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Molecular Phylogenetics and Evolution 57 (2010) 59–70
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Rare genomic changes and mitochondrial sequences provide independent support
for congruent relationships among the sea spiders (Arthropoda, Pycnogonida)
Susan E. Masta *, Andrew McCall, Stuart J. Longhorn 1
Department of Biology, Portland State University, P.O. Box 751, Portland, OR 97207, USA
a r t i c l e
i n f o
Article history:
Received 15 September 2009
Revised 15 June 2010
Accepted 25 June 2010
Available online 30 June 2010
Keywords:
Chelicerata
Arachnida
Mitochondrial genomics
tRNA secondary structure
Gene rearrangements
a b s t r a c t
Pycnogonids, or sea spiders, are an enigmatic group of arthropods. Their unique anatomical features have
made them difficult to place within the broader group Arthropoda. Most attempts to classify members of
Pycnogonida have focused on utilizing these anatomical features to infer relatedness. Using data from
mitochondrial genomes, we show that pycnogonids are placed as derived chelicerates, challenging the
hypothesis that they diverged early in arthropod history. Our increased taxon sampling of three new
mitochondrial genomes also allows us to infer phylogenetic relatedness among major pycnogonid lineages. Phylogenetic analyses based on all 13 mitochondrial protein-coding genes yield well-resolved relationships among the sea spider lineages. Gene order and tRNA secondary structure characters provide
independent lines of evidence for these inferred phylogenetic relationships among pycnogonids, and
show a minimal amount of homoplasy. Additionally, rare changes in three tRNA genes unite pycnogonids
as a clade; these include changes in anticodon identity in tRNALys and tRNASer(AGN) and the shared loss of
D-arm sequence in the tRNAAla gene. Using mitochondrial genome changes and tRNA structural changes
is especially useful for resolving relationships among the major lineages of sea spiders in light of the fact
that there have been multiple independent evolutionary changes in nucleotide strand bias among sea spiders. Such reversed nucleotide biases can mislead phylogeny reconstruction based on sequences,
although the use of appropriate methods can overcome these effects. With pycnogonids, we find that
applying methods to compensate for strand bias and that using genome-level characters yield congruent
phylogenetic signals.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
Pycnogonida, or sea spiders, are arthropods with anatomical
features so unique that their relationships to other members of
Arthropoda have been enigmatic. Pycnogonids possess a proboscis
for feeding, extreme abdominal reduction, reproductive organs on
their legs, and ventral appendages termed ovigers with which
males carry eggs. There are two main hypotheses regarding pycnogonid relationships to other arthropods (reviewed in Dunlop and
Arango, 2005). One hypothesis is that Pycnogonida is the sister
group to all other Arthropoda. Under this hypothesis the arthropods, excluding pycnogonids, have been named Cormogonida
(Zrzavý et al., 1998). A second hypothesis is that Pycnogonida are
members of Chelicerata, and are the sister group to the Xiphosura
(horseshoe crabs) plus Arachnida. Anatomical and developmental
* Corresponding author. Fax: +1 503 725 3888.
E-mail address: [email protected] (S.E. Masta).
1
Current address: Department of Biology, National University of Ireland, Maynooth. County Kildare, Ireland.
1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2010.06.020
data have each yielded results that support both of these hypotheses, and therefore, it is still not known which hypothesis, if either,
is correct. More data, including data from different sources, is
necessary to resolve the placement of Pycnogonida within
Arthropoda.
Pycnogonids typically possess anterior appendages termed
chelifores, and the lack of clear homology of these appendages
with other arthropod appendages has fueled debate over the closest relatives of pycnogonids. If sea spiders are chelicerates, then
chelifores should be homologous to the chelicerae of other chelicerates, as possession of chelicerae is one of the defining traits
for the Chelicerata. However, if chelifores are not directly homologous to chelicerae, then this calls into question whether sea spiders
are truly chelicerates. Some analyses based on developmental features have found evidence that these appendages are not direct
homologues of chelicerae, and support the hypothesis that sea spiders are sister to all other arthropods (Maxmen et al., 2005). However, more recent data based on developmental expression
patterns and neuroanatomy have shown that chelifores are likely
chelicerae homologues (Brenneis et al., 2008; Jager et al., 2006;
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Manuel et al., 2006), consistent with the placement of sea spiders
as sister to other chelicerates.
Analyses of molecular data are likewise inconsistent in their
support for these opposing two hypotheses. Some analyses have
found support for pycnogonids being sister to all arthropods (Giribet et al., 2001), or sister to all arthropods except chelicerates (Regier et al., 2008), or sister to the myriapod arthropods (Mallatt and
Giribet, 2006; Mallatt et al., 2004). However, most molecular analyses recover phylogenetic trees that are congruent with pycnogonids as a sister group to all other chelicerates, although often
with low bootstrap support (Bourlat et al., 2008; Mallatt and Giribet, 2006; Regier et al., 2008). Phylogenomic analyses with expressed sequence tags yielded a tree with more support for
pycnogonids as sister to other chelicerates, although taxon sampling was more limited (Dunn et al., 2008). Analyses of mitochondrial genome sequences have suggested a third hypothesis, that
sea spiders are derived arachnids. The mitochondrial genomes of
two species of sea spiders have previously been sequenced, and a
third has been partially sequenced. In phylogenetic analyses of
the protein-coding genes from these genomes, along with those
of other arthropods, pycnogonids have been recovered as derived
arachnids (Hassanin, 2006; Park et al., 2007; Podsiadlowski and
Braband, 2006). Given the contradictory results from different
sources of data, and the often sparse taxon sampling, it is necessary
to further evaluate the phylogenetic placement of sea spiders.
Despite the growing number of studies aimed at understanding
the relatedness of pycnogonids to other arthropods, very few studies have attempted to infer relationships among the major pycnogonid lineages. Hedgpeth and Fry classified sea spider taxa into
families (Fry, 1978; Hedgpeth, 1947), but did not use modern cladistic methods. More recent work by Arango has utilized both morphological and molecular characters to infer phylogenetic
relationships within Pycnogonida (Arango and Wheeler, 2007;
Arango, 2002, 2003). These phylogenetic studies have lent support
to some of the previously defined families of sea spiders, but have
also found relationships among pycnogonids that differ from the
traditional taxonomy. Additionally, although many relationships
among sea spiders were similar using morphological and molecular analyses, some relationships were supported only by molecular
data. Arango proposed that more characters were necessary to
clearly delineate the pycnogonid lineages (Arango and Wheeler,
2007; Arango, 2002, 2003).
