Table Of ContentTHE NAUTILUS 123(4):303-312, 2009 Page 303
Revising oc-taxonomy in shelled gastropods: the case of Rissoa
panJwrmensis Verdnin, 1985 (Caenogastropoda: Rissoidae)
Francesco Criscione*’^ Danilo Scucleri Francesco Paolo Palti^
Functional and Evolutioiian' Dipartiniento di Biologia Auiinale Functional and Evolutionarv
Ecology Lahoratoiy Universita degli Studi di Catania Ecolog)' Lahoratoiy
Stazione Zoological“Anton Dohrn" ViaAndrone, 81 .Stazione Zoological“Anton Dohrn”
P.ta S. Pietro, 1 95124 Catania, ITALY P.ta S. Pietro, 1,
S0077 Ischia (NA), ITALY [email protected] 80077 Ischia (NA), ITALY
[email protected] [email protected]
ABSTRACT European species belonging to six snbgenera of the ge-
nus Rissoa (Eremimdlle ms) Desmarest, 1814 (Gastro-
In one ot his landmark papers on the genus Rissoa (Caenogas- poda: Rissoidae). He took into account quantitative
tropoda: Rissoidae), Verdnin described the new species Rissoa (shell measurements, number of sculptural elements)
ptiaoini,linoonncnneswis.dabtaasoedn othneaspteecwieesmphatvyesbheelelsn.pAulbtleirsihtesddeasncdriipt-s and (jiialitative shell features (colour pattern elements),
taxonomic status has remained questionable. We studied the assigning to the latter mnnerical values based on pres-
hqve material of R. panlnn'incnsis and several living specimens ence/absence or degree of intensity Eor the species be-
whose shells resembled those ol 74 pauhonncnsis. Although longing to the snbgenera Li^xostoma Bivona-Bernardi,
we could ditferentiate R panhonnensis from R. giierhiii on 1838, and 71/3,sort Veidnin (1983, 1986) only provided
the basis ofqualitative visual obsenaitions and geometric mor- narrative descriptions, but for the snbgenera TurbocUa
phometric data, itwas not possible to separate botli taxa using (Leach ms) Gray, 1847, Rissostoinia Sars, 1878, Gonios-
bfjoudeyncceoldoautar.pWatetetrhnesreafnodre16s8ugg+esCtOtIhamtiRt.ocphaonnhdroinanlenDsiNsAmsaey- toOs)aUt(oi,(1A8l8e4g,erVleermdsn)inVi(l1l9a,76,184119,82a,n1d98A5p)icptrdeasreinateAdlomntoerre-
be a rare morpliohqie of R. guerinii and should be .s)mony- elaborate data. The shell characters examined in each
mized\\4th this latter species.
paper were the same, except for some variation accord-
Additional ket/words: Gastropoila, mitochondrial DNA, geo- ing to the main features of the group studied. In his
metric moiphometiy, variation, moiphotvpe papers, Verdnin (1976, 1982, 1983, 1985, 1986) provided
meticnlons measurements whicii were summarized in
classical representations (scatterplots or histograms) to
facilitate the species comparisons. Verdnin placed con-
INTRODUCTION siderable importance on the dimensions of the shell
apex. He observed that there was a larger and smaller
Alost older gastropod species descriptions wei'e limited t\pe of apex, witli no intermediates. According to Ver-
to shell diagnoses, which for a long time were consid- dnin (1986), shells sharing the same morphological fea-
ered to be sufficient to justify specific taxa. However, tures, but belongingto Rvo different groups accordingto
more recent molecular systematic tools (e.g. Knowlton, apex dimensions, mmst be considered members of sepa-
2000; Bickford et ak, 2007) have often lieen used to rate species. This statement was supported bythe obser-
invalidate species described purely on shell characters vation that, in some Rissoa .species (e.g. Rehfeldt, 1968),
alone. On the other hand, cases in which genetic difler- different tvqres of apex are linked to different lanal de-
entiation is hidden by similarity in shell moqrhology are velopmental strategy. A small apex is considered txqrical
not uncommon (e.g. references in Knowlton, f993, of a planktotrophic veliger and large apex is thought to
2000). Tims, the degree ofshell morphological differen- be linked to a lecithotrophic larva (e.g. Thorson, f95()).
tiation may not reflect genetic or anatomical differentia- It is commonly accepted for gastropods that there is no
tion even among congeneric species. evidence of intraspecific polymorphism in developmen-
A purelyconchological approach was followed byVer- tal strategy (Bonchet, 1989). Verdnin (1986) listed eight
dnin (1976, 1982, 1983, 1985, 1986) in his revision ofthe “pairs” of sibling species of Rissoa {scnsii Alavr, 1963)
that differed mainly in apex dimensions. In doing so,
^ Current address: Malacology Section. Australian Museum, Verdnin (1985) not only revised existing Rissoa species,
6 College Street, 2010 Sydney, NSWAustralia blit also described some as new In his paper on the
“ Correspondingauthors snbgenera Apictdaria and CxOtiiosfoma (Verdnin, 1985),
Pao;e 304 THE NAUTILUS, Vol. 123, No. 4
C*
one ol the new species, R. pcnihorn^cijsis, was based on Table 1. The composition ofthe studied lots oiRi.s.soa spp.,
seven empt\' sliells in the Philippe lhantzenberg collec- vvdth the inscription of the original label and their acronym.
tion (now housed in the Royal Belgian Institute ofNatu- No further data (exact locality', date, etc.) regarding these lots
ral Sciences, RBINS). were available.
