molecular systematics of the order anaspidea based on mitochondrial dna sequence (12s, 16s, and coi)
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Molecular Phylogenetics and EvolutionVol. 15, No. 1, April, pp. 41–58, 2000doi:10.1006/mpev.1999.0736, available online at http://www.idealibrary.com on
Molecular Systematics of the Order Anaspidea Based onMitochondrial DNA Sequence (12S, 16S, and COI)1
Monica Medina2 and Patrick J. Walsh
Rosenstiel School of Marine and Atmospheric Science, Division of Marine Biology and Fisheries, University of Miami,4600 Rickenbacker Causeway, Miami, Florida 33149
Received February 12, 1999; revised June 21, 1999
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Fragments from three mitochondrial genes (12S,6S, and COI) were sequenced to reconstruct a molecu-ar phylogeny of the opisthobranch order Anaspidea.he molecular phylogeny supports the placement of
he genus Akera, a taxon previously regarded by someuthors as a cephalaspidean, within the Anaspidea.ncongruence between the molecular data and thelassifications based on morphology suggests that somef the taxonomic characters (i.e., shell, parapodia fu-ion) traditionally used for the classification of seaares must be reevaluated, since they may be homoplas-ic. The ancestral nature of Notarchus based on theolecular evidence suggests that homoplasy may be
n explanation for the morphological resemblance ofhis species to the more derived sea hares with highlyused parapodia and concentrated nerve ganglia. Fi-ally, examples are given of how comparative studiesf the evolution of learning mechanisms in the anaspi-ean clade will benefit from the phylogenetic hypoth-sis presented in this paper. r 2000 Academic Press
INTRODUCTION
Recent developments in the field of systematics, thatake into account the evolutionary history of the traitsnder study (i.e., ancestral vs derived conditions), havellowed for more careful and explicit phylogeneticnalyses (Hennig, 1966). In some cases, morphologicalharacters traditionally used for classification are ofuestionable significance for resolving phylogenies be-ause they are not homologous, thus obscuring phyloge-etic associations between taxa. Previous attempts toetermine the phylogenetic relationships of the opistho-ranchs based on analysis of morphological charactersave been hampered by extensive homoplasy (Gosliner,
1 The sequence data reported here have been deposited in theenBank database under the Accession Nos. AF156109–AF156156.2 To whom correspondence should be addressed at present address:
osephine Bay Paul Center for Molecular Biology and Evolution,arine Biological Laboratory, 7 MBL Street, Woods Hole, MA
e2543-1015. Fax: (508) 457-4727. E-mail: [email protected].
41
981, 1991; Mikkelsen, 1993). Gosliner (1991) pre-ented comprehensive evidence of homoplasy for thehole subclass based primarily on reduction or loss of
haracters, and Mikkelsen (1993) presented evidence oforphological homoplasy, focusing on the relationshipsithin the primitive Cephalaspidea. Examples of traits
hat have been reduced or lost many times indepen-ently are the shell, the operculum, the ctenidum, theadula, and the gizzard plates. Euthyneury (or thentwisting of the lateral nerve cords) has evolvedeveral times from streptoneurous (or twisted) ancestors asconsequence of detorsion, i.e., the most recent Anaspidea
rom the primitive streptoneuran Akera (Gosliner, 1981,991). Thus, traditional opisthobranch taxonomists areaced with many homoplastic traits that are difficult tonterpret for phylogenetic reconstruction.
The Anaspidea is one of the smallest opisthobranchrders. It has, however, been a group of interest notnly to taxonomists (Eales, 1944; Beeman, 1968; Beb-ington, 1974, 1977; Marcus, 1972) but to scientistsrom other disciplines of biology. Neurobiologists havehosen sea hares of the genus Aplysia as model organ-sms due the ease of following synaptical pathwayshrough their large neuronal system (Kandel, 1979).ecently, comparative studies of related neurologicalnd behavioral traits within the Anaspidea have ap-eared in the literature (Nambu and Scheller, 1986;right et al., 1996). Ecologists have used sea hares to
tudy chemical defense mechanisms (Avila, 1995; Pen-ings and Paul, 1993; Pennings, 1994; Nolen et al.,996), feeding behavior (Carefoot, 1987; Pennings andaul, 1992), and physiological adaptation (Bedford andutz, 1992; Carefoot, 1991).The order has traditionally been composed of 9 or 10
enera (Boss, 1982; Eales, 1944; Marcus, 1972; Pruvot-ol, 1954), but relationships among these genera arenclear. The taxonomic classifications available atresent for the Anaspidea have been based upon fewharacters, without taking into account ancestral orerived conditions (Eales, 1944; Marcus, 1972). Anxception is the partial phylogeny developed by Wright
t al. (1996). Some major taxa, however, were not1055-7903/00 $35.00Copyright r 2000 by Academic PressAll rights of reproduction in any form reserved.
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42 MEDINA AND WALSH
ncluded in their analysis. The two most commonaxonomic classifications are presented below. In onelassification, the Anaspidea has one family (Table 1),he Aplysiidae, consisting of five subfamilies (Eales,944; Beeman, 1968; Boss, 1982; Willan, 1998). Thether classification, based on the length of the visceraloop (Pruvot-Fol, 1933, 1954; Marcus, 1972), places thearious genera into two suborders with one family eachTable 2). The suborder Longicommissurata presentsong visceral nerve cords and the suborder Brevicommis-urata exhibits short visceral nerve cords. Burn (1989)lso used the length of the visceral loop as the diagnos-ic character for separating taxa. His classification ishe same as that of Marcus (1972), except that he useswo subfamilies (Aplysiinae and Notarchinae) as taxo-omic ranks instead of the suborders Longicommissu-ata and Brevicommissurata. Note the absence of Akerarom one of these classifications (discussed below). Theenus Syphonota may be another Aplysia species (Gos-iner, pers. comm.; Willans, pers. comm. in Carefoot,987).The systematic placement of the Akeridae has been
ontroversial due to its streptoneurous appearance andther plesiomorphic features. Some workers place AkeraMarcus, 1972; Thompson, 1976; Schmekel, 1985; Burn,989) within the Cephalaspidea (the most primitivepisthobranch order) based on the possession of aontentaculate cephalic shield, a large external shell,nd free parapodia, in contrast to the Anaspidea, whichave tentacles and rhinophores, as well as differentegrees of parapodial fusion. Other workers (Mortonnd Holme, 1955; Pruvot-Fol, 1954; Ghiselin, 1965;eeman, 1968; Boss, 1982; Gosliner, 1991) suggest that
he Akeridae are better placed in the Anaspidea be-ause they appear to be an intermediate step betweenhe cephalaspidean ancestor and the rest of the seaares. Morton and Holme (1955) and Gosliner (1991)rgued that the authors that place Akera with theephalaspideans based their conclusions on plesiomor-hies shared between Akera and the Cephalaspidea as
TABLE 1
Taxonomic Classification of the Order AnaspideaProposed by Eales (1944), Modified by Boss (1982)
amily Akeridae Akeraamily AplysiidaeSubfamily Aplysiinae Aplysia Linnaeus 1767
aSyphonota Adams & Reeve 1850Subfamily Dolabellinae Dolabella Lamarck 1801Subfamily Notharchinae Notarchus Cuvier 1817
Stylocheilus Gould 1852bBursatella Blainville 1817
Subfamily Dolabriferinae Dolabrifera Gray 1847Petalifera Gray 1847Phyllaplysia P. Fischer 1872
a Synonym of Paraplysia (Eales, 1944).b
eSynonymized with Barnardaclesia by Gosliner (1987).pposed to apomorphic characters. Supporters of thelacement of Akera in the Anaspidea (Morton andolme, 1955; Ghiselin, 1965; Gosliner, 1991) present
everal synapomorphies shared by Akera and the Anas-idea, from the reproductive system, defensive glands,adula, gizzard, and the central nervous system. Mik-elsen (1996) included the anaspidean taxa Akera andplysia in her study of primitive Cephalaspidea. Sheoncluded that the Anaspidea is a monophyletic cladeupported by two autapomorphic traits of the digestiveystem (presence of a secondary gizzard and a cecumxtended from the stomach). Williams (1975) alsoresents the fusion of the right parietal ganglion withhe supraintestinal ganglion as a synapomorphy forkera and the remaining sea hares.