Using genomic-level characters to infer relatedness among taxa
has become possible with the increased capability of sequencing
large contiguous stretches of DNA. The use of data on gene
arrangements to help deduce phylogenetic relationships has been
particularly useful for inferring relationships among some metazoan taxa. Much of this work was pioneered with comparisons of
mitochondrial genome sequences, as the small size of mitochondria made them an early target for studying genome evolution
and making phylogenomic inferences (Boore, 2006; Boore and
Brown, 1998; Boore et al., 1995; Larget et al., 2005).
Moreover, as well as changes in primary sequence and genome
arrangements, the structures of molecules can evolve, subject to
the selective constraints imposed on them to function within the
cell. One of the most highly conserved molecular structures is that
of transfer RNA, which maintains a canonical cloverleaf shape
across the three domains of life, Eukarya, Bacteria, and Archaea.
However, metazoan mitochondrial tRNAs are typically much smaller than their nuclear counterparts, and in some cases may lack
either the DHU or TWC arms present in the canonical tRNA structure (see reviews in Soll and RajBhandary, 1995; Wolstenholme,
1992a). Even among metazoans, however, the evolutionary loss
of one of the paired stem regions in a mt tRNA gene is rare. Therefore, when organisms share similar non-canonical tRNA structures,
it is likely due to shared evolutionary history. An unprecedented
number of non-canonical tRNA structures have been found within
arachnid mt genomes (Masta and Boore, 2008), but their structures
have also proven to be phylogenetically informative within some
groups, such as ticks (Murrell et al., 2003) and spiders (Masta
and Boore, 2008).
Besides potentially providing new types of genome structure
characters for making phylogenetic inferences, mitochondrial genomes provide a rich source of sequences for analysis. Typical metazoan mitochondria encode the same 37 genes, consisting of 13
protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes.
Therefore, it is clear that each of these genes is homologous among
metazoans, making full-genome sequence comparisons possible
among animal taxa. However, while mt genome data have many
useful attributes, there are limits on which genes or gene regions
are useful. In part, this depends on the divergence times of the taxa
used in the analysis. The rate of evolution in mitochondrial genes
tends to be more rapid than in many nuclear loci, and therefore
mt gene comparisons tend to show the effects of phylogenetic saturation sooner than other loci. Additionally, arthropod mt genomes
tend to possess a pronounced A + T bias, exacerbating the effects of
saturation. Because saturation can mislead phylogeny reconstruction, it is necessary to try to correct for the possible misleading effects of saturation. Possible corrective measures for protein-coding
genes include omitting saturated third- and first-position nucleotides of codons, or using only translated amino acid data. Alternatively, coding third and first positions of codons as purines or
pyrimidines (RY coding) has been found to help reduce the misleading effects of saturation found in mtDNA phylogenetic analyses
(Phillips and Penny, 2003).
Another potential challenge when using mtDNA sequences in
phylogenetic analyses is uneven base composition among taxa included in the study. Genome-wide biases are present in metazoan
mitochondrial genomes in that genes encoded on the strand associated with initiating replication tend to have a pronounced A + C
bias (Reyes et al., 1998). However, within arthropods, and arachnids in particular, this bias is reversed in some taxa (Hassanin
et al., 2005; Masta and Boore, 2004; Masta et al., 2009), so that
mitochondrial genes of phylogenetically disjunct taxa can share
similar compositional bias. This has the effect of causing the reversed-biased taxa to cluster together in phylogenetic analyses,
unless measures are taken to ameliorate the misleading impact
of such biases (Hassanin et al., 2005; Jones et al., 2007; Masta
et al., 2009). Such measures include recoding some of the nucleotide sites in the codons of the protein-coding genes as purines or
pyrimidines (Hassanin et al., 2005; Jones et al., 2007; Masta
et al., 2009), or recoding amino acids into categories corresponding
to their physiochemical properties (Masta et al., 2009).
In this manuscript, we present new data to bear on the relationship of sea spiders to other arthropods, and on their relationships
to one another. We have sequenced the complete suite of 13 mt
protein-coding genes from three divergent sea spiders. This boosts
the taxonomic coverage of pycnogonids with complete mt proteincoding genes to 5 taxa. We conduct phylogenetic analyses using
the entire suite of 13 protein-coding genes, and include representative taxa from 8 arachnid orders. Previous analyses that concluded that pycnogonids were derived arachnids had
representatives from only three arachnid orders. We compare gene
arrangement of our three new pycnogonid mt genomes (one complete, and two missing sequence near the control region) to the
two published pycnogonid mt genomes and infer the phylogenetic
history of gene arrangements in this group. Gene order provides
independent phylogenetic characters that support our findings
based on sequences. We also compare secondary structures of
the transfer RNA genes in these 5 mt genomes, and use these structures as phylogenetic characters. All three types of data are in
agreement. Together, our data provide strong support for relation-
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
ships among the pycnogonids that have not been inferred based on
anatomical characters.
2. Materials and methods
2.1. Specimens used, DNA extraction, amplification, and sequencing
Specimens of the pycnogonids Tanystylum orbiculare (collected
from the coast of Massachusetts; gift from Jeffrey Shultz), Colossendeis sp. (collected from Antarctica; gift from Jeffrey Shultz), and
Ammothea hilgendorfi (collected from the coast of California; gift
from Bonnie Bain), were used in this study. DNA was extracted
from a leg, or part of a leg, from each specimen, following the protocol from the Qiagen™ DNAeasy kit.
Initially, short fragments were PCR amplified from both the CO1
and Cytb genes with the primers LCO/HCO and CobF/CobR, respectively (see Masta and Boore, 2008 for further details). Then, taxon
specific outward-facing primers (see Appendix) were developed
from these resulting fragments, and used for long-PCR amplification. Long-PCR amplification was performed using the Takara™
LA Taq DNA polymerase kit. A 100 ll reaction contained final concentrations of 0.16 mM of each dNTP, 0.4 mM of each primer, 1X
Takara™ polymerase buffer, 1 ll of mitochondrial DNA (concentration not determined) and 2.5 Units of Takara™ polymerase. The
reaction was cycled at 92 °C for 30 s, 50–58 °C (depending on the
primers) for 25 s, and 68 °C for 12 min, for 37 cycles, followed by
a final extension at 72 °C for 15 min. The PCR products were electrophoresed in a 0.8% agarose gel to estimate size and concentration, then cleaned by ethanol precipitation, and resuspended in
water.
The mitochondrial genome for Tanystylum was amplified in one
complete 15 kb piece using primers facing out from the Cytb
amplicon. The majority of protein-coding genes for Colossendeis
and Ammothea (about 9 kb) between CO1 and Cytb were amplified
in one fragment using specific primers from the two alternate
genes. These long-PCR fragments were sheared into 1.5–2 kB
pieces, cloned into plasmid libraries, and subject to automated
sequencing at the DOE Joint Genome institute (as in Masta and
Boore, 2008). This resulted in approximately 15 coverage depth
for Tanystylum and Colossendeis, and 5 coverage depth for
Ammothea.
