Gastropoda): an assessment based on partial 18S rDNA and cyt

¥A/ Molecular Marine Biology and Biotechnology (1997] 6(1), 1-20

Phylogeny and relationships of pleurotomariid gastropods (Mollusca: Gastropoda): an assessment based on partial 18S rDNA and cytochrome c oxidase I sequences

M.G. Harasewych* Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Waslvngton, DC 20560. U.S.A.

S. Laura Adamkewicz Department of Biology, George Mason University, Fairfax, Virginia 22030, U.S.A.

Judith A. Blake, Deborah Saudek, Tracy Spriggs, and Carol J. Bult The Institute for Genomic Research, Rockville, Maryland 20850, U.S.A.

Abstract

The phylogenetic position of the ancient family Pleurotomariidae within the Molluscan class Gas- tropoda, as well as the relationships of its Recent genera and species, were assessed using an iterative, two-gene (18S rDNA and cytochrome c oxidase I) approach to phylogeny reconstruction. In order to orient the Pleurotomariidae within Gastropoda, partial 18S rDNA sequences were determined for 7 pleurotomariid and 22 other gastropods that span the major groups within the class as well as for one cephalopod and two polyplacophorans, which serve as outgroups. Cladistic analyses of a sequence of approximately 450 base pairs (bp) near the 5' end of the 18S rDNA support the monophyly of the following higher gastropod taxa: Patellogastropoda, Vetigastropoda, Neritopsina, Apogastropoda, and its subclades Caenogastropoda and Heterobranchia. The 18S rDNA sequences and 579 bp of cytochrome c oxidase I (COI) analyzed separately and together, indicate that Pleurotomariidae are included within Vetigastropoda but comprise a clade that is the sister group to the other families referred to this order. Monophyly of the Pleurotomariidae is also supported by the unique presence of seven separate inserts (ranging in length from 1 to 68 bp) within the

'"'Correspondence should be sent to this author. *^ 1997 Blackwell Science, Inc.

V2 variable region of the 18S RNA. Relationships of the genera and species within Pleurotomariidae are fully resolved using "total molecular evidence" consisting of partial sequences of 18S rDNA and COI and including data on length variation within the inserts.

Introduction

The superfamily Pleurotomarioidea is recorded from Upper Cambrian deposits (Knight et al., 1960; Tracey et al., 1993) and comprises one of the oldest undisputed gastropod lineages. Its diversity at all taxonomic levels was greatest during the Paleozoic, when these animals were the most numerous, varied, and abundant of all gastropods. Pleurotoma- rioideans were conspicuous components of shal- low-water marine communities throughout the Paleozoic and Mesozoic, but Cenozoic fossils are rare, occurring in deep-water facies (for review, see Hickman, 1976). Of the approximately 1500 species that have been described, only 24, all members of the family Pleurotomariidae, are living today. The Recent species are limited to tropical and temperate latitudes, occurring along the margins of continental tectonic plates that bound the western shores of the Atlantic, Pacific, and Indian Oceans as well as along southeastern Asia, northwestern Australia, and intervening islands (for review, see Anseeuw and Goto, 1996). All living species are members of upper continental slope communities (approx. 100-1000 m), most are restricted to hard substrates and steep to vertical slopes, and tend to be the nu- merically dominant, large gastropods in these habi- tats (Harasewych et al, 1988).

Pleurotomarioideans are most readily identified on the basis of having dextral, conispiral shells with narrow, generally deep emarginations or "slits" along their outer lips that give rise to a spiral band or selenizone along or near the periphery of the shell (Figure 1). The discovery of the first living pleurotomariid in 1856 prompted an era of intense anatomic scrutiny of these rare "living fossils," with every new collection of specimens generating a se-

//

2 M.G. Harasenych et al.

Figure 1. Representatives of fossil and living Pleurotomariidae, (A) Pleurotomaria angUcus (Sowerby, 1818), type species of Pleurotomaria, lurassic. France. (B) Perotrochas quoyanus (Fischer and Bernai'di, 1856). Recent, Lesser Antilles. (C) En- teinnotrochus adansonianus (Crosse and Fischer, 1861), Recent, Lesser Antilles. Reproduced from Knight et al., 1960, pi. 131 (A) and from Ball, 1889, pi. 37 (B and C).

ries of publications (for review, see Bayer, 1965). Pleurotomariids liave since been regarded almost universally as the most primitive living gastropods (e.g., Thiele, 1929; Taylor and Sohl, 1962; Boss, 1982; Vaught, 1989) and appeared as such in para- digms of gastropod evolution (e.g., Bouvier, 1887; Yonge, 1947; Morton and Yonge, 1964; Graham, 1985; Salvini-Plawen, 1985; Brusca and Brusca, 1990; for a detailed review, see Bieler, 1992). The combination in pleurotomarioideans of an asym- metrically coiled shell and symmetrically paired palliai organs (including gills, auricles, kidneys, and hypobranchial glands) has been regarded as transitional between extinct, planispirally coiled, bilaterally symmetrical ancestors, generally be- lieved to be bellerophonts, and asymmetrical mod- ern gastropods.

Interest in gastropod phylogeny has had a renais- sance over the past two decades, prompted in large part by discoveries of primitive, previously un- known groups, primarily from deep-sea hydrother- mal vents, and fueled by developments of molecular techniques and software for cladistic analysis, as well as by the more widespread application of the principles of phylogenetic systematics. The first of the major works to reassess gastropod evolution was that of Golikov and Starobogatov (1975). These authors recognized that the patellogastropod limpets, together with some Paleozoic groups, manifested a series of archaic characters that were neither shared with nor could be derived from other gastropods. They placed these taxa in the subclass Cyclobran- chia to segregate them as an earl}', monophyletic offshoot fi-om the remaining gastropods, and considered the pleurotomarioideans to be the most primitive of the Recent forms among the remaining gastropod taxa.

Salvini-Plawen (1980) divided the basal gastropods into the Vetigastropoda, in which he included the Pleurotomarioidea (Pleurotomariidae + Scis-

surellidae -I- Haliotidae -I- Fissurellidae), Cocculi- noidea, Trochoidea, and the extinct Macluritoidea and Murchinsinoidea, as well as two other "early side-branches," the patellogastropod limpets and the Neritopsina. Following a rapid succession of articles providing data on new suites of anatomic and ultrastructural characters as well as on newly discovered taxa, Haszprunar (1988) published a hypothesis of gastropod phylogeny in which he pro- posed the successive emergence of 13 higher taxa (Figure 2A). More recently, Ponder and Lindberg (1996, 1997) published parsimony-based phylogenetic analyses of Gastropoda based on anatomic characters (Figure 2B) that differed from Haszpru- nar's hypothesis in the composition and placement of several key families and orders, but concurred in placing the patellogastropod limpets as the sister taxon to the remaining gastropods and the pleurotomariids as closely related to the trochids.

Few DNA sequence-based phylogenies containing more than five gastropods have been published to date (Emberton et al., 1990; Tillier et al,, 1992, 1994, 1996; Rosenberg et al., 1994, 1997), all based on small portions (approx. 150-700 bp) from various domains within the large ribosomal molecule (28S rRNA). Most focused on evolutionary relationships of higher taxa within Gastropoda, but several (Tillier et al., 1992, 1994; Rosenberg et al., 1994, 1997) spanned the class as a whole. These studies, for the most part, supported themonophyly of Vetigastropoda and Apogastropoda [sensu Pon- der and Lindberg, 1997 - Caenogastropoda + Het- erobranchia), but differed in the placement of such basal groups as the Neritopsina (with Bivalvia in Rosenberg et al, 1994, 1997) and the patellogastropod limpets. Relationships of the patellogastropod limpets have proved especially difficult to resolve, as they emerge as a sister group to all other Gastro- poda (Tillier et al., 1992), as a sister group to either Vetigastropoda, Neomphalina -I- Apogastropoda,

r

Molecular phylogeny of pleurotomarüd gastropods 3

Figure 2. Recent morphologj'-basecl hypotheses of gastropod phylogeny. (A) Phylogenetic reconstruction of Haszprunar (1988, Figure 5), with no- menclature and taxon rank modified/ updated to be comparable with that of (B) Ponder and Lindberg (1996a, Figure 11.3D). Taxa listed in upper- case ai'e represented in the present study (see Table 1). The "Hot Vent C" limpets reported by Haszprunar (1988) were subsequently described as family Neolepetopsidae by iVicLean (1990) and included in Patel- logastropoda by Ponder and Lindberg (1996, 1997),

P*TELl.DaASTPOPail*

Ci>]CULWIlOAE TEIELLOIÜEA

NEPiTOPSIHA

- POLVPi-rtCOPHLWA

• PISSUHELLOIDEA . SCis5ur4ttb<lH

- HÍLlOTO(DE> - PLEuADTOHAnoQ^

COCíUL íUfíf^MIft

ueTPGASrftûPD[>rt

- CEniTHllD4E

- MEDTÍE^IOQLOSSA

l'rRAlJiPELtOlCftA EUtHYMEURA

CAZWOÜBTÍÍOPDUA

HFTEROElñArJGHIA

- ríAUílLOIDÉA

- CATEUOÜASTUDPOQA

- LfPÇTÏLUOlDEA 1

- RSSüBELLOlDEA - PtEJPOTOWAfllCrtlEA uetlGASTBOPCOA

- TTICCMIDAE

- S99<i<Kiz»doe J

- NEmropsiHA

- OOCCHlNlQtó

- Vlfil idrd

- CyyímN3r.duif

CAEHDGAS1 Rf^POOA

- 10NNC}I[>LA

- KJfiJCOIDEA

- COMQ<íl£A

- 0PISTHüBJlAí4L^t^|.-,

- Pm.fd<ííATA

APOGASTBOPOOA

HFl£ftOBA4NCHm

or Heterobranchia (Tillier et al., 1994), or within Caenogastropoda (Rosenberg et al., 1997), de- pending on such variables as outgroup, included taxa, and the algorithm used for tree construction. In the only study to contain a pleurotomarüd (Ro- senberg et al., 1994), the single species emerged within Caenogastropoda at the base of the Neogas- tropoda. Sequences of the small subunit ribosomal RNA (18S rRNA) molecule have been determined for relatively few (<5) gastropods, generally in the context of broader studies such as the evolution of the Metazoa (e.g., Field et al., 1988; Ghiselin, 1988; Winnepenninckx et al., 1994).

The composition of the superfamily Pleurotoma- rioidea has also undergone considerable revision during the course of renewed scrutiny. Knight et al. (1960) included within the Pleurotmarioidea 20 extinct families as well as the living families Pleurotomariidae, Scissurellidae, and Haliotidae, a scheme followed in most contemporary classifications (e.g., Hyraan, 1967; Hickman, 1984; Vaught, 1989). Salvini-Plawen (1980) added Fissurellidae to Pleurotomarioidea, which he included in Vetigas- tropoda. Boss (1982) added Neomphalidae, but removed Fissurelloidea as a separate superfamily. In a reassessment of the Vetigastropoda, Salvini-Plawen and Haszprunar (1987) identified other anatomic characters to support the monophyly of Vetigast- ropoda, which they considered to include the superfamilies Fissulrelloidea, Trochoidea, and Pleurotomarioidea (containing the families Pleuro- tomariidae, Scissurellidae, and Haliotidae), with Neomphalidae appended as "incertae sedis." These authors noted that no synapomorphies could be found to unite the pleurotomarioidean families, and that Pleurotomarioidea was most likely polyphy-

letic. Most recently. Ponder and Lindberg (1996, 1997) added the superfamily Lepetelloidea and the family Senguenziidae to the Vetigastropoda, removed the Neomphalidae, grouping it with other "Vent ta:xa," and considered Pleurotomarioidea to be the sister taxon of Trochidae + Seguenziidae (see Figure 2B).

The family Pleurotomariidae is of Triassic (Ladi- nian) origin (Tracey et al., 1993). The majority of the dozen genera included in this family are extinct, and all have been defined typologically on the basis of the presence or absence of a few easily recogniz- able shell characters used sequentially. Most mod- ern authors concede that generic distinctions are not always clear and that many taxa are not readily assigned into existiiig genera (Cox, 1960; Hickman, 1976; Szabó, 1980). The genus Pleurotomaria De- france, 1826, is generally regarded as having ranged from the lower Jurassic to the Lower Cretaceous (Knight et al., 1960), while the genera Pemtrochus Fischer, 1885, Entemnotrochus Fischer, 1885, and Mikadotrochus Lindholm, 1927, have been pro- posed for living pleurotomariids. Some authors regard these as, at best, subgenera of Pleurotomaria (e.g., Dall, 1889; Hickman, 1976, 1984; Abbott and Dance, 1982), while most others (e.g.. Knight et al., 1960; Bayer, 1965; Benfrika, 1984; Vaught, 1989; Endo, 1995; Anseeuw and Goto, 1996) accord some or all of them generic rank. A synopsis of available information on living Pleurotomariidae has recently been published (Anseeuw and Goto, 1996). To date, there have been no published studies of phylogenetic relationships within the famil)'.