ossil Record
The outgroup taxa chosen for this study, Bulla andaminoea, have a distinct external shell. As a conse-uence of strong shell calcification they have a fairlyeliable fossil record. These bulloid cephalaspideansate back to the beginning of the Jurassic (200 millionears ago [Wenz, 1938; Tracey et al., 1993]). There isaleontological evidence that Akera appeared in theid-Jurassic (165 million years ago [Tracey et al.,
993]). The oldest Aplysia fossils date back to theiocene (25 million years ago [Tracey et al., 1993]).here is also shell evidence that the genus Floribella, a
ossil species closely related to Dolabella, has beenresent in the fossil record since the early Miocene (25illion years ago [Geiger and Jung, 1996]). The scat-
ered shell fossils of Dolabrifera have been found onlyrom early Pleistocene deposits (1.6 million years agoWenz, 1938]). No thorough taxonomic revision haseen conducted on these fossils because the opistho-ranch fossil record is incomplete as a result of shelleduction and poor fossilization (Mikkelsen, 1996).onsequently, the paleontological record of this group
s poorly known. Available fossil dates must be consid-
TABLE 2
Anaspidean Classification Proposed by Marcus (1972),Modified from Pruvot-Fol (1954)
uborder LongicommissurataFamily Aplysiidae
Subfamily Aplysiinae AplysiaSyphonota
Subfamily Dolabellinae Dolabellauborder BrevicommisurataFamily Notharchidae
Subfamily Notharchinae NotarchusSubfamily Dolabriferinae Stylocheilus
BurstellaDolabriferaPetaliferaPhyllaplysia
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43ANASPIDEAN mtDNA PHYLOGENY
argin of error. For instance, based on amino acidequence divergence for the egg-laying hormone gene,sing fossil evidence, Nambu and Scheller (1986) placehe appearance of the most primitive member of theenus Aplysia (A. parvula) in the Cretaceous (140illion years ago). Thus, the genus Aplysia may have
iverged shortly after Akera diverged from their mostommon ancestor.
olecular Markers
Because of the high levels of morphological homo-lasy and the tendency toward reduction of many of theraits traditionally used by molluscan taxonomists,dditional characters must be used to resolve phyloge-etic relationships within the opisthobranch clade.olecular markers are suitable for this purpose be-
ause they provide a large number of characters thatan be used in phylogenetic analyses. There are manyptions of molecular markers that are informative atifferent systematic levels. Mitochondrial DNAmtDNA) is a molecule that has proven to carry phylo-enetic signal at different taxonomic levels (Moritz etl., 1987). MtDNA has been widely used for phyloge-etic studies because it is maternally inherited andonrecombining and has higher rates of evolution thanuclear genes in many taxa. Perhaps most importantly,tDNA has an array of genes that can be useful at
ifferent phylogenetic levels (Brown, 1985; Avise et al.,987; Moritz et al., 1987; Simon et al., 1994). Thesenclude the ribosomal genes (12S and 16S), which aremong the most conserved regions in the mitochondrialenome. Because mitochondrial ribosomal genes (rRNA)volve at faster rates than their nuclear homologsMindell and Honeycutt, 1990; Hillis and Dixon, 1991;imon et al., 1994), these genes have been used primar-
ly at lower taxonomic levels, from genus to populationevel (Avise et al., 1987; Moritz et al., 1987; Simon et al.,994). It has been suggested, however, that mitochon-rial rRNA genes can be used to infer phylogeneticelationships to 300 million years old (Mindell andoneycutt, 1990) or 65 million years old (Hillis andixon, 1991). The radiation of characiform fishes wassed as an empirical test of the efficiency of these genesor accurately resolving phylogenetic associations. Thistudy suggested that mitochondrial rRNA markers areeliable for divergence times to 100 million years oldOrtı and Meyer, 1997). The earliest reliably identifiedkera shell fossils are mid-Jurassic (165 million yearsgo [Tracey et al., 1993]), but the earliest report of theemaining primitive sea hares (Aplysia and Dolabella)s Miocene in age (25 million years ago [Tracey et al.,993]). Thus, the window of application proposed byrtı and Meyer (1997) embraces the fossil appearancef most anaspidean taxa with the exception of the twoephalaspidean outgroup taxa.Although there are prob-ems with using fossil evidence to date the appearance
f the taxa in this study, the mitochondrial rRNA genes Eppeared to be appropriate markers for resolving phylo-enetic relationships within this clade.The cytochrome oxidase subunit I gene (COI) has
een reported as one of the most conserved protein-ncoding genes in the mitochondrial genome (Brown,985). Though third-codon positions are known toaturate quickly, first- and second-codon positions showntermediate-level resolution (family to genus levelomparisons [Folmer et al., 1994]). It has been sug-ested that, at even higher levels (class and phylum),he inferred amino acid sequences can be used to assesshylogenetic relationships (Folmer et al., 1994). Thus,he COI gene was chosen for this study because of thisotential to be phylogenetically informative at interme-iate and lower taxonomic levels, possibly complement-ng the rRNA sequences where these regions could lackesolution.The purpose of this research was initially to recon-
truct anaspidean phylogeny based on mtDNA se-uence data, then to compare it to the morphologicalvidence, and last to map the evolution of two behav-oral traits (swimming and inking) onto the molecularhylogeny.
