MALACOLOGIA. 2007, 49(2): 293-311

SPECIES DELIMITATION IN THE GENUS BYTHINELLA (MOLLUSCA: CAENOGASTROPODA: RISSOOIDEA): A FIRST ATTEMPT COMBINING MOLECULAR AND MORPHOMETRICAL DATA Jean-Michel Bichain'", Marie-Catherine Boisselier-Dubayle", Philippe Bouchet ' & Sarah Samadi'

ABSTRACT Within the springsnail genus Bythinella, few dlscontlnuous morphological characters allow to unambiguously delineate species-Ievel taxa . Opinions on the alpha-t axonomy of the group are divergent, with sorne authors recognizing every morphologically distinct local form as a species, while ethers interpreting such geographical forms as intraspecific variati on. Because the value of morphological characters was rarely contrasted with molecular data, such opinions remained untested. In thiswork, variation between populations was studied through genetics isozymes , phylogeny (DNA), and morphometrics. Eleven populations represenling rive putative specles were sampled from the french Pyrenees, a region where a high number of nominal species are classically recognized. Based on genetic and phylogenetic analyses, the mate rial clusters lnto three groups, of which one consists of several nominal species. Environmental factors , sexual dimorphism, and genetic factors contribute ta the significant morphological variation observed within the geneticgroups. Thus, the numberof species of Bythinella recognized in the Pyreneesappears probably overestimated, and thecharacters traditionally usedfor species delimitation should be re-evaluated. Keywords: hydrobioid springsnails, ITS-1 nucleargene, isozyme, phylogeny, morphometry, multivariate analyses.

INTRODUCTION Among gastropods, hydrobioid springsnails are one of those taxa in which the lack of obvious discriminating morphological characters makes species delimitation diffieult, resulting historically in the establishment of nurnerous nominal species and currently in a variety of specialist opinion on the taxonomie validity of these taxa (Kabat & Hershler, 1993; Herschler & Ponder, 1998; Wilke et al., 2001 ; Szarowska & Wilke, 2004). A consequence is that different speeialists have differentevaluationsof the magnitude of biodiversity. The difficulty of resolving sueh differing opinions is also hampered by the lack of a solid theoretical and methodological framework, de facto rendering many taxonomie opinions untestable hypotheses. However, and although delimitating

species boundaries is a central aim of aiphataxonomy, there ls no consensus coneerning the meaning of the term "species" (as instances of the extensive literatureon thisquestion , see, e.g, Howard & Berlocher, 1998 ; Wilson, 1999; Winston , 1999 : Wheeler & Meier, 2000; Hey, 2001a , b; Mallet, 2001; Noor, 2002). The objective of this paper is ta test the validity of the traditional delimitation of some species belonging ta the genus Bythinella Moquin-Tandon , 1856, based upon a species concept that permits testable hypothèses. For this purpose, we use the Hennigian inter-nodal species concept recently developed and formalized by Samadi & Barberousse (2006). This species definition is close ta the Evolutionary Species Concept (Simpson, 1961 , modified by Wiley, 1981; Wiley & Mayden ,

'Muséum National d'Histoire Naturelle, Département Systématique et Evolution, Unité Taxonomie et Collections (USM602UMS2 700), Case Postale 51, 55 Rue Buffon, F-75005 Paris CEDEX 05, France ' Systématique. Adaptation et Evolution, UM R 713 8 P6-I RD·MNHN-CNRS (U R IRD 148), Service de systématique moléculaire (CNRS, IFR 101 ), Département Systématique e t Evolution (USM 60 3), Muséum National d'Histoire Naturelle , Ca se Postale 26 , 57 Rue Cuvier. F-752 31 Paris CEDEX 05 , France *Correspondlng author: [email protected]

293

294

BICHAIN ET AL.

2000), the Internodal Species Concept (Kornet, 1993) and the General Lineage Species Concept (De Dueiroz, 1998, 1999) . For De Dueiroz (1998), ma st "species concepts" are ill-named , because they should rather be

regarded as recognition criteria that, associated with a formai definition of the "species " that is independent of the mechanism by which

species are recognized, permit delimitation of specles in practice . As suggested by these authors, the various techniques used in sys-

tematics can be set in a coherent framework starting tram species descriptions bas ad on morphology and/or behavioural and ecologi-

cal attributes . These descriptions are the primary hypotheses for species delimitation that correspond ta alpha-taxonomy. Second, these hypotheses can be tested against criteria de-

rived trom the varions so-called "species concepts ", such as the Phylogenetic Species Concept (PSC) and/or the Biological Species Concept (BSC), corresponding respectively ta the approaches of phylogenetic systematics (sensu Hennig , 1966) and evolutionary systematics (sensu Mayr, 1942). Finally, the results of these tests permit the selection of character sets corresponding to more robust primary descriptions. Here we attempt ta deIimit sorne Bythinella species, in a restricted geographical area, in coherence with the theory of evolution and ta justify why, as argued for example by Sites & Marshall (2003, 2004), we need an integrated approach in taxonomy. Bythinella species live in emerging groundwater springs and in the uppermost courses

of small streams, where they can form large populations (1,000-1 O,OOOs individuals).

marily delimited based on non-discrete characters. A consequence of this continuous variation of character states is a range of taxonomic opinions among the various taxonomie

authorities . For example, in Italy, based on shell morphology and reproductive anatomy, Alzona (1971) recognized elght species of Bythinelfa, whereas Giusti & Pezzoli (1980) regarded ail Italian populations as belonging to two species. Based on shell characters,