We were unable to amplify across the control region and rRNA
segments for Colossendeis and Ammothea with long-PCR. Instead,
for these two taxa remaining genomic segments of protein-coding
genes were amplified by primer-walking. In both cases, a 2 kb region containing ND2 was amplified in a single reaction using complementary primers that targeted tRNAMet and CO1. Additional
taxon specific primers were designed within this amplicon for
internal sequencing across ND2. To amplify between CytB and
ND1, we first amplified a short piece of the 16S rRNA in both taxa,
and then designed taxon specific primers amplicon to sequence
internally. In addition, we sequenced between 12S rRNA and 16S
rRNA in Ammothea using taxon specific primers.
2.2. Sequence annotation, analyses, and tRNA secondary structure
determination
Protein-coding genes were identified by similarity of inferred
amino acid sequences to those of other arthropod mtDNAs. Ribosomal RNA genes were annotated based on alignments with other
arachnid rRNA genes. After the protein-coding gene boundaries
had been determined, the remaining regions were searched for
tRNAs with the use of the program tRNAscan-SE 1.21 (Lowe and
Eddy, 1997), and by aligning the candidate sequences to the secondary structure-based alignments of other arachnids (see Masta
61
and Boore, 2008). Inferred secondary structures of tRNAs were
made by searching for conserved structural features. Anticodon
stems always consist of 5 pairs of nucleotides while acceptor stems
always consist of 7 pairs of nucleotides. However, the stems of the
TWC or DHU arms (also termed T-arm and D-arms) can vary in
length in the mitochondrial genomes of arthropods. These arms
were determined to be present if there were potential stems consisting of at least 2 nucleotides that could pair with the nucleotides
on the adjacent stem, and if the T- or D-loops consisted of at least 3
nucleotides. If either of these two conditions were not met, the
nucleotides present in these regions were determined to form TV
or D replacement loops, structures common in other chelicerates
(Masta and Boore, 2008). The secondary structures of the tRNAs
from the published mt genome sequences of the sea spiders Achelia
bituberculata (GenBank accession # NC_009724) and Nymphon
gracile (GenBank accession # NC_008572) were also inferred and
compared to those from the three newly sequenced sea spider
mt genomes.
Nucleotide composition was inferred with the aid of the program MacVector version 10.0.2. Strand asymmetry or skew was
calculated following Perna and Kocher (1995), where AT
skew = (A T)/(A + T) and CG skew = (C G)/(C + G). Skew was
calculated for the strand encoding the majority of the protein-coding genes in most arthropods. This strand is termed the alpha
strand in this manuscript, while the opposite strand is termed
the beta strand.
We compared the gene order of the mt genomes of these three
sea spiders to those of the published sequences of the sea spiders A.
bituberculata and N. gracile, and to the gene arrangement ancestral
for chelicerates, as found in horseshoe crabs, mesothele spiders,
amblypygids, and some scorpions and ticks (see Fahrein et al.,
2009; Masta, 2010).
2.3. Sequence alignment and phylogenetic analyses
Sequences from 34 additional complete arthropod mitochondrial genomes that span the diversity of arthropods were selected
for phylogenetic analyses, with the onychophoran Epiperipatus
used as an outgroup (see Table 1 for a list of taxa). We purposefully
excluded candidate mitogenomic data for several mites (e.g., Leptotrombidium, Steganacarus; Metaseiulus), as these taxa show many
substitutions that may cause branch attraction artifacts (e.g., Podsiadlowski and Braband, 2006, plus exploratory analyses not
shown). All 13 mt protein-coding genes from these 40 taxa were
translated with the Drosophila mitochondrial genetic code and
the amino acids initially aligned with MacClade (Maddison and
Maddison, 2000). Amino acid sequences were then aligned in Clustal X version 1.83 (Thompson et al., 1997) using the PAM350 protein matrix, gap opening equal to 10, and gap extension equal to
0.1. To increase confidence that our alignment accurately reflected
homologous amino acids, poorly conserved sites were subsequently excluded with the program Gblocks (Castresana, 2000)
version 0.91b. Regions of low sequence similarity were removed
by using the half gap setting and conserving alignment regions
with at least five consecutive conserved amino acids. The
Gblocks-trimmed amino acid sequences from the 13 genes were
then concatenated into a single sequence.
For analysis of nucleotide sequences, we used only nucleotides
that correspond to the amino acids kept by Gblocks for phylogenetic analyses. The output files from Gblocks (that list which amino
acids in the alignment were kept) were used to compare to the
nucleotide alignments in MacClade, using the ‘‘Nucleotide with
Amino Acid Translation” display option. The regions that were excluded from the GBlocks alignment were then manually excluded
from the MacClade file. The nucleotides at the first and third positions of each codon were then recoded as either purines or pyrim-
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S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
Table 1
Taxonomic sampling used in phylogenetic analyses.
Taxonomic lineage
Chelicerata
Species
GenBank No.
Thelyphonida
Opiliones
Tanystylum orbiculare
Ammothea hilgendorfi
Achelia bituberculata
Colossendeis sp.
Nymphon gracile
Limulus polyphemus
Ornithodoros moubata
Haemaphysalis flava
Ixodes hexagonus
Rhipicephalus sanguineus
Carios capensis
Varroa destructor
Phrynus sp.
Calisoga longitarsisb
Ornithoctonus huwena
Habronattus oregonensis
Nephila clavata
Hypochilus thorelli
Heptathela hangzhouensis
Pseudocellus pearsii
Buthus occitanus
Centruroides limpidus
Uroctonus mordax
Eremobates palpisetulosus
Nothopuga sp.