MATERIALS AND METHODS
amples and DNA Isolation
Most of the samples utilized in the present studyere provided by opisthobranch specialists from world-ide museums and laboratories (Table 3). Some of the
amples were frozen and some were collected alive andreserved in a solution that inhibits DNA degradationDMSO 20%, 250 mM EDTA, NaCl saturated) (Seutint al., 1991).Total DNA was isolated by standard SDS/Proteinasedigestion (Sambrook et al., 1989). Tissue was gently
omogenized in an Eppendorf tube in 700 µl of 13 NETuffer (150 mM NaCl, 10 mM Tris–HCl [pH 8.0], 10 mMDTA), with 1% SDS and 100 µg/ml proteinase K, and
ncubated at 65°C for 1–2 h. Nucleic acids were isolatedy successive phenol:chloroform (3:1) extractions re-eated until the interface was clear, followed by ahloroform:isoamyl alcohol (24:1) extraction and precipi-ation by the addition of Na–acetate to 300 mM and.5–3 volumes of absolute ethanol. Genomic DNA wasesuspended in TE buffer (10 mM Tris–HCl [pH 8.0], 1M EDTA) and RNase (50 µg/µl) treated at 37°C,
ollowed by a chloroform:isoamyl (24:1) extraction andthanol precipitation. The DNA samples were resus-ended in TE buffer.
CR Amplification and Sequencing
Amplifications were performed in 25 µl of a solutionontaining approximately 50 ng of DNA, 13 PCRuffer, 200 µM each dNTP, 1.5 mM MgCl2, 0.5 µM eachrimer, and 1.25 units of Taq polymerase (Perkin–
lmer/Cetus). After an initial denaturation step of 2
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in at 94°C, 30 cycles of 30 s at 94°C, 30 s at 45 or 50°C45°C for 12S and 50°C for 16S and COI), and 30 s at2°C were performed, followed by a final extension stepf 5 min at 72°C. The primers used were the universalrimers for the mitochondrial small ribosomal subunit12S), 12SA-L (58-AAACTGGGATTAGATACCCCAC-AT-38) and 12SSB-H (58-GAGGGTGACGGGCGGT-TGT-38), and for the large subunit (16S), 16sar-L
58-CGCCTGTTTATCAAAAACAT-38) and 16sbr-H (58-CGGTCTGAACTCAGATCACGT-38), developed byalumbi et al. (1991). The primers used for the mito-hondrial cytochrome c oxidase subunit I fragmentere the universal primers LCO 1490 (58-GGTCAA-AAATCATAAAGATATTGG-38) and HCO 2198 (58-AAACTTCAGGGTGACCAAAAAATCA-38) developedy Folmer et al. (1994).Double-stranded PCR products were purified with
he Gene Clean Kit (Bio 101). 12S and 16S primersere end-labeled with g-33P (NEN Dupont) and samplesere cycle-sequenced with both primers using the DTaq
equencing kit (United States Biochemical). Samplesere run in a 6% denaturing (50% w/v urea) polyacryl-mide gel (19:1 acrylamide to bis-acrylamide ratio).he COI fragments were cloned into a TA vector
Invitrogen) and clones were sequenced using thehermo Sequenase cycle-sequencing kit (Amersham)ith dye-labeled universal M13 forward and reverserimers in a LiCor automated sequencer.
TAB
Sample List, Distribution Range, Sampling L
Species Preserv.
ulla gouldiana Pilsbry 1893 Frozen Eaminoea virescens Sowerby 1833 Frozen Ekera bullata Muller 1776 Ethanol Nplysia cervina Dall & Simpson 1901 Frozen Wplysia punctata Cuvier 1803 Ethanol Cursatella leachi Blainville 1817 Frozen Colabella auricularia Solander 1786 DMSO Inolabella auricularia Solander 1786 DMSO Inolabrifera dolabrifera Cuvier 1817 DMSO Cotarchus indicus Schweigger 1820 Ethanol Inotarchus indicus Schweigger 1820 DMSO Inetalifera ramosa Baba 1959 Ethanol Cetalifera ramosa Baba 1959 DMSO Chyllaplysia sp Gosliner 1995 DMSO Phyllaplysia taylori Dall 1900 DMSO Ntylocheilus longicauda Quoy & Gaymard 1824 DMSO C
FIG. 1. 16S Structural model for Aplysia cervina. Highlighted rarge subunit molecule. Loop numbering follows Horovitz and Meyeetween the structural templates are discussed in the text. Lower grequences including the outgroup taxa. The vertical axis represents th
xis represents the position in the multiple alignment. Lower case lettersequence Alignment
The COI sequences were easily aligned by eye. The6S and 12S sequences were aligned using the defaultettings of the Clustal V algorithm (Higgins and Sharp,988) in the multiple alignment routine of the Dnastarrogram (version 3.06) for Macintosh computers. Subse-uently, the alignment was improved with the use ofecondary structure models because stem and loopegions were easily identified. Structural homologyllowed a more reliable assessment of sequence homol-gy. A composite alignment including the three frag-ents can be downloaded from the University of MiamiIH–Aplysia Resource Facility web site (www.rsmas.iami.edu/groups/sea-hares/); regions excluded from
he final analysis are marked by an asterisk belowhem. Regions of ambiguous alignment excluded fromhe final data set were present in the two ribosomalenes and were found only in loop regions. The criterionsed for the exclusion of regions of ambiguous align-ent was the following: in places in which more than
wo gaps were needed, all sites involving gaps werexcluded. There were two exceptions to this rule: thentire loop regions were excluded in helix L10 of 16SFig. 1) and in helix 31b of 12S (Figure 2). The wholeoop was excluded in both cases because large indels.9) and completely different nucleotide sequences inome taxa prevented a reliable alignment, and thentire helix 42 (both stem and loop regions) of 12S was
3
alities, Preservation Method, and Collector
stribution Locality Collector
rn Pacific Venice, California Marinus Marinern Pacific Venice, California Marinus Marine
h E. Atlantic Algoleran, Sweden J. M. Turbevilleern Atlantic Gulf of Mexico, Texas Ned Strenthmtropical Spain Jesus Orteamtropical Florida Bay, Florida Tom CapoPacific Guam Steve PenningsPacific Gulf of California, Mexico Monica Medinamtropical Guam Steve PenningsPacific Ryukui Is., Okinawa Terry GoslinerPacific Batangas, Philippines Terry Goslinermtropical Canary Islands Jesus Orteamtropical Batangas, Philippines Terry Gosliner
ppines Mindoro, Philippines Terry Goslinerh E. Pacific Friday Harbor, Washington Kadee Lawrencemtropical Okinawa, Japan Steve Pennings
ons represent homology with the human and fruitfly mitochondrial995). Roman numbers were assigned in this study. The differencesrepresents a sliding window analysis of the 16S molecule for all theariable sites in a sliding window of seven nucleotides. The horizontal
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45ANASPIDEAN mtDNA PHYLOGENY
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FIG. 2. 12S Structural model for Aplysia cervina. Highlighted rearge subunit molecule and the scallop nuclear large subunit. Helix ntructural templates are discussed in the text. Lower graph represe
gions represent homology with the chiton and nematode mitochondrialumbering follows Van der Peer et al. (1994). The differences between thents a sliding window analysis of the 12S molecule for all the sequences
ncluding the outgroup taxa. The vertical axis represents the variable sites in a sliding window of seven nucleotides. The horizontal axisepresents the position in the multiple alignment. Lower case letters (a–i) indicate the most variable regions in the structural model.