The principal objectives of this study are to re- construct the major outlines of gastropod evolution on the basis of partial sequences of the 18S rDNA

4 M.G. Harasewych et al.

gene and to assess the ph3'logenetic position and composition of the superfamily Pleurotomarioidea on the basis of these data. Corroboration and better resolution of the relationsliips of the genera and species within the family Pleurotomariidae are pro- vided by analyses of sequence data from the mitochondrial cytochrome c oxidase subunit I gene.

Results

Partial sequences comprising approximately 450 base pairs (bp) near the 5' end of the 18S rDNA gene were determined for 27 gastropods, one cephalopod, and one polyplacophoran, and aligned with homologous regions of the complete gene sequences of ope polyplacophoran and two gastropods obtained from GenBank (see Table 1). The portion of the gene used in this study corresponds to positions 60 to 515 of the ] 8S rRNA sequence of Onchidella céltica as reported by Winnepenninckx et al. (1994, p. 101). Among the ta3<a included in the present study, the length of the gene between these two positions varied considerably (430 bp in Xenopbom to 537 bp in Perotrochus midas) owing to the presence of insertions and deletions, especially in the regions of the helices 10, E-lO-l, and 11 (see VVin- nepenninckx et al. 1994, Figure 1). The large insertion in this region that was found in all Pleurotomariidae is of questionable homology to

the smaller E-10-1 insertions found in Nautilus and in the patellogastropod and cocculiniform limpets. Therefore, the alignment encompassing all taxa spans 653 positions (Figure 3), although the longest constituent sequence is 537 bp. Seven autapomorphic, single-nucleotide insertions were excluded from phylogenetic analyses, as were 10 regions, ranging in length from 1 to 171 bp, that could not be reliably aligned (Figure 4B). Of the remaining 392 alignable sites, 199 were constant and 73 were parsimony uninformative. Cladistic analysis of the 120 informative characters using the branch and bound algorithm of PAUP 4.0.0d42 produced 4656 most parsimonious trees with a length (L) of 433, consistency index (CI) of 0.621, retention index (RI) of 0.744, and rescaled consistency index (RC) of 0.481. The strict consensus of these trees is illus- trated in Figure 5A. Results of bootstrap and jackknife analyses (1000 replicates) are superimposed on the consensus tree. Characters were reweighted by the maximum value of the rescaled consistency (RC) indices using a base weight of 1, and reanalyzed using the branch and bound algorithm to yield 60 most parsimonious trees of length 229.95 (CI = 0.779; RI = 0.845; RC = 0.659). The strict consensus of these trees, with bootstrap and jackknife support for nodes, is shown in Figure 5B.

Quantitative comparisons of the 18S rDNA sequence-based phylogeny with morphology-based

Table 1, Locality data, tissues extracted, voucher specimen information, and sequence accession numbers for taxa used in this study.

Taxon

Class Polyplacophür:!

Acaí\thQ)]lt!í.na jirjioniro (Lischkr. 1ü"¿l

Cnviioc.'u/.ï" ~-írf,Vri (Miiidfiuioril. ia-17)

Ctaís Cßplialüpttdii

¡VaiFf/íus .ícioiinitoílí« L-jililluijl. 1786

Clj^s CüSliopuJ.i

PatcllojiïaBlFopQds

Acinoeu niüra Rntrlike. líi^rí

O Huno ni^yr-ürwula IReeve. 1854)

Cocriilinirorniia

CnucuJJnidiH!

Coi:(.:utinu ilics=ii!-J McU:.;n í^ il.i-.iscwych, 1995

Lepeltjlluidea

iVoíDLTrorpr /ioijbr>i;^í Mv.Lt;.iíi ^ H^rdíví\\y(li, 1995

NprdupHÍno

Nf^riio ver.Tj'rijMfGrnelin. 1791

Neritina recH^'íila {Say. IHZ^)

CoMeciicjn localiiv

y'iuir.Wrr

n-.iireijul

SíjqiiL^ni:e

accR&sion

riiimbrr 18S

Gmßauk ex \ViiLLirp<'¡;iimckx .\ ak. 1993

Bsiiiütjld, B.C., CanaJii

PajJLia, New GtLLr]GEi

BomfiFid, B.C., C.iiada

MiTjub+í. Jíipaii

üjj Pine Kay. Id , USA

iig Pille Key, FL, USA

EMBL X70210

Biuxid musLli- USNM 888657 CSDB L78876

imral rmisde USNjVI 8S5r,78 GSDB L78377

Hiici-.il niLisrliJ USNM 888640 CSDB L78878

Buuiinl miisili! USK.vl 888623 GSDB 1,8879

Huc«l iiiusde USNM 888655 GSDB L78880

Whuk iiiiimiil USNM üUSbjb GSDB L;888:

BUCCHI niu.icie L'SNM 888658 CSDB L78882

nm;i;ol raustlo USNM 888659 GSDB L78883

^rij'ii-ince

nrri ^s:na

i;,iiidrr GOl

Table 1. Continued

Molecular phylogeny of pieurotomariid gastropods 5

Tax on Colleclion locniitv

Sequence Sequenct;

Voucher accession 3CrA:ssinn

material number 18S number COI

Vetigaslropoda

Fissiirelloidea

Diodora cayenensis (Lamarck, 1822)

Halioloidea

Holiolis ítj/csrríis- Swainson. 1822

Trochidae

/\si(aeo coelato (Gmelin, 1791)

Citlarium pica [Linné. 1758)

P)eurrjloinarioidea

Eníemnoírochiis adonsonianus (Crosse & Fisc)ier. 1861)

Eníp;n;ioííodl(i5 rumphii (SciTepman, 1879)

Perolrochus quovanius (Fisc)ier K: F^ernardi, 1856)

Pecotrochus iucaya P.M. Bayer. 1964

Perohoclnis nwuieri Harase\vych & Askew. 1991

Perolrochus midas P.M. Bayer. 1965

Peroirochus Icremochii Kuroda. 1955

Caeaogastrûpoda

Cerithiidae

Cerilhium alivlum (Born. 1778)

Xenoplioroidea

Xenophora esiilum Reeve, 184,3

Tormoidea

Pusitrilon oregonense (Redfie)d. 1848)

Miiricoidea

Oliva sayona Ravene). 1834

Conoidea

Hosítdo cinérea [Born. 1778)

Heterobranchia

Pyiamide)]oidea

Fargoo busihona Bailsc):. 1909

Eulhvneiira

Opislhobianchia

Huniinoea onliHarinn (d'Oibignv, 1841)

Aplysia doclyloinela (Rang. 1828)

Pulnionata

Onchidella cellica (Cuviíjr. 1817)

Physa Iwleroslmpha Say. 1822

Limicoíaria konibeal (Bru^uière. 1792)

Liniax mo.viinus Liuní-'. 1758

Sebastian [n)(!t. FL. USA Buccai mustie USNM 888660 GSDB L78884 CSDB L78908

Bamfield, B,C.. Canada Bucea) muscie USNM 888642 GSDB L78885

Berrv (s., Bahamas

jamaica

Buccal mnsile USlNM 888603

Bucea) musc)e USNM 888661

Guadeloupe Buccal muscle USNM 888647

Aniami-O-Shima. )apan Bucea) nuiscle USNM 888698

Guadeloupe Buccal uuiscle USNM 888646

Bahamas Buccal muscle USNM 888619

Charleslon. se. USA Buccal muscle USNM 388662

Goulding's Cay. Bahamas Buccal muscle USNM 888645

lapan Buccal muscle USNM 888617

CSDB L78886

GSDB L78887

GSDB L78888

CSDB L78889

GSDB L7He90

GSDB L78891

GSDB L78892

GSDB L78893

GSDB L78894

Minabe. Japan

Dutch Haibor. AK, USA

Fl. Pierce. FL, USA

Pi. Pierce, PL, USA

Fl. Pierce. FL. USA

Ft. Pierce. FL. USA

Minabe. Japan

Buccal muscle USNM 888631 GSDB L78896

Buccal muscle USNM 888534 CSDB L78897

Buccal muscle USNM 888605 GSDB L78898

Buccal muscle l S\M 888611 GSDB L78899

Whole animal USNM S88638 GSDB L78900

Buccal musr:le

Buccal muscle

Genbank ex VViunepenninck,\ (-1 al.. 1994

llhaca. NY, USA Bu< ( .:! muscle

Genbank ex VVinnepenninckx et al.. 1992

Silver Sjirlnp. MD, USA Buccal muscle

USNM 888664

USNM 888624

USNM 888613

USNM 888604

GSDB L78901

CSDB L78902

EMBL X70211

GSDB L78905

EMBL X66374

CSDB L78906

GSDB L78909

GSDB L78910

GSDB L78911

CSDB L7Ö915

CSDB L78916

GSDB L78914

GSDB L78913

GSDB L78912

Sebaslian Inlel, FL, USA Buccal muscle USNM 888663 GSDB L78895 GSDB L78907

EN4BL indicates European Molecular Biology Laboratory Data Library; GSDB. Genome Sequence Data Bank; USNM, niollusk collection, National Museum of Natural History, Smithsonian Institution.

phylogenetic hypotheses are based on reanalyses of sequence data for subsets of shared taxa (shown in upper-case letters in Figure 2) and subsequent manipulation of tree structure using MacClade (Maddison and Maddison, 1992). Resulting increases in tree length and indices are reported in Table 2,

To better resolve the relationships among the Vetigastropoda, a subset of taxa that included the seven pieurotomariid and four nonpleurotomariid vetigastropods, as well as the two Neritopsina and Cerithiuin and Hastula (as representatives of Caeno- gastropoda), was reanalyzed. Although the alignment of nucleotides was not altered, elimination

6 M.G. Horasevvych et al.

ID ion

AcaíiClmpleuia Cryprt:cichitor,

Cellanö

¿kdansorjLan! i

QuoyanMS lucaya

midas P. U.erarnacîiii Cerithàum

Fljsitricon Oliva I-Lgscuid

Hamino^ Aplysia cnchidellö Lîltlicolaria

liITTflX

T.AAiïr.A'CTiGACTTT- " • - -Ly,-C"A[A01I.;"wv\Caï:AMTaX'rCAWAMIX3iïrrATCATTTCTTMaiCCTA" "O\C"T"0C''TAel

. . .T"

. . .•! ••

r. .T"

. . .T"

. . Y . '•

. .C.•.T

..G...GT cr,-

.T

.r

.G. .C

.c. .c -G. .C

"G. .C

.G

.G

ACG.A. CG^. . . A. .A.C..C. . .G A-O^.G. .C. . .G ....A....JC.G .CG.A. . . .Ain"; G..C. . . .

-c.CA""r.

"03W.AG.GQG.". T. .C. .T-.GO;.".

--.GA".- . rk.O\.'i -.cr-l'.G.

.C. ..".• . r.. . . 1 . . .

rCcmxXMGG. .G.G. 'OCC. " . . ronncrjxc- .G.COJ.TC. " .C

G

...."G..c.• G. .

••

".".CGGTia .A • •• •• .A..C..-"- .A...C.-"-

.A...C.

.-.:-i.c.

rr "" .TT'";ccc. "..

. • ç. ,-.(-• •'

"" .-G. •<X.. . ." . .

.G.-.G...."..

.T

.T .G. . . .G. . .

.C. .

.G. .

.G. .

G. .C. . .G G. .C. . .. G. .C... .

G C

. CG/Î . . " .• •-•: . • .

. ."i. . • •. .A-.".

..GA.." ..A'.". GA lA" "

. . .TA

. . .'1 . . .T" . . .T"

. .T"

. . : •

"T"

. . .'1- .T"

.•.cc.-

.•.cc.-

.CG.ce."

.CG.CC.-

.OG-CC."

.•.cc."

• ••• .GCY-.•.c."..

• .GG.".OG.C.". . ••'•• ". .av .•.. .T.. ""•" -"..C'".CG. . .". .

-"• . .OT".CG. . .T. . .G.".CG.C."..

.T

.T "T .T .T .T

YG. . . .G. . . "G. . . .G... YG. . . .G. . .

.Y....

.Y

.Y. . . .

.G. .

.G. .

.G. .

.G. .

.G. .

.G. .

G. .C. . .. G. .C. . .. G. .C. ... G. .C.. . . G. .C. . . , G. .C. . ..

.CG. . . '" .CJ\T. •• .

.GG. . . "".CLA-.".

-CG. . . "".CíA:'.".

.OG. . ."".G-AT.".

.CG. . .' .CJC".

.CG. . . "•• .GAT'. " .

" •-Trrv" rr: G CCA G C . . lA.C- •. .CT. • . . .V-A.G-". .AT.""

• [•. . .

"T".ce." .C..ce." .c..ce."

