Journal of The Malacological Society of London

Molluscan StudiesJournal o f Molluscan Studies (2013) 79: 1 9 -2 9 . doi:10.1093/m ollus/eys028 A dvance Access p u b lica tio n date : 29 N ovem ber 2012

A M O L E C U L A R P H Y L O G E N E T IC F R A M E W O R K F O R T H E E R G A L A T A X IN A E (N E O G A S T R O P O D A : M U R IC ID A E )

M A R T IN E C L A R E M O N T 12, ROLAND H O U A R T 3, SUZANNE T. W IL L IA M S 1 AND DAVID G. R E I D 1

d epartm en t o f foology, .Natural History M useum, London S [ I 7 5 B D , U K ;^Division o f Biology. Imperial College London, Si.lwood Park Campus, Ascot SL 5 7PT, U K ; and

fn s titu t Royal des Sciences .Naturelles de Belgique, Rue Vautier, Brussels, Belgium

Correspondence: AI. Claremont; e-mail: m .elaremontidnhm.ae.uk

(R eceived 2 A p ril 2012; accep ted 28 S e p te m b e r 2012)

ABSTRACTT h e va lid ity o f the m uric id subfam ily E rg a la tax in ae has recen tly been confirm ed w ith m olecu lar d a ta , b u t its com position an d th e re la tionsh ips am o n g its constituen t gen era rem ain unclear. In o rder to investigate this, we use four genes (28S rR N A , 12S rR N A , 16S rR N A a n d cy tochrom e c oxidase su b u n it I ) to construct a B ayesian phy logeny o f 52 e rg ala tax in e species in 18 genera , rep resen ting c.40% o f the cu rren tly accep ted species a n d 86% o f th e genera . T h is is the m ost com plete phy logeny of this taxonom ica lly confusing subfam ily yet p ro d u ced . O u r results in d ica te the po lyphy ly o f m any trad itio n a l genera, in c lu d in g Morula, Pascula an d Orania. In o rd e r to im prove the correspondence betw een classification an d phylogeny, we restrict the defin ition o f Morula, resurrect Tenguella and elevate Oppomorus to full genus, b u t describe no new genera . Several species in this analysis could not be identified a n d m ay be new, b u t we do no t describe them . F u rth e r m olecu lar a n d m orpholog ical analyses, in the con tex t o f this fram ew ork, should help to resolve th e rem ain in g am bigu ities in the classification o f this subfam ily. T h e oldest fossil m em b er o f the E rg a la tax in ae know n to us is o f E arly O ligocene age.

I N T R O D U C T I O NT h e M u ric id ae a re a large, taxonom ica lly com plex fam ily o f neogastropods. M an y species in this fam ily are well know n for th e ir charism atic shells (Murex, Chicoreus), as p red ato rs ( Ocenebra, Urosalpinx) o f com m ercially im p o rta n t oysters, as an an c ien t source o f p u rp le dye (Bolinus, Plicopurpura) o r as food item s (Concholepas, M urex). H ow ever, u n til recently , the p h y l­ogeny o f the fam ily was poorly know n; subfam ilies have been erected based large ly on superficial shell an d ra d u la r resem ­blances ra th e r th a n on rigorous phylogenetic analyses. O n e such subfam ily is the E rg a la tax in ae , the diagnosis o f w hich was based on som ew hat vague characters o f the shell, opercu lum , ra d u la an d egg capsule (K u ro d a & H ab e , 1971). I t has been criticised as “ the least w ell-defined [subfam ily] in th e en tire fam ily M u ric id a e" (Vokes, 1996a, 27). E ver since its in tro d u c ­tion, the tax o n o m y o f the g ro u p a n d its re la tionsh ip to a n o th e r m uric id subfam ily, the R a p a n in a e (= T h a id in a e o f au th o rs), has been controversial. Som e w orkers co n ten d ed th a t there was little m orpho log ical su p p o rt for the sep ara tio n o f the R a p a n in a e an d E rg a la tax in ae (T an , 1995, 2000). O th ers described new e rg ala tax in e gen era a n d transferred existing gen era to the subfam ily (e.g. H o u a rt, 1995a, b, 1997; Vokes, 1996a, b). R ecen t m olecu lar phylogenetic analyses have tested

these opposing views, establish ing the m onophy ly o f the E rg a la tax in ae an d su p p o rtin g its recognition as a subfam ily (C larem ont, R e id & W illiam s, 2008; B arco et al., 2010; C larem o n t et ed., in press).

Like the rest o f the M u ric id ae , ergala tax ines are carnivorous, p rey ing u p o n a w ide v a rie ty o f organism s in c lud ing corals, po lychaetes, barnacles an d o th er m olluscs (e.g. T ay lo r, 1980; T an , 1995; Ish ida , 2004; C larem ont, R e id & W illiam s, 2011a). E rg ala tax in es are m ain ly found in the tro p ical Indo-W est Pacific a n d occupy a w ide range o f h ab ita ts , w ith some species com m on in the h igh in te rtid a l, w hile o thers live m ore th a n 500 m deep (K u ro d a & H abe, 1971; H o u a rt, 1995b; T an , 1995). T h e subfam ily was in itially established for only three gen era (Ergalatax, Bedevina a n d Cytharomorula), b u t m an y t r a d ­itiona lly ‘th a id id ’ gen era have been transferred to it subse­q u en tly (e.g. Morula, Spinidrupa, Cronia, Drupella; T rö n d lé & H o u a rt, 1992; K ool, 1993; Vokes, 1996a; T an , 2000; V erm eij & C arlson, 2000; H o u a rt, 2004; C larem o n t et al., 2011a). New erga la tax in e gen era have also been described, based on c h arac ­ters o f th e shell a n d ra d u la (e.g. Habromorula', H o u a rt, 1995a).

C harac ters o f the shell, in te rn a l an a to m y a n d ra d u la have also been used to suggest affinities betw een various gen era (e.g. Fujioka, 1985; H o u a rt, 1995a, b, 2004), b u t cases o f p lasticity ,

iC) 77íe Author 2012. Published by Oxford Uniiersity Press on behalf o f The Malacological Society o f London, all rights reseroed

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p o lym orph ism a n d of p a ralle l a n d convergen t evo lu tion w ith in the E rg a la tax in ae have m ade phylogenetic reconstruc tion based on m orpho logy difficult. For exam ple, ra d u la r m o rp h ­ology has been show n to v a ry w ith in species acco rd ing to sex, g row th ra te a n d season (Fujioka, 1985; K ool, 1993; T an , 1995). Such is the p revalence o f hom oplasy in m orpholog ical c h a rac te r transfo rm ations th a t early cladistic analyses d id not even find su p p o rt for the m o nophy ly of the E rg a la tax in ae (K ool, 1993; V erm eij & C arlson, 2000). A lth o u g h m olecu lar studies have confirm ed its m o nophy ly (see above), so far they have suffered from lim ited taxonom ic sam pling . A co m p reh en ­sive m olecu lar analysis o f this im p o rta n t subfam ily rem ains to be done.

Previous m olecu lar phylogenetic analyses o f the M u ric id ae have in cluded only a few representatives o f th e E rg a la tax in ae (6 species in C larem o n t et id., 2008; 10 species in B arco et ed., 2010; 6 species in C larem o n t et ed., in press). Elere, we aim to prov ide a m ore com prehensive phy logenetic fram ew ork o f the E rg a la tax in ae for fu rth e r taxonom ic, ecological a n d phylogeo- g rap h ic w ork; to test m o nophy ly o f nom inal gen era cu rren tly defined by m orpho log ical characters a n d to p rov ide a new phylogenetic classification. T o do this, we construct a m olecu­la r phy logeny o f 52 erga la tax in e species using one n u c lear and th ree m ito ch o n d ria l genes.