46
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47ANASPIDEAN mtDNA PHYLOGENY
xcluded for the same reasons state above. The COIragment was translated into amino acid (a.a.) dataith the ‘‘invertebrate’’ mitochondrial genetic code in
he Dnastar program, and this alignment can also beound at the NIH–Aplysia web site.
The use of secondary structural models can improveomputer-generated alignments because stem, loop,nd bulge regions can be identified (Vawter and Brown,993). This understanding of the secondary structure,n turn, permits a more accurate determination ofomologous characters for phylogenetic analysis (Kjer,995). To obtain a secondary structure model for all thenaspidean taxa, both large and small subunit se-uences were compared to the models developed forplysia cervina (Figs. 1 and 2). The large subunit (16S)equence of Aplysia was compared to the mitochondrialodel for humans (Gutell, 1994; Horovitz and Meyer,
995) and Drosophila yakuba (Gutell, 1994 [Fig. 1]).he Aplysia small subunit (12S) secondary structureas refined with help of the model for the third domainf the 12S mitochondrial gene presented by Hickson etl. (1996). The rest of the molecule was compared to the2S model for the nematode Caenorhabditis elegansnd the 18S model for the scallop Placopecten magellani-us, both available from the ribosomal RNA structureeb site (Gutell, 1994 [Fig. 2]). All the new structuralodels for the remaining Anaspidea and the two out-
roup taxa are depicted in similar figures that can beownloaded from the NIH–Aplysia web site. Someutative stem regions could not be confirmed for theplysia model since the complementary strands of
hose regions were not sequenced. The only noncanoni-al (non-Watson–Crick) pairing allowed throughout thentire model was the wobble G · U pair, which formswo hydrogen bonds and is virtually as stable as an
· U pair (Chastain and Tinoco, 1991). On a fewccasions, in which other noncanonical pairings woulde necessary to maintain a structure similar to theemplate, symmetrical bulges (Hickson et al., 1996)ere invoked instead, and each region is explainedelow in detail. Alignment gaps placed by the computerlignment that disrupted stem pairings were moved toontiguous loop or unpaired regions of the model, andn one occasion an indel in a stem region was identifiedor 12S. A sliding window analysis (7 bp—following Ortınd Meyer, 1997) over the entire sequence alignmentas performed in MEGA (Kumar et al., 1993) to
dentify the most variable regions of each ribosomalolecule.
hylogenetic Analysis
Basic statistics (nucleotide composition, transitionnd transversion frequencies, number of variable andarsimony sites) were performed in MEGA (Kumar etl., 1993). Parsimony and neighbor-joining (NJ) analy-es were conducted in PAUP* version 4.0d64 (Swofford,
998) and maximum-likelihood analyses were per- (ormed in Puzzle 3.1 (Strimmer and von Haeseler,996, 1997). The sequence data were partitioned intowo data sets: the rRNA (12S and 16S) genes and theOI fragment. Subsequently, these two data sets werelso partitioned. The rRNA matrix was partitioned intotem and loop regions. The COI fragment was parti-ioned into first- and second-codon positions, and third-odon positions. The informative COI amino acid sitesere included in a combined data set with the rRNAata set for parsimony analysis. The partition homoge-eity test or incongruence length difference (ILD) test
Farris et al., 1995) implemented in PAUP* was used toetermine whether the different data sets could beombined. The settings were 10 random stepwise addi-ions with TBR branch swapping and 1000 randomiza-ions. Initially, rRNA pairwise sequence divergenceersus COI divergence was plotted (Fig. 3). Pairwiseequence divergence for both data sets clearly in-reased in correlation for ingroup comparisons, whereasor outgroup comparisons, the COI pairwise divergencealues tapered off at approximately 20%. To exploreurther saturation of the COI sequence, observed tran-itions (Ts) and transversions (Tv) were plotted againstequence divergence (Fig. 4). In the rRNA data set, Tsere more frequent than Tv and both increased lin-arly in number with increasing sequence divergence.here was an exception for the outgroup comparisons
n which Tv were more frequent. This observation is anndication of Tv covering Ts (Fig. 4a). The observedubstitutions at first- and second-codon positions of theOI gene follow a scattered nonlinear relationship,ith Ts more abundant than Tv (Fig. 4b). In contrast is
he even more scattered, nonlinear relationship ofubstitutions versus sequence divergence for more an-ient pairwise comparisons at third-codon-positionhanges (Fig. 4c), in which Ts abundance tends topproximate Tv values, indicating saturation at theseites. Two combinations of weights were used for theRNA data set (equal weights, Tv:Ts 8:1). For theeighted parsimony analysis, the Ts:Tv weighting was
alculated by averaging the Ts:Tv ratios of the pairwiseomparisons within the genera with more than oneample (Aplysia, Dolabella, Notarchus, Petalifera, andhyllaplysia). The same procedure was followed tostimate the weighting used with the stem (6:1) andoop (4:1) data sets, and the COI third positions (4:1).or parsimony analysis, gaps were treated as missingharacters. In all cases, heuristic searches were per-ormed, with 10 replicates of random stepwise additionnd TBR branch swapping. Consistency index (CI) wasalculated as a measure of fit between the data and theeported topologies (Kluge and Farris, 1969). In casesn which more than one tree was found, a 50% majorityule strict consensus is reported. Bootstrap analysis50% majority rule) with 1000 pseudoreplicates (Felsen-tein, 1985) in PAUP* and Decay analysis in TreeRot
Sorenson, 1996) were conducted to estimate branch
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upport. Neighbor-joining reconstructions were basedn the Hasegawa–Kishino–Yano (HKY85) distances toccommodate multiple hits. The logDet distance wassed to correct for nucleotide bias. Bootstrapping (1000seudoreplicates) was also performed for confidencestimation. The quartet maximum-likelihood approachmplemented in Puzzle 3.1 was utilized to explore theupport under the HKY85 model with 1000 quartetuzzling (QP) steps. The estimate of branch supporthat the QP procedure produces is similar to bootstrapupport values (Strimmer and von Haeseler, 1996,997).
RESULTS
itochondrial Data
The three gene fragments comprised a total of 1372ites, of which 564 were variable sites and 439 werearsimony informative. All three genes presented an-T nucleotide bias (61.4% for 16S, 63.9% for 12S, and2.6% for COI). The average A-T nucleotide bias variedithin codon positions of the COI gene (56.8% for firstositions, 57.5% for second positions, and 76.3% forhird positions). A 658-nucleotide fragment of the COIene was sequenced for all taxa. The average nucleo-ide composition on the coding strand was 23% A, 39%, 18% C, and 20% G. In this portion of the gene,
FIG. 3. Pairwise comparisons of sequence divergence (uncorrectedxis and rRNA (16S and 12S) divergences on the horizontal axis.