Radoman (1976) recognized about 12 species in the Balkans and Asia Minor. Based on shell morphology, soft part anatomy, and geographical distributions, Falkner et al. (2002) recognized 42 valid species in France, with several

applications of species names differing from those of Bernasconi (2000). Thirteen of the se species were restricted ta the Pyrenees and theirfoothills . However, in Poland analyses of enzyme polymorphism and morphometric studies performed on shell and anatomical

characters (Falniowski et al. , 1998, 1999; Mazan, 2000 ; Mazan & Szarowska, 2000a , b) questioned the ability of the shell and anatomical characters used in alpha-taxonomy ta de-

lineate species of Bythinelfa . Consequently, because of these various opinions, whieh recognize these taxonomie entities alternatively as "good species" or not, we have no clear

idea of the true diversity of this group. Because oftheir narrow ranges and speeialized habitat, hydrobioid springsnails in gen-

eral, and Bythinelfa species in particular, are vulnerable ta even small-scale habitat transformation , such as trampllng by cattle or artificial diversion of springs. Several hydrobiid species are considered extinct , and many oth-

but occaslonally with spiral keel(s) or one or

ers are categorized as threatened (IUCN 2005, http://www.redllst.org). Of the species occurring in France, nine are nationally protected (statutory order October 7,1992). Legal protection was bestowed upon them based on the state of taxonomie knowledge in the 1980s, and it is now fitting ta evaluate whether the names involved in the legal texts apply ta real units of biodiversity. Of the putative species

Sorne taxa live exclusively in groundwater

(Giusti & Pezzoli, 1980; Falniowski, 1987; Bernasconi , 2000; Bertrand , 2004; Bichain et al., 2004). The smail (usually around 2.5 mm , maximum 4 mm), nondescript shell is charac-

terlzed by a blunt apex, with an overall ovoid to conical-elongate shape, most often smooth, more axial varices on the last adult whorl. Spe-

analyzed in the present study, two are nation-

cies of Bythinelfa are gonochoristic. Males are characterized anatomically by a penis-flagel-

ally protected .

lum complex; females have a bursa copulatrix and a seminal receptacle. France is an area

BytlJinella speeies from the Pyrenees was explored according ta criteria derived from the PSC and the BSC , and our purpose was ta test if sets of morphological characters (traditian al or newly developed) correspond ta these criteria. For this purpose, we used populations

of high specifie richness for the genus. Of the species currently recognized in Europe, 62% occur in France , ofwhich 90% are endemic ta

France (Fauna Europaea, 2004). ln Bythinelfa , as in the maj ority of hydrobioid (sensu Davis , 1979) genera , species are pri-

Speeies delimitation in a number of nominal

representing nominal speeies sampled from their type loealities or from the nearest pos-

SP ECI ES DELIMITATION IN TH E GENUS BYTHINELLA sible area if the preci se type locality cou ld not be foun d or no longer exis led . We performed rnult lvar tate ana lyses on shel l parameters to evalua te whether the pattern of she ll variation

295

two springs . The the rmal spring is the type loc alil y of B. rubiginosa (Boub èe, 1833), a nominal

speci es consid er ed ta be res tricted

ta this locality; the applica tion of the species

within a nd amo ng populat ions permit s non-

nam e is thus unam biguous. Th e population

ambig uously att ributing each of the sampled population ta a putative spe cies, and we tested species delimitation resulting tram Iraditional morphological approaches aga inst BSC and PSC crite ria. The BSC crite rion am ounts ta delimitation of species as reprod uctively isolated groups that be cam e ge netically isolaled from other su ch groups. Isolation was ev aluated indirecUy thro ugh analysis of ge ne flow

sa mp led fro m Aud2 had been ide ntifi ed by Be rnasconi (2000) as B. eurystoma (Paladilhe , 1870). However, specimens do no t match the

among populations using electrophoretic variability of isozymes. In the mast comman view of the PSC , spec ies are mono ph yleti c gr oups. Mo nop hy ly of gro ups wa s identi fied using the pattern of variab ility of the ITS-1 nuclea r gene . Th e se three approaches were compa red in

arder ta evaluate the potential use of lhe morphological cha racters in th e de limitation of Bythinella species .

MAT ERIAL AN D M ET HODS Taxa and Populations Studied

original description of this nominal speciesweil (type locality is St J ean-de-Fos, Département Hé raull ); we co nsequentl y d o not foll ow Bernasconi in app lying this name ta that population, w hich we will from herson des ignate as B. cf. eurystoma . T he th ird po pu lalion (Aud3 ) was not inci uded in Be rn asco ni's dataset, and we co uld not easi ly place it in one of the nominal specie s. As a geographica l out-group within Bytflinella, we used B. vir/dis (Po iret. 1801). the type species of the gen us, whic h we samp led from the type localily (Che) in the east of the Paris basin .1l has an ovoid shell that cleariy sets it apart from ail the Pyrenean Bythinella species included in our study. Specimens were collected by washing small pebbles, aq uatic veg eta lio n and dead leaves ove r Iw o sieves (2 mm and 450 pm mesh).

For anatomicaJand biometrica! studies, specimens were fixe d in 70% ethanol; for molecu-

The taxa selected for the prese nt study essentia ily orig inate fro m the Dé par te me nt Ariège , in the cen tral/ northeaste rn Py ren e es. Th e area has extensive karsts , with nume rDUS springs w he re BythinelJa species live in

dense populations. Ba sed on shell and re produc tive anatomy, Bernasconi (2000) had pro posed an alph a-taxonomy of Byth inella from this area, and we followed his treatment in how we applied specifie names ta each of the popu lations sampled . Eleven popu lations were sampled that, based on Bern asconi (2000) and Falkner et al. (2002), correspond ta five speci es (Table 1, Fig. 1). Ten of these populations, representing four putative species, are from the Pyrenean foo thills, but B . reyniesil (Dupuy, 1851) is a high er ailitude species . Bythinella simoniana (MoquinTandon , 1856), a spe cies relatively weil ch aracterized by one or more axial varices on the body wharf , was sampled from lhree populatio ns (Eng , Ca l and Sou). Fou r populal ions (Td1 1, Td12, Suz and Roq) are attributable ta B. utricufus (Pa ladilhe, 1874) . At Aud inac, we sam pled Bythinella from a therm al spring (ca . 18-20'C; Aud 1), from a coId sp ring (ca . 13' C; Aud2) j ust 30 meters away, and from the co nfl uence (Aud3) of the

lar studies, specimens we re Fraz en alive al

-80' C. Given the very sma ll size of th e spec imen s, each of the differ ent analyse s (morphometric , isozyme and DNA seq uence analys es) could not be carried out on the same individuais. Isozyme Electrophoresis

Protein extraction was carried out on whole anim aIs (including the shell), and followed the protocol of Boisselier-Du bayle & Gofas (1999) . El e ctro ph or esis w as do ne usin g ve rtica l acryla mide gels on dis conti nuou s sys tems .