Ri.sf:oapanJiormciisis, accordingto its original descrip- Acronym Couteut Label
tion (Verdnin, 1985) was differentiated as follows: “The
shells ol R. pa)}]ionnensis strongly recall the normal Rpt 6R. panhormensis Rissoapanhormensis
colour \arietv' of R. guerinii (Dantzenberg and Durou- 1 R. violacea Verduin/Det,:Verduiu,1983
chonx, 1914), but have the larger type of apex, i.e. Rea 51 fi. gnerinii Pdssoa costnlata
6t0.e.0r2m6mi5mna<.lADrl(il)bsh+apv0ee.r7pw2uhdnocr<lt.a0t.Te2h9se0pirsmahmlel,sltsraimnaeedaoshnuarvteehefonlrlooywme5r4-.p6SartVto2 RRcehm 92125fi.ffii..viggonnleearrciiennaiiii RRii..Mssssooonaatsccoo.ssttnnllaattaa,, AMleddietr.
of the body whorl. For the remainder, the shells are Rsm 12 fi. gnerinii Rissoasnhcostnlata, Schw'arz
covered with fine, dense spiral striae, wiiich merge into Mediterranee
the punctate spiral striae. There are 7.6-8.2 whorls
and 314—4% ril)bed whorls. The ribs continue up to the
1814) were not further considered. Only five shells from
wliaibiitailshricb.oloTuhr,e alsabairale trihbe iosthwe^rellridbse.veAlsopiend,theanndoromfala lot Rpt were considered. These five shells offi. panhor-
caroelouorl a\arreimeWarkoafblRe. ggrueeyriisnhii,coltohuer.uTppheermeodsgte wohfortlhse mfernosmiseaacnhdo2f0trhaendfoomulrylocthso,sReena,sheRlclsb,ofRfie.mgnearnidniiRs(mfi)v,e
were selected for this study. The presence of a well-
aperture is pmplish.”The tvpe localityis Palernnj (Sicily, formed labial ril) in all the shells indicated that they
ThyTrenian Sea).
were all at terminal grow'th (Waren, 1996).
Together with the description, Verdnin (1985)
Each shell w'as placed in vertical position under the
provided a black and wiiite photograph ofthe holotype,
microscope by fixing the shell base w'ith plasticine. Total
w'hich until now' is the only knowai picture of Rissoa numbers of whorls, ribbed whorls and terminal ribs
panlionnciisis. After its description, the species slipped per w'horl, were counted according to Verduin's (1982)
into obscnritv with hardly a mention in the literature. methods. Then each protoconchwas photographed from
Currently, Rissoa panhormensis is considered to be an above using an Olympus® CAMEDIA C-7070 WZ digi-
endemic species ofthe Western-Central Mediterranean
(Bodon et ak, 1995). We examined the t\pe material of tal camera. Shells w'ere then positioned, with the help of
plasticine, w4th their v'ertical axes parallel to the obser-
R. panhormensis and other specimens of Rissoa in the
vation plane and pictures were taken ofthe teleoconchs.
mDaatnetrziaelnbweergwecroelleacbtlieonto(cRriBtIiNcaSl)l.y rOevnisethtehebaosriisginoafltdhea-t CDiSg2itiizmeadgeimeadgietsorvvs'oerfetwoarpee.neUdsiinngAmdeoabseu®remPehnottostohoolps
scription of the taxon and tov'erifyshell measurements.
\W also sampled living Rissoa spp., collecting some prov'ided bythe software (appropriatelycalibrated), total
shell length (L), the diameter of the protocoirch nucleus
specimens whose shells closely resembled the des-
cription of R. panhormensis. This alkwed ns to study (tl) aud the diameter of the first half w'horl (Do) w'ere
measured, according to Verduin's (1985) methods. Fur-
other features such as external soft bodyparts and mito-
chondrial DNA. Several empty shells corresponding thermore, the apex tv|?e (=Do + 0.72d) w'as calculated
according to Verdnin (1985). Using the statistical soft-
to R. panhormensis were also foimd washed ashore.
ware SPSS v'.15 (© SPSS Inc., 2006), a discriminant
Based on this mateiial w'e provide a morphological
function analysis was performed on the data matrix
and molecular re-evalnation of the ta.xonomic status of
obtained, in order to detect significant differences be-
R. panhormensis.
tw'een the tv'^^e specimens offi. panhonnei^sis and shells
offi. gnerinii (treating the four lots as a single group).
MATERfALS AN14 METHODS Liv'e-gollected M.vterial: Liv'ing material w'as sampled
on the rockwshore of SantaTecla, Sicily (Mediterranean,
Dautzenberg Coelegtion AIatekial: The tvqve material Ionian Sea) at 1-5 m depth in Apr 2006. About 0.03 in'"
of Rdssoa pa)ihormensis in the Dantzenberg collection ofthe red alga PterocladieUa capillacea (Gmelin) Sante-
comprises the holotv'pe and six paratvpes from Palermo lices and Hommersand w'as collected by SCUBA diving
(Sicily, Tyrrhenian Sea). Four other lots were selected, during each sampling. Collected material w'as immersed
wliich, according to present taxonomy belonged to in seawater and transferred to the laboratoiy The total
Fi. gnerinii Recihz, 1843. The specimens of these latter amount ofsampled material w'as partitioned into 20 sub-
lots closely resembled R. panhormensis tvqve material. samples that W'ere vv'ashed lor no more than 5 min in a
Table 1 summarises the data of the lour lots and the tank containing 5 I ol 50% seawater. The osmotic shock
acronyms used in this study to identify them. All the provided forced all vagile fauna to detach from the algal
shells ol the five lots were examined usingan Olympus® thalli and to fall on the bottom ol the tank, from w'here
SZXIO stereoscopic microscope. Damaged shells and specimens were easily collected and returned to sea-
misclassilied specimens (e.g. R. violacea Desmarest, water, After recovering from the osmotic shock, liv'e
F. Criscione et al., 2009 Page 305
inollusks were sorted and identified under a V^dld
Makroskop M420 stereoscopic microscope. Specimens
(d Rissoa g^iieri)tii and five specimens reterahle to ll. cd.
panhonncnsis were picked np from the sorted material.
Scnne of them were placed in miming seawater at 1S°C
and provided with fresh P. capillacea talli; others were
preseiwed in 80% ethanol. After each sampling, beached
detritus was also collected on the beach facing the sam-
pling site. This yielded f8 emptv shells belonging to
K. cf panhormcnsis. The relativeproportion ofspecimens
of R. cf ptntlionnensis and specimens of R. was
about 3/100 in samplings of both living and dry material.