••Hu•.,_, •^, Q-. G . . a"A.G .G ...

Gr" CG " . 'JGi\ G C . . . . .(JA.C"" . .iV-.. "" (JA C"" AA "• " -i^" oG 'Ï'LA G C

.c...G." . .GAC. "• .u«\ • . . . .&C. •'•.&.' .".

..GAC."•.GA .". CíA:'. " ''vv ".

.CCA.G..C....

"GA G C . .r...G." .c...G."

.c...c."

.c...G."

- .. (,, CGA f- C . ..iAC.-.'" " ...A .".

. .GAG. • .GA- . " . llllll'.l'î''.^:^.''''.'' .;... .OGA.G. .C. . . . .O:ä.G. .C ...

71

HO

93

9Ö

'U Slj

IB

n 95 81 ai 83 sa 81 ai

79

79

IfiO IBO 300

AcanciBple'jva Crypcochimn tûLLlua Acraea Ce: lara Ca^cnj ; u'A

Nericijia Diodora HalioCis AsLraea Citcarium E. ac3ansor"ü.¿y;ü E. rufipt'ii P. quoyanus P. Icicaya P. ireureri P. niiclss P. ceCBnsc.M i Cei-Lthiudi Xenophura Rjsicriton 01 i-^ rastula Fàrçfca H^tminoca f^lysiii Onchidella LuTiicoUria Physa Lirrew

""irrÄ 'A;-'.CTGTGr:TAATTCTAGiOrrAATA2ATGACGTTCA''GCTCCGa^ " " "TTT- *"' TT GC'- 'AGGÂ'^ -.AGi CC":'l l'.A'i'r ii5

C ... CAACGAT' ."TC. . , ' Ul.G .A'",".... • .J

AA. ... AA. . .W

CAACG. ."C.GTC.T. .CCC"' 'CIT. .CCTVAICT..Ç-TK!r.'i.y:r, "

M cmcG. ."".G."A. ..C. ••••c""GCii::.A'- • G;

A. CG

. . .G

.G. .

.A. .

.G. . . . .G

Ml 164 I7T . .A. .A C-M>\. .T. . - . . .T. .CXXiTi'-XTT. . .LXiGC"ai;.CÏCGT- .T"Ce. .GC

""C... c G. .CAAß^.. " . . . .T. . . . .C C l'îl 151

C-AG.TTO C . .Cec- -".=A- G .A. .

.G. . G

. . . C . . .C .. .c

; wi

C-AC.A.A C"- . . .C"T .A' G ist

C"AG.ATA C"' . .CQC" "" .A 'G 157

C".';.CAIA C . .Ö3Z~ .A"-G .G. . .. .c m

C OZ-^-ZC. . " " " "" C .CGC •".."" 15H •,. Q TG^'^'"' " - . .. - C.crcC ' " \5B

C OGAG':. . " H•H.,_^.,^ (-^Q-. i 57 .... C . ..•_'GACC.." .C.C•-"" ".."" 157

c ("SAIX.." "".CCTC""" .."" 15K

i- rG.AC^'.." .C.CGG"*" •'".,"" I5P, -i- U-CG " -"•* C CCC' • J5â

"-:::' C.,V-.C . ." C.CAC G. .A. . " . . C. A C . . " C" C. ce " " • G . . A. . C.TAC. ." G"-- C.• G. .A. .

. .R. . . .1' . . .Y

IS4 lie ist

c:r.AC.." C C.CG-" " G. .A. . 15-î

C. .AC. . " c c.•" "G. .A. . 151 •,. A.C C.TAC. .A ••- C.CG " C. .A. . T51

C C. :G.G.A ""C.• T. .A. . 151 •.. C CTKE. .A -C.• •- :'. .A. . 15;

G ... .C'A. . .A ""- "c.cr G 151

G .... i.-TT.AC. . A " " C. tr-." " • c. . A. . 15J

•• C .'i'V.-.C. .A C.CG T. .A. . iu

Figure 3. Aligneci partial sequences of the molluscan 18S rDNA gene spanning helices 6-17, and corresponding to positions 60-515 of the 18S rRNA sequence of OncbJdeUa céltica as reported by VVinnepennincLx et a!. [1994, p. 101). Among the included taxa the length of the gene between these two positions ranges from 430 to 537 bp owing to the presence of insertions and deletions, especially in the regions of the helices 10, E-10-1, and 11. All sequences were confirmed by at least two sequencing reactions in one direction and a third sequencing reaction in the opposite reaction. Ambiguous base assignments are noted using lUPAC symbols. Dots (•] represent nucleotides identical to those oí Acan- thopleum, dashes (-) represent missing data, and quotation marks (") represent gaps inserted during alignment.

Molecular phviogeny of pleiurotomariid gastropods 7

Cin/pLDcM tüEi ^tóut i iUE

Ceilans CoccjliiH NsLoctater

NeriCí.r^"L DiodoETa fialipt.iE

cittariim EnadansoniöTTLiti E.IMiphii P. qixi^siius p. lucayíj P.n«'uir¿i"i ?. midas P. terajvdchii Cerithiimi

PUSitirilQfi oliva lastula Fargoa ]-taminotía Äplyäie Onchidel 1 a Liinicoiariñ Physa LiffBX

230

?£;"ATCAñGA TCAíM- -fïïJX" "C" "TC" •GG"

- .C -C. .TC. . "C..T"".

"".G.., .A.

.A.

•'IC-

"CG.T.TA"

"C..G""A"

"". . . .GA.

-(•:.-".CC.

'C. .G

C, . . . " .

C.... "" .

G.Ac; c. .CC"

G.AG- -c. • Q-)" G.Ai:;- "C. .•" G.A3- -c. .GC"

,CA."

. .A.

• A." . . A. •

. .A."

. . A. "

, . A. •

. .A."

. -A."

. .A." . . A. "

"C.a'J" G. .G."

"C. .G.- G. .G. '

•C. . . . ' •f. . . . •

"C " "C...."

"C. . . . " "G...."

"C.,.."

. "1 .

.ce. . "T.

."T.

. "T.

. •T.'rC.A.AAAi:.CW:"A.

."T.TC.A.AAAC.CQL-A. . -T.YC.^.AAAG.CBG'A. ."T.TC.A.AAAr.CGG"A, ."T.TC.A.AaAC.CGC-A. ."T.YC.A.AAAC.G'Ct'A. . "T.'1G.A.AAAC.CGC"A.

.A, G. .G,-' .

G"--C AA. .-" , .GTTG"."VlCAAA. CGGTCGCICTGAIXXTATCTGGTT. . .GCCC^ A.' •CC'IG.GAÄETA CCGICAAAA. .Gf-G, IGTTCAATTTAATT rris"AA'[TQ«lACAGTCC"" . .

.'rit;ri'• arrj--AOI'

.AGC""GBOe3-"03• K- "OG-GT ••

"TGG.

C.G" C.G" ce- r.f.j"

C.G-

C.G" ,C.G"

G"

G

T-

• TCG"

. "C"

.GC"

.GC""

.GT"-

.•"-

.GC""

.GC-"

"C"

"AC C" "AA. . • 'AA.A" "AA.." "AA. ." •?A. . " "AA. . "

"TC.TTGGGACïriG-

.cin:'rcA..G--

.GCrTCTT""

.QfT'C.ljT"'

.GnT"C-OT""

.GCT"C.C?r""

.SCT"C.GC""

.KS1-C.0C-" GC- ""CCC- "T"G""

T CCG"-X,- aXÎJT.G" IDG"

'T"G "•\-~rjr." -TT' TXG ••• "ÏOX" -

T" .CCG""TGrT"GCTC'"' .TCG"TrGCC.TîrAG' . .OG"" .CGC. -TOGC' .TlDG-iTAOC.TTrGC'

.03"

.GG" "GOG"

"•OC "•ce. •"CC.

""GC..

""CT.

•-CC.

••OG.

--05.

•••. "-•. ""QG. ••-Ci).

•"GG.

•"CC. ""CC.

-•ce. ••ce. •"CC.

"TO3G

GC" GT'"

.GC"

.OC* i^C"

CÏ-"

.GG'

••GG"

•-GG- " "Q3-

••00- ""GG-

" iT

1C "TC'CA-^

1C TC "

-GG-iTnCTTCC" -Cr.-CrfirAíW;" "ÜG-GGITM^G" -•-ooTTCsr;' "GG"GGrTCM:;- ••ívrrriTCJí}- •«"G-TICAG"

K '

•. OGOG-CDGT are " "COOQcocrrr • • ••íTK-C- - •• T. TTCOG "GGGGGCGOCGnr • C • e " Qrí".jxGGGa7;t;"C"C" " ---COGGCGCQ:-OCTC-CA""

03"T COTC: CGGC IJK'-'C-C-

1B<!

1S5

234

246

?.n 151

18J

ISA

192

lea 1G9

211

?11

•¿í<)

209 21(j 2Û5 M 9 i a.? 174 lai lai 181 198 197 •JÜC)

19S 198 ir; lis

310

Cfypcochif."-:-! fsä'Jl;i lus Aomea Celläna Coccüllna NococraLer Nerita Nericijia Diodora Haliocis Asctaeñ C: f.l-xurl'jni E. .sclanscniarjui E. runîMlii P. quoysHLis P. liiGdyä P. maureri P, mida5 P, tieraiTQchii Cerlchiuni Xenophora FXjsitriton Oliva Mani-.:la

Hanr^noea f^lysja •Jnchidel lo Limicolai: j.a Physa

COT A. .C"" TTGCAA. •CGTT . GA " " - .GÄ"-"

--•c.

T. T.

•MTCQCAAATA-

?:07XAT"TCTCAATA

CAA"

• ••'-•«'•

"YT.G.;. ^ •pGG••GC^CCCACCA.Ca30CCCÏJrT^TTCGCrr:H^G(KIrTC^G^•,^•ÏV^CG.A. ;•:•, :-;.C :'lG"•cCCCAOCAC•CL\'TrijrTirxr"CGCOUCCrTTCCCGAACC. A.

"':s.GA YTa7r3a3.Acxix32"CcccccACCAccrrr•<TîGr"Acrc^xTccr,n7rrTTrr.AAAcc.A. "X. GA CirGTCaiAGGroC"CCCCX.CACCACÜGIOGC'G<Tr ACt^rrCTCOGOriTTTTCTAAACC.A. ••r.3 .GA ClXT::XX)G.ACC.'lGS"COXO::ACCAC001CGrn7r ACGCIXrTCOGOGTrriT-^AAArc\ A.

- tG.GACTIG•'TTCCICGraSGACTTGGXCCCCCACCACCGlCfï.CGr"AOGC7CT0CGaiTrr?.'TAAjYC.A. " TG. GACnaXTTGcrOJtCGGAC - " -CrrGCOCCCAOIACOGrrCGÜGQT • ACGCrcrCCGCGTTTKTTAAAa •. A.

"T. . "T. . •T. . "T. .

.G".

.AA.

.AA.

204 2(Jb 275 269 2Í6 2Da 332 202 203 211 2Û7 ¡se 221 2f!l 281 :ee 2iia ?.m 29 e 295 201 193 200 200 300 218 216 21* ";i7 2ii 217 216

Figure 3. Continued

of taxa that contained putatively nonhomologous insertions enabled the alignment to be condensed to 556 positions. All of the regions excluded from the original analysis could be unambiguously aligned, although not all were present in all taxa. All Pleurotomariidae were found to contain seven insertions in the V2 region (De Rijk et al., 1992) that

appear to be unique to the family. These insertions, which vary in length from 1 to 68 nucleotides, are concentrated in the helices 10 and E-10-1 (Table 3, Figure 4C). Two of the seven insertions vary in length among pleurotomariid taxa, each appearing to contain one or more insertions or deletions. Five autapomorphic, single-nucleotide insertions from

8 M.G. Harasewych et al.

500

Acanthopieura

Cfell^na. Cocculina NoCLOcrûLei-

fjerita

MeriLina

LJiocbira

iteliotis

Astraea Ciutârium E. ad^nspruanus

E. rurtphii P. qijqygnus

P. iucaya

P. rmureri.

p. midas p. tetanöChil

Cerithiuri

ACr-TTGIGCraATCGTAirJ'; Lr"Ac-"Ga:;' CCOGOSA ' aji'ATCTrrcHACTKTKTCCcci'ATrA" Acmrr ̂ •iX<-Tfv;.13K3ATf;iT.CCTACC

. . .T -.A. . .

..."G.."".C...

..."... .""C. . .

..-"CGC

. .C •" C. . .

. . . .G. .".GT-."". .

.. .GG.. "T.i-rr•.G.'xc.. .A. .. •" . .c."" .AJ-.m ..A.'

. .TA. . "".TTT--"T.C '" . .A. . . .

. . .GG.AGTTCTTAACA.GCTC.TA. . . .