T he running buffe r was Iris-glycine (4.95 mM , pH 8.3 ). Of lh e eight enz yme sy stems assay ed , thre e gave scorable band ing patterns and are used in this stud y (pho spho glucomutase : Pgm , EC 5.4 .2.2; glu cose-6-p hosph ate isomerase : Gpi, EC 5.3 .1.9; aspartate aminotransferase: Aat, EC 2.6.1.1 ). Because of lhe small size of the specimens , il was technically not feas ible ta study ail three enzyme systems on the sa me individual. We therefo re extracted be twee n 30 and 40 indi viduals pe r popu lation, giv ing approximately 20 for eac h loci. In talai, 439 individuals we re used for the analyses (Table 1).

.......- 'f--- -1-'-- l-l-t-

x

o

Wap2 W lw W sh

FIG. 2. Morphologieal parameters taken from shell of Bythinella in standard position: the collurnelar axis and the ape rture plane are parallel to the horizontal plane . Left - Landmarks locations. O r~

thonormal frame (Ox, Oy): the Oy axis Is paraliel ta Ihe collumelar axis and tangential ta the left edge of the shell. The Ox axis is perpendicular to Oy axis and tangenlial to the lower edge of the aperture. The aperture center ls the center of the E ellipse which is adjusted with the aperture edge. RighlMeasurements taken, Shell height (L,,), aperlure length (L,,, 2'[x,- x )' +(y,-y,,), D, aperture heighl (L" ,) , aperture diameler (W,,, 2, [x,,-x,,)' +(y,,-y,,),J, aperture widlh (\lJ"1 x,-x,,), shell widlh (W",), wldth of lasl whorl (W~ = x, ), widlh 01 penultimale whorl (W.. = ' [x, -x, '+( y,-y l'D, widlh of antepenultimate whorl (W lM' = ~[x:f XJ )2+(Y2-YJ)2]). Height measurements of the spire of each wholi were taken on the left and on the riqht sidas of the shell: left and right spire heights (LSP1 =YI-Y2' ~2 =YçYs). left and right helghls of penultimate whorl (L.... L..,), left and right heiqhts of last whori ( ~ " ~,) .

=

=

=

and

Analysis of Sexual Oimorphism - Sexual dimorphis m in Bythinella had been reported by Falniowski (1987) but never quanti fied. We assess ed first how sheli dlmorphism might interfere with specie s delimitation wl thin populations " For this. we analyzed sheli param eters in 59 individuals Irom Roq . The input dat a ln miilim eters were transformed lnta Log-sh ape Ratio (LSR) data in arde r ta limit an eventual size effe ct (Mosiman n, 1970 ). We first performed a Principal Component Analysis (PCA) ta explore how the range of sheli param eters was dis tributed between males and females . Then a Discriminant Function Analysis (OFA) was used ta test stat istically the differe nces betw een males and fema les and the n ta iden-

tily whic h shen paramelers reflecl sex ual dlmorphism . Wil ks' Lam bd a (W L) used in an ANOVA (F) lest of mean differences was used to test if the discriminant model as a whole

was slgnifica nt. Then , if the global F test was significant, each va riable was tested using

Wilks' lambda to determine which variable differed significantly in mean between discrimi-

nated groups. Second , we evaluated the effecl of sexual dimorphism a mo ng population s, taxonomi e

groups and genetic groups. For this purpose, an analysis of variance (ANOVA) with two fixed factors (population and sex) was ca rried out on the parameters reveaied by the DFA as significantly contributing to shell dimorph ism in

BICHAIN ET AL.

300

also be discriminated based on thelr shell. We then comparedthe reciassification scores of the discriminating linearfunctions of the twogrouping methods (i.e., taxon or genetic groups). Ali analyses were performed with the software package STATISTI CA 6.0 (Statsoft, 2001)

the population analyse d. This analysis permitted comparison of the relat ive elfects of sex and population location on she ll shape, and

identification of the interaction between these two factors. Tukey's HSD post hoc test and its assoc iated probability were used ta examine the statistical significance of the differences betwee n aI l pairs of means.

and the level of significance use d was 5% .

RESULTS

Analysis of Shell Variation Between Popu lations and Taxa - DFAs and assoclated Wilks' Lambda were performed on ail 15 measure-

Isozymes Polymorphism

rnents afte r LS R tra nsformation, and two discriminant factors we re tested: (i) the Taxon discriminati ng factor: popu lations referred a priori to a nominal species based on the col-

The three enzyme systems involving lour putative loci revealed Irom three ta five alleles per locus (Table 2), and the mean number of alleles per population ranged tro m 1.0 ta 2.25 (Table 3). The Aar-t locus was the least poiymorphlc, with a total 01three alleles overall and with each population being monomorphlc. The one population (taxon) used as the out-group (Bythinella viridis) was monomorphic for ail four loci, as was the Sou population. For the other populations , the mean observed heterozygosity (Ho) ranged lrom 0.015 ta 0.241 . Genotype

lection site were tested a posteriori ta check whether they couId be discriminated based on shell parameters (popu latio n Aud3 was exciudad tram the analysis because it was not

assigned ta a nominal taxon); (ii) the Genetic discriminating fac tor: clusters of populations resulting lrom the genet ic and phylogenetic ana lyses we re tested a posteriori ta check whether specimens grouplng together could

, - --

-

-

-

Che

I-- - - - Po,

1il. viridis -

Chc! -Chc3- -

1

-

-

Por~

rau

B. mr iculus

......