Head-Fool; Adult living .specimens ofboth R. cf j)an-
honue)isis and 21 guerhiii were placed in a Petri dish
with seawater under a Leica Z16 APO stereoscopic
microscope. Shells were held with forceps and the snails
attempted to crawl e.xtending their foot completely,
enabling the head-foot to he obsemed in detail. Images
of shells and head-foot w^ere taken and digitized using
a Leica DFC 300 FX video camera and Leica Applica-
tion Suite version 2.4.0 software. Color drawings w'ere
also made to better represent the color pattern of the
head-foot.
Geometric Moi-jihometry: We randomly choose
15 adult shells from ethanol preseived specimens of
R. oucrinii and 15 adult shells ol 24. cl. p(nihontieWiis
(three from ethanol-storerl material and the remaining
12 from collected emph' shells). Shells w'ere ohserx'ed
using a Leica Z16 APO stei'eoscopic microscope, and
color images were taken and digitized using a Leica Figure 1. A .sliell of a living Rissoa cf. pauhoniwnsis from
DFC 300 FX \ldeo camera and Leica Application Suite ,S. Tecia sliowing the placement of the 19 landmarks nscxl for
version 2.4.0 software. The sliells were always placed in geometric morphometric analysis. Scale bar = 1 mm.
the same position, with the coiling a.xis in vertical posi-
tion and the aperture on the same plane as tfie objective relative warps, RWs) shape components for each speci-
(Camfjal-Hodn'gnez et ak, 2005). Lfsing the software men. Classical parametric tests w?ere peidbrmed on the
wteprseDleGs2tabv.lis2h.e1d0 ((FRiolgiulrfe 210).07aL)A,M19is ltahnedmaaprekxsof(LAtIh)e obtained vaidables by the SPSS/PC package v. 15.0.
shell; LAI2, LAI4 and LAI6 are placed on the right Molecular Systematies: Thirty-five live 24. giuTinii
border of the profile at the beginning of the three last and five live 24. cf. pauhormcnsis, from the Santa Tecia
DNA
complete whorls. LAI15, LAI17 and LAI19 are the samples, w?ere used foi' analysis. Tlie color pattern
corresponding landmarks on the left border of the pro- of each specimen was recorded before processing. The
file. LAI3, LAI5, LAI16 and LAI18 mark the intermedi- sliell of each specimen w?as broken in a mortar and the
ate position respectively between LA12 and LM4, LAI4 entire organism w?a.s homogenized in a 1.5 ml eppendorl
and LAI6, LAI15 and LAI17, LAI17 and LAI19 along the tube using 150 pi 2xCTAR extraction buffer (50 niAI Tris
cmwatiire of the w'horf LAI8 is at the low'er sntnre of the I4C1 [pH"8.0], 0.7 M NaCl, 10 niAI EDTA, 1% CTAR,
last complete wdiorl and LAM marks the intermediate 0.4% p-niercaptoethanol) vyith the addition of 10 pi of
position between LAI6 and LAM along the cmwatnre Proteinase K. DNA w?as extracted using standard CTAR
of tlie wdiorl. LAI9 is the most external position in tlie protocol (Doyle and Doyle 1987) wdth one extraw?ash in
upper part of the outer lip; LAIlt) and LAI12 are the plienokchloroforimisoaniylalcohol (25:24:1) and one in
most external positions respectively in the external right chloroforimisoarnylalcohol (24:1) in order to eliminate
and left part oftlie outer lip; LAIl 1 is the lowest point at jiolysacchai'ides. Tw'o mitocliondrial DNA markers were
the base; LAI14 is the most e.xternal point in the last amplified by PCR: (1) a 337 bp fragment of 16S rRNA
whorl at the left profile oftlie sliell; LAI13 is the profile was amplified in a 20 pi final xolnme containing 1 pi
point between LAI12 and LAI14 (close.st to LAI7). As template DNA, 2 pi of lOX Roche diagno.stic PCR reac-
described in Caiwajal-Rodrignez et al. (2005) the matrix tion buffer, 2 pi dNTPs lOX (2 niAI), 0.8 pi of each
of raw? coordinates generated by tpsDIG2 w?as used primer (20 pnioPpl), 1 pi Riogeni Tacjpolymerase (3 n/pl),
in tpsRelw v.1.45 (Rohlf, 2007b) to compute shell size 0.2 pi BSA. The primers w?ere (designed w?ith Oligo v.
(CS), nnilorm (U1 and U2) and non-miiform (several 6.71 software), U37 (5'-AGACAATTACGCTGTTATCC
Page 306 THE NAUTILUS, Vol. 123, No. 4
CTGT-3 and L373 (5 -AGAGAATTACGCTGTTATCC degreeof shell degradation. Thehqies appearedtobeveiy
)
GTGT-3’) \\4th atarget length of360 bp; PGRconditions similar to specimens identified as R. guerinii from die lots
were: 94°G for5 min, 40cycles of94°G for 1 min, 50.1°G Rea, Rcb, Rsm and Rem (see Table 1). ParatiqAes were
for 1 min and 72°G for 1 min and a final elongation step rather slender with a reduced number of ribs often
of 7 min at 72°G; (2) a 372 bp fragment of GOI was showing a high degree of bluntness tow^ards the earlier
amplilied in a in 20 pi final volume containing 20 to whorls. This feature of the ribs was exacerbated on the
50 ng template DNA, 2 pi of lOX Roche diagnostic spire ofthe holotype, giringthis specimen the most pecu-
PGR reaction buffer, 2 pi dNTPs lOX (2 mM), 1 pi of liar aspect of the shells ofRqie series. The shell pigmenta-
each primer (20 pmol/ pi), 1 pi Biogem Taq polymerase tion was ty^Aical of R. guerinii: white with brown spaces
(2.5 n/pl), 0.2 pi BSA; the primers were, 59R-GOI betiveen ribs and a gray-\iolet apex. The tube with the
(fonvard: 5-ATTGGTGGGTTTGGAAATTG-3’) and lioloRqDecontainedalabelwithtwodifferenthandwiitings:
59L-GOI (reverse: 5-GATAGGGTGAGGAGGTGGTG-3’; Verdnin’s original note: “Rissoa panhonnensis Verduin/
PanicoandPatti,2005)with atargetlengthof450bp; PGR Det.: Verduin, 1983” and an additional indication added
conditions were: 94°G for 5 min, 40 cycles of 94°G for in handwriting: “7 paraRq^es”. Unfortunately, the staffof
1 min, 45°G for 30 sec and 72°G for 45 sec and a final the malacologicalsectionofRBINSwris unabletoidentify
elongation step of7 min at 72°G. thishandwriting. Theboxcontainedalargerlabel withthe
PGR products were separated liy gel electrophoresis text: “Rissoa panhonnensis VERDUIN/Palerme/Lemoro,
andpurifiedusingtheQIAquickgel e.xtractionkit (Qiagen, Monts./PARATYPES”. We were notable to find the origi-
GmbH, Hilden, Germany) following the manufacturer’s nal label with the inscription “Rissoa guerinii. Reel./
instructions. Purified products were sequenced on a Palerme, Lemoro Monts.” mentioned by Verduin (1985).