•. .ex: G..AC . .C.. '.T.TG.CA.G T

•ICC... A.G r •.AA. . .CAA,-,. . . .A '. .CGC.CGAA..AC

• A

. .C •

.."....".T..G.

..'!...." .T.'3A.

G-.. A' C...G..

• A

.G.

J'A.

.CG. .T.axj.. .c...

. CG. c. ce

.CG.C.CC-C

.ÍXJTC. .ce. .A.C. . .

.CG.aX.'.G c...

365 J62

294

351

. . • -.G". . .G. . .

..'-.CIA. .G. . .

. .-".CTA. .G. . . . . -".CTA. .G. . .

' G AT G

C. .

... .ce C

. ^ •.. . 392

. .T: .A.

..rn-'... 'G ^ r-- "" Ahi ' . . . .

•..CG. .CA. .. .A

• . .C. . . .A

' . .C... .A

•..CG. . .A

• . .CG A

' . .03 A A. .rr, ".A T.

". .CG A

". .CG A • . .CG A

' . .CG A

". .C A ". .C ".A

' . .r A

". .C A " . .:• A

" . .C A

G...G..

<-. . .G..

G...G..

A

-.A.GA.

-.A.GA. ".A.GA.

".A.GA. ".A.GA.

-.A.G..

.C

.C.

.C.

.C.

.C.

.C

.C.

. .A. . .c . .A.. .c •;....

. .A. . .'• T. . . .

. .T. . .C T. . . .

i-x:... .c... CJC. .. .c . . ;•;•:•... .c .. ACCC....C... ACCC....c... CCC...C... cx".r... .C... .CG c

300 2S6 257

31.'; 37?

..." .C.A'l'.G. . .

..." .CAT.G. . .

....C..G..C."".A.

. . . .C .G. .TT."".A • '" S.

C. .G. .TT. "• .A. •

372

177

379

" C AT G C G .••';'. " ' .A. 360

. . .".C.AT.G. . .

...".C.AT.G... . . . .C .

3.. T: . ' ' . A

3. .T"."" .A. -'

' . .TT .A.

389

3 S«

290 281

..." C. . . C

.."T."".A. •" • • . . -T. -" .A.

2ëii

Hflstiijla

Fargoa Í^OTiinoea .^Ç'iysia crchittelU ijimi col aria Phyp-";

c c

289

C • . . "T. "" .A. 2B9 3ÜB

- . .C A ".A.G.. -¡(¡6 ,. •. ::vvi- .T. " . .C A

". .C A

" . .C A

" .A.G..

T.".A.G".

".A.G.. c

309 • 107

306

" C A - .A.G . 306 • • • ,. ' . "i".ACT. ' " • . .C A -.A.G.. c 3(17

540 5^.0 SBO

CrypCochi ter;

Acnaea Cal lare Cûcculirfi pJotociraLer iiiericfi ^jtîriLirid iTíicdíiríi Hfâiiotis Astraea Cirtariijm E. adansorü-sri K- runphii P. quoyamJS P. lucaya P. iTBureri. p. inidcJä P. fierarrecñi i Ceritfiiurri Xenopbora FusitJficcn Olivs tíasculñ Farpoa Hamlnoaa Apiysia Cnchidel Ui Licnicolariâ

LLTTCIX

ATCCri • ITGTAACGGGTMC ' C =C"1AG=VA'ICA •• U.X?r! iIGATT-: -V.GGÍi.GAGGGAC^ATGí^GAAACEGr "AC 7\CATCCAAGG " AilTOTA :CAaXGr.GCAAATT

G QA. . . . ^••

C. .C C. G. . . . C • G "... C.-C C. G. . . . .c..

G. .. A. . . .C. .

C. T T .C. .

G " G C

G " G " " r

G "

GA • G " • G '• ..

G •' . . . .G. . C " r G G " ....G. C .. .C..

G ce G G " C .. .C. .

G ce G G C .C . .C. .

G. . . .ce . . . G. . " G ... . C .c.. C .C. .

r r-

. . .T.A.C. . . . .G '• G "

-•' .C. . r

. , .T.G.C . . . . . . .G. •• .G " c

G M r

. . .T G GV T T ... .T.... .c.. .c..

G " .. A. -" 1-

i-¡ ......•• -G •' .'•

3» 392 4Si 459 420 392 424 388 3B9 197 393 394 411 46& 469 474 476 477

43S 463 367 378 itiâ 386

3B6

405

4U3

40(î

4D.Í

401

403

Fisure 3. Continued

other portions of the sequence were excluded from all analyses. An initial maximum parsimony analysis (exhaustive search option, 94 informative characters) was performed on data from which the seven insertions (totaling 94 nucleotides) unique to Pleu- rotomariidae were excluded. Figure 6A illustrates the strict consensus of the resulting 36 most paj'si- raonious trees (L = 168; CI = 0.851; RI = 0.914;

RC = 0.778). Figure 6B shows the strict consensus of the six most parsimonious trees (L = 196; CI = 0.872; RI = 0.918; RC = 0.801) produced when the analysis was repeated with tlie seven insertions included in the data set (105 informative characters).

Figure 7 contrasts the phylogenetic information contained in the length polymorphisms of these in-

Molecular phylogeny of pleurotowariid gastropods 9

Figure 3. Continued t; • 620 630 640 650

Acêinchopieura Aca:?.CTCCT03:.H•«:GA•TA£r;GAa;ajAfATAACA'c-;j.; ri.a-'/.r- 441 Crvptochi-tín v .. 443 Nautil-js A.. .c - T.cr,.T,. ...••••... 'íi.o Acnse* A. . .C.A 'Ca':. . . .GOL'. . vil Cellam A. . .C.A.T T.-GCO-: Y.C. ,"2 Coccuiina A. . .C.A c. . . . C. . . '144 NotOcraCEr- A. .TC.A.T..A " ".-,.-.G C• 474 tterica A •• C. . . 440 Naritína T • C.. . 441 EHotora A. .•?:•.AT.. .A " c.. . 449 Haliocis A. .'.r.AT. . .A " •• C. . . 444 AstlBsa A. .TC.A. . . .A " C. . . 44f CittariLm A. .TC.A. . . .A " C. . . 463 E. udflnsoTLÍsriu^: " C, . . W.i K. nirrpiLLi " C. . . b2l ?. quoyanus " c. . . "^26 r". luc^ys " C. . . ^28 P. jTHLireri " " C. . . 528 P. [nidas " C. . . 537 P. tei-ämäch:. " '• C... 534 Oerithi'jT. ". . .AA.C. . . 439 Xencptotö '• • • • A.c... .-:3c FVjsicriton " A.C. . . 4j8 OUva "....A.C... 4J6 HastUla " A.c. . . •;3B Fargoa C " C... 457 Hamirioea c " C... 455 fplysía C " A C. . . 460 Onchidella C " C... 456 Liinicoiiuria " .c A.G " c.. . 45? Physa C • " C... 454 LiiIBX C " C. . . 456

Figure 4. Distribution of variable sites along tlie 18S rDNA gene among the 32 taxa in this study (Table 1). (A) Positions of helices (from Winnepen- ninckx et a!., 1994, Figure 1]. (B) Posi- tions of bases excluded from phylogenetic analyses due to uncertain homology or alignment (bars) or because they were autapomorphic insertions (*). (C) Location of the seven insertions unique to Pleurotomarii- dae. Asterisks (*) indicate single base insertions.

5 7 8 9 10 11 12 13 14 15 16 17

B C

50 100 150 200 250 300 350 400 450 500 550 600 650 SITE

sériions with that contained in their sequence. Of the 94 aligned positions in the inserts, only 11 are parsimony-informative, producing 9 trees (L - 27; CI = RI = RC = 1.0], A consensus tree is shown in Figure 7A. Figure 7B illustrates the strict consensus of three trees (L = 22; CI 0,864; RI = 0.885; RC = 0.764) based on an analysis of the distribution of the 19 insert segments (Figure 8) among pleurotomariid taxa.

Alignment of the partial sequences for the mitochondrial cytochrome c oxidase I gene (Figure 9) produced an unambiguous alignment correspond-

ing to positions 32 to 611 of the COI sequence of DrosophUa yakuba (Folmer et al., 1994, Table 2), Of the 579 nucleotides, 333 were constant and 59 were parsimony-uninformative. Analysis of the 187 informative cites using the exhaustive search algorithm produced a single most parsimonious tree (L = 540; CI = 0.669; RI = 0.581; RC = 0.388), shown in Figure lOA with bootstrap and jackknife support for nodes superimposed.

The "total molecular evidence" data matrix con- sisted of 1154 characters (556 18S rDNA -I- 579 COI + 19 insert segments), of which 755 were constant

10 M.G. Harasewvch et al.

U^ POLYPiACOPHORA

• NAUTILOlDEA

PAT EL LOUAS T ROPODA

COCCUilNIFORMIA

NERlTOPSIWA

«^LELinOTOMARIIDAE

VETtGAËTnOPODA

CAEN0GASTF1ÖP0DA

APOGASTHOPODA

HETEHOB RANCHEA

J

Figure 5. Phylogenetic relationships among major groups of Gastropoda, Nautilus, and two Polyplacophora based on approx. 450 bp of 18S rDNA sequence from which autapomorphic insertions and regions of uncertain alignment have been removed. (A) Consensus of the 4656 shortest trees (L = 443; CI = 0.621; Rl = 0.744; RC = 0.481) resulting fxoni a branch and bound search. [B] Consensus of 60 shortest trees (L = 229.95; Cl = 0.779; RI = 0.845; RC = 0.659) produced by a branch and bound search of a data set in which characters were reweighted by the maximum value of the rescaled consistency index (base weight = 1). Bootstrap proportions given as percentages above the nodes and jackknife proportions given as percentages below the nodes. An asterisk (*) indicates that the node was not supported in a 50% majority rule bootstrap or jackknife consensus tree.

Table 2. Comparisons of 18S rDNA sequence-based phylogeny of the class Gastropoda with contemporary, morphology- based hypotheses.*

Phylogenetic hypothesis length CI RI RC Taxa [N)

Haszpruirar (Figure 2A) 18S rDNA tree+

Ponder & Lindberg (Figure 2B) 18S rDNA tree*

360 338

371

338

0.65 0.53 0.34

0.69 0.61 0.42

0.63 0.50 0.32

0.69 0.62 0.43

14 14

15 15

*TQ facilitate compaiisons. trees were pruned to contain ttie same sets of taxa. When t-vigt\er taxa were represented by oMiltiple species in the 18S data set. a single representative was selnired with the objective of minimizing the number of deletions and ambiguous base as.signments. Patellogastropoda is represented by Cellana: Neritopsina by Nerita: Pteurotomariidae by Perotrochus lucaya: Trocliida« by Astraea: Euthyneura and Opisthobranchia by Haminoea: Pulmonata by Oncliidnllo. Polyplacophora were excluded from the data set, and Naulilus was selected as the

outgroup. Comparisons are based on taxa identified by Upper Case in Figure 2,

'A single most parsimonious tree resulted from an anahsis using the reduced subset of taxa.

'Three equally parsuironious trees differing only in the topology of taxa within Caenogastropoda resulted from an analysis \ising the reduced subset of taxa. One of tlies(i frees, which agieed with the generally accepted topology of Caenogastropoda included in Ponder and Lindberg (Figure

2B), wa.s used as the basis for comparison.

and 275 were parsimony-informative. An exhaustive search yielded a single most parsimonious tree (L - 712; CI = 0.729; RI = 0.658; RC = 0.479), shown in Figure lOB. Bootstrap and jackknife val- ues are given for each node.

Discussion

Maximum parsimony analyses of the conservative, unambiguously aligned portions of the 18S rDNA

sequence data resuhed in a phylogenetic hypothesis for the class Gastropoda (Figure 5) that is, for the most part, similar to those based on morphologic data (Figure 2], Quantitative comparisons (Table 2] indicate that the phylogenetic hypotheses advocated by Haszprunar (1988) are, as a whole, more congruent with the molecular data than those published by Ponder and Lindberg (1996, 1997). On the basis of molecular data, living Gastropoda represented in our study are divided into four groups.

Molecular phylogeny of pleurotornariid gastropods 11

Table 3. Insertions within the 18S rDNA gene tliat are unique to Pleurotomariidae.*

Insertion Length (bp) Position in aligned sequence (Figure 3) Segment

1 2 3a 3b 3c 4 5 6a 6b 6c

6d 6e 6f

6g 6h 6i

6j 6k 7

3 1 3 1 4 7 6 1 1 8

10 1 2 1 1

20 1

23 1

211-213 220

247^249

250

251-254

276-277, 284-285, 290-291, 293 296-299, 310

314 315

316-323

324-333

334

335-336

337

338

339-358

359

360-382

422

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16 17

18

19

•Positions are referenced to llie aligned sequences stiown in Figure 3. Insertions 3 and 6 vary in lengLli among pleurotornariid taxa, and tiave been subdivided into segments that could be scored as either present or absent in individual taxa (Figure 8) and sen.'e as the basis for the cladogram in Figure 6B.

