An integrative approach to the taxonomy of thepigmented European Pseudomonocelis Meixner, 1943(Platyhelminthes: Proseriata)

MARCO CASU*, TIZIANA LAI, DARIA SANNA, PIERO COSSU andMARCO CURINI-GALLETTI

Dipartimento di Zoologia e Genetica Evoluzionistica, Università di Sassari, Via F. Muroni 25,Sassari 07100, Italy

Submitted 2 March 2009; accepted for publication 11 June 2009bij_1316 907..922

Only a few species belonging to the Proseriata (Platyhelminthes) show a parenchymatic pigmentation, which mayaid identification. Among these, Pseudomonocelis agilis has a yellowish body and is provided with a reddish–browngirdle in front of the statocyst. The species is known for limited areas of northern Europe and the Mediterranean.The present study was conducted to assess both the taxonomic status of populations attributed to the species acrossthe unusually wide range for an interstitial flatworm, which lacks an obvious means of dispersal, and the levelsof genetic variability within and among populations, by employing an integrative approach that included theanalyses, on six populations, of three molecular markers (small subunit ribosomal 18S-like gene, inter-simplesequence repeat, allozymes), karyotypes, and 11 morphological characters. Furthermore, crossbreeding experi-ments were carried out on the Mediterranean populations. The results obtained revealed the existence of fourhighly divergent genotypic clusters, accompanied by karyological differences, with complete intersterility amongthe clusters tested. The combination of approaches adopted strongly supports the conclusion that the wide-rangingEuropean pigmented species P. agilis is actually composed of four species: P. agilis in the Baltic area; Pseudomono-celis cetinae in the Adriatic; and Pseudomonocelis sp. nov. A and Pseudomonocelis sp. nov. B in the westernand eastern Mediterranean, respectively. The latter two species are morphologically indistinguishable for theparameters essayed. Reconstruction of the phylogenetic relationships of these taxa, including congeneric andconsubfamilial outgroups, showed that pigmentation is a plesiomorphic condition for the genus Pseudomonocelisand that Pseudomonocelis sp. nov. A shares a previously undetected, sister-group relationship with species of theunpigmented P. ophiocephala complex. The present study thus depicts complex speciation processes in a mesop-sammic species, which involves allopatric divergence operating on different scales and ecological shifts, andhighlights that the contribution of microturbellarians to marine biodiversity may be seriously underestimated.© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 907–922.

ADDITIONAL KEYWORDS: biodiversity – cryptic species – meiofauna – phylogeny – speciation.

INTRODUCTION

Evidence is accumulating indicating that the inven-tory of marine biodiversity is far from being exhaus-tive (Mathews, 2006). The state of knowledge isparticularly unsatisfactory for meiofaunal orga-nisms, comprising an assemblage of marine benthic

metazoans, intermediate in size between macro-fauna and microfauna, whose morphology, physiology,and life-history characteristics have evolved to exploitthe interstitial matrices of marine soft sediments(Warwick, 1984). Meiofaunal organisms, as a resultof their size and diversity, are known to be taxono-mically challenging (Kennedy & Jacoby, 1999).Interstitial Platyhelminthes (‘microturbellaria’) areparticularly problematic because their study neces-sitates observations on both living and sectioned*Corresponding author. E-mail: marcasu@uniss.it

Biological Journal of the Linnean Society, 2009, 98, 907–922. With 5 figures

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specimens (Cannon & Faubel, 1988). Furthermore,numerous microturbellarian taxa are morphologically‘austere’, lacking features that can be effectivelyused to distinguish species (Rieger, 1977; Larsson,Ahmadzadeh & Jondelius, 2008). These problemsmay not be alien to the fact that species with wide,circumpolar or cosmopolitan distribution are reportedeven in the recent literature (Norenã et al., 2007;Ax, 2008), despite a lack of planctonic larval stages(Swedmark, 1964). Although short range dispersal ofadults has been reported for flatworms (Armonies,1988) and meiofauna in general (Committo & Tita,2002), it is unclear how this process may activelymaintain an adequate level of gene flow among widelyallopatric populations.