M A T E R IA L AND M E T H O D S

T a x on sam pling and identification

A to ta l o f 109 erga la tax in e specim ens were o b ta in ed for m o­lecu lar analysis (S u p p lem en tary M ateria l, T ab le S I) . T hese w ere iden tified m orpholog ically by the au th o rs (R .H ., D .G .R . an d M .C .) as belonging to 52 species in 18 genera, rep resen t­ing 40% of the c. 129 accep ted species in the subfam ily and all b u t th ree o f the 21 cu rren tly accep ted gen era (H o u a rt, 1986, 1987, 1991, 1995a, b, 1996a, b, 1997, 1998, 2000, 2002, 2004, 2008; T rö n d lé & H o u a rt, 1992; T an , 1995; Vokes, 1996b; V erm eij & C arlson, 2000; T ab le 1). W e have sequenced the type species o f 15 gen era (H o u a rt, 1995b; V erm eij & C arlson, 2000; T ab les 1, S u p p lem en tary M a te ria l T ab le S I). Based on m orphology , n ine ad d itio n a l gen era have been in cluded in the E rg a la tax in ae by some au th o rs (T rönd lé & H o u a rt, 1992; H o u a rt, 1995b, 2004; Vokes, 1996b). O f these n ine genera , one is u n availab le ; one is a possible synonym of a n o th e r e rga la tax in e genus; th ree have been rem oved from the subfam ily on th e basis o f th e ir m orpho logy (Ponder, 1972; R a d w in & D ’A ttilio , 1976; R . H o u a rt, 2010, u n p u b h ); and one has been rem oved based on m olecu lar analyses (Barco et al., 2010; see S u p p lem en tary M a te ria l, T ab le S2). T h e th ree e rg ala tax in e gen era no t in cluded in ou r analysis (Daphnellopsis, Lindapterys, Uttleya) are poorly know n an d lack clear m o rp h o ­logical sim ilarities to o th er E rg a la tax in ae , so have been p laced in th e E rg a la tax in ae only ten ta tively (R . H o u a rt, u n p u b h ; S u p p lem en tary M a te ria l, T ab le S2). R ep resen tative species o f these gen era should be in cluded in fu tu re m olecu lar analyses.

W e used 66 previously p u b lished e rg ala tax in e sequences (S u p p lem en tary M ateria l, T ab le S I; C larem o n t et ed., 2008, 2011a; B arco et ed., 2010). O u tg ro u p species w ere selected front the R a p an in ae , as this is the sister subfam ily to the E rg a la tax in ae in som e analyses (C larem ont et ed., 2008; b u t see B arco et ed., 2010). All ra p an in e ou tg ro u p sequences have p re ­viously been p ub lished (C larem ont et ed., 2008; B arco et ed., 2010). N ew sequences were su b m itted to G enB ank (S u p p lem en tary M a te ria l, T ab le SI ). L oca tion of v oucher m a ­te ria l is no ted in S u p p lem en tary M ateria l, T ab le S I. G eneric assignm ents in the follow ing text a n d figures a re valid , based on the conclusions o f this study (sum m arized in T ab le 1).

W here generic assignm ents rem ain u n c lear (because o f poly- phy ly a n d /o r lack of type species in the analysis), we have fol­lowed those o f H o u a r t (e.g. H o u a rt, 1986, 1987, 1991, 1995a, b, 1996a, b, 1997, 1998, 2000, 2002, 2004, 2008), except for 'T ha is’ castanea (w here we have followed Steyn & Lussi, 1998). Single q u o ta tio n m arks have been used to in d ica te these cases o f uncerta in ty .

D .N A sequencing and alignm ent

F or all sam ples, th ree m ito ch o n d ria l genes [cytochrom e c oxidase su b u n it I (C O I), 16S rR N A a n d 12S rR N A ] an d one n u c lear gene (28S rR N A ), know n to be in form ative for p h y lo ­genetic analysis in the M u ric id ae (C larem o n t et al., 2008; Barco et al., 2010), were sequenced, following th e protocols o f C larem o n t et cd. (2011b). Polym erase ch a in reactions (PC R s) am plified c. 1500 bp o f 28S, 750 bp o f 16S, 700 bp o f C O I and 650 bp of 12S. P rim ers a n d P C R conditions for all genes were as described by B arco et ed. (2010), except for some forw ard fragm ents o f 16S th a t were o b ta in ed using a new in te rn a l p rim er, 16S-In t56F (5 '-A A C R G C C G C G G T A C T C T G -3 ') an d some C O I sequences th a t were o b ta in ed using p rim ers C O IF an d C O I-M LTR (C larem ont et ed., 2011b). W e were u n ab le to am plify all genes for all specim ens. T h u s, the 12S a lig n m en t consisted o f 115 sequences, the 16S a lig n m en t o f 106 sequences, the 28S a lig n m en t o f 95 sequences an d the C O I a lig n m en t o f 78 sequences (S u p p lem en tary M ateria l, T ab le SI ).

Sequences were assem bled an d ed ited w ith Sequencher (v. 4.8; G eneC odes C o rpora tion , A n n A rbor, M I) . C lear h e t­erozygous peaks in b o th th e fo rw ard an d reverse sequence of 28S w ere coded as polym orphism s. A fter rem oval o f p rim er regions, 28S sequences were 1497 bp an d 12S sequences were 573 bp . C O I sequences o b ta in ed w ith C O IF /C O I-M L T R were 703 bp ; those sequences o b ta in ed using un iversal p rim ers (Folm er et ed., 1994) were 658 bp . Sequences o f 16S ob ta in ed w ith C G L eu R (H ayashi, 2005) a n d 16S-In t56F (this pap er) were 705 bp, w hile those o b ta in ed w ith C G L eu R (H ayashi, 2005) a n d 16SA (P alum bi et ed., 1991) were 825 bp.

R ib o so m al (28S, 16S an d 12S ) sequences were a ligned using the Q T N S -i m eth o d of M A F F T (M ultip le A lignm en t using Fast F o u rie r T ransfo rm ; v. 6.847b; K a to h & T oli, 2008). Based on p re lim in a ry analyses, we d id no t expect long gaps in ou r a lignm ents, so the offset v a lue was set to 0.1. T h e resulting a lignm ents were ad justed by eye in M acC lad e (v. 4.06 O S X ; M ad d iso n & M ad d iso n , 2003). Gblocks (v. 0.91 beta; C astresana, 2000) was th en used to rem ove poorly a ligned sites (m in im um n u m b er o f sequences for a conserved position: 70% ; m in im u m n u m b er o f sequences for a flank ing pos­ition: 9 0% ; m ax im u m n u m b er o f contiguous non-conserved positions: 3; m in im u m len g th o f a block: 5; all gap positions allow ed). E lim in a tio n o f am biguous regions reduced the 28S a lig n m en t by 5% to 1432 bp , the 12S a lig n m en t by 10% to 518 bp an d the 16S a lig n m en t by 14% to 620 bp. C O I sequences were a ligned by eye in M acC lad e. T h ere were 91 p hylogenetically in form ative base pa irs in 28S, 232 b p in 12S, 173 bp in 16S an d 272 bp in C O I.