Beckman Geq 2000 automatic sequencer, using a Dye- This hasprobablybeen lost.
terminator cycle sequencing kit (Beckman) according to
Morphometiy: The eigenvalue of the disciiminant
manufacturer’s instructions. Sequences were assembled
using the DNASTAR computer package (Lasergene), function between the two species was 0.683, the canoni-
supplied with the Beckman sequencer. Sequences cal correlation 0.673 and Wilks' lambda (0.594) was not
obtained for different marker from the same indiUdnal significant (p > 0.05), indicating alackofanystatistically
valid separation between the two groups. As shown by
wteedreascosnicnagtleenasteeqdueinncBeioaendidt av.li5g.n0e.d6 (wHiatlhl,G1o99d9o)n,Gtoredae- Table 2, of the variables employed, the number of
AVlIiA)g,neursivn.g G1.l6n.s3ta(lGWo(dTohnoGmopdseonCoeitqa^ho,ra1t9io9n4,) aDleidgnhmaemn,t gruiibsbheidngwhtohrelsWowagsrotuhpes.mMoosrteoivmepro,rtiatnwtasonteheionnldyistoinne-
revealing a consistent degree of correlation w4th the
method. The alignment was refined by eye. For all sam-
discriminant function (0.772).
ples, both fom^ard and reverse strands were analysed.
Genbank accession numbers range from GUI77879 to Using the values ofthe discriminant function ofeach
GU177963 for the 16S gene and from GU177964 to individualtopredict itsaposteriori species membership,
GUI78011 for the GOI gene. 21 (84%) indi\iduals, out ol the total 25 used in the
The concatenated se(|uences were subjected to Maxi- analysis, w^ere attributed to the correct species and only
mum Parsimony and Vlaximum Likelihood tree recon- four (16%) w^ere erroneously a posteriori classified.
struction using PAUP* V. 4.04 (Swofford, 2003). Rissoa sLiosokwienrgeactosrpreeccitelsy satsastiisgtnicesd, atlol sthheilslsspoefciRe.s.paOnnhloynnfeonu-r
labiosa (Montagu, 1803) was used as the outgroup (Gen-
bank accession numbers: AY676128 for GOI and (20%) specimens of R. guerinii were erroneously
AY676117 for mtlOSrRNA). The program Modeltest assigned to R. panhonnensis, wdiereas 16 (80%) were
assigned to the correct taxon.
version 3.06 (Posada and Grandall, 1998) was employed
to selected HKY + I model for VIL analysis. Trees were Li\e-collegted Material: Head-foot: The intensity of
computed with 1000 bootstrap replicates. Bremer sup- the jrigmentation ofthe shell and head-foot enabled two
port values (Bremer, 1994) were used in conjunction color tAqies of Rissoa guerinii to be distinguished, \4z.
with bootstrap. A reduced median joining network (M[) ty^iical R. guerinii and R. guerinii “var. conspersa" (Dant-
(Bandelt et ak, 1999) was obtained with the software zenberg and Dnronchoax, 1914; Figure 4 and 5). In both
Network v. 4.5 (Flu.xns Technology).
Table 2. Shell variables in Rissoa ,spp. and their coefficients
ami correlation with the discriminant fnnetion.
RESULTS
DF Correlation with
Coelhcients DF
Daut/enbergGollegtion VIatehial:'Vi.siialOb.sei-vation:
Thety|Ae specimens ol Rissoaf>anltoniicnsiswere keptin a Hihhet! whorls 1.020 0.772
glass tube in a small cardboard box. The holoty}re was Terminal rihs per -0.0.52 -t).27S
iscdated from the pararipes and enclosed in a small plastic w'hoi1
case. Tliebad stateol presemition ol thepeiiostracnm and Do -0.075 -0.236
d -0.292 0.151
the consistent presence ol mineral concretions, visible on
thesurlaceand in the insideol someshells, indicatetlsome Lc'iigth -0.404 0.031
F. Criscione et al., 2009 Page 307
Figures 2-5. Pigmentation ofthe soft bodyparts ol livingspecimens. 2. Darksmudge of the middle part of the sole. 3. Rissoa cf.
panhonnensi.s. 4. R. guerinii “var. conspersa." 5. R. guerinii (typical pigmentation). Scale bar = 1 mm. Drawings by Danilo Scuderi.
t)q5es, the footwaswhitish and the middlepart of the sole analysis forshell shape measurements conductedbystep-
was stained brown (Figure 2), this latter feature being wise multtple regression analysis for centroid size (as
lighter in ty|3ical R. guerinii than in “var. conspersa.” The dependentvariable) andtwo uniform and 29non-nniform
snout was light browai in R. guerinii and darker browm in measurements, as independent variables. The F-test of
“var. conspersa The margin of the distal portion of the tlie regression analysis was significant (p < 0.05) and only
snout and the rest ofthe headwas yellowish in R. guerinii one relative waip, RW3, contributed significantly to die
andlightbrown in“var.conspersa."Thecephalictentacles regression model on tlie centroid size (Beta = -0.406).