B. stmonùnm

Eng Sou

35

AUd2

"t,dl 74

1

Roq

1

-

-

-

-

1--

95 3

100 12

cm3 ~

70

,

-

_-_._-- --- -_._-- -_.....

Cut

54

-

Suz5

SU l.

Ion

ruu.z

SU,, ~

Roq

64

-

Tdll .1

Tdll

60

-

100 15

B. reynies ii

60

_ 0._' _

-

1B. cf. eurystoma

Soul

Audl .3

1

Aud2.6

1

A"dll ~ Aud!.:!

1B. rubiginosa

Aud3. 1

Alld] . Bythinclia sp .

Aud3.2

77 1

. .. .. . - .. .. . . . . __. .. -

FIG. 3. NJ tree constructed trom isozyme data on the 0 cha rd distance (Cavalli-Sforza & Edwards,

1967) between populations of Bythinella (left) with the Bootstrap values and parsimony tree (strict consensus) computed from ITS-1 sequences(right)with a heuristic search (Boatstrap values above, Bremer index below).

SPECIES DELIMITATION IN THE GENUS B YTHINELLA

301

TABLE 2 . Allele frequencies observ ed at 4 loci on 12 populations of Bythinella. N ;; number of indlviduals ana lysed for each locus.

Aud1 Aud2 Aud3 Pgm-I

pgm-2

Gpi- l

Aat-I

Tdl1

Tdl2

Roq

(24)

Suz

Par

Che

(24)

(22) 1.000

(7)

(N)

(24) (24) (18) (24) (18) (28) (21) (20) (19) (19) (17) 1.000 1.000 1.000 0.646 0.889 1.000 0.262 0.225 0.029 0.354 0.111 0.738 0.775 1.000 1.000 0.97 1

(N) 1 2

(23)

Sou

(22) (28 ) (25) (25) (24) 0.023 0.417 0.458 0.674 0.250 0.068 0.140 0.100 0.563 0.458 0.326 0.688 0.886 1.000 0.340 0.300 0.854 0.021 0.083 0.520 0.600 0.146 0.063 0.023

(N) 1 2 3

(24)

Eng

(N) 1 2 3 4 5 1 2 3 4

(24)

Cat

0.063 0.208 0.729 1.000 (9)

1.000 (24)

(25)

(22)

(27)

(22)

(30)

(27)

(30)

(26)

(26)

(24) (19) 1.000 1.000

0.958 1.000 0.955 0.981 0.932 1.000 1.000 1.000 1.000 1.000 0.042 0.045 0.019 0.068 (8) (1) (1) (9) (12) (12) (17) (16) (10) (14) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

(14)

(10)

1.000 1.000

TABLE 3. Genetic variability computed on the four ana lysed loci on Bythinel/a populations. Niot ;;

total number of individuals used for isozyme actlvlttes: n ;; mean number of individuals analysed per locus; P ;; percentage of polymorphie loci; A ;; mean number of allele per locus; Ho ;; observed heterozygosity; He ;; expected helero zygosily; HWE ;; Hardy-W einberg equilibrium te sts (Fischer's

melhod for test of significance. · significant at 0.05 level). Population

Aud1 Aud2 Aud3 Cal Eng Sou Tdl1 Tdl2 Roq Pa r Suz Che

Nlo t

n

P

A

Ho

He

HWE

35 38

20.3 20.3 18.8 21.8 19.8 25.5 20.8 22.3 17.5 19.3 17.5 11.3

0.5 0.25 0.5 0.75 0.75

1.75 1.5 1.5 2.0 2.25 1.0 1.75 1.75 1.25 1.25 1.5 1.0

0.104 0.146 0.164 0.134 0.124

0.150 0.146 0.135 0.244 0.137

0.22 1 0.712 0.003" 0.146

0.241 0.162 0.052 0.015 0.073

0.251 0.227 0.064 0.0 15 0.107

0.179 0.158 0.398 1 0.089

34

36 38 40 37 42 33 38 34 25

0.5 0.5 0.25 0.25 0.25

302

BICHAIN ET A L.

TABLE 4. F-statistics indices (Weir &Cockerham, 1984) calculated for the Bythinefla populations analysed (excluding out-group and Par popula-

tions. see text for exp ianations ). Locus

Pgm-l Pgm-2 Gpi-l afl

F,s

FST

FIT

0.209 0.179 -0.033 0.189

0.332 0.718 0.020 0.481

0.472 0.769 -0.012 0.579

frequencies generally matched Hardy Weinberg expectations aftar a sequenUal Bonferroni correction, except for the Cat population, which had a heterozygosity deficil. No genotypic linkage disequilibrium between pairs of loci was round. These four loci were together diagnostic far two of the six presumptive taxa : (i) individuels

from the type-Iocality of B. viridis had diagnostic alle les for the two loci Pgm-l and Pgm2; and (ii) individuals from Por, attribu ted to B. reyn iesii, had a diagnostic allele for Pgm- 2. Furthermore, individuals attributed to B. viridis and B. reyniesii together differed from ail other Byth inefla specim ens by the same diagnostic alleles at the two loci Gpi- l and Aat -1. Ai l other populations sampled from the Pyrenean foothills shared alleles wit h differ ent frequ enci es at ail the loci scored. Th us, gene flow between any ofthese three groups and any other population from our sa mple can be excluded , whereas gene flow among populations fram Ihe Pyrenean foothills cannot be exclud ed. The NJ tree cons lructe d on the chord distance illustr ates Ihe genetic relation ships betw e e n po p u latio ns (F ig . 3, left ). Tw o pop ulations, B. viridis and the Por popul atio n (attributed to B. reyniest î; were highly divergent from ail others . For other populatio ns, the topology of the tree revealed two main genetic grou ps. First, the four popul atio ns Td11 , Td12 , Suz, and Roq (attributed to B. utricu/us ) elustered together, with values of 0 ranging from 0.001 (Td11 vs. Td12) to 0.1 (Td11 vs. Roq ). Seco nd, the populations Aud1 (B. rubiginosa), Au d2 (B. cf. eurystoma ), Aud3, Sou, Eng , and Cat (the last three attributed ta B. simonia na ) clustered together, wit h 0 ranging from 0.011 (Aud l vs. Aud2) to 0.139 (Aud3 vs. Sou ). Because the patt ern of sha red alleles indicated that gene flow cou ld not be excluded among the Pyrenean foothill populations, we analysed the genetic structure among them using F sta tistics (Table 4) . The out-grou p and