F or each gene p a rtitio n , 24 different m odels o f nucleo tide su b stitu tio n were tested w ith M rM o d elT est (v. 2.2; N y lan d er, 2004). A dditionally , we tested a site-specific m odel o f evolution o f C O I using PALTP* (v. 4 .0 b l0 ; Swofford, 2002). T h e m odel chosen by b o th th e h iera rch ical ra tio test an d A kaike’s In fo rm atio n C rite rio n in M rM o d elT est was G T R + I + G for 28S, 16S an d 12S, a n d H K Y + I + G for C O I. C om parison of log-likelihood values in d ica ted th a t H K Y + I + G was a significantly b e tte r fit to the d a ta th a n a m odel th a t allowed site-specific ra te v a ria tio n across codon p a rtitions .

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M O L E C U L A R P H Y L O G E N Y O F E R G A L A T A X IN A E

T ab le 1. S ta tu s o f g en e ra o f E rg a la ta x in a e in c lu d ed in th is study.

Genus Type species First placed in

Ergalataxinae by

Type species

analysed herein

Validity

Bedevina Habe, 1946 Trophon birileffi Lishke, 1871 Kuroda & Habe, 1971 Yes Valid; could include Spinidrupa

Cronia H. & A. Adams, Purpura amygdala Kiener, 1835 Tröndlé & Houart, 1992 Yes Valid; could include Maculotriton and

1853 Ergalatax

Cytharomorula Cytharomorula vexillum Kuroda, Kuroda & Habe, 1971 Yes Uncertain; not monophyletic in any analysis

Kuroda, 1953 1953

Drupella Thiele, 1925 Purpura elata Blainville, 1832

[=Drupa cornus Röding,

1798]

Claremont e ta /., 2011a Yes Valid

Ergalatax I redale, Ergalatax recurrens Iredale, Kuroda & Habe, 1971 No (although included Uncertain; not monophyletic in any

1931 1931 [ = Murex pauper E. contracta is analysis. In clade with Cronia and

Watson, 1883] possible synonym;

Houart, 2008)

Maculotriton; potential synonym of Cronia

Habromorula Houart, Purpura biconica Blainville, 1832 Houart, 2004 Yes Not a valid genus; within Morula clade;

1995 monophyly of Habromorula not strongly

contradicted, so potentially valid

subgenus

Lataxiena Lataxiena lataxiena Houart, 1995b Yes Valid; composition unclear due to

Jousseaume, 1883 Jousseaume, 1883

[ = Trophon fimbriata Hinds,

1844]

uncertainty surrounding Orania

Maculotriton Dali, 1904 Triton bracteata Hinds, 1844

[= Buccinum serriale Deshayes, 1834]

Tröndlé & Houart, 1992 Yes Valid; potential synonym of Cronia

Morula Schumacher, Morula papillosa Schumacher, Houart, 2004 Yes Valid; definition restricted (cf. Houart, 2002,

1817 1917 [=Drupa uva Röding,

1798]

2004)

Muricodrupa Iredale, Purpura fenestrata Blainville, Tröndlé & Houart, 1992 Yes Valid; definition restricted (M. fiscella

1918 1832 excluded)

Oppomorus Iredale, Morula nodulifera Menke, 1829 Houart, 2004 Yes Valid genus (subgenus of Houart, 2004)

1937

Orania Pallary, 1900 Pseudomurex spadae Libassi,

1859 [=Murex fusulus Brocchi, 1814]

Houart, 1995b No Uncertain; type species not included;

species previously assigned to Orania form clades with members of Lataxiena,

Usilla, Cytharomorula and Pascula.

Pascula Dali, 1908 Trophon citricus Dali, 1908 Tröndlé & Houart, 1992 No Uncertain; type species not included;

paraphyletic as currently defined

Phrygiomurex Dali, Triton sculptilis Reeve, 1844 Tröndlé & Houart, 1992 Yes Valid

1904

Spinidrupa Habe & Murex euracantha A. Adams, Houart, 1995a Yes Uncertain; some species in clade with

Kosuge, 1966 1851 Bedevina, which has priority

Tenguella Arakawa, Purpura granulata Duelos, 1832 This study Yes Valid; synonymized with Morula by Fujioka

1965 (1985), but shown here to be distinct

Trachypollia Woodring, Trachypollia sclera Woodring, Vokes, 1996a No Probably valid, although sampling limited

1928 1928 and type species not included

Usilla H. Adams, 1861 Vexilla nigrofusca Pease, 1860

[= Vexilla fusconigra Pease,

1860 = Purpura avenacea

Lesson, 1842]

Vokes, 1996b Yes Valid, but type in clade with type of

Lataxiena and some Orania-, retained as

genus because morphologically distinct

(following Vokes, 1996b; Houart &

Tröndlé, 2008)

See Supplementary material, Table S2 fo r status of unsampled, excluded and doubtful genera. Valid genera are those confirmed as monophyletic, with type species included. Potential synonymy and other uncertainties should be resolved by further sampling and analysis.

Phylogenetic analysis ( single gene)

All single-gene a lignm ents were analysed using B ayesian in fer­ence a n d the M arkov C h a in M o n te -C arlo M e th o d (M C M C ; M rB ayes v. 3.1; H uelsenbeck & R onq u ist, 2001). M odel

p a ram ete rs for each gene were set acco rd ing to the m odel selected by M r M odel Test a n d were free to v a ry am o n g gene pa rtitio n s . T h ere were tw o in d ep en d en t runs o f the M C M C analysis, each using four chains. C onvergence betw een the in ­d ep en d en t M C M C runs was tested by exam in ing the average

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M. C L A R E M O N T E T A L .

deviations o f split frequencies a n d the p o ten tia l scale-reduction facto r (P S R F ), as well as th e traces in T rac e r (v. 1.5; D ru m m o n d & R a m b a u t, 2007). T h e M C M C analysis was ru n for five m illion generations u n til the M C M C runs converged. In all cases, we used a sam ple frequency o f 1,000 a n d a b u rn -in o f 1,501; o th er p a ram ete rs w ere d e fau lt values. C lades w ith posterio r p ro b ab ility (PP) > 9 5 % were considered well supported , w hile P P of 9 0 -9 5 % was tak en as m arg in a l support. B ranches in the consensus tree su p p o rted by P P < 5 0 % were collapsed. All P S R F values for M rB ayes analyses w ere 1.00, w hile average dev ia tions o f split frequencies converged on zero, in d ica tin g th a t all trees h a d reached sta tionarity .

Construction and analysis o f concatenated alignments

In spection o f in d iv id u a l gene trees d id no t reveal an y well- su p p o rted clades (PP > 95% ) in conflict. C onflict am ong strongly su p p o rted clades (PP > 95% ) can be in te rp re te d as evidence o f genetic incongruence an d d iv ergen t phy logenetic histories, w hile conflict am o n g w eakly supp o rted clades (PP < 50% ) m ay be due to stochastic e rro r (W iens, 1998; R eeder, 2003; W illiam s & O zaw a, 2006). W e therefore c reated con ca te ­n a ted datasets. As m ore sequences were availab le for 28S and 12S th a n for 16S an d C O I, we co n structed tw o datasets: a four-gene d a ta se t (28S + 12S + 16S + C O I) a n d a tw o-gene d a ta se t ( 2 8 S + 1 2 S ) . T h e tw o-gene d a ta se t consisted o f 94 sequences, w hile the four-gene a lig n m en t consisted o f 59 sequences.

B ayesian analysis o f the tw o-gene a n d four-gene da tase ts was perfo rm ed exactly as above; M C M C analyses o f b o th datase ts w ere ru n u n til s ta tio n arity was reached a t nine m illion generations.