hird-codon positions were the most variable sites, a
ccounting for 78% of the overall variation, with first-nd second-codon positions accounting for 19 and 3% ofhe variable sites, respectively. Nucleotide sequencesere translated into amino acids (alignment can beownloaded from the NIH–Aplysia web site). Sevenmino acids were common to all Anaspidea (a.a. 31, 59,03, 104, 126, 155, and 175); a phenylalanine and aaline (a.a. 30 and 178) distinguished the genus Phyl-aplysia. Akera shared one isoleucine with the out-roups (a.a. 96). Aplysia shared an a.a. with theutgroup (a.a. 32). Akera and Notarchus shared aeucine at position 33. The rest of the variable sitesere uninformative characters.From the 16S ribosomal gene, a fragment of approxi-ately 432 bp was sequenced, of which 48 bp were
xcluded from the phylogenetic analysis. The second-ry structure model for Aplysia cervina is depicted inig. 1. The average nucleotide composition of all taxa
or 16S was 30% A, 31% T, 16% C, and 23% G. Thelignment required two to nine indels (insertion/eletion events) per sequence (0.7–5.3% of the aligned6S sequence length). Most indels comprised up to fourucleotides, except for a larger indel of nine nucleotides
n the outgroup taxa.Finally, a fragment of approximately 377 bp was
equenced from the 12S gene, of which 47 bp werexcluded from the final analysis. The proposed second-
for all taxa including outgroup taxa. COI divergences on the vertical
-p)ry structure model for Aplysia cervina is presented in
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49ANASPIDEAN mtDNA PHYLOGENY
ig. 2. Average nucleotide composition of all taxa for2S was 39% A, 25% T, 16% C, and 20% G. Thelignment required 12–18 indels per sequence (4.7–2.4% of the aligned 12S sequence length). Indels werearger than in the 16S region, the largest being 19ucleotides long.The rRNA data set was separated into two structural
ategories, stems and loops, using the same criteria asrtı et al. (1996). Only regions that were paired in the
econdary structure models were considered stems.oop regions included hairpin loops, multibranched
FIG. 4. Plot of observed number of transitions (Ts) and transver-ions (Tv) versus percentage sequence divergence (uncorrected-p). (a)RNA data set; (b) first and second positions of COI; (c) third positionsf COI.
oops, and internal loops in stem regions. Bulges and m
utative stem and loop regions that could not beerified due to lack of complementary sequences werexcluded from this analysis. No compositional bias wasbserved in stems (50% A-T content), whereas loopegions showed a great A-T bias (69.8% of all nucleo-ides).
Sequence divergence (uncorrected-p) among taxa forhe ribosomosal and the COI data sets can be down-oaded from the NIH–Aplysia web site. For the rRNAata set, variation within the ingroup ranged from 0.3o 14.8%, and between Anaspidea and Bulla and Hami-oea it ranged from 24.6 to 28.1%. For the COI data set,hese values ranged from 0.3 to 19.9% between anaspi-ean taxa and from 18.2 to 23.1% for outgroup compari-ons.
econdary Structure Models
The nomenclature proposed by Kjer (1995) was usedo mark different structural helices on the multiplelignment (available at NIH–Aplysia web site). Theecondary structure model for the 16S gene of Aplysiaervina is presented in Fig. 1 (the models for the rest ofhe anaspidean taxa and the outgroup can be down-oaded from the NIH–Aplysia web site). Loop regionsere identified following the numbering of Horovitznd Meyer (1995), with some stem regions additionallyssigned roman numerals (I, II, III) to locate them onhe multiple alignment. The model differed from theuman and Drosophila models more noticeably in thehortening of loop regions L7 and L11. The loop region9 from the model of Horovitz and Meyer (1995) couldot be identified in the sequences in this study. The L10
oop was smaller in the outgroup taxa and the L11 stemairings were not clearly identified in some taxa. Oncehe structural model was developed and the sequencesere aligned, a sliding window analysis (seven nucleo-
ides) similar to the one presented by Ortı et al. (1996)as followed to identify variable regions (Fig. 1). Theost variable regions (identified by lowercase letters)ere found in loops, in agreement with the analysis foriranhas of Ortı et al. (1996), except for the stem regionf L13 (Fig. 1). Regions b, c, d, e, f, and g were alsomong the most variable in the piranha study for the6S molecule.The model of the 12S third domain presented byickson et al. (1996) and the 12S and 18S models for
he nematode Caenorhabditis elegans and the scalloplacopecten magellanicus (Gutell, 1994) were used toeconstruct the Aplysia cervina 12S secondary struc-ure (Fig. 2). The models for the rest of the anaspideanaxa and the outgroup are available at the NIH–plysia web site. Helices were numbered following Vane Peer et al. (1994). Helices 40, 42, 47, and 48 were theegions that differed the most in sequence compositionrom the template structural models; however, thesetructures were identified once the backbone of the
olecule was reconstructed. In the loop of helix 31,
tA(itbfi1stsNbtrwtmm
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secttccwttittpv(is
mitmb(wDovavt8tg
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50 MEDINA AND WALSH
here was an indel of variable size (13–17 bp) in the twoplysia sequences that was absent in the outgroup
Fig. 2). An indel of one nucleotide was also found in allngroup taxa in helix 33. In the outgroup taxon Bullahe internal loop regions between helices 35 and 36 andetween helices 36 and 38 present two large indels ofve and four nucleotides long. A slippage event (Kjer,995) was necessary for helix 39 to maintain theequence alignment in Bulla with the other outgroupaxon Haminoea (see alignment at NIH–Aplysia webite). Helix 42 could not be identified in the genusotarchus (NIH–Aplysia web site). Stable symmetricalulges (Hickson et al., 1996) were necessary to main-ain the structure in helices 47 and 48 (Fig. 2). Variableegions are highlighted in Fig. 2, also using a slidingindow of seven nucleotides. Loop regions were again
he most variable, but some stems also exhibited poly-orphism. Regions b, c, e, f, and g were also among theost variable in the piranha study (Ortı et al., 1996).