the Par population of B. reyniesii were not inciudad in this analysis. The genetic variance amon g populations (F ST = 0.48 1) represented the princ ipal comp onent of the total gen etic variance of the pop ulation s analysed (FIT = 0.579); the genetic variation wi thin populations was relatively low (FIT = 0.189). Thus, these Bythinefla populations are highly differentiated genetically. The e va lues estimated between pairs of populations were significantly different trom zero, except between populations collec ted in the same locality (Aud 1, A ud2, Aud3; Td1 1, Td12 ) or nearby (Td12 with Suz). The genotypic differentiations calculated for each pair of populations and for ail loci reveal significant P values , except between population s located in the same locality (Aud1 to 3 and Tdl1 and 2) . Phylogenetic Analysls

Of the 360 bp analysed , 76 posi tions were va riable and 48 wer e phylogenetically informative. Figure 3 (right) displ ays the strict consensus tree based on 2,700 equi-parsimonious trees resulting fram the heuristic sea rch. The tree lenglh was 96 steps wi th CI 0.96 and RI 0.96. The topology revealed three distinct clades. The first clade (clad e R) includ ed the individuais fro m Por referred to B. reyniesii. Thi s clade was supported by strong Bootstrap values (= 100) and Bremer inde x (= 15). The seco nd clade (clade U) included the individu als from popul ations referred to B. utricu /us (TdI1, Suz and Roq). The thi rd clad e (clade S) includ ed the individuals referred 10B. rubiginosa (Aud1), B. cf. eurys toma (A ud2) , B. simoniana (Cat and Sou), and the Aud3 popuia tion. Clade U and S constituted a monophyletic group , which was also supported by strong Bootstrap values (; 100) and Bremer index (= 12). The phylogenetic ana lysis corrobora ted the enzyme polymorph ism studie s. Inde ed , the three monophyletic groups revealed corresponded ta the B. reyn ies! population and the two m ain gr oups revealed by the isozyme analyses. Therefore, in the following morphological analy sis, we consider these gro ups as a hypothesis alternati ve to the classi cal taxonomy, which can be formulated as follows: four popu lation gro ups of our datas et constitute four distinct species: Group V = Che (used as out-group ); Group R = Por; Group S ; Aud1, Aud2, Aud3, Cat, Sou and Eng; Group U ; Tdl1, Td12; Suz and Roq.

=

=

SPECIES DELIMITATION IN THE GENUS BYTHINELLA Morphometrical Analysis Sexual Dimorphism - Overall 358 specimens were successfully sexed, 55% males and 45 % females . Within a single population , 59 specimens from Roq included 36 (61%) males and 23 (39%) females. PCA was carried out on the 15 shell parameters LSR transformed. The first (PC1) and second (PC2) principal components respectively account for 63.5% and 13.4% of the total variation (Fig . 4). Three groups of strongly correlated parameters were detected: (i) global shell size parameters (L"" W"" W,,,, L w1 ' Lpw2 ' L,wl' L'W 2)' (ii) spire parameters (Wnw'

WfWlI L~Pl andLsP2) and(Hi) aperture parameters

(L ~Pl ' L..P2 ' W flP1 • W IlP2 ) · The firsttwo sets ofvariables, which are Iinked ta global shell size , were primarily associated with PC1 . This first axis was thus interpreted as a size factor. The

third set of parameters, related to shell aper-

ture, were prlmarily assoclated with PC2. The projection of the individuals in the PC1 x PC2 factorial plane revealed that, whereas females and males had similar distribution patterns along PC1, they differed along PC2 (Fig. 4). The DFA computed with sex as the discriminate factor was slgnificant (WL = 0.4241. P = 0.0002) . Scores ofWL with the probability associated with each variable indicated that the two sexes differed significantly in one aperture paramet er (W" , with WL =0.501 and p < 0.008). The global reclassification score of the discriminating linear functions was 86.44%

(78.26% for females and 91.67% for males). These results suggest that females differ from

303

males by having a larger aperture width (W" ,), but not by shell size. 8etween populations , the same DFA analyses were then performed on ail populations referred , taxonomically and genetically, ta the group utriculus: 136 specimens were sexed, 62% males and 38% females. This DFA was significant (WL = 0.725, P = 0.0004 ), but the two sexes differed significantly in the parameter Lap1 ' another aperture parameter (LIIP 1 with WL =0.755, P =0.028). Finally , a DFA was performed with sex as the discriminate factor on the global data set of 358 sexed specimens. The analyses was globally significant (WL = 0.914 , P = 0.011), indicating that the set of variables chosen allowsdiscrimination between the sexes. Scores of WL with the probability associated with each variable suggested that the two sexes differed significantly onJy by one aperture parameter (L'O, with WL =0.927 and p < 0.030). ln order ta test for the respective effects of sexual dimorphism and taxonomie differenees, two analyses of variance (ANOVA) were performed on the LSR global data set on the two aperture parameters W ap2 and LIIP1 that signifieantly differed between males and females. First , we tested the effects of the two factors taxonomie group and sax and their interaction , and then the effect of the factors genetic group and sax and their interaction. Thesa two ANOVAs revealed that bath effects of taxonomie group and genetic group were significant for the two parameterswhereas the effeet of sax was not significant in any analysis.

l'C I fiJ.5%

• •

.;

~

• •

o

0

•

•

• FIG. 4. Sexual dimorphism in Roq population of Bythinella: results of PCA. Left Correlation of the 15 parameters measured on the shell to the first two principal components PC1 and PC2. Right Projection of the individuals in the first factorial plan PC1 x PC2. Open circles are females and filled eireles are males.