RESULTSIn general, the single-gene analyses were less well resolved th a n tw o-gene an d four-gene analyses (Figs 1—3), w ith 28S being p a rticu la rly poorly resolved a t the suprageneric level (Supplem entary M ateria l, Fig. SI ). W e found th a t the two-gene an d the four-gene analyses were most useful for d e te rm in ing the relationships am ong genera (Figs 1 an d 2) while, because it had the most available sequences, the 12S analysis was most useful for d e term in ing relationships am ong species (Fig. 3). For this reason, we will no t discuss the single-gene trees o th er th a n 12S in detail; none of the rem ain ing single-gene trees have any w ell-supported branches in conflict w ith the trees discussed (Supplem entary M ateria l Fig. SI ).

Suprageneric relationships

T h ree w ell-supported suprageneric clades were found (Figs 1 an d 2; C lades A —C; P P = 1 0 0 % ) , w ith a single lineage (Trachypollia lugubris) in an am biguous position (Figs 1 — 3). R ela tionsh ips am o n g the m ajo r clades were no t well supported in m ost trees, a lth o u g h B a n d C form ed a clade in the all-gene tree (Fig. 1; P P = 9 8% ).

W ith in clade A, we recovered species th a t have been previous­ly assigned to the genera Morula sm.su stricto, Muricodrupa and Tenguella (Figs 1 an d 2; P P = 100% ). Inc luded in clade B were species th a t have been previously assigned to Morula s. s., Muricodrupa, Morula (Habromorula) an d Cytharomorula (Figs 1 -3 ; P P = 100% ). W ith in clade C fell species th a t have previously been assigned to Orania, Cronia s. s., Cronia (U silla), Lataxiena, Drupella, Ergalatax, Maculotriton, Bedevina, Spinidrupa, Morula s. s., Morula (Oppomorus), Pascula, Cytharomorula an d Phrygiomurex (Figs 1 -3 ; P P > 95% ).

Genus-level clades

Tenguella granulata, T. ceylonica, T. musiva an d T. marginalba form ed a w ell-supported subclade FT w ith in m ajo r clade A, to g e th e r w ith a n a p p a re n tly undescribed species ( Tenguella n. sp.; Figs 1 -3 A ; P P = 1 0 0 % ) . Muricodrupa fenestrata and 'M orula’ anaxares form ed a poorly su p p o rted clade in the tw o-gene a n d four-gene analyses (Fig. 1, P P = 93% ; Fig. 2, P P = 8 5 % ). A sister re la tionsh ip betw een 'M orula’ nodulosa an d all o th er species in m ajo r clade A was well supported (Figs 1 -3 A ; P P > 9 8% ).

W ith in m ajo r clade Ba w ell-supported subclade V consisted o f Morulauva an d 10 o th e r Morula species (Aí. aspera, M . bene­dicta, M . biconica, M . chrysostoma, M . coronida, M . japonica, M . nodicostata, M . spinosa, M . striata a n d M . zebrina', Figs 1 -3 A ; P P > 9 9 % ). 'M orula’ rumphiusi a n d 'M orula’ fiscella form ed a w ell-supported clade in all analyses (Figs 1 -3 A ; P P = 100% ). A n un iden tified species ('M orula’ n. sp.) fell w ith in m ajo r clade B, b u t its position was no t resolved (Figs 1 -3 A ).

F o u r w ell-supported subclades W - Z were observed w ith in m ajo r clade C (Figs 1, 2 an d 3B; P P > 95% ; b u t Z u n su p p o rt­ed in 12S analysis Fig. 3B). Usilla avenacea, Lataxiena fimbriata, 'Orania’ bimucronata, ' 0 . ’ gaskelli an d ' 0 . ’ serotina form ed sub­clade W (Figs 1, 2 an d 3B; P P = 100% ). W ith in subclade x Drupella cornus, D . eburnea, D. fragum, D . rugosa, D . margariticola ‘C o n tin e n ta r an d D . margariticola 'O cean ic ' form ed a well- su p p o rted clade in the tw o-gene a n d four-gene analyses (Figs 1 an d 2; P P > 9 5 % ). A sister re la tionsh ip be tw een this clade an d one consisting o f Ergalatax contracta, 'E .’ junionae, Cronia amygdala, C. aurantiaca an d Maculotriton serriale was well sup­p o rted in the four-gene analysis (Fig. 1; P P = 100% ). W ith in subclade Y, a clade of Oppomorus nodulifera, 0 . purpureocincta and 0 . funiculata was well su p p o rted in the four-gene analysis (Fig. 1; P P = 100% ). Also w ith in subclade Y, a clade consist­ing o f 'Spinidrupa’ infans, 'S .’ cf. infans, 'Spinidrupa’ sp. and Bedevina birileffi was well su p p o rted in b o th the tw o-gene and four-gene analyses (Figs 1 an d 2; P P > 9 8 % ), b u t th e position o f Spinidrupa euracantha was no t resolved w ith in subclade Y (Fig. 3B). W ith in subclade Z, a clade consisting o f Cytharomorula vexillum, 'C .’ cf. gravi, 'C .’ springsteeni, 'Orania’ orna­mentata, ' 0 . ’ mixta, ' 0 . ’ pacifica, ' 0 . ’ fischeriana, ' 0 . ’ cf. rosea and 'T ha is’ castanea was well su p p o rted in the tw o-gene a n d four- gene analyses (Figs 1 an d 2; P P = 100% ). 'Pascula’ ochrostoma an d 'P .’ muricata form ed a clade (Figs 1 an d 2; P P = 100% ), as d id 'P .’ darrosensis a n d 'P .’ submissa (Figs 2 a n d 3B; P P = 100% ). 'M orula’ parva was sister to all th e o th er m em bers o f subclade Z in the tw o-gene a n d four-gene analyses (Figs 1 and 2; P P > 9 5 % ). T h e position o f 'Cytharomorula’ paucimaculata in subclade Z was unresolved (Fig. 3B). T h e positions o f Phrygiomurex sculptilis an d 'M orula’ echinata (only 12S sequence availab le for the la tte r) w ith in m ajo r clade C were no t resolved (Figs 1, 2 an d 3B).

Species-level relationships

T his study was no t designed to test species b o u n d aries o r to resolve relationships betw een species com prehensively. N evertheless, some results suggest the need for fu rth e r investi­ga tion . W e found evidence in d ica tin g tw o possible new species (Tenguella n. sp. an d 'M orula’ n. sp.; Figs 1 -3 ; and S u p p lem en tary m ateria l, Fig. S I) . Sm all genetic distances are suggestive o f possible synonym y o f species; for exam ple, there was < 1 % d ivergence am o n g C O I sequences for the p a ir Morula uva an d M . aspera, a n d sim ilarly for the p a ir Cronia amygdala a n d C. aurantiaca. In tw o cases, m orphospecies w ere no t m onophy le tic ('Cytharomorula’ cf. grayi an d 'Pascula ochrostoma’; Figs 2 a n d 3B).