Figure 6. Phylogenetic relationships among Vetigastropoda, Neritop- sina, and Caenogastropoda based on 18S rDNA sequences. Five autapomorphic, single-nucleotide insertions are excluded from analyses. (A) A consensus tree of 36 most pai-simo- nious trees (L = 168; CI = 0.851; Rl = 0.914; RC = 0.778) resulting bom a branch and bound search of the data set ft-om which the seven insertions [94 nucleotides) unique to Pleuroto- mariidae have been excluded. [B] Consensus of the six most parsimonious trees (L = 196; Cl = 0.872; RI = 0.918; RC = 0.801] produced when the analysis was repeated with the seven insertions included in the data set. Bootstrap proportions given as percentages above the nodes and jackknife proportions given as percentages below the nodes. An asterisk (*) indicates that the node was not supported in a 50% majority rule bootstrap or jackl<Jtife consensus tree.

100

^- ^

iCO

60 98

eo 96

IDO

100 EA

ICO '•'

100

ICO I A

CUtarium

P.C¡'j<jyanus

CL-ciiiniuni

l-!ir';-jia

dicdofo

HjfröiiS

AS{T:!C.I -^ Cillariiim •

TROCHIDAE

E.adansonianus -n

F:uij:rhu Enteninolrochus

r-^u-.jyanL/s -^ P\.C.l.>^ Peroirochus

"Group A' P. maurQii - P.midas •

Pero!rochus

P ¡rrs.-n'Cb:: "Group B"

B Ci

the limpets, the Neritopsina, the Vetigastropoda,

and the Apogastropoda, each representing radia-

tions within an extinction-resistant clade (Erwin

and Signor, 1991) with early Paleozoic origins (Tra-

cey etal., 1993). Relationships between these clades

are not robustly resolved, suggesting that diver-

12 M.G. Harase\v)'ch et al.

A B

Eníemnoírochus

Perotrochus Group "A"

Perotrochus Group "B'

Figure 7. A comparison between the phylogenetic information contained in the length polymorphisrns of the insertions unique to Pleurotomarii- dae with that contained in their sequence. (A) Consensus of 9 trees (L = 27; CI = RI - RC = 1.0) based only on sequences of the inserts. (B) Consensus 3 trees (L = 22; CI = 0.864; Rl = 0.885; RC = 0.764) based on an analysis of the distribution of insert segments among pleurotoma- tiid taxa. Bootstrap proportions given as percentages above the nodes and jaclclsniFe proportions given as percentages below the nodes. An asterisl< (*) indicates that the node was not supported in a 50% majority rule bootstrap or jackknife consensus tree.

Insert Segment

Asiratia E. adansonianus E. rumphii P. quoyanus P. lucaya P. maureri P midas P. leramachii

1 111111111

1234567890 123456789

+ + + + + + + + + - -- + + - + + + + + + + + + + ++-Í-- -- + + - + + + +

+ + + - + + + + -- + + + + - + - + + + + + - + + + + -- + + + + - + - + + + + + - + + + + -- + + + + - + - + + + + + - + + + + + + + + + ++ + - + + 4-J. + - + + + + + + +-- + + + - + +

Figure 8. The distribution of 19 insert segments among Pleurotomariidae. These insert segments (characterized in Table 3) are absent in Astraea and all nonpleurotomariid taxa within our study. Inserts are scored as present (-I-) or absent (~) without regard to their sequence.

gences were ancient and rapid. Philippe et al. (1994) calculated that rapid (S40 Million-year) radiations are beyond the limits of resolution of the entire 18S

rDNA gene. Perhaps the most anomalous result of our study

is the grouping of the cephalopod Nautilus in a clade together with the patellogastropod and cocculiniform limpets. Although the node connecting Nautilus to the limpet taxa is not strongly corrobo- rated by either bootstrap or jackknife proportions, moving Nautilus to its accepted position as a sister group to the Gastropoda increases the length of the tree by 9 steps. The monophyly of patellogastropod limpets is strongly supported (bootstrap and jackknife proportions of 100). The two species in our study contain uniquely shared insertions (e.g., positions 16-27, 32, 86, 643) and a deletion (position 62) that provide additional, independent support

for their monophyly. Our data do not strongly support the monophyly of Cocculiniformia, but do indicate that Cocculinidae and Lepetelloidea are more closely related to each other and to the patellogastropod limpets than to Neritopsina or Vetigastro- poda, respectively, as suggested by Ponder and Lindberg (1996, 1997).

Manipulation of the tree to produce a monophyletic Cocculiniformia emerging basal to Neritopsina (ITaszprunar hypothesis) increases the tree length by 9 steps. Placing Cocculina as a sister taxon to Neritopsina lengthens the tree by 11 steps, while making the lepetelloidean limpet Notocrater the basal vetigastropod increases the tree length by 17 steps. The most parsimonious placement of Noto- crater within Vetigastropoda is as sister tajxon to the nonpleurotomariid vetigastropods, which increases tree length by 12 steps. Although data are lacking for Cocculina, Notocrater contains a portion of the large patellogastropod insert (positions 16-20), adding support for its relationship to the patellogastropod limpets.

It should be noted that all the taxa grouped in this clade are anomalous in containing insertions that are highly variable in sequence and length, difficult to align, of uncertain homology, and are absent in other gastropods as well as in the chitons. Although these insertions were excluded from the data set and did not contribute to the structure of the tree, we conjecture that these ta^a may have undergone inversions, translocations, or changes other than point mutations in this region of the gene that are not homologous among all taxa. Branch

Molecular phylogeny of pleurotomariid gastropods 13

i'rAf.r-íxr.íAciíU.'i'it;^;; nj-x; 1 ï vrrrriÄ;!:;: wr.raes c.T.'Tr.. . .G. .AC.A. G.A. . . .A. . />^-. i .A. .A. . / •.. .cr.AG.G. .G. .c:. . . .1- . .G .A t'-J £:. acfensoniñiíus c.c.(7rG. . . .G.CTC.A. .-rc. . . .A. . . .A. ,A. .A. .A. .C. ..Ar. :. .A. C.7. ... .c .."G.G.. C •íí

. . . .GTT, .

A...O!'... . .G.CC. .A. . .A.r-. .A.

.c..

.c.. . .A. . . . A . . . .A. .C.

..AC. . .

.A, . . .c. .A. .A. .T.

..T.O.^.,

. .':-.ü. .G. .A. .0. .C. c. :. G.T.

.G.. . . . i.. A, . "

...G. ...'• "• C 99 P. iiiufes' A,..cTr.., . .A.r?. .A. .r . . .A. . . .A. r^. .A, . . .c. .A. .T. . .7.r;..G. .G. .c. C.T. . . .G. . . .• • C 99 p. TOijster; A. . .GT. . . •. .A.CT. .A. . r.. . .A. . . .A. .c. .A. .(• .A. .A. .A. .0. ..• ... .G. .c. .a:, i . . . .G.C. .' •. .C, . . 100 p. quoyoniis C...AT... '. .ce:-. .A. . .A. . . .A. .c. .A; .r. .A. .A. .A. .A. .C. .AC. .. .G. .C. .,A.'.T. . . .G. . . .• ..C... 10Ö

P. lucays C...GT... -. .;;.C7. .A. . .A. . . .A. .c. .;c.G. .A. .A. .A. .A. .C..AC... .G. .C. .C'.T. ...G.... . .C. . . lOC CeriLhiíJir, . .G.A:T. . •crc.c. .TC. . .A. . . . " . .c. .A. . . . .A. .T. . C. . . C. T. ... .O':... . .A . .G. .A J9

110 120 130 140 170 180 190 200

DicóDra Ascraea E, fdar.sQ'n i ,ín;i E. fim^î ÎV ccrsrrachjl P. niidas P. [íBueret 1 P. cfucyaniis P. ] ^jcays Cerithium

CA'rGC^H'iTnT..va'AATiTi':^!';'-U7irr.^G\;GCxGrATGATAA'rixKaKATiTQ3GAACiGA':"AGiTCG'^ .Ai'•rwr . .C 'I' C. .A. .A C. .T T T. .T. .C.íG. .A. . .C.'í'. .A ' C. .C. .ACT. G C A. .A. .A. .A. .A A. .T 'i' ( TG GG. . . .AC. . . .A. .C , .ACG. A A. .A. .A T. .T r. .T. . .GlC. .G. .(G 'IG.C.. .A A. .1 . .AG.G.

C..T C G. .G A. .A A A. .T. .(JCl. . .A. .CC.T. . .C. ; . .G. .C. .G. .G. .AG. . C. .T C G. .G A. .A A A. .T. .GCl. . .A. .GC.T. . .G. ; ..G. .G. .G. .G. .W3.T.

C C. . . .A. . .A

.A. -A. .A. .A ...A.. . .C. . .C

.A. T. .aT. . .A. .te.T. ..AG.G.

.Ai'.G.

.Ai-.C.

.... G.

.A.

.G.

.G.

.A.

.G.

.G.

.G.

.C.

.G.

.C.

.G.

.AC.T.

C. . . .A. .fC7. .(T.\ . C.. • • c.. T.A. . .A. .A. .A.... . . .T. . .C. T. . . .'.-G ..-•G. T.

200 195 199 200 300 ino 199

1D[)

Diodorra Astraea E. adansoniâiUi;: E. r^jnçhii P. CfîraJivichii P. miitis p. rrtîLêrErL p. quQyanUi P. lucays cerlthium

TTCCTCmc "TAATAA " hTmXV -T03aGrrGCClX;GTrCV>i •• TAA " TprcTTATAIlC 'TCKiGViQiaTriGiiAaGGQaïGCTCaA' i Ci ; ITTi .V .T. .G. .T.IT. .AC.ce.T. .G. . .G.AC. .TIG"" .C.r'TT.CT ITT. .T. .A. .T.T" .G.

.Y. .C.

.G..G.

TC. ..AT.AC.A. . . . .G. .A.T;'. .A:\AC . . . .A.Ti . .Al'. . . .A. .A. . . . .A.T. . .AV. . . .A. .A. .G. .A.'iG .... .AG. : . .G.

.cr.

.AT.

.AT.

.AT.

.G.G.A. . . .T.GTJG. .A. .T. .A. .A A.T~ G. .A.

.OCC.AC. . .T.GCA. .G. .G A A. . . . 1. . .T

.G.. .AcrnroGA T. .A. .G •..'ic. .A. .G. .G.

.G. . .AC. IGIGÜCA T. .A. .•."; A.'". .A. .C. .C. ;CT.GC. .AC.G.- .Gcr. .T. .A. .A. .G. .T-

.G. .A.TT. .AG.l. .G. .GC. .AC.C.G.GCG T. .A. .A. .G. .T. .G.

.T.TG. .AC.T. .A.

299 3 DO

299 JOB 30(1

310 320 J30 340 JÜO 3 Si 390

E. 3íÍLnson.L¿ijiu¿ E. rijnpliii 1^. tcraiTïich ;• i P. nudas P. rauerer;. P. quciyonuE P. lucai'a

': • : ; ATTG-XC; •• '?CJPG1TJ1AAA•GGGTCATGCTCK " CGTlGTCmGAWPrAACTAl-.' • A. .T ....-•.. .G.:G"A C..TCGC. . ". .A-.TC.ÜG. . .TG

.G CT.AG. .A.C. .ce C. .C. .T. .A. .A. .G. .T. . .G. . .Tl A A

A. .A. .G. .A. .AT.A.G. .A.C. . .C C. .G. .C. .A. .A. .A. .¡C. .G.G.'ri C.T C. .G A.'..%,GAA. . . .C. .A A. .C A. .A. .T. , .G.A.TC A. .A. .C.

C. A. A. A.

•'P.'TCTTIGQ'. : " TGQ'^. • GiGTG"! CTTGT.ATTT: AGCr: . .A-GG^.'". . . • A. .G G

.G. . .G. . . .G. .A A. .T. .G. .G. . .C. . . .G A. .A. .A. . A. .A. .AA. .G.

.G.

...AT.AG.AA....G..A A..G A. .A. .T...G.A. G A..A..C..A..A..AA.T G A

. . .AT.AG.AA C. .A. .•:• G. .A. .T. . .G.A. .:'; OC.A. .G. .A. .A. ,AA,T G. . .G. . . .G . . .AT,A,G.GA A. .G G. .A. .T. . .G.A.-G.' A. . A . . ". . A. .G. . AA. 1' G. . .G. . . .G . . .Ar.A,G.GA A. .G G. .A. .T. . .G.A. ]'; . .G. .A. .A. .G. .A. .G. ..AA.T G. . .G. . . .G . . .TT.AG.A. .G. . .C.A C. .A. .GGï.Gi. .A. .C. .G. . .G. . .¡T. .G. . .G..¡ . .G;:.A G. .T G

397

.19.1

399

4D(J 399 399 400 ÎOO <tOD 159

430 4R(; 490

D' octari Ai E.