hylogenetic Analysis
Based on the ILD test, the rRNA and COI data setsupported significantly different phylogenetic hypoth-ses (P 5 0.001). The COI gene, though one of the mostonserved protein-coding genes of the mtDNA, ishought to evolve overall at a much higher rate thanhe ribosomal genes. Saturation, especially at third-odon positions, was considered a possible cause of theonflicting signal. To evaluate possible saturation, pair-ise sequence divergence values were plotted for the
wo data sets (Figs. 4a–4c). Because saturation athird-codon positions seemed to be one of the contribut-ng factors to the conflict in phylogenetic signal be-ween the rRNA data set and the protein-coding gene,hey were excluded in a posterior analysis. In a secondartition homogeneity test, the rRNA data set was runersus the COI first and second positions onlyP 5 0.004). This test also indicated conflicting signaln the two data sets; so, the data sets were analyzedeparately.The equal-weights rRNA analysis produced a singleost-parsimonious tree of 619 steps with a consistency
ndex of 0.688 (Fig. 5a). This tree differed from theraditional classifications in two ways: (1) the place-ent of Notarchus as sister taxa with Dolabella and
asal to the rest of the taxa with short visceral loopBrevicommissurata) and (2) Petalifera as sister taxaith Bursatella and Stylocheilus rather than witholabrifera and Phyllaplysia (see Table 1). Monophylyf the Anaspidea was supported by a 100% bootstrapalue. Monophyly of the genera Aplysia, Notarchus,nd Petalifera was also well supported with bootstrapalues of 98–100% and decay indices of 6–29, except forhe Phyllaplysia node with a relatively high support of4% and a decay index of 3. The NJ analysis with bothhe HKY85 and the logDet distances produced topolo-
ies that diverged from the equal weights parsimony cnalysis in the placement of the taxa Notarchus, Dola-ella, and Aplysia but bootstrap values were low forhese nodes with all the analyses (Fig. 5a). The maxi-um-likelihood analysis performed by quartet puz-
ling with the HKY85 model of evolution, under thessumption of rate homogeneity, also revealed resultsimilar to those of all the previous analyses, given thathe relationships of Aplysia, Dolabella, and Notarchusere not clearly recovered. The branch support giveny this method is also depicted in Fig. 5a. When theRNA data set was analyzed with rate heterogeneitygamma distribution) most branches were collapsed athe base of the tree and the support for the remainingodes was low, except for the genus-level nodes. Theeighted parsimony analysis produced a single tree inhich the genus Notarchus is basal to the remaining
axa (Fig. 5b).It has been suggested that rRNA stem and loop
egions contain different phylogenetic signals; conse-uently, special care should be given to any analysissing rRNA data (Wheeler and Honeycutt, 1988; Dixonnd Hillis, 1993; Vawter and Brown, 1993; Simon et al.,994). The objective of partitioning the rRNA data setnto stems and loops was to identify at what systematicevel the two structures were more informative. Thequal-weights parsimony analysis of the stem data setroduced 5 shortest trees (TL 5 141, CI 5 0.816); atrict consensus tree is presented in Fig. 6a. The 6:1eighting produced 9 most-parsimonious trees of length46 (data not shown). The stem data set recovered theopology of deep nodes with high bootstrap support;owever, some of the genus-level relationships wereollapsed into polytomies with other taxa. The parsi-ony analysis of the loop regions produced 47 shortest
rees (TL 5 217, CI 5 0.664); the bootstrap consensusree is presented in Fig. 6b. When a 4:1 Tv:Ts weightingas used, 2 shortest trees were produced (TL 5 514,ot shown). The loop data set failed to recover theeeper nodes in the tree, but it was useful for lower-evel comparisons. Both distances in the NJ analysesroduced the same tree with similar bootstrap values.he NJ results were congruent with the findings of thearsimony analysis (Fig. 6b).Initially, the COI fragment was analyzed completeith three different weightings for third-codon posi-
ions (equal weights, 4:1, and Tv only); then, third-odon positions were excluded. When equal weightsere used, a single tree was produced (TL 5 957,I 5 0.437), with no bootstrap support for any node.he monophyly of the ingroup was disrupted and the
opology was completely different from the rRNA dataet (tree not shown). Notarchus was the most basalaxon, and Haminoea appeared as sister taxa withkera in a terminal node in the tree. The monophyly of
he genera was maintained except for Aplysia, whichecame a grade ancestral to the Haminoea–Akera
lade. Different weighting schemes for third positions
dpaad
gBtw
dqW an
51ANASPIDEAN mtDNA PHYLOGENY
id not improve phylogenetic signal. Two trees wereroduced for Tv:Ts 4:1 (TL 5 1880), and there wasgain no bootstrap support for any node. The Tv-onlynalysis produced four trees (TL 5 454); it also pro-
FIG. 5. (a) rRNA most-parsimonious tree produced by a heuristicepicted above each branch and bootstrap values and branch support auartet puzzling). In shadow are the bootstrap values for the equeighting of 8:1 Tv:Ts produced a single shortest tree (TL 5 2314). (C
uced topologies that disrupted monophyly of the in- d
roup and placed Akera as a recently derived taxon.ecause these additional weightings did not improve
he resolution of the data set, no bootstrap analysesere performed. In the NJ (HKY85, logDet) analyses, a
rch under equal weighting (TL 5 619, CI 5 0.688). Decay indices aredepicted in boldface below the branches (parsimony/neighbor-joining/
eights combined analysis of COI amino acid and rRNA data. (b). Is., Canary Islands; Phil., Philippines; Jap., Japan).
seare
al w
ifferent tree was obtained, with no support for the
mptwwosirmega
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va
52 MEDINA AND WALSH
onophyly of Anaspidea and low or no bootstrap sup-ort for most nodes, except at the genus level. Becausehird-codon positions contained misleading signal, theyere excluded in a subsequent analysis. An equal-eights analysis (first- and second-codon positionsnly) produced two most-parsimonious trees of 135teps and a consistency index of 0.452. Even though thengroup did show monophyly, the topology did notesemble the relationships produced by the rRNA andorphological data. First- and second-codon positions
xhibited low substitution rates (Fig. 4b), which sug-ested no saturation at these sites. Therefore, no
FIG. 6. (a) Strict consensus of five shortest trees with equal wealues are depicted below each branch. (b) Bootstrap consensus of 47nd NJ bootstrap values are depicted below branches. (Can. Is., Cana
dditional weightings were used for these positions. s
esults similar to those of the parsimony analysis wereroduced by the NJ (HKY85) analysis, but the logDetistance produced a slightly different tree. These analy-es showed that the first- and second-codon positionsad low signal, which was also misleading, but in thisase was due to lack of informative sites.
ombined Analysis
The COI amino acid data set contained 24 variableites, and only 12 were parsimony informative charac-ers. These characters were also combined with theRNA data set and an equal-weights parsimony analy-
ing for the stem data set (TL 5 141). Parsimony and NJ bootstraps under equal weighting for the loop data set (TL 5 217). Parsimonyslands; Phil., Philippines; Jap., Japan).
ighttreery I
is was performed. A single shortest tree was produced
(os
P
ht5sfnagpnwNwswsrcrococdeatlTscthmwsq
wtcdtblumcb
wlidhtlsslBtbt
udtccbidppatdntaetaPumatcwwtdbrul
T
rtcmi
53ANASPIDEAN mtDNA PHYLOGENY
TL 5 638, CI 5 0.694) with the same topology as thene obtained with the rRNA data set alone. Bootstrapupport was increased for most nodes (Fig. 5a).