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Concholepas concholepas - Dicathais orbita

Rapana bezoar

28S rRNA + 12S rRNA + 16S rRNA + COI 0 .0 5 s u b s titu tio n s /s ite

Thais nodosaThalessa aculeata

Mancinella intermedia

Trachypollia lugubris (C osta Rica) io o l Trachypollia lugubris (Panam a)

'Morula'nodulosa (C osta Rica)— 'Morula'anaxares (M ozam b iq u e) r Muricodrupa fenestrata (M ozam b iq u e)

T Muricodrupa fenestrata (N ew C aled on ia)Tenguella musiva (M alaysia)Tenguella granulata (N ew C aled on ia)

- Tenguella n. sp . (G uam )Tenguella ceylonica (M alaysia)Tenguella marginalba (Australia)

'Muricodrupa' fiscella (N ew C aled on ia) r 'Morula rumphiusi (Japan)1— 'Morula' rumphiusi (M alaysia)

'Morula' n. sp . (P h ilip p in es /8 8 2 8 )Morula coronata (M ozam b iq u e)

Morula japonica (Japan)Morula nodicostata (G uam )

Morula spinosa (Jap an /0148) lo o 1 Morula spinosa (Jap a n /0 3 5 1 )

98l r Morula striata (Japan/YK)100 Morula striata (Jap a n /0 3 4 6 )

Morula uva (G u a m /0 7 7 5 )Tööjr Morula uva (Japan)

8 4 Morula aspera (Egypt)82 Morula uva (G u a m /0 8 3 1 )

Phrygiomurex sculptilis (V anuatu)'Orania' bimucronata (M alaysia)

r Usilla avenacea (H awaii)Usilla avenacea (Japan)

Lataxiena fimbriata (Australia)‘Orania'gaskelli (V anuatu)

‘Orania'serotina (P h ilippines)- Drupella fragum (Japan)Drupella cornus (N ew C aled on ia)

Drupella margariticola 'C on tin en ta l' (H ong Kong)— Drupella margariticola 'O cean ic' (M ozam b iq u e)— Drupella rugosa (H ong Kong)

'Ergalatax'contracta (Japan)100 'Ergalatax'contracta (Malaysia)

t Ergalatax'junionae (UAE)ïoo~ 'Ergalatax'junionae (Kuwait)

Maculotriton serriale (Japan) f Cronia amygdala (Australia)1 Cronia aurantiaca (Australia)

'Spinidrupa ' infans ( Ma da g a sca r)Bedevina birileffi (Japan)

'Spinidrupa'cf infans (M ozam b iq u e)— Spinidrupa sp. (M ozam b iq u e)Oppomorus nodulifera (A ustralia /! 257)

t Oppomorus purpureocincta (Japan)looi Oppomorus purpureocincta (M alaysia)

r Oppomorus funiculata (J a p a n /0 1 40)Oppomorus funiculata (Jap an /0355)

93 Oppomorus funiculata (Jap a n /0 3 4 0 )'Morula'parva (P h ilippines)

- ‘Pascula’submissa (M o za m b iq u e /5 4 4 0 )'Pascula'ochrostoma (P h ilippines)

'Pascula' muricata (M o za m b iq u e /0 9 5 0 )— 'Cytharomorula'springsteeni (P h ilippines) 'Cytharomorula' cf grayi {New C a le d o n ia /8 2 2 5 )

Cytharomorula vexillum (N ew C a le d o n ia /8 1 74)loo1— 'Orania'ornamentata (M ozam b iq u e)1QÛ1— 'Orania'fischeriana (M adagascar)

I ’ Thais'castanea (Sou th Africa)'Orania'pacifica (P h ilippines)'Orania mixta (P h ilip p in es /8 2 0 1 )

F igure 1. Bayesian phylogeny of the Ergalataxinae and rapanine outgroups, based on the four-gene analysis of 28S rRNA, 12S rRNA, 16S rRNA and cytochrome c oxidase subunit I (GOI) sequences. Posterior probabilities >50 are shown at the nodes. Generic assignments are based on our revised classification (Table 1); uncertain assignments are indicated by single quotation marks. Type species of valid genera are indicated in bold. Locality is shown in parentheses after species name; when more than one sample of a species is from a given location, the last four digits of the voucher numbers are given (see Supplementary material Table SI). M ajor clades are indicated by letters A—C and subclades by U —Z.

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r Trachypollia lugubris (Costa Rica) i1 Trachypollia lugubris (Panama)

arula' nodulosa (Costa Rica)'Morula'anaxares (Mozambique)

r Muricodrupa fenestrata (Mozambique)L Muricodrupa fenestrata (New Caledonia)

Tenguella ceylonica (Malaysia)Tenguella marginalba (Australia)

( Tenguella musiva (Malaysia)J <ftrTenguella granulata (New Caledonia)I ! — Tenguella granulata (Seychelles)iooI r Tenguella n. sp. (Vanuatu)

loo1- Tenguella n. sp. (Guam) i- 'Muricodrupa' fiscella (Australia)

raoi- 'Muricodrupa' fiscella (New Caledonia) i - 'Morula' rumphiusi (Japan)1— 'Morula'rumphiusi (Malaysia)

■ 'Morula'n. sp. (Philippines/8828)Morula benedicta (Hawaii)

— Morula coronata (Mozambique)------------- Morula japonica (Japan)- Morula nodicostata (Guam)Morula uva (Guam/0831 )Morula uva (Japan)Morula aspera (Egypt)

85l Morula uva (Guam/0775) r Morula spinosa (Vanuatu)

loolr Morula spinosa (Japan/0148)50 Morula spinosa (Japan/0351 ) r Morula zebrina (Moorea)

lôa r Morula striata (Japan/YK)94] Morula striata (Japan/0346)

Morula biconica (Philippines)'Orania'gaskelli (Vanuatu)

Lataxiena fimbriata (Australia)'Orania'serotina (Philippines)

'Orania'bimucronata (Malaysia) j Usilla avenacea (Hawaii)

1001- Usilla avenacea (Japan)'Morula'parva (Philippines)

'Pascula'darrosensis (Philippines)__r 'Pascula'submissa (Mozambique/5440)100L 'Pascula' submissa (Mozambique/5609)

— ‘Pascula'ochrostoma (Japan)9*Lr 'Pascula'muricata (Mozambique/0435)I L 'Pascula'muricata (Mozambique/0950)

100| p ‘Pascula' ochrostoma (Tahiti)îooLr 'Pascula'ochrostoma (Guam)

w1- 'Pascula'ochrostoma (Philippines) j- Cytharomorula vexillum (New Caledonia/8174)

'Oö1— 'Orania'ornamentata (Mozambique)88]— 'Cytharomorula'c f grayi (New Caledonia/8225)1— 'r "tharomorula'springsteeni (Philippines)

tharomorula'cr grayi (New Caledonia/8205) r 'Orania' mixta (Pnilippines/8708)100Lf 'Orania'mixta (Philippines/8209)

7? 'Orania'mixta (Philippines/8201 )'Orania' fischeriana (Madagascar)— Thais'castanea (South Africa)

'Orania rosea' (Philippines) loo Lr 'Orania' pacifica (Australia)

53 'Orania'pacifica (Philippines)

28 S rRNA + 12S rRNA 0.1 substitutions/site

F igu re 2. B ayesian p h y logeny o f th e E rg a la ta x in a e , C onven tions as in F ig u re 1.