Mastigoproctus giganteus
Phalangium opilio
GU370074
GU370075
NC_009724
GU370076
NC_008572
NC_003057
NC_004357
NC_005292
NC_002010
NC_002074
NC_005291
NC_004454
NC_010775
NC_010780
NC_005925
NC_005942
NC_008063
NC_010777
NC_005924
NC_009985
NC_010765
NC_006896
NC_010782
NC_010779
NC_009984
NC_010430
NC_010766
Lithobiidae
Scutigeridae
Spirobolidae
Harpagophoridae
Lithobius forficatus
Scutigera coleoptrata
Narceus annularus
Thyropygus sp. DVL-2001
NC_002629
NC_005870
NC_003343
NC_003344
Hexapoda
Collembola, Poduridae
Insecta, Diptera Drosophilidae
Insecta, Diptera
Neoptera, Hemiptera
Podura aquatica
Drosophila yakuba
Anopheles gambiae
Triatoma dimidiata
NC_006075
NC_001322
NC_002084
NC_002609
Crustacea
Maxillopoda, Cirripedia,
Malacostraca, Paguridae
Branchiopoda, Anostraca
Diplostraca
Pollicipes polymerus
Pagurus longicarpus
Artemia franciscana
Daphnia pulex
NC_005936
NC_003058
NC_001620
NC_000844
Peripatidae
Epiperipatus biolleya
NC_009082
a
Pycnogonida
Ammotheidae
Xiphosura
Arachnida
Colossendeidae
Nymphonidae
Limulidae
Acari, Ixodida
Acari, Mesostigmata
Amblypygi
Araneae, Mygalomorphae
Araneae, Araneomorphae
Araneae, Mesothelae
Ricinulei
Scorpiones, Buthidae
Scorpiones, Chactidae
Solifugae
Myriapoda
Chilopoda
Diplopoda
Pancrustacea
Onychophora
a
Tanystylum was formerly placed in the family Tanystylidae, but found by Arango (2002) to possess morphological characters that placed it within Ammotheidae, and was
subsequently considered to be in the family Ammotheidae (Arango and Wheeler, 2007).
b
This mygalomorph spider was formerly labeled as Aphonopelma sp. in GenBank, but it has now been identified as Calisoga longitarsis (although it is currently placed in the
genus Brachythele, until the nomenclature is revised).
idines (RY coding), following Phillips and Penny (2003) as this has
been found to increase the phylogenetic signal in analyses of mitochondrial genomes (Delsuc et al., 2003; Phillips and Penny, 2003).
Phylogenetic analyses of amino acids and nucleotides were performed using maximum likelihood with RaxML, version 7.0.4 (Stamatakis, 2006). For analyses with amino acids, the MtREV model of
evolution was used, with gamma plus invariant sites model of rate
heterogeneity. The MtREV model of evolution is the best model for
mtDNA analyses that is available in RaxML, as determined by the
use of ProtTest (Abascal et al., 2005). For analyses of nucleotides,
the GTR with gamma plus invariant sites model of evolution was
used on the RY recoded dataset. One thousand bootstrap replicates
were performed for both of these analyses using the same models,
except using the CAT approximation instead of gamma for the amino acid analysis. RaxML using the CAT approximation has been
shown to perform well on large datasets (Stamatakis, 2006). Figures of the recovered phylogenetic trees were made with the aid
of FigTree v1.2.3.
Because some of the pycnogonid mt genomes were found to
possess a reversed nucleotide strand bias, as were those of some
arachnids (Masta et al., 2009), we also recoded amino acids into
six physiochemical groups. We categorized amino acids into the
following six groups: sulfhydryl (cysteine); small/neutral (serine,
threonine, alanine, proline, and glycine); acidic/amides (aspartate,
glutamate, asparagine, and glutamine); basic (histidine, arginine,
and lysine); hydrophobic (valine, leucine, isoleucine, and methionine); and aromatic (phenylalanine, tyrosine, and tryptophan). This
six-character-state data matrix was then analyzed by parsimony
using PAUP* (Swofford, 2000) with a heuristic search with 10 replicates, ACCTRAN, and tree-bisection-reconnection branch-swapping. One thousand bootstrap replicates were performed using
these same parameters.
3. Results
3.1. Genome composition
We obtained the entire mt genome sequence of T. orbiculare, a
circular genome of 15,251 nt (GenBank accession number
GU370074). For the mt genome of A. hilgendorfi, we obtained
14,026 nt of sequence (GenBank accession number GU370075),
and for Colossendeis we sequenced 12,776 nt (GenBank accession
number GU370076). The regions not sequenced in Ammothea and
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
Colossendeis are located near the where the control region is typically found in arthropods. It is not uncommon to have difficulty
amplifying and sequencing across this region in arthropods, possibly due to the high A + T content, or the stem-forming stretches of
nucleotides that create secondary structures and can inhibit function of polymerase enzymes. As a result, some of the mt tRNA and
rRNA genes were not sequenced for Ammothea and Colossendeis;
however, we obtained full coverage of all 13 of the mt protein-coding genes for each of the three pycnogonids.
The mt genome of Tanystylum contains all 22 tRNA genes and
the SSU and LSU rRNA genes that are typically found in metazoan
mt genomes. The partial mt genome of Ammothea lacks sequences
for the genes for tRNAIle and tRNAGln and has only the 30 ends of the
SSU rRNA and tRNAMet genes. The sequence for the putative control
region is lacking. Presumably the control region plus the two missing tRNA genes are present in the unsequenced portion of the mt
genome. The partial mt genome of Colossendeis possesses 16 tRNA
genes, as we did not obtain sequence for tRNAPro, tRNAAla, tRNAAsn,
tRNASer(AGN), tRNAGln, and tRNAVal genes. The partial genome of Col-
Table 2
Nucleotide composition and skew of the alpha strand of pycnogonid mt genomes.
Taxon
Tanystylum
Ammothea
Achelia
Nymphon
Colossendeis
Proportion of nucleotides
A
C
G
T
0.389
0.414
0.379
0.454
0.363
0.097
0.117
0.119
0.141
0.116
0.103
0.081
0.110
0.085
0.127
0.410
0.388
0.391
0.319
0.388
AT skew
CG skew
0.0263
0.0324
0.0156
0.1746
0.0333
0.03
0.1818
0.0393
0.2478
0.0453
63
ossendeis also lacks sequence for the SSU rRNA gene, and has only
the 30 end of the LSU rRNA and tRNAMet genes. Sequence coding for
the putative control region is also missing.
Surprisingly, there are pronounced differences in the nucleotide
skews in these pycnogonid mt genomes. Ammothea and Nymphon
have a biased composition with positive AT and CG skews on the
alpha strand, while Tanystylum and Colossendeis have the opposite
pattern, with negative AT and CG skews (Table 2). Achelia shows a
third pattern, with a negative AT skew and a positive CG skew.
3.2. Gene order comparisons
The mitochondrial gene arrangements of all 5 pycnogonid taxa
with complete protein-coding data from the mt genome are depicted in Fig. 1. Tanystylum has a gene order identical to that of
Achelia, which differs from the ancestral chelicerate gene order
only in the location of a single tRNA gene. In these mt genomes,
the tRNAGln gene is located between the SSU gene and the large
noncoding region, instead of between the genes coding for tRNAIle
and tRNAMet as it is in the ancestral chelicerate genome. With the
exception of the missing region, the gene order for the Ammothea
mt genome is identical to that of the ancestral chelicerate, or Tanystylum, and Achelia.
The genome of Colossendeis shows a gene order quite different
from that of the ammotheid sea spiders, in several ways (Fig. 1).
