analyses). Additional sequence characteristics are shown in table 2
and further discussed below.
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Phylogenetic congruence between variable sites of the exon and intron sequences was assessed using the ILD test (Farris et al. 1995
). This test suggested that the data were congruent (P = 0.38), justifying combined phylogenetic analyses. Heuristic parsimony analyses of the combined data resulted in many thousands of MP trees (N = 14,207, searches swapping to completion). Despite this large number of MP trees, a strict-consensus tree was reasonably well resolved (fig. 6
), with many clades found in a high proportion of bootstrap replicates (e.g., more than 80% of replicates). Monophyly of species groups united by multiple morphological synapomorphies (i.e., the agilis, amicus, americanus, and dorotheae groups; see fig. 1
) was strongly supported by the EF-1 data. The coecatus, viridipes, and decorus groups were never recovered as monophyletic in MP trees, although coecatus group monophyly could not be convincingly rejected by the data (see below). Most "straggler" taxa did not fall into obvious groups, aside from some well-supported sister relationships (e.g., Habronattus texanus plus Habronattus altanus). An unexpected result was the placement of the Pellenes clade within Habronattus, arising near the deep-diverging amicus and agilis species groups.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Multiple Copies
The presence of multiple copies of EF-1
in Habronattus is consistent with similar findings in other arthropod taxa. Danforth and Ji (1998)
provide evidence for two EF-1 copies in the genus Apis, with each copy containing multiple introns that vary in position. Similarly, two copies (with and without introns) have been found in Drosophila melanogaster (Hovemann et al. 1988
). Although some intron insertion positions are shared between Drosophila and Apis (Danforth and Ji 1998
), the insertion positions of the Habronattus introns do not overlap with those reported for either insect (fig. 4
), arguing against a shared ancient duplication. Instead, it is almost certainly the case that duplication events have occurred independently in these divergent arthropod lineages, consistent with the idea that such events have occurred multiple times independently within insects (Danforth and Ji 1998
).
Although both paralogous copies appear to be functional in Drosophila and Apis, the limited amount of data available for the intronless copy suggests that this is not the case for Habronattus. One of the intronless copies includes a single nucleotide deletion that ultimately results in a stop codon, and all copies are characterized by relatively high rates of amino acid substitution as compared with copies with introns (figs. 4 and 5
). This evidence for relaxed functional constraints suggests that the copy without introns may be evolving as a pseudogene. Alternatively, the copy with introns appears functional. The relative proportion of nucleotide changes at first and second versus third codon positions is as expected (table 2
), and amino acid variation is nonexistent in these sequences. In addition, the intervening introns are within the range of expected sizes, include functional 5' and 3' splice site sequences, and are higher in AT content than adjacent exons (see Mount et al. 1992
).
The distribution and phylogenetic affinities of multiple EF-1 copies sampled from the same Habronattus species (see fig. 5
) might be explained in one of two ways. First, it might be that derived, independent duplication events account for the relationship of copies within species being closer than that between species. We view this possibility as unlikely for the following reasons. We have detected smaller PCR products, corresponding in size to the intronless copy, in many other species and species groups of Habronattus. Although we have not sequenced these products, we believe they are homologous to the intronless copy. Also, we note that although multiple bands are not apparent in all PCR experiments, this is likely due to primer mismatch or competition rather than to copy absence. Both sets of observations are consistent with a broader phylogenetic distribution of multiple EF-1 copies in Habronattus.
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This broader phylogenetic distribution is most parsimoniously explained by a common duplication at the base of Habronattus (i.e., the presence of paralogous gene copies is shared among all Habronattus), with concerted evolution maintaining high copy similarity within species. A more comprehensive perspective on this question, provided via phylogenetic analysis of all exon sequences (i.e., the three intronless copies plus all others), suggests that the rate of this concerted evolution approximately keeps pace with the rate of speciation in the genus (fig. 7 ). Based on functional considerations, we hypothesize that the directionality of concerted evolution is from "with intron" to "intronless" at a rate high enough to promote concerted evolution but perhaps not high enough to maintain the functionality of the intronless copy. Future studies directed at testing this hypothesis should include not only more intronless sequences, but also sequences from sets of more closely related Habronattus, thus providing finer resolution of rate dynamic issues.