were whibsh, but sometimes dark brown in “var. con- Centroidsize, uniformcomponents andonlythe firsteight
spersa.” Awhitish spot behind the base ofcephalic tenta- relative waips were considered in the analysis of variance
cles was always present. The body pigmentation ofR. cf (ANOVA) performed to evaluate die significance ofdiffer-
panhormensis was similar to that offi. guerinii “var. con- ences in size and shape variables. Shells of R. guerinii
spersa” with a shght tendency to be darker (Figure 3). and R. cf. panhormensis differed significantly in U1 (p <
Table 3 summarizes the comparison among the different O.OOf), RW2 (p < O.OOf and RW6 (p < 0.05). The cumula-
pigmentation patterns. tive results of the analysis are shown inTable4. The signif-
icancelevel obtainedforthecorrectedanalysis (ANCOVA)
Geometric Moiijhometry: Table 4 shows the per- with centroid size as covariate was not maintained for die
centages and a descriptive statistical summaiy ofthe rela- difference in RW6, but was onlyslightly affected for R\\^2
tive score for CS, the two uniform components and the and for U1 (botli with p < 0.05). In addition, a significant
first 8 RWs, explaining more than the 91% ofthe overall difference was found between the two groups also for
variation. Table 5 shows the results of the allometric RW3 (p < 0.05). Table 4 shows these results.
Table 3. Sumniai-y ofthe obseivation on the pigmentation features in tlie specimens ofPii.s.soa spp. considered.
Feature R. guerinii R. g. “var. conspersa" R. cf panlionnensis
Foot colour whitish whitish whitish
Sole stain colour light brown darkhrov\ui darklirown
Snout colour light brown dark brown dark brown
yellovrish distally light brown distally light brown distally
Head colour yellowish liglit brown light brown
Cephalic tentacles colour whitish whitish ordarkbrown whitish ordark brown
Tentacularspot colour whitish whitish whitish
1
Page 308 THE NAUTILUS, Vol. 123, No. 4
Table 4. Descriptive statistical siimmaiyand results ofANOVAand ANCOVAforthe main shell size and shapevariables between
Rissoa otieriiui and R. of. panhoniicusif;. p < 0.05, p < 0.001, ns = non significant.
Measure CS U1 U2 RWl R\V2 RW3 RW4 RW.5 RW6 RW7 RWS
\'ariance explained .39.3% 27..5% 7..3% 5.1% 4.1% 3.2% 2.5% 2.0%
R. guerinii Mean 1021 -0.010 -0.002 0.002 -0.017 -0.005 0.000 -0.002 0.004 0.001 -0.001
St. dev. 122 0.014 0.010 0.0.39 0.025 0.017 0.014 0.010 0.011 0.01 0.009
R. cf. jHinljormensis Mean 996 0.010 0.002 -0.002 0.017 0.005 0.000 0.002 -0.004 -0.001 0.001
St. dev. S9 0.013 0.007 0.0.38 0.029 0.014 0.014 0.014 0.008 O.OOS 0.008“
ANOVA 0.41 18.26”* 1.47 0.06 11.80*** 2.49 0.04 "" 0.64 5.52** 0.38 0.34
“
ANCOVA 9.09** 3.14 0.56 5.69** 3.84** 0.50 '" 0.89 3.31 0.30 1.65
The eigemalue of the stepwdse discriminant function values of70-75 (MP and ML respectively). The M| net-
between the two species, calculated for all 29 RWs, was work (Ligure 5) confirmed the presence of a common
4.033, the canonical correlation 0.895 andWilks lambda haploUpe occurring in 19 specimens (n = 19) of both
(0.199) was highly significant (p < 0.001), indicating a R. guerinii (n = 15) and R. cf. panhonnensis (n = 4), one
good separation between groups. Seven shape variables haplotyj^re occurring in tw'o specimens of R. guerinii, 17
contributed to the discriminant function (RW2, RW6, unique haplot)qres, and one haplotype that occurred in
RW13, RW3, RW27, RW16, and RW25). The standar- onespecimenofeachspecies. 14haplotyj^res areseparated
dized coefficient matrix (Table 6) shows the relative im- by 1-5 differences from the main one, but four showed
portance of the independent variables in determining largerdistances (27, 25, 23 and 15 respectively).
the standardized canonical discriminant function. The
mean values of the discriminant function for the two
groups were -1.940 for R. guerinii and 1.940 for R. cf DISCUSSION
I'lanhormci^sis. Using the individual values of the dis-
criminant functions to predict a posteriori species mem- Dautzenberg Collection AI.aterial: VisualObservation:
berships, 26 (86.7%) individuals out of30, were assigned Monterosato (1884) considered R. costulata Alder, 1844,
to the correct species, lea\4ng only 4 (13.3%) that were and R. subcostulofa Schwarz, 1864, as synonyms of
erroneously classified. Looking at species statistics, 13 R. guerinii, so we checked if the original label for
(86.7%) specimens ofR. guerinii were correctlyas,signed R. panhonnensis might be found accompanying a lot of
to this species and only two (13.3%) were assigned to those two tcLxa. Onlytwo labels were found to have these
Re.rrcoln.epoaunsJlnynicnleansssiifsi.edThspeecsiammeenspeorfceRn.tacfg.espaonfhcoonrnreenctsliys/ cRhearmact(eRriissstoiacsc:ostthuahtitao/fMetchliet.a/lMroenatdsy.)meanntdioonneedfosuanmdplien
were obseiwed. In Ligure 3 the thin plate spline repre- the bottom ofa box containing lots ofR. guerinii coming
sentation allowed us to interpret in geometric terms the mainly from Lrench coasts, whose inscription was:
positive (characteristic ofR. cf. panJiormensis) and neg- "Rissoa costulata, A/f/cr/Palerme/Lemoro Monts.”. While
ative deviations (characteristic of R. guerinii) values for it is possible that one ofthese could be the original label
the most significant non uniform shape variable, RW2, accompanying the t)qie lot ofR. panhonnensis, it is un-
between the two species. likely as it does not correspond exactly to the wording
given by Verduin. We are unsure if the tyjre material
Molecular Phylogeny: After combining the COI and originallyconstituted a single separated sample orwheth-
16S rRNA sequences, aconcatenated sequence of709 bp er it was a part of a larger sample from which Verduin
was obtained, yielding20differentbaplohq^es 18ofwhich isolated seven specimens. However, there was a strong
in\’olved exclusively R. guerinii, while the two remaining resemblance behveen R. panhonnensis t)q)e material and
ones were shared by both R. guerinii and R. cf. panhor- the four lots referable to R. guerinii (Rea, Rcb, Rem and
mensis. Thetopologies ofthe MPand MLtrees (Ligure4) Rsm) suggesting that these lots may at least share a com-
were comparable: fcnir haploty|ies of R. guerinii occupy mon geographic origin.