— Phrygiomurex sculptilis (Vanuatu) Drupella eburnea (Japan) Drupella cornus (New Caledonia)

- Drupella fragum (Japan)Drupella rugosa (Hong Kong)

Drupella margariticola 'Oceanic' (Hong Kong)- Drupella margariticola 'Continental' (Mozambique) 'Ergalatax'junionae ((JAE)'Ergalatax'junionae (Kuwait)

69j- 'Ergalatax'contracta iJapan)C- 'Ergalatax' contracta (Malaysia)

I Cronia amygdala (Australia)rao1 Cronia aurantiaca (Australia)

Maculotriton serriale (Japan)Maculotriton serriale (Philippines)

Bedevina birileffi (Japan)' oinidrupa' infans (Madagascar)

'Spinidrupa' cf infans (Mozambique) 'Spinidrupa'sp. (Mozambique)

Oppomorus nodulifera (Australia/1257) r Oppomorus funiculata (Japan/0355)îol; Oppomorus funiculata (Japan/0140)73L Oppomorus funiculata (Japan/0340)

r Oppomorus purpureocincta (Guam) lööLr Oppomorus purpureocincta, (Japan)

941 Oppomorus purpureocincta (Malaysia)

based o n th e tw o-gene analysis o f 28S a n d 12S rR N A . R a p a n in e o u tg ro u p s n o t show n.

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100

58

j Trachypollia lugubris (Costa Rica)L Trachypollia lugubris (Panama)

r 'Morula' nodulosa (Costa Rica)ÍL1001

98

'Morula' nodulosa (Senegal)¡- 'Morula'anaxares (M ozambique)

1001 'Morula'anaxares (Vanuatu) Muricodrupa fenestrata (M ozambique)

56

100

1 oo1 Muricodrupa fenestrata (New Caledonia)— Tenguella ceylonica (Malaysia)— Tenguella marginalba (Australia) Tenguella musiva (Malaysia) r Tenguella n. sp. (Vanuatu)

100 Tenguella n. sp. (Guam)841 i Tenguella granulata (Seychelles)

9 9 |j Tenguella granulata (M ozambique)9 ? Tenguella granulata (New Caledonia)

r 'Morula' rumphiusi (Japan)1— 'Morula' rumphiusi (Malaysia)

B100

100100

100

67\

99

100

— 'Muricodrupa' fiscella (Philippines) 'Muricodrupa' fiscella (Australia) 'Muricodrupa' fiscella (New Caledonia)

_________ r 'Morula' n. sp. (Philippines/8828)’Sol 'Morula'n. sp. (Philippines/8827)

— Morula nodicosta (Guam) ________ Morula benedicta (Hawaii)

Morula chrystosoma (UAE)— Morula coronata (M ozambique)

Morula spinosa (Japan/0148)Morula spinosa (Japan/0351 )

L Morula spinosa (Vanuatu)100 r Morula striata (Japan/YK)

Morula striata (Japan/0346)100 Morula biconica (Philippines)

L Morula zebrina (Moorea) Morula japonica (Japan)Morula uva (Guam /0780)Morula aspera (Egypt)Morula uva (M ozambique)Morula uva (Guam/0762)Morula uva (Guam /0775)Morula uva (Japan)Morula uva (Guam /0821)Morula uva (Guam/0831 )

65

91

64

96< major clade C

12S rRNA— 0.05 substitutions/site

F igure 3. Bayesian phylogeny of the Ergalataxinae, based on single-gene analysis of 12S rRNA. Rapanine outgroups not shown. Conventions as in Figure 1. A. M ajor clades A and B, with subclades U and V. B. M ajor clade C, with subclades W and Z. Note that subclade Z includes ‘Morula? parva in Figures 1 and 2.

D ISC U SSIO N

Phylogenetic relationships and classification o f the ergalataxine genera

O u r results ind ica te po lyphyly in n early all th e e rg ala tax ine genera , as h ith e rto defined by m orpho log ical characters . For exam ple, species previously assigned to Morula are found in each of the th ree m ajo r clades, w hile species o f Orania are found in each subclade w ith in m ajo r clade C. H ere, we use a m olecu lar d a ta se t to construct a new phylogenetic classification th a t reflects evo lu tionary re la tionsh ips ra th e r th a n superficial sim ilarities in shell m orphology. A phylogenetic classification requires th a t gen era are clades th a t co n ta in th e ir designated type species, b u t some decisions a b o u t n a m in g an d rank ing

rem ain subjective. W h en m ak ing such decisions, we have p re ­served cu rren t usage (based on m orpho log ical definitions o f genera) w here possible. W e em phasize th a t o u r study provides only a n in itia l fram ew ork w hich should be refined an d tested by fu rth e r m olecu lar a n d m orpho log ical analyses a n d conse­q u en tly we n am e no new gen era a t this stage.

T h e genus Trachypollia is here represen ted by only one o f its four cu rren tly recognized m em bers, all o f w hich occur in the easte rn Pacific a n d A tlan tic O ceans (EPA) (Figs 1 -3 , T ab le 1; Vokes, 1996a; H o u a rt, 1997). F u rth e r species, in c lud ing the type, should be investigated w ith m olecu lar techniques, no t only to test the m onophy ly o f the genus, b u t also to resolve its position w ith in the E rg a la tax in ae . I f the in cluded species T. lugubris is represen tative o f the genus, it is c lear th a t the

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BW

96

5 6 '

100

Lataxiena fimbriata (Australia) 62i— 'Orania'gaskelli (Vanuatu)

— 'Orania'bimucronata (Malaysia) 'Orania'serotina (Philippines)

r Usilla avenacea (Hawaii)ÖL

57L

52

100

100 Usilla avenacea (Japan)'Morula'parva (Philippines)

Phrygiomurex sculptilis (Vanuatu) 'Morula'echinata (Vanuatu)

Drupella rugosa (Hong Kong)— Drupella eburnea (Japan)— Drupella cornus (New Caledonia)

Drupella margariticola 'Continental' (Hong Kong) — Drupella margariticola 'Oceanic' (M ozambique) Drupella fragum (Japan) i ‘Ergalatax’junionae (UAE)'ööl ‘Ergalatax’junionae (Kuwait)

72

79

99

82

74r 'Ergalatax'contracta (Japan) ̂ 'Ergalatax'contracta (Malaysia)

Cronia amygdala (Australia) Cronia aurantiaca (Australia)1001

70

loo r Maculotriton serriale (M ozambique) i oor Maculotriton serriale (Japan)53 Maculotriton serriale (Philippines)

Spinidrupa euracantha (M ozambique)Bedevina birileffi (Japan)

'Spinidrupa' c f infans (M ozambique) 'Spinidrupa'sp. (M ozambique)

89100

100

5 5 '

5 6 '

7 0 '

12S rRNA— 0.05 substitutions/site

'Spinidrupa'infans (Madagascar) ngi- Oppomorus purpureocincta (Guam)

Lj Oppomorus purpureocincta (Japan)85* Oppomorus purpureocincta (Malaysia)

Oppomorus nodulifera (Australia/1257) Oppomorus nodulifera (Australia/03918.1 ) Oppomorus nodulifera (Australia/03918.2)

72 p Oppomorus funiculata (Japan/0140)! Oppomorus funiculata (Japan/0355)

Oppomorus funiculata (Japan/0340)'Pascula' darrosensis (Philippines)

tool r 'Pascula'submissa (M ozam bique/5440)z) lOOL 'Pascula'submissa (M ozam bique/5609)

loo i— 'Cytharomorula'c f grayii New Caledonia/8225)H i— ‘Orania'ornam entata (M ozambique)

io o L Cytharomorula vexillum (New C aledonia/8174)98* Cytharomorula vexillum (New Caledonia/8203)

i— 'Cytharomorula' c f grayi (New Caledonia/8205)Töo' 'Cytharomorula' springsteeni (Philippines) r 'Cytharomorula'paucimaculata (Philippines/8202)

Too*- 'Cytharomorula'paucimaculata (Philippines/8204) r 'Orania'mixta (Philippines/8708)

0n 'Orania' m ixta (Solom an Islands)74l ‘Orania’mixta (Philippines/8209)

'Orania'mixta (Philippines/8201) i— ‘Orania' fischeriana (Madagascar)

q Thais'castanea (South Africa)' 'Orania'pacifica (Australia)

'Orania'pacifica (Philippines)'Orania rosea' (Philippines)— 'Pascula'ochrostoma (Japan)

i oor 'Pascula'muricata (M ozam bique/0435)89 'Pascula'muricata (M ozam bique/0950)

S 'Pascula'ochrostoma (Tahiti)'Pascula'ochrostoma (Guam)

'Pascula'ochrostoma (Philippines)

7597

89

F igu re 3. Continued

A tlan tic 'M orula’ nodulosa m ust be excluded front the genus, in ag reem en t w ith Vokes ( 1996a).