1 raea odariËOiiiani

E. V. P.

teraiTöchii

p. fTHtícrer] P. quoyarLu£ P. luqaya Ceri cûm

' \ T.'^. .7. .A. "A. .AG.ff: 'G ... •... :. .G. .a:.. . . ¡GGA T. .G. . • A. ..." . .^ . .A. . . .A. ./; "" ^^r .• ;.. . .AG. .7 G. .•.G. .AL.A. . . .T. .A. ,s. .G G. .G.

. .C. .T. .C. .G. .C. .G. .AA.'^. .G. .AG.,;...- . .A. . .G. .GC.A. .AV.A. . . .T. ,G. .A. .T. . A-- .G. . .C .T. .G. .G. ..AA.G. :r.. .AC.G .A. . . .AG. .1 . .A. . .'i-.A. .AC.A. . . .T. .A. .A . GG .T. .G. .G. .A.A.C. .T. . .AG... .A. . . .AG. .T. .G. . .'i .A. .A.'.A. . . .T. .A. .A. .( :•

.T. .AA.T. .T. . . .A. .AG.A, .G. . . .AG. .T . .'l'.A. .G;.A.. . .T. .G. .G. .A. .Gi. .A. .AC..AG.G. . . .AG. .7. .A. . .7.A. .G: .A T. .G. .A A G. .A. .00 .A. .AG..AG.G .-..G. .-1 . .A. . .'V.A. .GT.A T. .G. .A A G. .A. .CC

.A. .AGG..-.G.A AG. .G. .G. .GC. . . .TC.T G. .A AA.G. .T. .CG. .C

497

49-1 499 son •;59 •199

'l'Jlî

500 500 499

540 'JÉO

•J.cdois.

Ascraea E. fldansorianu.-

runphii cerflTTïîcli 1 i

TATK7n7i'L'IG;CGTlGGCGGríGTTC<.C'?G0a:CT.ATT.ACr.A7Gn'TÜrJÍ«»3A71GT.A¡iTrr.AAT.A7:'T>;

qijnyariJJ,

lucaya Gerithr^ïïTi

.A. . . . .AL'.ce.A A. . .C. ". .G. .A. .GC.T A. .A. .C. .A. .A A. . .G.-: . .A. . . . .A. . .G.G. .AC.i . .A A. .C. .A. .A A. .ACT. .A. .T. .AC.T. .A. . .lA ¡'. .A. .AC.A. .G. .A. .A A. .,AC. .C.T. .T. .AC.T. .A. . .G. r. .A. .AG.A. .f;. .A. .A A. .AC. .C.T.

.G. ..V:.T. .A. . .G. /. .T A A. .G. .G. .A. .A. .AG.A.

.T. . .C.T. .A. . .C.T A G. .C. .A. .A. ..AG.A.

.T. . .C.T. .A. . .C. i ....".. .A G. .G. .A. .A. .AG.A.

.T. .AG.TC.T. .ce. . . .T. .AC.G. .A. .T G. iG.T.

.G AG.

. . . .'.'). .G. .A. .

....G..G..A..

.C C

.G G

T. .G. .G. .G.

.G G.

.C C. . . . .AG. . . .G.

;-.'9

578

Figure 9. Aligned partial sequences of the moUuscan mitochondrial cytochronie c oxidase subunit I gene corresponding

to positions 32 to 611 of the COI sequence of Drosophila yakuba (Folmer et ah, 1994, Table 2). All sequences were

confirmed by at least t-wo sequencing reactions in each direction. Ambiguous base assignments are noted using lUPAC

symbols. Dots (•) represent nucleotides identical to those of Diodora, and quotation marks (") represent gaps inserted

during alignment.

14 M.G. Harasev\ych et al.

• F JUiflSûntBfiiK

P. 'ff.',l'mcnn

B

Figure 10. Phylogenetic relationships among Recent pleurotomariici species and genera. [A) The single most paisimonious tree resulting from an analysis (exhaustive search option) of 579 bp fi-om the mitochondrial cytochrome c oxidase 1 gene (L = 540; CI 0.669; RÍ = 0.581; RC = 0.388). Bootstrap proportions given as percentage above the node and jackknife proportions given as percentage below the node; (*) indicates that the node was not supported in a 50% majority rule bootstrap or jackknife consensus tree. (B) The single most parsimonious tree resulting from an analysis (exhaustive search option) of the "total molecular evidence" of 1154 characters (556 18S rDNA + 579 COI + 19 insert) (L = 712; CI = 0.729; RI = 0.658; RC = 0.479). Bootstrap proportions given as percentages above the nodes and jackknife proportions given as percentages below the nodes.

lengths for these taxa are two to three times as long as for other species in the tree, raising the possibiHty that they may be grouped on the basis of long branch attraction. Several authors (e.g., Termier and Ter- mier, 1968; Shileyko, 1977; Ponder and Lindberg, 1997; TiUier et al., 1992) have speculated that the patellogastropod limpets may not be gastropods, and suggested that the monophyly of the Gastro- poda requires further corroboration. However, cocculiniform Umpets, which together with the patellogastropod limpets comprise a monophyletic group in our study, have been regarded as unambiguous gastropods in all previous classifications.

The monophyly of the Neritopsina is strongly supported, perhaps in part because both representatives in our study are members of the same subfam- ily (Neritinae). The position of the Neritopsina within Gastropoda is less certain and strongly influ- enced by the outgroup. When Polyplacophora served as the outgroup, Neritopsina emerged basal to Vetigastropoda, as hypothesized by Haszprunar

(1988) and Hickman (1988), and supported by limited 28S RNA sequences (Rosenberg et al., 1994). However, when Polyplacophora were excluded from the data matrix and Nautilus was specified to be the outgroup, Neritopsina emerged between Vetigastropoda and Apogastropoda as advocated by Ponder and Lindberg (1996, 1997). Thus, 18S sequence data appear insufficient to resolve conclu- sively the relationships of Neritopsina.

The monophyly of the Pleurotomariidae is strongly supported by 18S sequence data as well as by the presence of seven insertions unique to the family. Contrary to the predictions of the morphology-based phylogenies, the Pleurotomariidae emerge as the sister group of the remaining vetigastropods, which also comprise a highly robust clade, rather than as a sister group to the family Trochidae. Remarkably, the monophyly of the Vetigastropoda, which is well supported by morphologic evidence (Salvini-Plawen and Haszprunar, 1987; Ponder and Lindberg, 1996, Figure 11.2), is but weakly supported by sequence data (bootstrap and jackknife proportions < 50% with both raw and reweighted characters), suggesting an ancient divergence between the two clades. Although Pleurotomarioidea date to the Cambrian, the remaining taxa appear later•Trochoidea first appear in the Ordovician, and earliest records of Haliotoidea and Fis- surelloidea are from the Triassic (Tracey et al., 1993).

Reanalyses of the 18S rDNA sequence data from a subset of taxa for which the sequences could be reliably aligned in their entirety (Figure 6) resulted in an identical division of Vetigastropoda into pleurotomariid and nonpleurotomariid clades, with each clade strongly supported by bootstrap and jackknife proportions of 100. However, monophyly of the Vetigastropoda remains weakly supported. The relationships of the pleurotomariid taxa to each other as well as to other vetigastropods, predicted by the 18S sequences, were confirmed by sequence from the mitochondrial gene for cytochrome c oxidase I (Figure lOA). The phylogeny based on this gene sequence confirms the monophyly of the Pleu- rotomariidae and supports the monophyly of the nonpleurotomariid vetigastropods, specifically re- futing a close relationship between Pleurotomarii- dae and Trochidae.

There have been differing opinions among mor- phologists about the relationships of vetigastropod taxa. McLean (1984] commented that the Tro- choidea "share so many features with the Pleuroto- mariidae that their derivation from the group is readily perceived." However, Hickman (1984)

Molecular phylogeny of pleurotomaríid gastropods 15

noted that the radula of Pleurotomariidae "had little in common with any living gastropod, including the [then included] two other pleurotomariodean families [Scissurellidae, Haliotidae] of more recent geological appearance." While McLean (1984) hypothesized that the Haliotidea are the "limpet-like derivatives" of the Pleurotomarioidea, Haszprunar [1985) noted that Haliotidae and Trochoidea are further linked by shared aberrant chemosensory structures.

In the light of the molecular evidence, the morphologic data and analyses of vetigastropod phylogeny should be reappraised with special attention to the precise composition of higher taxa and the resulting distribution of characters. The usage of Pleurotomarioidea has been inconsistent in molecular as well as morphologic studies, Tillier et al. (1994), for example, concluded that "Fisurelloidea is the sister group of Pleurotomarioidea and Tro- choidea" based on 28S rDNA sequences, because they used Haliotis as the exemplar of Pleurotomari- oidea. Thus, what appears to be a contradictory find- ing is, in fact, concordant with our resrdts.

Within Pleurotomariidae, the monophyly of the genus Entemnotrochus was well supported by 18S sequences, even without data contained in the insertions. The insertions were required to corrobo- rate the monophyly of Perotrochus, as well as its small-shelled (Perotrochus "Group A," Bayer, 1965) and large-shelled [Perotrochus "Group B," Bayer, 1965) clades. Information contained in the sequences of the seven inserts unique to Pleurotomari- idae was sufficient to distinguish Perotrochus and to resolve it into small-shelled and large-shelled groups, while length polymorphisms of these inserts were able to differentiate Entemnotrochus from the large-shelled Perotrochus (Figure 7). By contrast, the nonpleurotomariid vetigastropod families could not be resolved by 18S sequence data. Cittarium, which emerged anomalously at the base of the clade, could be returned to its predicted position as sister taxon to Astraea by increasing tree length by only one step.

Cytochrome c oxidase I sequence data strongly support the monophyly of the genus Perotrochus and of its constituent subclades Peroirocii us "Group A" and Perotrochus "Group B," and are capable of resolving closely related, parapatric species. Ivlanip- ulation of tree topology to make the genus Entemno- trochus monophyletic increases tree length by one step. The single most parsimonious tree resulting from the exhaustive search analysis of the "total molecular evidence" is extremely robust and en-

tirely concordant with the morphology-based hypotheses of Bayer (1965). The inclusion of both Japanese [P. teramachii) and Caribbean (P. midas) large-shelled species in a clade separate from the small-shelled species indicates that the divergence between Perotrochus "Group A" and "Group B" oc- curred before the closing of the Tethys during the Oligocène.

The clade comprising Apogastropoda, with each of the constituent groups (Caenogastropoda and Heterobranchia) broadly represented, is well supported by the 18S rDNA sequence. Monophyly of the Caenogastropoda is more robustly supported than that of the Heterobranchia. Although unsup- ported in the strict consensus tree, Heterobranchia emerges as a weakly supported clade in bootstrap and jackknife analyses (61% bootstrap and 61% jackknife proportions). The seven heterobranch taxa in this study contain 15 to 20 more nucleotides in the E-10-1 helix than do any of the caenogastropods, providing further evidence for their monophyly in the form of unique insertions. Resolution of phylogenetic relationships within Apogastropoda is the subject of ongoing research, and will be reported in a separate paper.

The gene coding for the small subunit ribosomal RNA (18S RNA) was the first to be used for sequence-based phylogenetic inference (Field et al., 1988), and has subsequently been applied primarily to resolve relationships of phyla and classes. WiÜiin Mollusca, this gene has been applied to studies of bivalve evolution (Rice et al., 1993; Kenchington et al. 1994), in which it has successñüly resolved family and in some cases genus-level taxa. The present study, which represents the most dense and widespread sampling of taxa within a single molluscan class published to date, reveals the 18S gene to be capable of broad but uneven levels of taxonomic resolution, in some instances being able to resolve genera, in others failing to resolve superfamilies and orders. An empirical observation is that higher taxa that contain insertions are more finely resolved than those taxa that do not, even when the insertions are not included in the analyses. It further appears that larger insertions produce finer resolution than smaller insertions, but this hypothesis requires con- firmation. A corollary of this conjecture is that the 18S gene should prove more useful for resolution of phylogenetic relationships among cephalopods, patellogastropod and cocculiniform limpets, and heterobranchs, all of which contain insertions, and less informative about relationships among Caeno- gastropoda and nonpleurotomarioidean vetigastro-

16 M.G. Harasewvch et al.

pods, which lack insertions. It is possible that Caenogastropoda and nonpleurotomarioidean vetigastropods may have insertions in other variable regions of the 18S gene that would increase the resolving abilitj' of those regions.