Recently, on the basis of molecular information,wide-ranging putative species have been shown toconsist of discrete clusters of populations, sugges-tive of the presence of cryptic species (Casu &Curini-Galletti, 2004, 2006). However, the molecularapproach by itself is not free from difficulties asso-ciated with exploring the limits of species (Moritz &Cicero, 2004). For example, the sequencing of themitochondrial cytochrome c oxidase subunit I (COI),usually performed to make inferences on the geneticrelationships of different species, is not suitable forseparating species younger than 3–4 Mya (Bouzidet al., 2008). Furthermore, in the case of meiofaunalorganisms, limited dispersal capabilities may resultin a marked divergence among allopatric populations,and the degree of intraspecific versus interspecificdifferentiation should be tested on extensive taxo-nomic and geographic samplings.

An integrative taxonomy approach, which considersspecies boundaries from multiple, complementaryperspectives, was recently advocated as the best wayto overcome potential caveats of any species delimi-tation methods (Padial et al., 2009). In the presentstudy, we applied an integrative approach for thestudy of Pseudomonocelis agilis (Schultze, 1851)(Proseriata: Monocelididae) as a test case of a puta-tive species with a wide range, which includes theNorth-West Atlantic and most of the Mediterranean(Ax, 2008), aiming to obtain insight into the actualdegree of cohesion of populations, both on a broadallopatric scale (Atlantic versus Mediterranean) andon a smaller, intra-Mediterranean scale. In parti-cular, we used two independently evolved molecularmarkers: nuclear small subunit (SSU) ribosomal 18S-like gene and inter-simple sequence repeats (ISSR).

Allozyme electrophoresis was used to support theISSR analysis of population genetic structure bydefining the extent of Hardy–Weinberg disequilib-rium, if any.

Additionally, we performed multivariate analysesof morphological and karyological characters to test

whether the occurrence of genetic diversity amongpopulations was accompanied by detectable pheno-typic differentiations, allowing for the recognition ofpotential diagnostic characters. Finally, crossbreedingexperiments were conducted in the laboratory todetermine the degree of reproductive compatibilityamong allopatric populations.

TAXONOMIC BACKGROUND

Pseudomonocelis agilis was originally described onspecimens from the western Baltic by Schultze (1851).The species is characterized, among other features,by the presence of diffuse yellowish parenchymaticpigment in the whole body and a conspicuous girdleof concentrated reddish–brown pigmentation in thecephalic area, and, hence, it is peculiar among thevast majority of unpigmented European mesop-sammic flatworms. Subsequently, Meixner (1943)described the similarly pigmented Pseudomonoceliscetinae from the Mediterranean (type-locality: Omiš,Croatia, Adriatic Sea), apparently unaware ofSchultze’s species, which was not mentioned in thediscussion. The two taxa were later synonymized byAx (1959) and by most subsequent studies (Schütz& Kinne, 1955; Luther, 1960; Den Hartog, 1964;Karling, 1974; Sopott-Ehlers, 1993; Schütz, 2007; Ax,2008), with the exception of a study by Schockaert &Martens (1987), who suggested that the disjunctionof the range may hint at species distinction. Becausepopulations attributed to P. agilis are found inbrackish-water habitats, which are fragmented andoften heavily affected by human activities, the asse-ssment of the taxonomic status of the Europeanpigmented Pseudomonocelis populations, besides con-tributing to the catalogue of marine biodiversity, mayhave implications for conservation.

MATERIAL AND METHODSSAMPLING

The study was conducted on six populations ofP. agilis: one from the Oresund (HE) (Helsingør,Denmark) and five from the Mediterranean [PortoPozzo (PP), Giglio Island (GI), Omiš (OM) (type-locality of P. cetinae), Maliakós (MA), Charaki (CH)](Fig. 1). Samples were collected manually in lowerintertidal habitats by scooping up the superficiallayer of the sediment or from algae (Fucus sp.) foundin the upper subtidal (i.e. in the case of the Oresundpopulation). The animals were extracted with theMgCl2 decantation technique described by Martens(1984), and maintained alive in containers filled withsea water, stored at 18 °C.