First, four of the six unsequenced tRNA genes (tRNAAla, tRNAAsn,
tRNASer(AGN), and tRNAPro) should have been obtained had they
been in the same location as in the ancestral chelicerate; thus,
these must have been translocated. Second, the relative positions
of tRNAGlu and tRNAArg in Colossendeis are opposite to their posi-
Fig. 1. Mitochondrial genome arrangement of 5 pycnogonids and ancestral chelicerate arrangement. The circular mt genomes are illustrated as linear by depicting CO1 as the
beginning of each linearized genome (except the highly rearranged Nymphon). The putative control regions are designated with ‘‘lnr” (large noncoding region), while tRNA
genes are abbreviated with their one-letter amino acid code. The two leucine and two serine tRNA genes are further differentiated by numerals, where L1 = tRNALeu(CUN),
L2 = tRNALeu(UUR)), S1 = tRNASer(AGN), and S2 = tRNASer(UCN). Shaded regions indicate that the genes present in that location differ from those present in that location in the
ancestral chelicerate. Genes are not shown proportional to size.
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S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
tions in the ancestral chelicerate and in ammotheid sea spiders.
Third, the tRNATyr gene is located next to the tRNAPhe gene on
the beta strand of Colossendeis. The gene order between the ND3
and tRNAPhe genes in Colossendeis is therefore the same as in Nymphon, except that the control region of Nymphon is located between
the tRNAArg and tRNATyr genes (Fig. 1). Likewise, Colossendeis and
Nymphon have identical gene arrangements between ND4L and
ND6, as both lack the gene for tRNAPro that is located here in the
other pycnogonids. Finally, a unique gene arrangement found in
Colossendeis but not other sea spiders, is the location of the tRNAMet
gene immediately upstream of the tRNAIle gene.
3.3. tRNA structure comparisons
Most of the 22 tRNA genes in the mt genomes of these 5 pycnogonids have inferred secondary structures that are typical of
metazoan mt tRNAs. In some cases, we reached different interpretations of secondary structure for Achelia and Nymphon than had
been previously published (Park et al., 2007; Podsiadlowski and
Braband, 2006). While most of the tRNAs possess cloverleaf structures, some tRNA genes lack the sequence to form the DHU or Darm that is part of the canonical tRNA (see Fig. 2). All pycnogonids
lack D-arm sequences in their tRNAAla genes, with the most extreme nucleotide reductions occurring in Tanystylum, Ammothea,
and Achelia (Fig. 3), although Colossendeis was unsampled. Nymphon and Colossendeis lack well-paired D-arm stem sequences in
their tRNAArg genes. Like other metazoans (Wolstenholme,
1992b), all pycnogonids lack D-arm sequence in their tRNASer(AGN)
genes. All 22 of the tRNAs present in the mitochondrial genomes of
these pycnogonids possess T-arms (Fig. 3).
Another structural difference is present in the tRNAIle gene in
Nymphon and Colossendeis. These taxa, but not the ammotheids,
possess a variable loop that is only 4 nt long, instead of the 5-ntlong loop found in Tanystylum and Achelia.
Differences also exist in the anticodons present in two tRNA
genes within sea spiders. The anticodon for tRNALys is UUU in all
five pycnogonids, whereas other arthropods possess a CUU anticodon. The anticodon for tRNASer(AGN) is UCU in all four pycnogonids
for which this tRNA gene was sequenced. This differs from the typical GCU anticodon found in most other chelicerates.
Some mispaired nucleotides are present in the anticodon and
acceptor stem regions of 8 tRNA genes within pycnogonids. While
most of these are confined to only a single individual, tRNAIle and
tRNALeu(CUN) both possess mispaired nucleotides in their acceptor
stem regions in Nymphon and Colossendeis, whereas the other pycnogonids have well-paired stems. The anticodon stems of the
tRNACys genes have mispaired nucleotides in Nymphon and Colossendeis (Fig. 3).
3.4. Phylogenetic analyses of amino acids
There were 3894 amino acids in our initial alignments of the
protein-coding genes. Of these, 2313 amino acids from all 13 concatenated protein-coding genes were retained after GBlocks trimming of the dataset. This reduced dataset was used in
subsequent phylogenetic analyses.
The maximum likelihood analysis of amino acids yielded a phylogenetic tree in which a clade of chelicerates was recovered with
95% bootstrap support (Fig. 4A). Nested within the chelicerates, a
clade of pycnogonids was recovered with 100% bootstrap support.
While there is not strong support to infer which group of arachnids
may be the sister group to pycnogonids, there is ample support to
place them within the clade Micrura, which traditionally consists
of the orders Acari, Ricinulei, Araneae, Thelyphonida, Schizomida,
and Amblypygi (see review in Shultz, 2007).
Relationships among the other major lineages of arthropods are
similar to those that have been recovered in other analyses of
molecular data, with high support for a clade of Pancrustacea. Myriapoda was not recovered as a clade, but instead the centipedes
and millipedes were each found to be monophyletic, with very
weak support for a sister-group relationship between the Diplopoda (millipedes) and the chelicerates.
The interrelationships among the pycnogonids were all strongly
supported, with two divergent lineages recovered. One lineage
consists of Tanystylum, Ammothea and Achelia, while the other consists of Nymphon and Colossendeis.
The parsimony tree that we recovered from analyses of amino
acids that had been recoded into 6 functional groups yielded a tree
with unresolved relationships among the major lineages of arthropods, and therefore is not shown. However, this tree yielded identical relationships among the pycnogonids, with 100% bootstrap
support for a clade consisting of Tanystylum, Ammothea and Achelia,
and 100% bootstrap support for a clade consisting of Nymphon and
Colossendeis. There was 76% bootstrap support for a sister-group
relationship between Tanystylum and Ammothea.
3.5. Phylogenetic analyses of RY-coded nucleotides
A
B
tRNA-Alanine
tRNA-Arginine
aminoacyl
acceptor arm
D-arm
T
T
T AG T T
A TCA A
anticodon arm
A
T
A
5'
T
A
G
T
A
A
G
T
A
T
T
A
T
T
C
T-arm
CCG AT
G
GGT G T
A
T
TT
T
A
A
C
T
T
C
T
A
TCG
A
T
T
G
A
3'
variable
loop
3'
A
T
C
T
G
T
T
C
C G A T A TT
A
T
G
C TAT A
A
A
T
T
T TA T
T A
T A
A T
A T
A
T
A
T
TGC
5'
A
G
G
C
A
A
G
Fig. 2. Secondary structure drawing of the two types of tRNAs found in pycnogonids. The tRNA structures from Tanystylum orbiculare are illustrated. (A) A canonical
tRNA, as illustrated with tRNAArg. Note the 5 regions of the tRNA: acceptor stem, Darm, anticodon arm, variable loop, and T-arm. (B) A tRNA that lacks a D-arm, and
instead has only a D-loop, as illustrated with tRNAAla.