nested basal positions in the tree, while the renitiining 16
haplotypes, belonging to R. guerinii and R. cf. pr/zihor- Table 6. Standardizetlcoefficient matrixshowingtherelative
niensis, form a terminal clade supported by bootstrap importance ofthe shape variables in Ri.ssoa spp.
Tabic 5. Multiple regression model to test allometiy (or the Discriminant function
iioii-niiilorm shell shape variables in Rissoa spp.
RV\'2 1.145
RW6 -0.929
Multiple regression Variables in the moilel RW13 -0.702
1*0“ F Name Beta RW3 0.6S6
0.165 5.6** RW3 -0.406** RRWW2176 00..568813
< 0.05, < 0.001. RW25 0.460
.
F. Criscione et al., 2009 Page 309
G2
G4
G5
G8
G9
G11
G13
G14
G16
G19
G22
G27
G28
G3 G34
Figure 6. Thin plate spline representations for RW2, G18 + P4
showing the deformation ofthe grid for the average values of
Rissoaguerinii (left) and R. cf. panhonnensis (right). G1 * +(P1 P2 P3 P5)
G35
Another important obsemition was the unrepresenta-
tive nature ofthe holoty|3e with respect to the paraty|:>e G29
series. Tliis shell, rather than summarizing the average
G20
characteristics ofthe tyi^e series, represents instead tlie
most extreme variant with a nearlytotal lack ot ribs. The
G25
illustration provided by Verdnin for this shell is not of
good quality and this has contributed to perpetuating R. labiosa
the idea that R. panhormcnsis is a ribless species. This Figure 7. .Vlaximuni Likelihood treeobtained from concate-
characteristic is reflected in the shells of our sampled nated sequences of 16S and COl genes obtained lor Rissoa
specimens, here referred to as R. cf panhormcnsis guerinii and R. cf panhonnensis. A number of1000 bootstrap
Moi-phonietiy: Verdnin 1985) stated thatthe samples replicates were performed and its values (if above 50%) are
( shown on the nodes. Bootstrap values for Parsimony are in
containing the type specimens were proliably dredged, bold; in brackets the Bremer index values. Letter G refers to
because Monterosato used to obtain detritus from fishing R. guerinii baplotvpes, letter P to R. cf panhonnensis haplo-
nets and then pick out and classify the interesting shells. tqres. Gl* = GL' G6, G7, GIO, G12, G15, G17, G21, G23,
Using this method he selected the samples that he G24, G26, G30, G3L G32, G33. Specimens ofB. guerinii "var.
rettrined for his collection. In view ol this possible lack of conspersa”are underlined.
randomness in the samples obtained from Monteiosato,
we could not use them to relialrly infer inteq^opnlational observedapextv[:)es exceedingVerduin’s arbitrarv?, species
or interspecific differences betw^een samples. Our mor- specific “cut-offvalues” witliin R. guerinii (Criscioire and
phometric investigation arose from the obseivation that Patti, submitted.). Hence, if only the dimensions of the
the type material oi Rissoa panhormcnsis strongly resem- apexwere used to distinguish R. panhormcnsis (tvqje ma-
bles R. guerinii, despite the claim ofa morphological (and terial) from Dantzenbergs R. guerinii samples, then this
morphometric) distinction between both taxa (Verdnin, would not result in a clear separation of both taxa. In
1985). This claimwas mtiinlybasedon alleged differences otherwords: the R. panhonnensis typeswere not the only
in the size ofthe apex and differences in the number of specimens among Dantzenbergs R. guerinii material t<r
shell ribs. However, our discriminant analysis ot three have the larger type ofapex.
protoconch variables (d. Do and At) did not support sig- Although number of whorls (N) and shell length (L)
nificant differences l)etween the size of the apex ot show considerable intra.specific variation, they nevei'the-
R. panhonnensis and R. guerinii, and both belong to tlie less often reveal consistent interspecific differences (F.C.,
larger apex category (At > 0.235 mm). Moreover, we pers. obseiv.). However, our statistical analysis revealed
Page 310 THE NAUTILUS, Vol. 123, No. 4
R.guerinii(35)
R.patihormensis(5)
R.labiosa
medianvector 1mutation
Figure 8. Median Joining Network drawn from concatenated sequences of16S and COI genes obtained for Ris.soa guerinii and
R. ct. paiihormensis. In round brackets tire number ofsequences enrployed. Dashed lines represent higher number ofmutations
(values reported nearby).
that the dilferences between the samples for these tw^o the analysis for CS (ANCOVA), wdiile that of RW2 de-
characters were not significant. Finally, although Verdnin creased to significant (p < 0.05) and that of RW6 was
(1985) emphasized the low number of radial ribs of not significant, indicating that the shape difference ex-
R. panhormoisis, it is unclear to ns as to whether this plained by those variables was dependent on size. The
related to a smaller number of ribbedwhorls or a smaller difference in RW3, was not significant in the ANOVA,
number of ribs on the last whorl. In this study only the but became significant (p < 0.05) in the ANCOVA.
total number of ribbed whorls (RW) can be used to dis- Despite these variations, RW2 always showed the lower
criminate the two groups and this may be what Verdnin p value, which means that the two groups mostly differ
really meant as it appears to be the only difference by on this shape variable independently from the correla-
which R. panhonnensis and R. giierinii can be separated. tion between shape and size.