BICHAI N ET AL.

304

TABLE 5. Correlations rnatrlx between the variables and the first two discriminate functions (F1 and F2) for the two alternative Bythine/la hypotheses (Taxon group and Genetic group).

Taxon group hypothesis Shell parameters

F1

F2

F1

F2

-0.027

-0.058

0.523 3.308

-0.243 -0.216 0.327 0.265 -0.008 -0.267 -0.289 -0.267 -0.264 0.256 0.145 0.256 0.230 1.318

63.3%

88.5%

Lsh Wsh Lap2 Wap1 Wlw Lpw1 Lpw2 L1w1 L1w2 Waw Wpw Lsp1 Lsp2 Eingenvalue

0.001 0.076 -0.504 -0.068 -0.090 -0. 108 -0. 188 0.263 0.166 0.368 3.756

0.189 0.160 -0.300 -0.217 0.084 0.209 0.228 0.200 0.184 -0.184 -0.069 -0.179 -0.147 1.383

Cum. Eigenv.

53.8 %

73 .7%

-00460

00465

For each of the two ape rture parame te rs, the

taxonomie group x sex interaction was not significan t whe reas the genetie group x sex interaction was significant but only fo r the W op,

-8

-4

Genetic grouphypothesis

-00495 0.017 0.103

-00407 -0.102 -0.125 -0.147 -0.237 0.307 0.207

00418

pa rameter (L'P' F-test = 1.32 w ith P = 0.267 and W'P' F-test = 4 .226 wit h P = 0.006). For this parame ter, the post hoc test revealed that the geneticgroup x sex inte raction was caused

4

-8

-4

4 F2 25.2 %

-4

_. .

FIG. 5. DFAtasting the two alternative hypotheses Taxon groups vs Genetic groups within Bythinella species. Left Taxon groups hypothesis. Filled circles: B. viridis (one population), open clrcles : B. utrieu/us (four populations), filled triangles: B. reyniesii (one population), open triangles: B. simoniana (three populations), filled squares: B. cf. eurystoma (one population), open squares: B. rubiginosa (one population). Right Genetic groups hypotheses. Filled circles: group V (B. viridis, one population), fill ed triangles: group R (B. reyniesii, one population), open squares: group U (B. utrieu/us, four populations), filled rhombus: group S (B. simoniana, B. rubiginosa, B. cf. eurystoma and Aud3 population, total six populations).

SPECIES DELIMITATION IN THE GENUS B YTHI NELL A

305

TABLE 6. Global reclassifi cation scores of the discriminating linear functions for the two Bythinella hypotheses. Each line gives the number of specimens of a given taxon classifie d as any of the taxa. and the percentage of each that are correctly classified.

Taxon group hypothesis

aythinefla viridis reyniesii utriculus simoniana

cf. eurystoma rubiginosa

Total

% correct 88.9% 90 .0%

87.2% 82.2% 83.3% 83 .3% 85.6%

viridis reyniesii

24 0 0 0 0 0 24

0 27 5 0 0 0 32

utriculus

simoniana

cf. eurystoma

rubiginosa

3 3 129 13 1 1 150

0 0 13 74 0 2 89

0 0 1 1 25 2 29

0 0 0 2 4 25 31

Genette group hypothesis

Group V

Group R Group U Group S Talai

% correct

Group V

G roup R

Group U

8 1.5% 90.0% 86.5% 92 .8%

22 0 1 0 23

0 27 5 0 32

5 3 128 13 149

89.4 %

by Por (group R, attribuled ta B. reyniesiJ), ln which the sex ratio was 24% males and 76% females. Ta summarize, the analysis shows that variation of aperture parameters is mainly explained by differences between "spec ies" (defined on either the taxonomie or genetie dei imitation hypotheses ), and that sex ua l dlmorphism within a population is manifest in aperture size. Ta minim ize the effect of the sexual dimorphism, the Iwo aperture parameters were removed in the subsequent analyses. Shefl Variation Among Pop ulations and Taxa - Ta explore the pattern of shell variation, a PCA was carried out on the whole LSR datasel (385 individuals of bath sexes). The first factorial plane accounted for 92.1% of the global variation with 80 .3% by PC1 and 11 .8% by PC2 (graph not presented here). Three groups of strongly correlated paramete rs were detected: (i) spire and aperture parameters negatively correlated with the PC1, (Ii) helght of the various whorls positlvely correlated wilh PC1, and (ili) shell widt h and width of the last whorl negatively correlated with PC2. The projection of the ind ividuals in this faclo rial plane reveaJ ed a strong overlap between the various populati ons. On the PC2 axis, only individu-

Group S

0 0 14 167 181

ais of B. viridis had a distinct position. Thus, except for this taxon, the parameters used in this analysis dld not reveai any differen ces among groups of individuals when using either the taxonomlc or the genetic hypotheses. Taxon Hyp othesis - The DFAon the LSR global data set (Iess the taxonomically doubtful Aud3 population and L, 1 and W, ,) performed with Taxon as discriminats factor was significant(WL ~ 0.023, P < 0.05). Scoresof WL wilh proba bili ty ass oc iated w ith e ach va ria ble showed that ail parameters significantly discriminate the taxonomie groups. Projection of the individuais on the first Iactorial plane F1 x F2 (each axis respectively accounting for 53.8% and 19.8% of the total variance) allowed discrimination, mainly on the F1 axis. of a cluster ofindividuals attributed ta B. viridis. The F1 axis was mainly correlated wilh spire parameters (W w' L 1 and L. ), whereas the F2 axis was ma'inly ëxplaine;rby a size effect (Table 5). Indeed, for this axis, ail variables conlributed the same weight and were strongly correlaled (Fig. 5). In this F1 x F2 faclorial plane, other taxenomic groups we re poorly resolved with an extensive overlap among them. For example, individuals referred toB. simoniana. B. cf. eurystoma and B. rubiginosa were not separated.

306

BICH A IN ET AL.