DISCUSSION
hylogenetic AnalysisRibosomal genes. The rRNA data set resolved with
igh confidence the monophyly of the Anaspidea andhe relationships of the most derived sea hares (Fig.a). The low support for the intermediate nodes (Aply-ia, Dolabella, and Notarchus) might reflect the needor additional data or might be due to saturation ofucleotide changes. A third possibility is that there wasrapid radiation of these three lineages after diver-
ence from Akera. Weighting of Tv 8:1 over Ts in thearsimony analysis did not improve phylogenetic sig-al and reduced support for most nodes (Fig. 5b). Thiseighting scheme changed the topology by placingotarchus as ancestral to Aplysia and Dolabella, whichould force one to invoke unlikely morphological rever-
als, such as (a) elongation of the visceral loop once itas concentrated around the anterior nerve ring, (b)
eparation of previously fused nervous ganglia, and (c)eappearance of the adult shell once it was lost. Nerveord shortening, ganglionic fusion, and adult shelleduction and loss are common in other opisthobranchrders (Gosliner, 1991, 1994). Thus, even though theseharacters do exhibit high levels of homoplasy in otherpisthobranch clades, the evolution of these morphologi-al features would have to have occurred in the oppositeirection in the anaspidean clade. Consequently, thequal-weights parsimony topology seems to be a betterpproximation to the phylogenetic relationships withinhe Anaspidea. This topology was also supported by theogDet NJ and the maximum-likelihood QP analysis.he assumption of rate homogeneity for the QP analy-is had more phylogenetic resolution and gave resultsongruent with those of the other methods. Evenhough the maximum-likelihood analysis with rateeterogeneity is considered a more realistic model ofolecular evolution, most of the phylogenetic signalas lost when this approach was used because it
uggested some star-like evolution of the rRNA se-uences.Stems and loops. Only part of the rRNA data setas used in the analyses of these structures, because
he structural model for some stem and loop regionsould not be corroborated due to incomplete sequenceata. From the stem data set we were able to producehe basal nodes in the tree with high confidence levels,ut this tree lacked resolution for some of the genus-evel comparisons (Fig. 6a). When equal weights weresed, the loop data set contained high support for theonophyly of Anaspidea but the basal nodes were all
ollapsed into a polytomy (Fig. 6b). Recent nodes are
etter supported by the loop data set than by the stems, vhich is an indication of higher substitution rates inoop regions. There was some indication of homoplasyn the loop data set (CI 5 0.664) compared to the stemata set (CI 5 0.816). However, to account for multipleits in both data sets, two weightings were used. Forhe stem data set, a 6:1 weighting was used. For theoop regions, a 4:1 weighting was used (trees nothown). Stem regions seem to have been evolving atlower rates than loop regions and have not accumu-ated enough changes for resolving recent divergences.oth structural domains had complementary informa-
ion and the use of the complete rRNA data set hadetter resolution for resolving the relationships withinhe Anaspidea.
Cytochrome oxidase c I gene. The COI fragmentsed for this analysis failed in recovering with confi-ence both the monophyly of the Anaspidea and any ofhe phylogenetic relationships within the anaspideanlade (tree not shown). This is illustrated by the lowonsistency index (0.437) of the data set and the lowootstrap support for all nodes. Different weightings,ncluding the different distances in the NJ analyses,id not improve phylogenetic signal because thirdositions were saturated (Fig. 4c). Exclusion of thirdositions did not enhance the consistency index (0.452)nd only helped recover with relative confidence bothhe monophyly of the Anaspidea and some of the recentivergences. The translated amino acid sequences didot contain sufficient phylogenetically informative siteso resolve most relationships within the Anaspidea (a.a.lignment available at NIH–Aplysia web site). How-ver, there were seven synapomorphic amino acid siteshat also confirmed monophyly of the order and somemino acids that confirmed monophyly of the generahyllaplysia, Notarchus, and Aplysia. Therefore, whensing nucleotide data, the COI gene is not a reliableolecular marker for ancient divergences such as the
naspidean taxa. Analysis at the nucleotide level withhis gene should be restricted to genus- or species-levelomparisons. In contrast, the amino acid sequencesere highly conserved and little phylogenetic signalas recovered. Despite the low variability at the pro-
ein level, the fact that monophyly of the order Anaspi-ea was supported by seven amino acids, increasing theootstrap confidence in a combined analysis with theRNA genes (Fig. 5a), suggests that this gene could beseful in resolving relationships at higher systematic
evels when amino acid data are used.
axonomic Implications of the rRNA Evidence
The concentration of the nerve ganglia in the ante-ior part of the head associated with the shortening ofhe visceral loop (Brevicommissurata) is known asephalization (Gosliner, 1994). Cephalization is com-on in several opisthobranch lineages and has evolved
n parallel from different ancestral taxa with long
isceral cords. However, the visceral loop has been a
kada1wDps
tftorttm
M era
54 MEDINA AND WALSH
ey character in taxonomic revisions of the Anaspidea,nd in one of the common classifications it is used toivide the order into the suborders Longicommissuratand Brevicommissurata (Pruvot-Fol, 1954; Marcus,972). The molecular evidence presented in this studyeakly suggests an association between Notarchus andolabella (Fig. 5), which could be explained by twoossible scenarios (Fig. 7a). (1) The most parsimonious
FIG. 7. Morphological characters rendered homoplastic by theacClade 3.06. Equivocal cycling was used. (a) Shortening of the visc
cenario would be that Dolabella is ancestral to No- s
archus and they diverged almost at the same timerom their Aplysia ancestor. Subsequently, the No-archus lineage would have given rise to the remainderf the Brevicommissurata. This hypothesis would notequire one to invoke the shortening of the visceral loopo occur twice in the anaspidean clade. (2) An alterna-ive hypothesis would be that cerebralization (Brevicom-issurata) might have occurred in two separate in-
olecular phylogeny (rRNA). Character tracing was performed inl loop, (b) shell loss, and (c) fusion of parapodia.
m
tances in the anaspidean clade instead of only once.
NepsofwTtmaplopiFBlD
poabsbumcrspcsMpsttra(SdcNrtNs
T
ws[
Pstp1wusegnbatDt8gtptdmsae
T
fi(gaheminsAdiw1lfli8npAtts
55ANASPIDEAN mtDNA PHYLOGENY
otarchus, a taxon with short visceral loop, would havevolved from a lineage that also gave rise to therimitive Longicommissurata Dolabella. Dolabellahows a pronounced fusion of ganglia which is alsobserved in Notarchus. This would suggest that theusion of the nerve ring ganglia and the visceral gangliaas the initial step of cerebralization in this clade.hen, the lateral nerve cords were shortened in No-
archus, whereas in Dolabella the visceral loop re-ained elongate after the fusion of the anterior ganglia
nd the visceral ganglia. This evidence opposes therevious belief that Notarchus diverged from the sameineage as the rest of the Brevicommissurata in thisrder. The molecular evidence suggests the possiblearallel evolution of cephalization, which would rendernappropriate the separation of the sea hares by Pruvot-ol (1954) into the suborders Longicommissurata andrevicommissurata (Table 2). However, additional mo-
ecular data are needed to confirm support for theolabella and Notarchus clade.Other morphological characters would require homo-
lastic changes if the molecular tree is preferred. Mostf these characters are part of the external anatomynd tend to be diverse characters in other opistho-ranch groups, as well. For example, the evolution ofhell morphology in the Anaspidea, as in all opistho-ranch orders, is toward shell loss. Taxonomists havesed shell characters to identify the different Brevicom-issurata families, but shell loss seems to have oc-
urred in parallel in several lineages of the sea haresather than only once (Fig. 7b). In fact, it has beenhown that in the genus Phyllaplysia there is greatlasticity in shell morphology, such that in some spe-ies there can be adult individuals with and without ahell at the same time in a population (Beeman, 1963;arcus, 1972). Another example is the degree of para-
odial fusion, which is variable within the genus Aply-ia and possibly a plastic trait in the other anaspideanaxa. The morphological trend in sea hares is towardight parapodial fusion, which started with the poste-ior fusion in Aplysia from the free parapodia in Akerand the outgroups. The subfamily Notarchinae of Eales1944) contains the genera Notarchus, Bursatella, andtylocheilus (Table 1). The anterior fusion of the parapo-ia has been interpreted as a synapomorphy of thislade. The molecular data indicate that the subfamilyotarchinae is not a monophyletic clade. Therefore, the
esemblance in parapodial fusion between these threeaxa might be explained by convergence of this trait inotarchus, possibly due to its specialized mode of
wimming (Fig. 7c [see below]).