The discriminant function calculated from all the non
LiviNC AIaterial: Geometric Morphometiy: The uniform shape variables, was successful in morphome-
ANOVA did not reveal any significant difference in size trically discriminating the hvo groups. RW2 was the
(CS) between the twogroups, confirmingonr non-casnal most important variable in determining the distinction
obsemitions of the Dantzenberg collection samples. A behveen Rissoa Guerinii and R. cf panhonnensis. The
higli level of significance was instead observed for the mean values ofthis variable for each ofthe two groups
first of the uniform shape components, Ul, wdiich, ac- (positive for R. cf panhonncnsis and negative for
counting lor compression-dilation deformations, may be R. piterinii) were plotted in a tps representation (Fig-
inteqireted as indicating that Rissoa cf. panlioniicnsis ure 3). The plot showed that that variable RW2 is a
has a more slender shell compared to R. guer'Diii. Analy- rellection of the most obvious discernable shell shape
sis ol variance showed a highly significant difference dilference. Tins comprised the slenderer aspect of 71.
(p < O.(K)l) between die two groups in the second non cf ])aiihonm'mis, contributed byaconsistently narrower
nnilorm shape varialiles (RW2) and a significant diller- penultimate whorl (represented by the contraction of
ence (p < 0.05) for the sixth relative warp (HVV6). The the corresponding zone ofthe grid) than that of71. guer-
signilicance for Ul remained unaltered when correcting iuii. A similar interpretation of a single relative warp
F. Criscione et al., 2009 Page 311
resulting in shell slenderness has also been reported by CONCLUSIONS
Ccaasmel,jahio-wReovderri,gutehze eatbsael.nc(e20o0r6)reidnucNtaisosnariofus.axiaIln rtihbiss We find that Rissoa cf j)anhonnensis is a rare morpho-
may have affected the representation of horizontal t\pe of R. guerinii characterized by reduced develop-
dimensions, resulting in this visual difference. Tlie flat- ment of radial ornamentation, and that, as such, it
ter aspect of the whorls of R. cf. paiihormcnsis is also should be considered to be a junior synonym of this
linked to the lack ofribs. Also noticeable is the opposite latter species.
relative displacement of landmarks 8 and 9 (Figure 6)
and the subsequent modifications of the grid. In R. cf. ACKNOWLEDGMENTS
LM
panhonneusis, 9 is overlapped to a greater extent by
LVl S than in R. giierinii. This is because ofthe presence We would like to thank Dr. Winston Ponder and Prof.
of ribs on the penultimate whorl of R. guerinii, which Thieny Backeljau for having read and commented the
partlyoverhang the posteriorpart of the outer lip, gbdng manuscript and for the precious suggestious and
the impression of a less protruding peristome. In con- improvement provided. We would also like to thank
trast, the lack ofI'ibs in R. cf panhormenfiis accounts foi- Prof Jackie Van Goethem (RBINS) for the possibility to
the more protruding peristome ofthis moqrh. study Dautzenberg collection material. We also want to
Head-foot: In the genus Rissoa, the pigmentation of express gratitude to the Molecular Biology Seiwice of
head-foot is often an important species-specific charac- DStNazAione Zoologica “Anton Dohrn” (Naples, Italy) for
ter (Fretter and Graham, 1978). In addition, Rissoa sec|uencing.
species also often differ in the relative proportions of
head-footcomponents (cephalic tentacles, snout andante-
rior part ofdie foot) (D.S. pens, obseiw). One cannot use
these characters to distinguish R. cf panhonnemis from LITERATURE CITED
R. gtieiinii “van couspersa.”
Bandelt, H.J., P. Forster, and A. Rohl. 1999. Median-joining
Molecular Sy.steiiiatics: The combined 16S and COI networks for inferring intraspecific phylogenies. Molecu-
sequence data showed that R. giierinii and R. cf pan- lar Biolog)'and Evolution. 16: 37—48.
honnensis cannot be separated. Bickford, D.,‘d.J. Lolnnan, N..S, Sodhi, P.K.L. Ng, R. Meier,
K. Winker, K. Ingram, and I. Das, 2007. Cnptic species
Radial Ornamentation and Species Distinction: as a \rindow on diversirt and consemition. Trends in
Our results indicate that reduced radial ornamentation Ecologyand Evolution. 22:148-155.
is the only distinguishing feature between R. panhor- Boncliet, P. 1989. A reviewofpoecilogony in gastropods. Jour-
mensis and R. gtierinii. Problems with usingthe number nal of Mollnscan Studies 55: 67-78.
Bremer, K. 1994. Branch support and tree stability. Cladistics
ofribs for species distinction in the genus Rissoa are uot 10: 295-304.
new. There is evidence to show that rib number can be Can'ajal-Rodriguez, A., P. Conde-Padin, and E. Rolan-Alvarez.
influenced byenvironmental conditions. Forexample, in 2005. Decomposing shell form into size and shape by
fluctuating salinitv, R. parva (Da Costa, 1778) can shf)w geometric morphometric methods in two sympatric eco-
reduction in radial ornamentation (Wigham, 1975; Ver- types of lAftorina saxatili.s. Journal of Mollnscan Studies
dnin, 1976; Waren, 1996). Aloreover, several unpub- 71: 313-318.
lished field obseiwations made by the authors on other Cawajal-Rodriguez, A., J. Gnerra-Varela, B. Fernandez,
species of Rissoa, living in low salinity emdronments E. Rolan, and E, Rolan-Alvarez. 2006. An example of
(sheltered bays, coastal seagrass meadows), revealed the application of geometric morphometric tools to the mor-
tendency in entire populations (R. similis) or a small pliological diagnosis of two sibling species in Nas.sarius
(Mollnsca, Prosobrancliia). Iberns 24: 81-88.