T he globa l reclassification score (Table 6) of the disc rimina ting linear functions was 85.6%. with speci fie scores ran ging trom 83.3 % (i ndividuals attribu led 10 B. cf. eU/y stoma and B. rubiginosa ) to 90 .O% (individu als allribuled to B. reyniesil).

Genetic Hypofh esis - DFAs performed on the LSR global data sel (Iess L. , and W, , ) with Gene tic groups as discrim~n ate fa ct~r we re also signi fic ant (W L ; 0.062 , P < 0 .05). Scores of W L with prob ability ass ociated for each variabl e showed that ail param eters significan tly di scrim inate d the genetic groups. Th e F1 x F2 facto rial pl an accounted fo r 88.5% of the global variance, wi th 63.3 % for F1 and 25 .2% for F2 (Table 5). The spire paramete rs are positively co rrelated wi th F1, whereas W h and W tw we re negatively correlat ed with the sa,me axis. Th e. W aPI a nd L "P2 paramete rs co nl nb ule d ess entia lly to F2 . Th e projec lion of individuats in lh is facto rial plane revealed three distin ct groups on lhe F1 axis, correspo ndi ng to individuals assig ned to gro ups V, U and S defined beiow (Fig . 5). On the other hand , F2 allowed sepa ration of group R from ail other groups . Th e global reclassificatio n scores (Table 6) we re 89.3% under the Genetic hy pot he sis , wit h re classifi cali on scores ranging from 8 1.5% (group V) to 92.8% (group S). F ro m this re sult , we ca n id ent if y four morphogroups basad mainly on spire parameters : (i) group V wit h a sma ll spire and a large last whorl that gives the she ll an ovoid outli ne; (ii) group S wi th a iarge overall size and a high spire that giv es lhe shell an elo ngate outl ine ; (iii) group U, inle rmediate between V and S, with an oulline that can be described as ovoid elo ngate ; (iv) group R wi th a spire heig ht equa l to the height of the last whorl and a large apert ure , tha t giv es the shell a pupoid outline. DISCUSSION AND CONCLUSION Our an alys es co nfirm ed that ail spe cimen s atlributed to B. viridis (Fig. 6A) , wh ich are used as out-g roup , we re morp hologic ally and genetically distincttrom ail Pyrenean specimens. W ithin the Pyrenean sample, two species delimitations resulting trom our analysis confirmed earHer tradilional species deiimitatians (Bernasconi, 2000). Specimens att ribut ed to B. reyniesii (Fig. 6B ) we re both morp holog ically and ge nelically di stin ct fro m ail oth er specimens included in our study. The morpho-

metric analysis de scribes the shell as having a spire equal to the height of the last whorl and a large apertu re, resulting in a pupoid overall shape. However, since a single population was includ ed in the study, this description lacks robustness and we do not know how the characters ob served may be subject to phenolypic plas ticity or to genelic variability. The second vatidated species was B. utricufus (Fig. 6F ), in which speci men s from ali populations c1ustered both as a monophyletic group (DNA sequences) and as a gene tic group (isozymes) . We hypoth esize that the entir e clade can be classified as a single species. As several populatio ns are included within this species, our morphometrie characterisation integrates at least part of the phenolypi c plasticity component of shell shape variability. Ali other population s formed a single geneti c and phylogenetic group. Morphometric analysis describes the shell within this group as having a large ove rail size and a high spire thal gives il an elongate shape. This group included the nom inal B. simoniana (Fig . 6D) , characterized in traditional taxonomy by axial varices on the last wharl - a character not confirmed in our analysis as of specifie diagnostic value - and also included specimens Irorn the two springs complex at Audin ac, the co le sprin g (Fig . 6C ) and the therm al spring type iocality of B. rubiginosa (Fig. BE), and their con fluence . Thus, this c1uster groups at least two nomi nal taxa that shou ld be cons idered a singl e specles , to be nam ed B. rubigin osa bas ed on the Principle of Prior ity (ICZN Code Art . 23 .3). T he high quantita tive (size) or qualitative (varic es) shell variabil ity observed in this gro up may be due either to phenotypic plaslicity or to intraspecific genetic variation. H ow ever , althoug h DFA ana ly ses allow separation of these ge netic groups, the PCA resu lts highligh ted overlaps among mosl populations. indicating that a priori determinations at the species level rank in Bylhinella are still diflicull even when using the shell characlers here identifie d. More over, these shell charseters canno t be validated for alpha-ta xonomy without a robust test at the scale of the genus. We show here that it is possible to give an alternative speci es de limita tion hypoth esis to the traditional view in an evolutionary framewo rk and that these newly defined ent ities exh ib it distinct morphological feature s. We agree w ith Wilke et al. (2002) about the necessityto use molecular markers and morphomet rical approaches in concert to stud y cryptic or cJosely relaled species.

SPECIE S DELI MITATION IN THE GENUS B YTHfNE LLA

307

FIG. 6. Shells of lhe nominal species of By/hinel/a studied. A: B. viridis (Che): 8 : B. reyniesi; (Par): C: B. cf . eurystome (Aud2): D: B. simoniana (En9); E: B. rubiginosa (Aud1); F: B. utriculus (Roq) (Scale 1 mm). Genetic studi es of Poli sh populal ions revealed weak genetic diffe renlialion primarity corre lale d wilh ge o gra p h ic di st ance (Falniowski et al., 1998, 1999). The former suggesled Ihal ",.. Ihe superspecies status of Ihe genus (Giusti & Pezzoli, 1977) seem s the mostjuslified", The lerm superspecies was firsl used by Mayr (193 1) and subse quenlly defined by Mayr & Ashlock (1991) as "a monophyletic group of closely related and enlirely or largely allopatric species that are tao distinct to be included in a single species or l hal demonstrate their reproduction isolal ion in a zone of contact. " Our data don't refute this opinion within the Pyrenean area where a mosaic of populations probably belonging to

different species more or less morphologically differentiated seems ta coexist in allopatry.