he Evolution of Swimming
Two different types of swimming have been identifiedithin the Anaspidea: the metachronal or ‘‘butterfly’’
wimming (anterior to posterior parapodial oscillations
Hamilton and Ambrose, 1975; Von der Porten and tarsons, 1982]) present in Akera (Morton, 1955) andome Aplysia species (Eales, 1960; Carefoot, 1987) andhe ‘‘somersaulting’’ swimming, which is driven by jetropulsion (Pruvot-Fol, 1954; Martin, 1966; Willan,998). Akera tends to swim in an upward direction,hereas Aplysia moves horizontally in the water col-mn and Notarchus swims in cycles of one or severalomersaults. The topology produced by the molecularvidence seems to indicate that swimming has a phylo-enetic component. Even though the swimming mecha-isms in the three taxa are somewhat different, thisehavior seems to have appeared in the ancestralnaspidean and then subsequently to have been lost inwo instances (Fig. 8a), once in the lineage that led toolabella after splitting from Notarchus and again in
he ancestor of the remaining Brevicommissurata (Fig.a). If the two swimming mechanisms are not homolo-ous, then an alternative scenario for the evolution ofhis behavior would be that parapodial flapping is alesiomorphic state and it was lost in more derivedaxa, and swimming by jet propulsion evolved indepen-ently in the genus Notarchus. Homology of swimmingechanisms could be tested at the neurological level
ince the neural network that controls swimming haslready been identified in Aplysia species (Gamkrelidzet al., 1995).
he Evolution of Defensive Glands
The defensive glands of sea hares have been identi-ed as unique features of this opisthobranch group
Gosliner, 1994). Even though the opaline and purplelands are called defensive glands, there is controversybout their function (Nolen et al., 1996). For instance, itas been suggested that the purple gland has anxcretory function rather than a defensive role (Chap-an and Fox, 1969). The purple gland has been studied
n more detail than the opaline gland, especially at theeuronal level (Carew and Kandel, 1977). Most of thesetudies have been performed on the model organismplysia californica under artificial stimuli, which in-uces inking behavior (Carew and Kandel, 1977). Ink-ng behavior (release of purple ink) has been detectedidely in other sea hares of the genus Aplysia (Eales,960) and in several sea hares of other genera (Gos-iner, 1994; Kandel, 1979). The ability to release inkrom the purple gland appeared after the anaspideanineage diverged from the cephalaspidean ancestor, andt has been lost in several lineages independently (Fig.b). Therefore, the evolution of inking behavior doesot seem to be phylogenetically constrained after ap-earing in the Anaspidea. It has been shown thatplysia species extract the purple ink components from
heir seaweed diets (Nolen et al., 1995); thus, the abilityo ink might be correlated to the availability of redeaweed or the dietary habits of each species, rather
han to an evolutionary constraint.
R
bgf(aitmeWn
niifcetoelaw
c
56 MEDINA AND WALSH
elevance of This Study to Neurobiology
Because of the vast amount of information on neuro-iology and behavior of sea hares, especially from theenus Aplysia, the need for a phylogenetic hypothesisor this group has been pointed out by neurobiologistsKandel, 1979). Kandel (1979) gave an overview of thevailable knowledge on different learning mechanismsn Aplysia and emphasized the importance of compara-ive studies in order to understand the evolution of suchechanisms. These types of studies have started to
merge in the literature (Nambu and Scheller, 1986;right et al., 1996). The available information on the
FIG. 8. Mapping of behavioral traits onto the molecular (rRNA) pycling was used. (a) Swimming; (b) inking (purple ink).
ervous system of Aplysia includes maps of a great n
umber of the giant neurons in this taxa. The behav-oral function of many of these neurons has beendentified. This knowledge creates ample opportunityor comparative studies because neuronal homologyan be determined with high levels of certainty (Wrightt al., 1996). For instance, Wright et al. (1996) looked athe effects of a neuromodulatory transmitter, serotonin,n spike duration (time span of an action potential) andxcitability (number of action potentials for an intracel-ular depolarizing current) in tail sensory neurons thatre involved in defensive withdrawal reflexes. Theyere able demonstrate that learning-related mecha-
ogeny. Character tracing was performed in MacClade 3.06. Equivocal
hylisms common to most taxa in the anaspidean clade
wotislenoct
Sslahpaopaf
A
A
B
B
B
B
B
B
B
B
C
C
C
C
C
D
E
E
F
F
F
G
G
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H
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57ANASPIDEAN mtDNA PHYLOGENY
ere lost in Dolabrifera by mapping these charactersn a partial phylogeny based on morphological charac-ers (Wright et al., 1996). Wright et al.’s (1996) pioneer-ng study demonstrates the potential for evolutionarytudies of behavioral mechanisms at the neurologicalevel in the well-known sea hare system. The molecularvidence presented in this study provides a phyloge-etic hypothesis for the evolution of the anaspideanrder which can now be used as a framework foromparative studies of neurological traits in this opis-hobranch group.
ACKNOWLEDGMENTS
We are especially grateful to T. Gosliner, J. Ortea, S. Pennings, N.trenth, K. Lawrence, and T. Capo, who collected some of thepecimens used in this study. J. Silberman assisted in variousaboratory procedures. We are also thankful to D. Swofford forllowing the use of the beta version of PAUP*. We thank R. Willan forelpful comments on anaspidean taxa and for sharing material inress. We also thank K. Rosen, T. Collins, J. Fell, D. Olson, and twononymous reviewers for their helpful comments on a previous draftf the manuscript. This paper was prepared by Monica Medina inartial fulfillment of requirements for the Ph.D. in Marine Biologynd Fisheries at the University of Miami. Support for this project wasunded by NIH RR 10294.
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urn, R. (1989). Opisthobranchs (Subclass Opisthobranchia). In‘‘Marine Invertebrates of Southern Australia’’ (S. A. Shepard andI. M. Thomas, Eds.), Part II, pp. 725–788. South AustralianGovernment Printing Division, Adelaide, Australia.arefoot, T. (1987). Aplysia: Its biology and ecology. Oceanogr. Mar.Biol. Annu. Rev. 25: 167–284.
arefoot, T. H. (1991). Blood-glucose levels in the Sea Hare Aplysiadactylomela: Interrelationships of activity, diet choice and foodquality. J. Exp. Mar. Biol. Ecol. 154: 231–244.
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