The maximum likelihood analysis of nucleotides with the first
and third positions recoded as purines or pyrimidines yielded a
phylogenetic tree that was largely congruent with the aminoacid-based tree. A clade of chelicerates was recovered with 81%
bootstrap support, with a clade of pycnogonids nested within
Chelicerata (Fig. 4B). Unlike the amino-acid-based tree, however,
there was only very weak support for placing Pycnogonida within
the micrurans. Additionally, all micrurans were not included in this
clade, as Amblypygi fell outside of it.
The RY-coded analysis recovered a high level of support (97%
bootstrap) for a clade of Pancrustacea, as was also found in the
amino acid analysis. While there was generally lower support for
most groupings in the recoded nucleotide based tree, there was
high support for a monophyletic Myriapoda. Support for a sistergroup relationship between Myriapoda and Chelicerata was weak.
Pycnogonida was recovered with high support, as were the
same two divergent lineages found in the amino acid analysis (a
clade consisting of Tanystylum, Ammothea and Achelia, and a clade
consisting of Nymphon and Colossendeis). However, in the RY-recoded nucleotide analysis, Tanystylum and Achelia were recovered
as sister taxa with 68% bootstrap support (Fig. 4B).
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
65
Fig. 3. Secondary structure alignments for the 22 tRNAs found in the mt genomes of pycnogonids. The 5 regions of the canonical tRNA structure are given at the top of the
figure (see Fig. 2 for an illustration of these regions). Dashes separate these structural features in the alignments. Underlined nucleotides indicate they form base pairs with
adjacent underlined nucleotides in the same region of the tRNA.
3.6. Rare genomic changes mapped onto the phylogenetic tree
The gene translocations and tRNA structure differences that we
inferred were mapped onto the consensus phylogenetic tree of
pycnogonids. The pycnogonids are united by three tRNA features
that distinguish them from other chelicerates: the loss of a Darm in tRNAAla, and changes in the anticodon identities of tRNALys
and tRNASer(AGN) (Fig. 5). Within pycnogonids, the ammotheids
Tanystylum and Achelia are united by two rare genomic changes.
One is the translocation of tRNAGln between SSU and the large noncoding region, while the other is a reduction in the size of the Dloop in tRNAAla. In contrast, the clade consisting of Nymphon and
Colossendeis shows at least 12 changes in genome or tRNA structure that unite it as a monophyletic group. Five of these changes
occur within the tRNA genes, while the other seven changes are
based upon genome rearrangements (Fig. 5).
At least seven mt tRNA translocations must have occurred in the
lineage leading to Nymphon and Colossendeis. These are mapped
66
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
Fig. 3 (continued)
onto the phylogenetic tree of pycnogonids (Fig. 5). An eighth translocation, that of tRNAVal moving away from its ancestral location
between the LSU and SSU rRNA genes, cannot be placed with confidence onto the phylogeny, since we lack sequence data for this
region in Colossendeis. Four unique gene translocations distinguish
Nymphon, and therefore must have occurred after its divergence
from a common ancestor with Colossendeis (Fig. 5).
4. Discussion
4.1. Variable patterns of nucleotide bias
Arthropod mt genomes tend to have an AT nucleotide bias, with
a positive AC skew on the major or alpha strand. The strength of the
skew varies among taxa, and some arthropod taxa have been found
to show the opposite pattern; that is, a negative AC skew, or a positive TG skew (Hassanin, 2006). We found both of these two extreme patterns of skew present within the five pycnogonids we
analyzed. Although reversals in skew can impact phylogenetic
reconstruction and lead to erroneous conclusions (Hassanin,
2006; Jones et al., 2007; Masta et al., 2009), we used methods de-
signed to overcome the effects of saturation and opposite patterns
of mutational bias. Within pycnogonids, we found no evidence that
altered patterns of nucleotide bias affected phylogeny reconstruction. Biased nucleotide composition may be expected to cause taxa
with similar biases to group together. However, Tanystylum and
Colossendeis show patterns of skew opposite to that of the other
pycnogonids, and indeed to that of most arthropods. However,
these taxa were not reconstructed as sister taxa, despite their
shared biases. Likewise, other taxa with similar patterns of skew
(e.g., opisthothele spiders and scorpions; see Masta et al., 2009)
also did not group closely with these pycnogonids. This gives us
confidence that our phylogenetic methods have adequately accounted for different mutational biases in this dataset.
4.2. Pycnogonids as derived arachnids
Our analyses strongly support the placement of Pycnogonida
within Chelicerata, and therefore would reject the hypothesis that
sea spiders are the sister group to all other arthropods. Our phylogenetic analyses also consistently place sea spiders as derived
chelicerates. These findings are in agreement with other recent
67
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
Ixodes
100
100
100
100
Rhipicephalus
Haemaphysalis
Ornithodoros
Carios
Varroa
Tanystylum
Ammothea
Achelia
100
57
100
80
100
100
54
100
100
52
95
Limulus
56
100
100
55
100
57
93
58
97
Centruroides
Buthus
Uroctonus
Narceus
Thyropygus
Scutigera
Lithobius
Pollicipes
Pagurus
98
Scorpiones
Myriapoda
Podura
Artemia
98
Araneae
Amblypygi
Solifugae
Opiliones
Xiphosura
Phrynus
Nothopuga
Eremobates
Phalangium
100
Pycnogonida
Nymphon
Colossendeis
Pseudocellus Ricinulei
Mastigoproctus Thelyphonida
Heptathela
100
Ornithoctonus
Calisoga
Habronattus
100
78
Nephila
Hypochilus
69
73
Acari
Chelicerata
A
Pancrustacea
Daphnia
Triatoma
100
Anopheles
Drosophila
Epiperipatus
Onychophora
0.2
B
100
100
Ixodes
100
Rhipicephalus
Haemaphysalis
Ornithodoros
Carios
100
100
Acari
Varroa
68
100
Ricinulei
Pycnogonida
Nymphon
Colossendeis
100
Mastigoproctus
Heptathela
92
100
81
100
98
100
Phrynus
Limulus
84 98
97
77
98
100
Thelyphonida
Ornithoctonus
Calisoga
Habronattus
Nephila
Hypochilus
100
Nothopuga
Eremobates
Araneae
Solifugae
Amblypygi
Xiphosura
Centruroides
Buthus
Uroctonus
Phalangium
100
Narceus
Thyropygus
Scutigera
Lithobius
Pollicipes
Pagurus
93
Daphnia
Artemia
Podura
Triatoma
Anopheles
100
Drosophila
Epiperipatus
100
Chelicerata
100
Pseudocellus
Tanystylum
Achelia
Ammothea
Scorpiones
Opiliones
Myriapoda
Pancrustacea
Onychophora
0.1
Fig. 4. (A) Maximum likelihood tree based on amino acids from 13 mt protein-coding genes, using the MtREV substitution matrix. Bootstrap values greater than 50% are
shown above or below the corresponding branches. Likelihood = 86273.619981; Alpha = 0.857; invariant sites = 0.194572. (B) Maximum likelihood tree based on RY-coded
nucleotides from 13 mt protein-coding genes, using the GTR substitution matrix with gamma and invariant sites. Bootstrap values greater than 50% are shown above or below
the corresponding branches. For both trees, the bars colored medium gray highlight the three main arthropod groups: Chelicerata, Myriapoda, and Pancrustacea, while the
other colored bars are used to highlight the chelicerate groups for which multiple taxa were sequenced.