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Intron/Exon Dynamics and Utility
To date, most phylogenetic studies incorporating EF-1
have relied on exon variation and, accordingly, have considered systematic problems that are generally deeper than those considered here (e.g., among genera or tribes). Reported levels of exon variation within genera indicate limited divergence (e.g., up to 1% within moths; Cho et al. 1995
), with essentially all nucleotide variation occurring at third-position sites. Mitchell, Mitter, and Regier (2000)
estimate likelihood-based relative rates of 4:1:81 at first : second : third codon positions, respectively. Likelihood parameter estimates for the Habronattus exon data suggest similar patterns (to facilitate comparison with intron data [see below], some taxa were excluded from likelihood analyses; estimates using the full exon matrix are very similar). Using a best-fit GTR+
maximum-likelihood model (model fit assessed using MODELTEST; Posada and Crandall 1998
), relative-rate parameters estimated over a randomly chosen MP tree correspond roughly to 6:1:112 at first : second : third codon positions, respectively. A low estimated value of the gamma distribution shape parameter over all sites indicates a distribution with a strong left skew ( = 0.14), reflecting a combination of many invariant sites with fewer, more variable, sites (see fig. 13 of Swofford et al. 1996
). These variable sites appear to be evolving at approximately the same rate, given that the GTR+ model explains the data as well as both the GTR+%I and the GTR+%I+ models. Most exon site variation is accounted for by transitions (see table 3
).
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In considering both intron and exon data, are we simply adding additional sites that are evolving approximately like third position exon sites, or are we adding sites with substantively different evolutionary dynamics? To address this question, we independently estimated a best-fit likelihood model for the intron data. As for exon sites, this corresponds to a GTR+
model. Because a large fraction of intron I aligned sites include at least one indel in the full taxon matrix (and thus are ignored in likelihood estimates), we increased the total number of sites considered by excluding some gap-rich sequences (see table 3
). The estimated R matrix for intron sites differs markedly from that estimated for exon sites (table 3
). The transition bias is much less extreme, with some transversion rates (C<>G) approximating that of transitions. A higher estimated value of the gamma distribution shape parameter indicates greater symmetry ( = 3.09), reflecting more equal rates across all sites. Comparison of relative rates of evolution suggests that, as a site class, introns are evolving about twice as fast as exon third positions (relative-rate ratio = 2.1:1).
The differences in rate and substitution dynamics in EF-1 introns versus exons suggest the possibility that these sequence partitions may contribute in different, and perhaps complementary, ways to phylogenetic resolution in Habronattus. For example, we might expect the more rapidly evolving introns to provide more phylogenetic signal at shallow levels, complemented by deeper signal contributed by exons. Intron sites that include gaps may potentially confound such an interpretation, as such sites make up a large fraction of intron sites, and we cannot be sure that these sites are evolving like the "typical" intron site (i.e., they may be evolving more slowly). To address this issue, and to look for complementary phylogenetic signal at different divergence depths, we reconstructed changes of three separate site classes (i.e., exon sites, intron sites without gaps, and intron sites with at least one gap) on a randomly chosen MP tree (fig. 8
). A visual scan of the two right-hand boxes on each branch in figure 8
shows that of 93 branches showing change in either intron site class, 70 boxes remain the same or get darker as one moves to the right, whereas only 23 boxes get lighter. This would support the general conclusion that as a site class, the "with gaps" class changes as much as or more than the "without gaps" class (note that the reconstructed changes in the "with gaps" class are not all nucleotide <> indel changes; see fig. 8
legend). Patterns of intron : exon complementary resolution are less obvious. Some species groups show a pattern in which most exon changes occur basally, with internal resolution coming from introns (e.g., the americanus group), but, in general, a simple "exon deep, intron shallow" dynamic does not adequately explain the Habronattus data.
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Congruence with Prior Phylogenetic Hypotheses
Some Habronattus species groups are supported by enough character evidence to perhaps be considered as areas of "known phylogeny." Examples include the dorotheae, americanus, amicus, and agilis groups, defined not only by multiple morphological synapomorphies (see fig. 1 ; Griswold 1987
), but also by other characters not formally analyzed in a phylogenetic context (e.g., agilis group characters discussed in Maddison and Stratton [1988
]). These four clades are recovered and strongly supported by the EF-1 data (see fig. 6
). At least some character support comes from exon and intron sites without gaps (fig. 8
), although this pattern is less obvious for the dorotheae group. Here, intron sites with gaps provide most group support (fig. 8
), but visual inspection of intron matrices does not indicate that such support is obviously overestimated by strings of nonindependent gaps (see figs. 2 and 3
).