W ith in m ajo r clade A, we propose th a t th ree o r four genera can be recognized: Tenguella, Muricodrupa an d one or tw o u n d e ­scribed genera . S ubclade LT (Figs 1—3A) con tains the type species o f Tenguella, T. granulata, a n d we therefore assign to

Tenguella all species in subclade LT (Figs 1—3A). Tenguella has previously been synonyntized w ith Morula (Fujioka, 1985) and its species have no t previously been recognized as a m o rp h o ­logically d istinctive group . In c lu d ed in this g ro u p is an u n id en ­tified species (Tenguella n. sp. in Figs 1—3A); this has been illu stra ted as 'Azumamorula sp f (D h arn ta , 2005) an d as 'Morula

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mutica’ (W ilson, 1994). T h e shell shows some sim ilarities to M . mutica, w hich is th e type species o f Azumamorula, an d it is therefore possible th a t this genus m ay prove to be a synonym o f Tenguella (T ab le 1). T h e o th er species in clade A are m o r­pholog ically dissim ilar to species o f Tenguella. W e recom m end th a t Muricodrupa be re ta in ed for its type species, M . fenestrata. In th e all-gene analysis (Fig. 1), 'M orula’ anaxares form s a m a r­g inally significant clade w ith M . fenestrata, w hich is no t c o n tra ­d ic ted in o th er analyses. LTnworn shells o f b o th species show sim ilar scu lp ture an d , if fu ture studies confirm th e ir sister re la ­tionship , 'M orula’ anaxares m ay be transferred to Muricodrupa. A new genus is req u ired for 'M orula’ nodulosa, the sole rep resen ta ­tive o f clade A in the A tlan tic , well su p p o rted as sister to the rem ain in g Indo-W est Pacific (IW P ) m em bers (Figs 1—3A). T h e p a tte rn o f one or m ore E PA lineages sister to a m ore diverse IW P clade is com m on in tro p ical gastropod groups (e.g. W illiam s & R eid , 2004; C larem o n t et ed., in press).

E leven species th a t are com m only assigned to Morula form a m onophy le tic g roup w ith in m ajo r clade B. Since this includes M . uva, the type species o f Morula, we propose th a t the generic n am e should be restric ted to this subclade (Figs 1 -3 A : subclade V ). T h is clade also con tains M . biconica, the type species o f Habromorula, b u t species previously assigned to this g ro u p [as a subgenus o f Morula'. M . (H .) biconica, M . (H .) coronata, M . (H .)

japonica, M . ( H .) spinosa, M . ( H .) striata; H o u a rt, 2004] do no t form a clade in an y analysis (Figs 1 —3A). H ow ever, m onophy ly o f this m orpholog ically d istinctive g roup is no t strongly c o n tra ­d icted , so th a t fu rth e r sam pling an d analysis could yet su p p o rt Habromorula as a m onophyle tic subgenus o f Morula. Because they are m orpho log ically d issim ilar to species o f Morula s. s., o th e r species in clade B ('M orula’ rumphiusi a n d 'M orula’ fiscella) should be assigned to a new genus. T h e p lacem en t o f a n u n id en ­tified species ('M orula’ n. sp.) is no t certain .

M a jo r clade C is the largest an d m ost m orpholog ically d isp a ra te o f the e rga la tax in e subgroups a n d ou r sam pling includes the type species o f 10 described genera . M ost o f these a re poorly defined m orpholog ically an d some (Phrygiomurex, Usilla, Maculotriton, Bedevina) m ay be m onotyp ic . T h e affinities o f Phrygiomurex sculptilis an d 'M orula’ echinata are no t resolved.

Species in subclade W (Figs 1—3B) are m orpholog ically h e t­erogeneous, in c lud ing b o th the sm ooth-shelled Usilla an d foliose Lataxiena, to g e th e r w ith some Orania species. T h e genus Orania is strongly po lyphy le tic in ou r analyses, w ith m em bers in subclades W an d Z (Figs 1 —3B). Since the type species o f Orania (O. fusulus from the M e d ite rran ean ; T ab le 1) was no t included , it is im possible to d e te rm in e to w h ich o f the th ree to five clades o f 'Orania’ the n am e should be app lied . C learly a revision of the genus is necessary. M eanw hile , in view o f the m orpho log ical d isp a rity in this g roup , we suggest th a t Orania, Usilla an d Lataxiena should be re ta in ed acco rd ing to th e ir accustom ed usage.

W ith in subclade X (Figs 1 —3B) the genus-level classification o f Drupella has been investigated in d e ta il elsewhere (C larem o n t et al., 2011a). T h e genus is well su p p o rted (Figs 1 a n d 2 ), consisting o f D . cornus, D . fragum, D . eburnea, D . rugosa a n d tw o cryp tic species o f D. margariticola sensu lato (D. margari­ticola 'O cean ic ' an d D . margariticola 'C o n tin e n ta l’). O th e r species in subclade X form a w ell-supported clade in the all-gene analysis (Fig. 1), in clud ing the type species o f Cronia a n d Maculotriton. T h e type species o f Ergalatax, E. pauper, was no t sam pled, b u t th e in cluded E. contracta is p ro b ab ly close to it a n d has been considered synonym ous (H o u a rt, 2008). O f these th ree generic nam es, Cronia has p rio rity (T ab le 1). A lth o u g h Ergalatax is no t m onophy le tic in any analysis (Figs 1—3B), o u r sam pling o f this genus was lim ited . T herefore, we conservatively suggest th a t all th ree gen era are re ta in ed u n til relationships am o n g th em can be m ore th o r­oughly investigated w ith increased species-level sam pling .

S ubclade Y (Figs 1—3B) also con tains m orpholog ically diverse species. In the all-gene analysis (Fig. 1), th e type species o f Oppomorus, O. nodulifera, form s a w ell-supported clade w ith O. funiculata an d O. purpureocincta an d we therefore propose th a t Oppomorus should be acco rded full generic ran k (h ith e rto a subgenus o f Morula, e.g. H o u a rt, 2004). O f the rem ain in g tax a in subclade Y, th ree species previously assigned to Spinidrupa form a clade ( 'S .’ infans, 'S .’ cf. infans an d 'S .’ sp.), b u t th e ir re­la tionsh ip w ith the type species, .S’, euracantha, is unresolved in the only analysis in w h ich the la tte r was in cluded (Fig. 3B). F u rth e rm o re , in th e all-gene analysis the 'Spinidrupa’ c lade also includes Bedevina birileffi, th e type species o f Bedevina, w hich has p rio rity . P end ing the inclusion o f a d d itio n a l species, we conser­vatively recom m end the re ten tio n o f b o th Spinidrupa an d Bedevina.