68
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
A
9
1 2 3
Tanystylum
Ammothea
Achelia
N
I J K L
Nymphon
4 5 6 7 8 B C D E F G H
M
Colossendeis
Fig. 5. Phylogeny of pycnogonids with rare genomic changes depicted on the tree. Boxes that are shaded and with letters depict inferred gene translocation events, and
unshaded boxes with numbers depict inferred changes in tRNA structure or anticodon identity. Letters for tRNA gene translocations are as follows, using the one-letter amino
acid abbreviation: (A) Q translocated between SSU and the large noncoding region (lnr), on same beta strand as original location. (Not assessed in Ammothea); (B) I (or M)
translocated between M and ND2, on same alpha strand as original location; (C) Y translocated next to F, on same beta strand as original location; (D) N translocated to near
control region; (E) S1 translocated to near control region; (F) A translocated to near control region; (G) either R or E translocated such that E is directly upstream of R on alpha
strand; (H) P translocated to near control region; (I) translocation and inversion of K and D to near control region; (J) translocation of region coding for I-ND2-W-C-CO1-CO2
onto opposite strand, upstream of ATP8; (K) M translocated to beta strand, between P and S1; (L) control region translocated to between R and Y; (M) translocation of I to
between M and ND2, on same alpha strand and (N) V translocated away from location between LSU and SSU rRNA genes. Numbers for tRNA changes are as follows: (1) loss of
the D-arm in tRNAAla; (2) anticodon for tRNALys changes to UUU; (3) anticodon for tRNASer(AGN) changes to UCU; (4) variable loop in tRNAIle decreases to 4 nt in Nymphon and
Colossendeis, from 5 nt long in Tanystylum and Achelia; (5) loss of paired D-arm sequence in tRNAArg; (6) mispaired acceptor stems in tRNAIle; (7) Mispaired acceptor stems in
tRNALeu(CUN); (8) mispaired anticodon stems of tRNACys and (9) reduced number of nucleotides in D-loop in tRNAAla.
analyses of mitochondrial genome data (Jeyaprakash and Hoy,
2009; Park et al., 2007; Podsiadlowski and Braband, 2006). However, unlike those analyses, which had representative taxa from
just three of the arachnid orders, our analyses included taxa from
8 arachnid orders that span the diversity of Chelicerata. Our phylogenetic analyses support Pycnogonida as members of micruran
arachnids, a group that includes the Acari (mites and ticks) and
Araneae (spiders). Pycnogonids have been found to be nested within a clade of micrurans in combined analyses of morphological and
nuclear rRNA data (Giribet et al., 2002), but the authors cautioned
that they did not find high support for these relationships.
The possibility that sea spiders are derived arachnids or chelicerates has not been supported by developmental or anatomical
studies. However, most studies that have tried to determine the
taxonomic placement of pycnogonids have not included a diversity
of micruran arachnids in their analyses (but see Wheeler and Hayashi, 1998). Therefore, it is not clear which, if any, anatomical or
developmental features may be shared among pycnogonids and
other arachnids. Indeed the idea that sea spiders are derived arachnids may seem unlikely, not only because of sea spiders’ unique
and highly derived anatomy, but also due to their strictly marine
habitats. Most arachnids are strictly terrestrial, with anatomical
features that allow them to live in terrestrial environments.
The ecology of sea spiders, however, does not necessarily help
us understand their phylogenetic affinities. Although they are restricted to marine environments, pycnogonids are found in a diverse array of ecological niches, from the intertidal zone to deepsea hydrothermal vents to the cold waters of Antarctica. Although
it is tempting to think that their marine habitats may imply a close
relationship to the (marine) horseshoe crabs, sea spiders are not
the sole marine chelicerates. Some mites in the arachnid order
Acari inhabit marine environments, whereas others inhabit terrestrial and freshwater environments. Clearly, transitions from sea to
land and back again have occurred in the evolutionary history of
the chelicerates.
Paleontological data are consistent with an aquatic origin of
pycnogonids, and suggests they emerged during the Cambrian, before the other extant chelicerate lineages (Dunlop and Selden,
2009). Fossils of horseshoe crabs (Rudkin et al., 2008) exist from
the Ordovician, consistent with an early divergence of xiphosurans.
Because these horseshoe crab fossils possess derived anatomical
features, it was suggested that xiphosurans likely diverged even
earlier, in the Cambro-Ordovician (Rudkin et al., 2008). The wellsupported nodes in our phylogenetic trees (e.g., >70% bootstrap
support) are not in conflict with this paleontological data. Instead,
they suggest a scenario of an ancestral chelicerate diversifying in
the Cambrian into at least two lineages. One of these lineages could
then have subsequently diversified into pycnogonids and the
ancestor of micrurans, while the other lineage diversified into
xiphosurans and the ancestor of dromopodan arachnids (i.e., scor-
pions, solifuges, harvestmen). Under this scenario, the group
Arachnida would be polyphyletic, with two separate origins. Further fossil discoveries and developmental and molecular data with
stronger phylogenetic signal will help elucidate if this is a likely
scenario.
4.3. Rare genomic changes as phylogenetic markers
Although there are no gene arrangements that group sea spiders
with any single chelicerate lineage, pycnogonids are supported as
chelicerates by their similar gene order. The ammotheid sea spiders share the same basic gene arrangement as other chelicerates,
and differ only in the location of a single tRNA gene. Therefore, we
can infer that the translocation of this tRNA occurred after the
divergence of sea spiders from their common ancestor with other
chelicerates. Pycnogonids lack the derived mt gene order found
in Pancrustacea, in which the tRNALeu(UUR) gene is located between
CO1 and CO2, and not between ND1 and the LSU rRNA genes as in
chelicerates (Boore et al., 1995, 1998). Therefore, both our phylogenetic analyses and our genome arrangement data are consistent
with pycnogonids being chelicerates.