The distribution of variable and conserved regions in the 18S RNA molecule was described by De Rijk et al. (1992) and later refined by Winnepen- ninckx et al. (1992). Kenchington et al. (1994) identified variable regions on a finer scale that were useful for resolving relationships within the class Bivalvia and noted a region of potential utility for studies of Gastropoda. They emphasized the neces- sity of determining the entire gene sequence for phylogenetic studies in order to capture data from all variable regions throughout the gene. Recog- nizing the inevitable conundrum of molecular sys- tematists, Lecointre et al. (1993) assessed the comparative benefits of adding sequence length ver- sus those of adding taxa and concluded that the impact of species sampling on tree topology and robustness is greater than the impact of sequence length. These authors recommended that sequencing efforts be modified to sequence fewer nucleotides per species while increasing the number of representatives of presumed monophyletic taxa and to include several outgroups, an approach that we have followed in the initial phase of this study. A dense sampling of taxa also allowed us to recognize patterns in the distribution and lengths of insertions and deletions among related taxa and to interpret these variations in a phylogenetic context.

The resolution achieved by combining data sets from multiple genes demonstrates the utility of an iterative, multistep, multigene approach to phylogeny reconstruction. In the initial step, the phylogenetic relationships of the taxon of interest (Pleurotomariidae) were assessed in a broad context. The 18S rDNA gene proved to be sufficiently variable to resolve many of the relationships among higher taxa within the molluscan class Gastropoda, especially in those taxa that contain one or more large insertions. Both sequences and length polymorphisms within the insertions fmther improved resolution. After monophyly of the Pleurotomarii- dae had been established and appropriate sister taxa identified, finer-scale relationships on the order of genera and species were resolved with supplemen- tal sequence data from the more rapidly evolving mitochondrial gene. "Total molecular evidence," consisting of partial sequences of IBS rDNA and COI and including data on length variation within the inserts in the IBS rDNA gene, was sufficient to resolve fully the relationships of the genera and

species within Pleurotomariidae. A similar approach may be fruitful for resolving relationships within groups such as Caenogastropoda that have piovedrefractory to resolution by IBS data. Alterna- tively, the addition of data for one or more slowly evolving genes may enhance the resolution of deeper branches within Gastropoda, Mollusca, and Metazoa.

Experimental Procedures

Sample collection and preparation

All of the species sequenced were collected ex- pressly for this study, frozen while living, trans- ported to the laboratory on dry ice, and maintained at - 80°C until DNA was extracted. Specimens were collected by a variety of techniques including hand collecting, trapping, dredging, trawling, or use of the Harbor Branch Océanographie Institution's JOHNSON-SEA-LINK and CLELIA manned submersibles. A list of taxa, collection localities, tissue extracted, and voucher specimen information are given in Table 1.

DNA extraction

Specimens were thawed and maintained on ice while the buccal muscles were dissected out. Small shreds of muscle (totaling 1-2 mm=') were placed in a 1.5-ml Eppendorf tube containing 300 ¡xl of CTAB buffer at 60°C and ground using a disposable pestle (Kontes catalog no. 749520) in an electric drill. When the animal was minute, the shell was crushed, shell fragments were removed with for- ceps, and the entire remaining tissue mass was used for extraction. Individual animals were used for all extractions. The extraction protocol follows Adam- kewicz and Harasewych (1996), with the following modifications. The initial incubation at 60°C was extended to between one and two hours, and the tissue was ground again after 30 to 45 minutes. A second chloroform-isoamyl alcohol extraction was added. All centrifugation steps were extended to 10 minutes at 10,000 rpm. Absorbances at 260 and 280 nm were measured for all DNA preparations. When the ratio of absorbances at 260/280 nm was less than 1.5, the preparation was treated with proteinase K and reextracted.

Primer selection

Sequence data for taxa in Table 1 were obtained from a region of approximately 450 bp at the 5' end of the small subunit of ribosomal DNA (IBS rDNA) that contains the variable regions Vl and V2 (De

Molecular phylogeny of pleurotowaaid gastropods 17

Rijk et al., 1992], reported b^' Kenchington et al. (1994) to be useful in inferring molluscan phylogeny. The primers (forward, 5'-GCCAAGTAGCA- TATGCTTGTCTC-3', and reverse, 5'-AGACTTGCC- TCCAATGGATCC-3') were from Holland et al. (1991). Two additional, internal primers were de- signed by the authors for sequencing (forward, 5'- GGCGGCGACG(T/C)ATCTTTC-3', and reverse, 5'- GAAAGAT(A/G)CGTCGGCGGC-3']. Sequence data for pieurotomariid taxa and selected outgroups were also obtained from a 579-bp fragment of mitochondrial cytochrome c oxidase subunit I (COI) using the "universal" primers for COI of Folmer et al. (1994) (forward, 5'-GGTCAACAAATGATAAA- GATATTGG-3', and reverse, 5'-TAAACTTCAGGG- TGACCAAAAAATCA-3').

PCR amplification

PCR amplifications were performed using a Perkin- Elmer 480 thermal cycler. PCR reaction mixtures had a total volume of 50 |JL1, containing 200 to 500 ng of genomic DNA, 1.25 U of Amplitaq Taq polymerase (Perkin-Elmer), 200 |xm of each dNTP, 0.25 |JLM of each primer, 1.5 raM MgClz, and 5 ¡xl of a lOX Perkin-Elmer PCR reaction buffer. All PCR reactions were performed as "hot start" reactions with the following thermal cycler parameters: five minutes at 95°C, addition of Taq polymerase, 30 cycles of 45 seconds at 94°C for denaturing, two minutes at 50°G for annealing, and one minute at 72°C for extension. Following the 30 cycles, a five- minute final extension at 72°C was performed and the PCR reaction mixtures were then held at 4°C until use. For each PCR product 5 ¡xl was evaluated on a 1% (w/v) agarose gel. If a PCR reaction was imsuccessful, 1 ^x.\ of the initial reaction mixture was used as template for a second round of amplification. The thermal cycler profile for reamplifica- tions was the same as for the initial PCR with two exceptions: the number of cycles was decreased from 30 to 20 and the annealing temperature was raised from 50°C to 52°C. If a single band was ob- served, the reaction mixture was transferred to a Microcon-100 microconcentrator (Amicon, Inc.] for the removal of primers, unused dNTPs, and salts. An initial wash with 200 |xl of 40% (v/v) isopropa- nol was performed prior to a wash with 450 |j,l of sterile, distilled water. Samples were concentrated to a final volume of 10 to 20 jxl, and had a DNA concentration of 50 to 800 ng/[xl.

DNA sequencing and data management

Purified PCR products (100-200 ng DNA per reaction) were sequenced on Applied Biosystems 373A

automated DNA sequencers using fluorescence Dye Terminator Cycle Sequencing kits. Sequence data were edited and uploaded to a relational database called PHYLO. 18S rDNA target regions were sequenced and confirmed by at least two DNA sequencing reactions in one direction and a third reaction in the opposite direction. The COI target regions were sequenced and confirmed by at least two DNA sequencing reactions for each primer. Re- gions of ambiguity are noted using the lUPAC symbols. Sequence fragments for each gene were downloaded from PHYLO and assembled into a consensus sequence using modified TIGR Assem- bler software (Fleischmann et al., 1995). The consensus sequence was edited using a modified ABI Sequence Editor software. Sequences have been posted to the Genome Sequence Data Bank (URL http://www.ncgr.org), and accession numbers are included in Table 1.

Multiple sequence alignment

The confirmed consensus sequences from each or- ganism were dov\mloaded from PHYLO in multiple FASTA file format (Pearson and Lipman, 1988) and viewed with Genetic Data Environment (GDE) version 2.0 (Smith et al., 1994). The sequences were aligned using the multiple sequence alignment algorithm (MSA) of Sutton (Bult et al., 1997). For the protein coding gene, COI, the "protein assisted" feature of MSA was employed, which uses an opti- mal alignment of amino acid translations of the genes to guide the nucleotide alignment. Aligned sequences are available in nexus format via FTP at ftp.tigr.org/pub/data/gastropod_18s.aln (18S rDNA sequences) and ftp.tigr.org/pub/data/gastro- pod_col.aln (cytochrome c oxidase I), or from the corresponding author.

Phylogenetic analyses

Phylogenetic analyses using parsimony were con- ducted using PAUP 4.0.0d45 (Swofford, 1996; beta test version) on a Power Macintosh 6100/66. All characters were treated as unordered. For 18S rDNA sequences, autapomorphic insertions (present in a single taxon) and regions of uncertain homology, including insertions not present in all taxa (Figure 4B], were excluded from the data set prior to maximum parsimony analysis using the branch and bound search algorithm. In subsequent analyses, subsets of taxa with fewer regions of uncertain homology were analyzed using maximum parsimony and the exhaustive search option. Bootstrap and jackknife analyses (lOOO replicates) were performed using the "fast" stepwise addition option.

18 M.G. Harase\'\ych et al.

To compare 18S rDNA sequence-based gastropod phylogen)' with prevailing morphology-based phylogenetic hypotheses, subsets of the data matrix were created by pruning to contain taxa overlapping with the morphology-based trees under consider- ation (taxa appearing in upper case letters in Figure 2). When higher taxa were represented by multiple species in the molecular data set, a single representative containing the minimum number of deletions and ambiguous base determinations was selected. Following analysis (branch and bound option] of the reduced subset, the most parsimonious tree that most closely resembled the topology of the morphology-based tree to which it was being compared was imported into MacClade 3.01 (Maddison and Maddison, 1992), where branches were rearranged to correspond with their position in the morphology-based tree. Resulting changes in tree length and character indices were noted.

Phylogenetic signal contained in the seven insertions unique to Pleurotomariidae was assessed by analyzing only the information in these insertions. A phylogeny of the seven Recent Pleurotomariidae was calculated from these isolated insertion sequences. Individual insertions and deletions were then defined in terms of 19 segments (ranging from 1 to 22 nucleotides) that varied between the pleurotomariid taxa and scored as present or absent in each taxon regardless of sequence. A second analysis, based only on the presence or absence of these segments, was performed.

Cytochrome c oxidase I sequences of pleurotomariids and representative outgroups were analyzed using the maximum parsimony algorithm and the exhaustive search option. Bootstrap and jacknife analyses were performed as for 18S data. Prelimi- nary analyses based on amino acid sequence, as well as first and second codons only, yielded poor resolution and were not pursued further.

Finally, the 18S sequences, cytochrome c oxidase subunit I sequences, and the presence/absence matrix for the 19 inserts were combined into a single "total molecular evidence" data matrix and analyzed using the exhaustive search option.

Acknowledgments

We are grateful to the crews of the JOHNSON-SEA- LINK and CLELIA submersibles and their support vessels for assistance in collecting pleurotomariid and cocculiniform limpet samples. We thank the following colleagues for providing frozen samples of taxa used in our studies: Drs. Rafael Lemaitre and

Jon Norenburg, Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution; Dr. Peter Fankboner, Simon Fraser Uni- versity; Dr. Bruce Saunders, Bryn Mawr College; Dr. John B. Wise, Houston Museum of Natural History; Dr. Paula Mikkelsen, Delaware Museum of Natural History. Special thanks are due Mr. Yoshihiro Goto and Mr. Paul Callomon for providing frozen specimens of Japanese pleurotomariids as well as for making possible the senior author's field work in Japan. The assistance of Ms. Molly Ryan with the illustrations is gratefrdly acknowledged. We are especially gratefrd to Dr. David L. Swofford of the Laboratory of Molecular Systematics, Smithsonian Institution, for providing access to various beta test versions of PAUP 4.0. This is Smithsonian Marine Station at Link Port Contribution Number 421.

References

Abbott, R.T., and Dance, S.P. (1982). Compendium of Sea- shells. New York: E. P. Duttoa.

Adamkewicz, S.L.,and Harasewycli.M.G. (1996). Sj'stematics ajid biogeography of the genus Donax (Bivalvia: Donaci- dae) in eastern Nort)i America. Am Malacolog Bull 13(l/2):g7-103.

Anseeuvv, P., and Goto, Y. (1996). The Living Pleurotomarii- dae. Osal<a, Japan: Elle Scientific Publication.s.

Bayer, RM. (1965). New pleurotomariid gastropods from the western Atlantic, with a summary of the recent species. Bull Mar Sei 15(4):737-796.

Benfi'ika, E. (1984). Systématique, biogéographique et écolo- gie des Pleuvol;omiidés post-paleozoiques. Ph.D. disserta- tion. Université de Paris-Sud, Centre D'Orsay.

Bieter, R. (1992). Gastropod phylogeny and systematics. Annu Rev Ecol Systematics 23:311-3,38.

Boss, K.J. (1982). Mollusca. In: Synopsis and Classification of Living Organisms, vol. 1. Parker, S.P. (ed.). New York: McGraw-Hill Book Go, 945-1166.

Boss, K.)., and Jacobson, M.K. (1979). Soviet contributions to malacology in 1977. The Ke/jger 21(4):490-500.