number of individuals in a population (R. auriscalpiinn, Criscione, E. and F".P. Patti. F.P. Shells cannot tell much about
R. labiosa) to lack axial ribs. It appears that this latter phylogenv: Ris.soa guerinii Recluz, 1843 and Ri.s.soa Jin
situation is the case with R. guerinii. There is an abun- (.Monterosato), 1884(Gastropoda: Rissoidae)areonlyadop-
dant inflow offreshwater in our sampling site as well as tivesiblings?The Nautilus, [submitted, underreview]
other parts ofthe Sicilian Ionian coast. Where those con- Dautzenberg, P. and P. Donronchonx. 1914. Les mollnsques
ditions occur, it seems possible that R. guerinii might de la Baie de Saint-Malo. Fenille des Jennes Natnralistes
develop “smooth" morphopqres, as in onr sample locality. 44 (Snpp.): 25-64.
This view is in agreement wth the idea ofR. guerinii Desmarest, A.G. 1814. Description des coqnilles univalves dn
as a highly polymorphic anchor plastic ta.xon. Indeed, genre Rissoa de M. de Frenhnville. Nouvelle Bulletin des
bsepesciideess R.ofpaRnihsosnonaens(ei.sg., tRh.eredearceomotthaerPMhieldiiptpie,rra1n8e4a6n, DoyledScuJir.eJe.nclaeonrsds4m:Ja.7lL.l-9Dquoaynltei.ti1e9s87o.t AfrersahpitlleafDNtiAssuie.solPahtyitonocphreomcie--
R. torcfuilia, Pallary, 1912, and R. frauenfeldiana Bru- cal Bulletin 19: 11-15.
sina, 1868) whose shells are cpiite similar to R. guerinii. Fretter, V. and A. Graham. 1978. The prosobranch molluscs of
It would not be suqrrising if further studies show that Britain and Denmark. Part 4. Alarine Rissoacea. Journal
those species are also ecophenotvpes ofR. guerinii. ofMollnscan Studies, Supplement 6: 151-241.
.
Page 312 THE NAUTILUS, Vol. 123, No. 4
Hall, T.A. 1999. BioEdit: a user-friendly biological sequence Rohlf, J.F. 2007b. TpsRelw version 1.45. Available via http://
alignment editor and analyses program for Windows life.bio.sunysb.edu/mo1q3l1/soft-tps.html [Accessed July
9.5/98/NT. NucleicAcids Symposium Series 41: 9.5-98. 2008)].
Knowlton, N. 1993. Sibling species in the sea. Annual Review Swofford, D.L. 2003. PAUP*: Phylogenetic analysis usingpar-
ofEcologyand Systematics. 24: 189-216. simony (*and other methods), version 4.0b 10. Sinauer
Knowlton, N. 2000. Molecular genetic analyses of species Associates, Sunderland.
boundaries in the sea. Hydrobiologia. 420:73-90. Thompson, D.,D.G. Higgins,imdTJ. Gibson. 1994."GLUSTAL
J.
Mayr, E. 1963. Animal Species and Evolution. Cambridge, W:improvingdiesensitivityofprogi'essivemultiplesequence
Mass.: Ilan’ard University Press. ahgnmeiit through sequence weighting, position-specific
Monterosato, T.A. di, 1883-1885. Conchiglie littorali mediter- gappemilties andweight matrixchoice." NucleicAcids Re-
ranee. Naturalista Siciliano. 3(3): 87-91 (1883); 3(4): 102- search22: 467,3-4680.
111; .3(5): 1.37-140; .3(6): 1.59-16,3; 3(8): 227-231; 3(10): Thorson, G. 1950. Reproductive and laiwal ecology ofmarine
277-281; 4(1-2): 21-25; 4(3): 60-63 (1884); 4(4): 80-84; 4 bottom invertebrates. Biological Reviews 25: 1-45.
(8): 200-204 (188,5). Verduin, A. 1976. On the systematic of recent Rissoa of the
Panico, M. and F.P. Patti. 2005. Poecilogenesis in RLssoa aur- subgenus TurboeUa Gray, 1847, from the Mediterranean
iscalpiiim complex (Coenogastropoda: Rissoidae). Poeci- and European Atlantic coasts. Basteria40: 21-73.
logonv engine of speciation. Congresso Internazionale Verduin, A. 1982. On the taxonomy and variabilit)' of recent
delle Societa Europee di Malacologia, Naples, October European and NorthAfrican marinespecies ofthesubge-
200,5. nus Rissostomia Sars, 1878, of the genus Rissoa Desniar-
Posada, D. and K.A. Crandall. 1998. Modeltest: Testing the est, 1814 (Mollusca, Gastropoda, Prosobranchia). Basteria
model of DNA substitution. Bioinformatics 14: 817-818. 45: 143-166.
Rechiz C.A. 184,3. Catalogue descriptifde plusieurs nouvelles Verduin, A. 1985. On the taxonomy and variability of recent
especes de coquilles de France suivi d’obseiwations sur European and North African marine species ofthe sub-
quekpies autres. Rewiezoologiqueparla Societe CuMeri- genera Apicularia and Goniostoma of the genus Rissoa
enne 6: 5-12, 104-112, 228-2.38. 257-261. (Gastropoda, Prosobranchia). Basteria49: 10.5-1.32.
Rehfeldt, N. 1968. Reproductive and moiphological variations Verduin, A. 1986. On the systematics of some recent Rissoa
in the prosobranch Rissoa membranacea Ophelia 5: (Gastropoda, Prosobranchia). Basteria,50:13-21.
157-173. Wigham, G.D. 1975. Environmental influences upon the ex-
Rohlf, J.F. 2007a. TpsDig2 version 2.10. Available via http:// pression of shell form inRissoa paroa (daCosta) (Gastro-
life.bio.sunysb.edu/mo1q3l1/soft-tps.html [Accessed July poda; Prosobranchia). Journal of Marine Biological
2008]. Association ofUnited Kingdom ,55: 42,5^38.