The three tRNA structural characters that unite the pycnogonids
(see Fig. 5) show some degree of homoplasy when evaluated across
arthropods. A change in the anticodon from GCU to UCU in tRNASer(AGN)
has been found in some other arthropod groups. For example, all beetles appear to possess tRNASer(AGN) genes with UCU
anticodons, an apparent synapomorphy for this lineage of insects
(Sheffield et al., 2008). Likewise, a change in anticodon identity
for tRNALys has occurred multiple times in arthropods, and is correlated with changes in tRNASer(AGN) anticodon identity (Abascal
et al., 2006). Changes in anticodon identity and remolding of tRNALeu
has occurred multiple times in the evolution of metazoan
mtDNA (Rawlings et al., 2003), and can lead to incorrect inferences
about gene rearrangements if not correctly identified. However,
tRNA remolding cannot explain the translocations of whole blocks
of tRNA genes to new locations in the genome, as found in Nymphon and Colossendeis. The loss of the D-arm from tRNAAla has also
occurred independently in amblypygids (Fahrein et al., 2009).
Therefore, while these changes have occurred only rarely in evolution, they do exhibit some degree of homoplasy within arthropods.
Searching for other rare evolutionary changes that pycnogonids
share with other taxonomic groups may help to more definitively
infer their closest relatives.
In contrast, many of the genome rearrangements that we inferred to occur in the common ancestor of Nymphon and Colossendeis are unique among arthropods. For example, the gene
arrangement of the genes for ND3, tRNAGlu, tRNAArg on the alpha
strand, followed by the tRNATyr and tRNAPhe genes on the beta
strand, is not found among other arthropods. This gene order required multiple translocations of tRNA genes, and therefore, find-
S.E. Masta et al. / Molecular Phylogenetics and Evolution 57 (2010) 59–70
ing evolution convergent with this same arrangement seems
highly unlikely. However, the shared changes seen in the tRNA
genes among Nymphon and Colossendeis are likely more evolutionarily labile, and may occur on a time scale that allows them to be
useful for delimiting pycnogonid taxa, although not at deep phylogenetic levels. Together, though, the inferred gene translocation
and tRNA structural characters are synapomorphies that further
support our phylogenetic analyses of amino acids and that group
Nymphon with Colossendeis.
4.4. Phylogenetic relationships within Pycnogonida
A traditional classification of pycnogonids divided them into 8
families (Hedgpeth, 1947), but did not make inferences as to how
these may be interrelated. The taxa in our study included representatives from what have been considered to be quite divergent lineages: Ammotheidae, Nymphonidae, and Colossendeidae. A
cladistic analysis of morphological characters (Arango, 2002) found
that Ammotheidae was a diverse and paraphyletic group, with Colossendeidae derived within the ammotheids. Taxa from the genera
Achelia and Tanystylum were found to be more closely related to
one another than they are to Ammothea. This paraphyletic group
of ammotheids plus colossendeids was found to be sister to a clade
that contained Nymphonidae.
Our analyses provide support for a group of ammotheids, but
only phylogenetic analyses of RY-coded nucleotide data give support for the closest relationship being between Achelia and Tanystylum. Analyses of amino acids yield trees with high support for a
closer relationship of Tanystylum with Ammothea. We do not find
rare genomic changes that support or refute either of these scenarios. However, we do find very strong support for a close relationship between Colossendeis and Nymphon, in contrast to inferences
made based upon morphological characters (Arango, 2002) and
morphology plus six loci (Arango and Wheeler, 2007). We also find
Nymphon to possess a highly derived gene arrangement that
clearly shares its origins with those of Colossendeis.
Our findings are consistent with those of Arango (2003), who
found that 18S and 28S rRNA sequences yielded a tree with the
ammotheids sister to a clade that contains Nymphon and Colossendeis (which were reconstructed as sister taxa). However, our findings differ from those of Nakamura et al. (2007), who found that
18S rRNA sequences resolved the clade containing Colossendeidae
as sister to a clade containing Ammotheidae, with Nymphonidae
sister to that large clade. Moreover, our findings differ from those
of Arango and Wheeler (2007), who conducted a comprehensive
analysis built on the earlier nuclear rRNA study of Arango (2003)
by adding many more taxa and using four additional loci, including
3 mitochondrial genes (CO1, SSU rRNA, and LSU rRNA) and histone
3. Arango and Wheeler (2007) recovered Nymphonidae in a clade
with Ammotheidae, with Colossendeidae sister to that larger clade
containing most families of sea spiders.
Why our results should differ from those of two of the three
previous molecular analyses is not clear. Arango and Wheeler
(2007) analyzed six loci in conjunction with morphological characters, so it is not possible to determine which of these other loci, if
any, conflicted with our complete mt protein-coding gene analyses
and the prior nuclear rRNA analyses. However, given the high support we found for a clade of Nymphon plus Colossendeis in all of our
analyses – with both sequence data and rare genomic changes –
our data strongly indicates that these two specimens are much
more closely related to one another than either are to the ammotheids Tanystylum, Achelia, and Ammothea. One possibility is that
the Colossendeis specimen could have been originally misidentified,
and in fact is closely related to nymphonid sea spiders. This specimen has been used extensively for analyses of nuclear loci (Regier
et al., 2008, 2010), and little remains of it to aid in re-identification.
69
Both the analyses of Arango and Wheeler (2007) and Nakamura
et al. (2007) consistently recovered Callipallenidae as sister to
Nymphonidae. Our findings would be consistent with these previous analyses if our specimen were actually a member of the family
Callipallenidae, and not Colossendidae. However, both our sequence data and our rare genomic change data indicate that the
specimen is quite divergent from the N. gracile mt genome, and
that rare genomic changes are useful for resolving sea spider
relationships.
Our analyses provide strong support for the placement of sea
spiders as chelicerates, and moderate support for sea spiders as derived chelicerates. There are deep divergences within sea spiders,
resulting in phylogenetic trees with lineages separated by long
branches. Additionally, we find multiple cases of changes in mitochondrial strand bias among these lineages. Our analyses attempted to overcome the misleading effects of changes in strand
bias by recoding data as amino acids or as purines and pyrimidines.
However, it is more difficult to overcome potential problems associated with long branches causing the recovery of artifactual relationships. Future sampling of additional sea spider mitochondrial
genomes may help break up the branch leading to sea spiders, as
well as branches among the major lineages of sea spiders. Rare
changes in the mitochondrial tRNA genes and in mitochondrial
gene arrangements hold much promise for helping to resolve relationships among this enigmatic group of animals.
Acknowledgments
This work was funded by NSF award no. DEB-0416628 to S.E.M.
The manuscript was improved by the comments of Claudia Arango,
Alfried Vogler, and an anonymous reviewer.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2010.06.020.
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