Bouvier, E.L, (1887). Système nerveux, morphologie générale et classification des Gastéropodes Prosobranches. Ann Sei Nat Zoo¡ 7(3):1-510, table 1, pis. 1-19.

Brusca, R.G., and Brusca. G.). (1990). Invertebrates. Sunder- land, Mass: Sinauer Associates.

Bult, C.J., Adams, M., White, O., Sutton, G., Glayton, R., Kerla- vage, A,, Fields, C., and Venter, J.C. (1997). The impact of rapid gene discovery technology on studies of evolution and biodiversity. In: Biodiversity IL Understanding and Protecting our Natural Resources. Wilson, E.O., Wilson, D., and Realka-Kudia, M. (eds.) Washington, DC: Joseph Henry Press, 289-299.

Cox, L.R. (1960). The British cretaceous Pleurotomariidae. Bull Br ¡Vluseum (Nat Hist) 4(8):388-423, pis. 44-60.

Dall, W.H. (1889). Reports on the results of dredging, under the supervision of Alexander Agassiz, in the Gulf of Mex- ico (1877-78) and in the Caribbean Sea (1879-80), by the U.S. Coast Survey Steamer "Blake," Lieut.-Commander CD. Sigsbee, U.S.N., and Commander J.R. Barllett. U.S.N.,

Molecular phylogeny of pleurotomariid gastropods 19

commanding. 29. Report on the MoDusca, Part 2. Gastro- poda and Scaphopoda. Bull Museum Comp Zöol Harvard 18:1-492, pis. 10-40.

De Rijk, P., Neefs, J-M., Van der Peer, Y., and De Wächter, R. (1992). Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 20 (suppl):2075-2089.

Emberton, K.C, Kuncio, G.S., Davis, G.M., Phillips, S.M., Monderevvicz, K.M., and Guo, Y.H. (1990). Comparison of recent classifications of stylommatophoran land-snail families, and evaluation of large-ribosomal-RNA sequencing for their phylogenies. Malacologia 31:327-352.

Endo, H. (1995). Slit Shells: Pleurolomariidae. ivianazuru, Japan: Endo Museum.

Erwin, D.H., and Signor, P.VV. (1991). Extinction in aji extinction-resjstajitclade: the evolutionary history of the Gastro- poda. In: The Unity of Evolutionary Biology. Proceedings of the Forth International Congress of Evolutionary and Syslematic Biology, l Dudley, E.G. (ed.). Portland, Ore: Dioscorides Press, 152-160.

Field. K.G., Olsen, G.|., Lane, D.J., Giovanonni, S.]., Ghiselin, M.T., Raff. E.G., Pace, N.R., and Raff, R.A. (1988). Molecu- lar phylogeny of the animal kingdom. Science 239: 748-753.

Fleischmann, R.D., Adams, M.D., White, O., Clayton, R., Kirk- ness, E.F, Kerlavage, A.R., Bult, G.J. et al. 1995. Whole- genome random sequencing and assembly of Haemophi- lus influenzae Rd. Science 269:496-512.

Folmer, O., Black, M., Hoeh, W., Lutz, R., and Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Mol Mar Bid Biotechnol 3(5):294-299.

Ghiselin, M.T. (1988). The origin of molluscs in the light of molecular evidence. In: Harvey, P.H., and Partridge, L. (eds.) Oxford Surveys in Evolutionary Biology, vol 5. New York: Oxford University Press, 66-95.

Golikov, A., and Starobogatov, Y.I. (1975). Systematics of pro- sobranch gastropods. Malacologia 15(l):185-232.

Graham, A. (1985). Evolution within Gastropoda: Prosobran- chia. In: The MoUusca, vol 10, Evolution. Trueman, E.R., and Clarke, M.R. (eds.) Orlando, Fla: Academic Press, 157-186.

Harasewych, M.G.. Pomponi, S.A., and Askew, T.M. (1988). Spongivory in pleurotomariid gastropods. Nautilus 102(3):92-98.

Haszprunar, G. (1985). The fine morphology of the osphradial sense organs of the MoUusca, I: Gastropoda•Proso- branchia. Philos Trans R Soc Land B 307:457-496, 1-V.

Haszprunar, G. (1988). On the origin and evolution of major gastropod groups, with special reference to the Strepto- neuva. / Mollusc Stud 54:367-441.

Hickman, C.S. (1976). Pleurotomaria (Archaeogastropoda) in the Eocene of the northeastern Pacific: a review of cenozoic biogeography and the ecology of the genus. / Paleontol 50(6):1090-1102.

Hickntan, C.S. (1984). Pleurotomaria: pedigreed persever- ajice? In: Living Fossils. Eldridge, N., and Stanley. S.M. (eds.). New York: Springer Verlag, 225-231.

Hickman, C.S. (1988). Archaeogastropod evolution, phylogeny and systematics: a réévaluation. Malacolog HevSuppl 4:17-34.

Holland, P.W., Hacker, A.M., and Williams, N.A. (1991). A molecular analysis of the phylogenetic affinities of Sacco- glossus cambrensisBramhell &Cole (Hemicordata). Philos Trans R Soc Load Biol 332:185-189.

Hyman, L.H. (1967). The Invertebrates, vol 6, MoUusca 1. New York: McGraw-Hill Book Co.

Kenchington, E.L., Roddick, D.L., Singh, R.K., and Bird, Cf. (1994). Analysis of small-subunit rRNA gene sequences from six families of molluscs. / Mar Biotechnol 1:215- 217.

Knight, J.B., Cox, L.R., Keen, A.M., Batten, R.L., Yochelson, E.L., and Robertson, R. (1960). Molhisca-Gastropoda, sys- tematic descriptions. In: Treatise on Invertebrate Paleon- tology, part I, MoUusca 1. Moore, R.C. (ed.). Lav\Tence, KS: University of Kansas Press and the Geological Society of America, 169-331.

Lecointre, G., Philippe, H., Le, H.L.V., and Le Guyard, H. (1993). Species sampling has a major impact on phylogenetic inference. Mol Phylogenet Evol 2(3):205-224.

Maddison, W.R, and Maddison, D.R. (1992). MacClade. Anal- ysis of Phylogeny and Character Evolution, version 3. Sun- derland. Mass: Sinauer Associates.

jVlcLean, J.H. (1984). A case for the derivation of the Fissurelli- dae from the Bellerophontacea. Malacologia 25(l):3-20.

McLean, J.H. (1990). Neolepetopsidae, a new docoglossate limpet family from the hydrot)iermal vents and its rele- vance to patellogastropod evolution. / Zool Land 222:485- 528.

Morton, J.L., and Yonge, CM. (1964). Classification and structure of the moJIusca. In: Physiology of the MoUusca, vol 1. Wilbur, K.M., and Yonge, CM. (eds.). New York: Academic Press, 1-58.

Pearson, W.R., and Lipman, D.J, (1988). Improved tools for biological sequence comparison. Proc Nat Acad Sei USA 85:2444-2448.

Philippe, H., Chenuil, A., and Adoutte, A. (1994). Can the Cambrian explosion be inferred through molecidar phylogeny? Development 1994 suppi: 15-25.

Ponder, W.F, and Lindberg, D.R, (1996). Gastropod phylogeny•challenges for the 90s. In: Origin and Evolutionary Radiation of the MoUusca. Taylor, f.D. (ed.). Oxford, UK: Oxford, University Press, 135-154.

Ponder, W.F,, and Lindberg, D.R. (1997). Towards a phylogeny of gastropod molluscs•an an analysis using morphologi- cal characters. Zool ] Linnean Soc (in press).

Rice, E.L., Roddick, D., and Singh, R. (1993). A comparison of molluscan (Bivalvia) phylogenies based on palaeonto- logical and molecular data. Mot Mar Biol Biotechnol 2:137-146.

Rosenberg, C, Kuncio. G.S., Davis, CM., and Harasewych, M.G. (1994). Preliminary ribosomal RNA phylogeny of gastropod and unionoidean bivalve mollusks. In: Molecu- lar Techniques and Molluscan Phylogeny. Harasewych, M.G., and Tillier, S. (eds.) NautUus SuppJ 2:111-121.

Rosenberg, C, Tillier, S. Tillier, A., Kuncio, G.S., Hanlon, R.T., Masselot, M., and Williams, C.J. (1997). Ribosomal RNA phylogeny of selected major clades in the MoUusca. / Mollusc Stud (in press).

Salvini-Piawen, L. von (1980). A reconsideration of systematics in the MoUusca (phylogeny and higher classification). Malacologia 19:249-278.

Salvini-Plawen, L. von (1985). Early evolution and lire primitive groups. In: The MoUusca, vol 10, Evolution. Trueman, E.R. and Clarke, M.R. (eds.). Orlando, Fla: Academic Press, 59-151.

Salvini-Plawen, L. von, and Haszprunar, G. (1987). The Veti- gasti'opoda and the S)'stematics of streptoneurous Gastro- poda (MoUusca). ¡Zool Land 211:747-770.

20 A'/.G. Harasenych et al.

Shileyko, A.A. (1977). The symmetry of the Docoglossa and the problem of the origin of the order. Bull Moscow Nat Socß/o/Se;-81 (3):60-65 (English summary). See Boss and lacobson, 1977:494, for EngUsh summary.

Smith, S., Overbeelv, VV.R., Woese, C.R., Gilbert, W., and Gib level, P.ÎVI. (1994). The genetic data environment: an ex- pandable GUI for multiple sequence analysis. Comp AppI Biosci 10:671-675.

Swofford, D.L, (1996). PAUP, Phylogenetic Analysis Using Parsimony, version 4.0.0d42 (beta test version). Washing- ton, DC. Laboratory of Molecular Systeixiatics, Smithson- ian Institution.

Szabó, ). (1980). Lower and Middle Jurassic Gastropods from the Bakony Mountains (Hungary), Part II: Pleurotomaria- cea and Fissurellacea (Archeogastropoda). Annal Histor- ico-Naturales Musei Nat Hungarici 72:49-71.

Taylor, D.W., and Sohl, N.F. (1962). An outline of Gastropod classification. Malacologia 1(1):7~32.

Termier, H., and Termier. G. (1968). Evolution et Biocinèse. Les ¡nvértebvés dans l'histoire du Monde Vivant. Paris: Masson & Cie.

Thiele, ¡. (1929). Handbuch der systematischen Weichtier- kundo. Jena: Gustav Fischer, l(l):l-376.

Tillier, S., Masselot, M., Hevré, P, andTillier, A. (1992). Phylo- génie moléculaire des Gastropoda (MoUusca) fondée sur le séquençage partiel de l'ARN ribosomique 28 S. Comptes Rendus Acad Sel (Paris) Series 3, 134:79-85.

Tillier, S., Masselot, M., Guerdoux, J., and Tillier, A. (1994). Monophyly of major gastropod taxa tested ft'om partial 28S rRNA sequences, with emphasis on Euthyneura and Hot-Vent Limpets Peltospiroidea. In: Molecular Tech-

niques and Molluscan Phylogeny. Harasewych, M.G., and Tillier, S. (eds.). Nautilus SuppI 2:122-140.

Tillier, S., Masselot, M., and Tillier, A. (1996). Phylogenetic relationships of tlie pulmonate gastropods from rRNA sequences, and tempo and age of the stylommatophoran radiation. In: Origin and Evolutionary Radiation of the Mollusca. Taylor, J.D. (ed.). Oxford, UK: Oxford University Press, 267-284.

Tracey, S.,Todd,J.A,,and Erwin, D.H. (1993). Mollusca: Gas- tropoda. In: The Fossil Record 2. Benton, M.J. (ed.). Lon- don: Chapman and Hall, 131-167.

Vaught, K.C. (1989). A Classification of tlie Living Mollusca. Melbourne, Fla. American Malacologists, Inc.

Winnepenninckx, B., Backeljau, T., Van de Peer, Y., and De Wächter, R. (1992). Structure of the small ribosomal subunit RNA of the pulmonate snail Limnicolaria kambeul, and phylogenetic analysis of the Metazoa. FEES Lett 309:123-126.

Winnepenninckx, B., Backeljau, T, and De Wächter, R. (1993). Complete small ribosomal subunit RNA sequence of the chiton Acanthopleurajapónica (Lischke, 1873) (Mollusca, Polyplacophora). Nucleic Acids Res 21:1670.

Winnepenninckx, B.,T. Backeljau, and R. De Wächter. (1994). Small ribosomal subunit RNA and the phylogeny of Mol- lusca. In: Harasewych, M.G., and Tillier, S. (eds.) Molecu- lar Techniques and Molluscan Phylogeny. Nautilus suppl 2:98-110.

Yonge, CM. (1947). The Palliai Organs in the Aspidobranch Gastropoda and their evolution throughout the Mollusca. Philos Trans R Soc Lond B 232(591):443-518, pi. 18.

Gastropoda): an assessment based on partial 18S rDNA and cyt

Documents

Transcript of Gastropoda): an assessment based on partial 18S rDNA and cyt