Nevertheless, we show that sorne shell characters thal were lr aditionally used 10 segregale species of Bythin ella (see, for exa mple, Berna sconi, 200 0) do not necessarily have taxonomiesignificance, a result agreeing with Ihal of olh ers working on ol her hydrobioid group s (Falniowsk i & Wilke, 2001 ; Wilk e & Falniowski, 200 1; Szarow ska & Wilke, 2004). The high inlra speclfic shell variabilily may be linked 10 biotic facl ors, such as food availability, parasile-induced gigantism (Jourda ne, 1979; Gorbushin, 1997; Probsl & Kube, 1999), or sexua l dimorphi sm (Ponde r el al., 1999; Kurala & Kikuchi, 2000; Velecka & Jüllner,

308

BICHAI N ET A L.

2000; Chiu et al., 2002). Our study carraba rates Faln iawski 's (1987 ) observation that sexua l dimorphism is rather law in Bythinella, females differi ng from males by their generally larger shell ape rture. Hawever, as sex ratio appe a rs nat lixed in po p ulatio ns 0 1 Byt hinell a, superficia l marphal agical differe nces among populations may also be a con-

sequence 01dlfferent sex ratios. and cantribute ta fuzzy specie s delimilatians. Shell varlabllity may alsa be linked ta abiatic pararneters, such as temperature (Brown & Richard son, 1992). In the three Audinac populati ons of B. rubiginasa. specimens fram the thermal spring are large st, spe cimen s from the cold spring

are sma llest, an d speci mens from the connuence are intermediate. To conclude, the present work offers an examp le of a three-ste p integ rative taxanamy starting tram (i) primary hypotheses (curre nt spe cies delimitatians based on phenetic char-

acie rs), ta (ii) the test 01 these hypatheses by criteria lhat are ca nceptually sound. Population geneties appraaches allaw ta idenlify repr adu cti vel y isal ated gr o up s . Malec ula r phylageny allaws the recognition of manaphyletic gro ups and thus the detectia n 01 groups thal share ancestry. These Iwo criteria, derived lrom the BSC and the PSC . permit new species delimitatian, and finally (Iii) a leedback ta a new taxonomie hypoth esis associated with

new phenetic descriptars. Therefare, the final description corresponds ta the mast filt ing hypathesis given the data at lh e time of the description. A correct delim itatian of species has imparlant consequences bath for a reliable estimal ion of biad ive rsity bu t alsa when draw ing conservation priorities. Two of the nominal species in the present study (B. viridis and B. reyniesil) are categarized as Vulnerable in the IUCN Red Lisl and are alsa protected under French law. Bath emerged fram the present study as probabl y dis tinct evalutianary units. Our study was restricted ta a small part of the geag raphical range of Bythinella including a subset of ils nominal species and used a law number of isazym e loci and a short gene Iragment. Thu s, our results are of preliminary nature. In arder ta validate them, we need ta expa nd this approa ch ta a global revisian of the genus, including suppleme ntary mitachandrial and nuclear marksr s. Hawever, lhe challenges to expanding thismodel are immense. T he Iiterat ure on E uropean hydrobioids is repiete w ith nam e s of unkn ow n significance

(Davis, 2004 ) that blur evaluatians of regianal

species richness , conservation priorities and evolut ionary history. Th is is because man y nom inal spe cies we re estab lished by 19 11' and 20~ century authars that (i) worked on a local or regianal basis, and/or (ii) worked autside a

context of evo lutionary syste matics. Th e ir ap-

proach la alpha-tax anamy has alsa accasianally injecte d in the literature names based on few, sometimes single. specimens only weakly

diff erentiated can chalagicall y. Because the present stud y shows that geneti c or phylage netic distinctiveness cannat be inferred from

Ihe degree of shell distinctiveness, the challe nge to a co mp rehe nsive re vi s ion of Bythinella is that the se nominal species have ta be re -evalu aled based on pa pula l ia ns sampled alive fram their type lacalily. At this mom ent, we do nat knaw whethe r an integralive revisian of Bythinella will result in just a few or se ve re ! dozen spe cle s .

Haw do Ihe present results impact the systematics of fassil Bythine/ia, and daes Ihe resuit of the prese nt study preclude naming any new Bythinella species base d on shell charac ters only? De spite the numerou s nominal

species of Bythine/ia described by 19'" and 20· ce ntury a utha rs , new exploratio n 01 littl eknow n aqui fers, in partlcular hypogean en vironments in sauthern France, stilil eads ta the discavery 01 new marphatypes. Sam etimes , anly empt y shells are knawn and living specimen s e sca pe collection de spit e inten siv e

searche s. We un derstand that il may be necessary, for com municatio n or con se rva tion purpose s, ta name such taxa; howev er, it is

esse ntial thatthe primary description is based on shell characters Ihat have been tested for their robustness regard ing spec ies delimitatian. St abil ity in t h e a lpha-taxanamy 01 hydrobiaids will mast likely be reached thraugh an inlegralive taxanamy approach (Sites & Marshall, 2003 , 2004), A possible appraach wauld be Ihe Henn igian inter-nodal specie s concept derived from the Theary of Evolution (Sa ma di & Ba rberou sse, 20 06) th at he re proved an efficient taal in the study 01 Pyrenean Bythine/ia.

ACKNOWLEDGM ENTS Th e se quenci ng was done in the Service de

Sys lématique Molécu laire atthe Muséum national d'Histoire nat urelle of Par is with the techn ical hel p of Annie Ti ll ie r, J osie Labaurdiére and Céline Banilla. We are grate-

SPECIES D ELIM ITATIO N IN T HE G ENUS 8 YTHI NEL LA fui ta Ahmed Abdau, Ala in Be rtrand , Ben oit Fo nta ine and Ol ivier Gargamin y for the ir co llaboratio n during the malerial sampiing and ta Phi lip pe Deliat for his he lp in the marphametric ana lyses. Sp ecial thanks ta Rob ert Cawie and Sim on Tillier for their hel pful camments.

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Revised ms. accepted 29 September 2006