S ubclade Z (Figs 1—3B) contains species o f Pascula, Orania a n d Cytharomorula, b u t includes the type species o f only the last o f these. Based on the in cluded species, all th ree genera , as c u r­ren tly defined, are po lyphyletic . I t m ay be th a t th e en tire clade should be assigned to one genus. O f these th ree generic nam es, Orania has p rio rity (T ab le 1), b u t its m em bers also a p p e a r in clade W an d the type species was no t included . P en d in g a m ore com plete analysis, in c lu d in g the re levan t type species, we have conservatively re ta in ed all species in the gen era to w h ich they have been previously assigned. Also in cluded here is the en igm atic S o u th A frican species 'T ha is’ castanea', clearly, this is no t a m em b er o f the rap an in e genus Thais, b u t its ap p ro p ria te generic assignm ent rem ains to be de te rm in ed . A tem p o ra ry p lacem en t in Orania is suggested. In the tw o-gene a n d four-gene analyses (Figs 1 an d 2), 'M orula’ parva is sister to all o th er m em bers o f subclade Z, suggesting th a t a new genus m ay be requ ired for this species.

F ossil record and age o f the Ergalataxinae

O w in g to sim ilarity a n d convergence o f shell form am o n g e rg a ­la tax in e a n d rap an in em u ric id s an d lack o f unam b ig u o u s syn- apom orph ies for m an y clades (V erm eij & C arlson, 2000), unequ ivocal iden tification o f fossils can be p rob lem atic. V erm eij & C arlson (2000) gave th e earliest occurrences o f several e rg a la tax in e gen era as L ate M iocene, w hile L an d au , H o u a r t & D a Silva (2007) rep o rted th a t the g ro u p ap p eared in th e A tlan tic in the E arly M iocene. T h e oldest c erta in e rg a ­la tax in e know n to us is a specim en o f 'Taurasia’ sacyi C ossm ann & Peyrot, 1923 from the E arly O ligocene (G aas, Espibos, F rance; R u p e lia n stage, 2 8 .4 -3 3 .9 M a; C ollection o f R . H o u a rt). T h is fossil resem bles R ecen t species o f Pascula, Ergalatax a n d Cytharomorula, im ply ing a m in im u m age for m ajo r clade C. T his, how ever, is insufficient for a reliable tim e ca lib ra tio n o f o u r phylogeny. Palaeon to log ica l evidence sug­gests a C retaceous orig in for the M u ric id ae as a w hole (V erm eij, 1996). Som e recent m olecu lar studies have a tte m p ted to estim ate ages o f e rg a la tax in e clades, g iving figures o f 5 M a for Drupella (C larem o n t et al., 2011a) an d 82 M a for the sep ara tio n o f the E rg a la tax in ae an d R a p a n in a e (C larem o n t et al., in press). D etailed study of e rg ala tax ine fossils is req u ired before these estim ates can be refined.

C O N C LU SIO N SA lth o u g h this fram ew ork represents the m ost com plete p h y l­ogeny o f the E rg a la tax in ae yet constructed , m uch w ork rem ains to be done to achieve a robust phylogenetic classification of this taxonom ica lly difficult subfam ily. W e have clarified the classifi­cation o f 'M orula’ s. L, defin ing as d istinct clades the genera Morula s. s., Tenguella an d Oppomorus, a n d have confirm ed the va lid ity o f Muricodrupa, Phrygiomurex a n d Drupella. T h e de fin ­itio n an d va lid ity o f several trad itio n a l genera , includ ing

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M. C L A R E M O N T E T A L .

Orania, Pascula, Cytharomorula, Cronia, Ergalatax, Maculotriton, Usilla, Lataxiena, Spinidrupa a n d Bedevina rem ain to be confirm ed (see su m m ary in T ab le 1). A t least th ree new gen era (for 'M orula’ nodulosa', for 'A í.’ fiscella an d 'A Í .’ rumphiusi; an d for 'A Í .’ parva) a p p e a r to be requ ired . Several liv ing gen era th a t have been in cluded in E rg a la tax in ae by some au th o rs were no t ava il­ab le for inclusion in ou r analyses an d th e ir p lacem en t should be tested by m olecu lar analysis (Azumamorula, Daphnellopsis, Lindapterys, Uttleya; S u p p lem en tary m ateria l: T ab le S2).

SUPPLEM EN TARY M A TERIA LS u p p lem en tary m ate ria l is availab le a t Journal o f Molluscan Studies online.

A C K N O W L E D G E M E N T SW e are very gratefu l for the assistance an d loans p rov ided by the staff o f m useum s a n d research in stitu tions, especially P. B ouchet, V. Eléros, B. Buge an d N . P u illan d re o f M useum N atio n a le d ’H isto ire N atu relle , Paris (M N H N ); G. Pau lay , J . S lapcinsky a n d M . Bemis o f the F lo rid a M useum o f N a tu ra l Elistory: E. N a ran jo o f th e LTniversidad N acional A u tó n o m a de M éxico: J .E . M ichel-M orfin an d V. L an d a -Ja im e ofLTniversidad de G u a d a la ja ra ; Y. C am ach o of the LTniversity o f C osta R ica ; S. S lack-Sm ith an d C. W hisson o f the W estern A u stra lian M useum , Perth ; M . O liverio a n d A. B arco o f ‘L a S ap ien za ’ LTniversity o f R om e; A. K e rr an d B. Sm ith o f the LTniversity o f G u am M arin e L ab o ra to ry ; G. W illiam s o f the Swire M arin e L ab , Elong K ong; an d I. L och an d A. M iller o f the A u stra lian M useum , Sydney. W e w ould also like to th an k m an y others w ho prov ided specim ens or w ho assisted us in the field in c lud ing L. Alsayegh, C. B ennett, C. Bird, C. C arlson, T . P laga, T . L lam ada, G. H e rb ert, Y. Ito , R . Jo n es, Y. K ano , P. K uklinski, T . N ak an o , B. N g, S. N ielson, N . R aza lli, K .S . T an , S .H . T an , J .D . T ru e , R .C . W illan , an d Z. Yasin. T h e M N H N m ate ria l used in this study was collected d u rin g the follow ing expeditions: P A N G L A O 2004 M arin e Biodiversity P roject [a jo in t p ro jec t be tw een LTniversity o f San Carlos, C ebu C ity (LTSC; co-P I D an ilo L argo) a n d M N H N (co-PI Ph ilippe B ouchet), funded by the T o ta l F o u n d a tio n an d the F ren ch M in istry o f Foreign Affairs]; the ALTRORA 2007 cruise [M /V D A -B FA R associated w ith the N a tio n a l M u seu m o f the Ph ilipp ines (N M P, co-P I M arivene M a n u e l) , M N H N (co-PI Ph ilippe B ouchet) an d B FA R , funded th ro u g h a g ra n t from the L ounsbery F o u n d a tio n ]; the M N H N -IR D -P N I Santo 2006 exped ition (funded by g ran ts from , am o n g others, the T o ta l F o u n d a tio n a n d the Stavros N iarchos F o u n d a tio n ); E B ISC O , N O R F O L K 2 a n d C O N C A L IS cruises [R /V Alis dep loyed front N o u m ea by th e In s titu t de R echerche p o u r le D éveloppem ent ( IR D )] . M .C . was su p p o rted by a studen tsh ip from the N a tu ra l H isto ry M useum , L o n d o n a n d by an Im p eria l College D ep u ty R e c to r’s studen tsh ip . W e th a n k the A cting E d ito r E llen S trong, M arco O liverio a n d one an o n y m ­ous review er for helpful com m ents on a previous version o f this m an uscrip t.

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