Aside from the recovery of well-established groups, what do the EF-1 data suggest that is unexpected and potentially interesting? One well-supported, perhaps cryptic, species group is composed of a subset of "decorus" group taxa (Habronattus cockerelli and Habronattus cf. decorus) allied with a trio of species (Habronattus venatoris, Habronattus carolinensis, and Habronattus ocala) also previously thought to be related (see fig. 1
; Griswold 1987
). This clade is recovered in a high proportion of bootstrap analyses (fig. 6
) and is supported by all three classes of character change (fig. 8
). Sequences of other members of the "decorus group" (Habronattus banksi and Habronattus cf. sugillatus) are never placed with the above taxa, suggesting that this species group may not be a natural group. This would not be surprising, as the morphological support for this hypothesized clade is tenuous, based on the absence of three correlated characters (ventral fringe on femur, patella, and tibia I) that are homoplasious elsewhere in the genus (Griswold 1987
).
The species-rich viridipes and coecatus groups include taxa with males possessing striking first- and third-leg modifications used in courtship display. If Griswold's (1987)
phylogeny is correct, it would suggest that third-leg ornaments have arisen only once in Habronattus, with the absence of such modifications in Habronattus moratus, Habronattus orbus, and Habronattus trimaculatus (plus other species not sampled) representing cases of secondary loss (fig. 1
). In this case, the EF-1 data present a mixture of ambivalence, novel confirmation, and unexpected placements. Sequences from members of the coecatus group are allied with those of the viridipes group in MP trees, but because of the placement of Habronattus zebraneus with Habronattus divaricatus, the coccatus group itself is not monophyletic. This latter sister relationship may be an artifact of treating gaps as fifth states (see figs. 2 and 8
), and trees including a monophyletic coecatus group are only six steps longer than MP (733 vs. 727). The DNA data appear to confirm Griswold's (1987)
hypothesis regarding ornament loss in H. moratus, H. orbus, and H. trimaculatus, as these species are placed with otherwise morphologically similar taxa in a well-supported clade (fig. 6
). One of the most surprising results of this study is the placement of three viridipes group sequences (Habronattus notialis, Habronattus jucundus, and Habronattus calcaratus calcaratus) into a very distinctive clade far separated from other group members. These taxa are very similar to Habronattus viridipes and Habronattus calcaratus maddisoni (H. calcaratus calcaratus is classified as a member of the same species!), sharing several morphological, ecological, and behavioral characteristics (Griswold 1987
; unpublished data). Their placement in the EF-1 tree might indicate a potential orthology/paralogy problem, but intriguingly, mitochondrial DNA sequences indicate the same separation for the same taxa (unpublished data).
Phylogenetic relationships among species groups of Habronattus, particularly deep in the tree, are currently ambiguous (see fig. 1
). The EF-1 data suggest some potential resolution of this ambiguity, indicating that the agilis and amicus groups are basal lineages in the genus, with a well-supported branch separating these groups from more derived Habronattus (fig. 6 ). The character support for this separation comes from all three site classes (fig. 8
). Relationships among these basal groups, including species currently placed in the genus Pellenes, remain ambiguous. Pellenes is embedded within Habronattus in MP trees (fig. 6
), but character support for these deep relationships is weak and based in large part on intron sites with gaps (see fig. 8
). Given the strong morphological evidence for Habronattus monophyly (Griswold 1987
), we would be surprised if Pellenes were truly nested within Habronattus.
Habronattus is a large and complex genus, with over 90 species described and perhaps half that number waiting to be described. Fully understanding this diversity from a phylogenetic perspective is a nontrivial task and will ultimately require multiple lines of character evidence for a large number of species. Given the generally favorable results presented here, we feel that EF-1 sequence data should represent one such line of character evidence. Whether or not EF-1 will behave similarly in other spider taxa requires more study, but we believe that the gene has clear potential, particularly given the generally positive results coming from studies of other arthropod groups.
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Several persons helped to collect Habronattus, including Eileen Hebets, Gita Bodner, Jennifer Hedin, G. B. Edwards, and David Maddison. Tila María Pérez helped to obtain Mexican permits and specimens, which were collected with the assistance of Ricardo Ayala, José Luis Castelo, Fernando Alvarez, and Gita Bodner. Lab work was carried out in the LMSE, Arizona Research Labs, University of Arizona. This work was supported by NSF grant DEB-9707368 to W.P.M.
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Footnotes |
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Keith Crandall, Reviewing Editor
1 Present address: Department of Biology, San Diego State
University. 
1 Keywords: molecular phylogeny
Salticidae
phylogenetic utility
Elongation factor–1
paralogy
2 Address for correspondence and reprints: Marshal C. Hedin, Department
of Biology, San Diego State University, San Diego, California
92182-4614. mhedin{at}sciences.sdsu.edu
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