For the SSU analysis, the pigmented Pseudomo-nocelis cf. cavernicola Schockaert & Martens, 1987,

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from Dongwe, Zanzibar (Tanzania) (GenBankAccession no. FJ416061), and the unpigmentedPseudomonocelis ophiocephala (Schmidt, 1861),from Porto Torres (Sardinia) (GenBank Accessionno. FJ416065), and Pseudomonocelis caputdraconisCasu & Curini-Galletti, 2006, from Patrás (Greece)(GenBank Accession no. FJ416066) were used as con-generic outgroups. Monocelis lineata (Müller, 1773)from Helsingør (Denmark) (GenBank Accession no.FJ416062), Minona ileanae Curini-Galletti, 1997 fromGreat Bitter Lake (Al-Buhayrah Al-Murra Al-Kubra),Suez Canal (GenBank Accession no. FJ416063),and Peraclistus sp. nov. from the Grotte di Nettuno(Sardinia) (GenBank Accession no. FJ416064) (Pros-eriata: Monocelididae: Monocelidinae) were used asconsubfamilial outgroups.

MOLECULAR ANALYSIS

DNA extractionGenomic DNA was extracted from entire individualsusing the Qiagen DNeasy Tissue kit. After extraction,the DNA was stored as a solution at 4 °C.

SSU sequencingThree to six individuals belonging to each populationwere sequenced at the SSU partial region. Ampli-fications were carried out using primers, whosesequences are reported as ‘A’ (Forward) and ‘B*’(Reverse) in Littlewood, Curini-Galletti & Herniou(2000). The polymerase chain reaction (PCR), carriedout in a total volume of 25 mL, contained 5 ng mL-1

of total genomic DNA on average, 2.5 U of Taq DNAPolymerase (Eurotaq, Euroclone®), 1¥ reaction buffer,1.25 mM of MgCl2, 0.4 mM of both A and B* primers,and 200 mM of dNTPs. PCR amplification was carriedout in a MJ PTC-100 Thermal Cycler (MJ Research)programmed as follows: one cycle of 3 min at 94 °C,45 cycles of 40 s at 94 °C, 45 s at 54 °C (annealingtemperature), and 1 min and 40 s at 72 °C. At theend, a post-treatment for 5 min at 72 °C and a finalcooling at 4 °C were carried out. Both positive andnegative controls were used to test the effectiveness ofthe PCR reagents. Electrophoresis was carried outon 2% agarose gels (0.5 ¥ TBE buffer) at 4 V cm-1

for 20 min and stained with ethidium bromide(10 mg mL-1). PCR products, purified by ExoSAP-IT(USB Corporation) were sequenced using an externalsequencing core service.

Estimates of haplotype diversity (Hd) were ob-tained using the software DNASP, version 4.10 (Rozaset al., 2003) to characterize the genetic variationwithin each population. Phylogenetic relationshipsamong individuals were assessed by maximum parsi-mony (MP), with and without the Goloboff (1993)correction (K = 2), applied to look for the effect ofhomoplasy on tree topology. Heuristic searches werecarried out using a 1000 bootstrap branch-swappingtree-bisection–reconnection, with random additionsequences. MEGA 4 software (Tamura et al., 2007)was used to compute the consensus tree. Further-more, the phylogenetic relationships among popula-tions belonging to the genus Pseudomonocelis were

Figure 1. Sampling localities of the populations referred to Pseudomonocelis agilis. PP, Porto Pozzo; GI, Giglio Island;MA, Maliakós; CH, Charaki; OM, Omiš; HE, Helsingør.

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inferred by a median-joining network, using the soft-ware NETWORK, version 4.2.0.1 (http://www.fluxus-engineering.com).

Allozyme electrophoresisThe analysis was carried out on 50 individuals perMediterranean population of P. agilis, according tothe procedures outlined in Casu & Curini-Galletti(2006). Eleven enzymes yielded interpretable band-ing patterns (Table 1). The proportion of polymorphicloci (0.95 cut-off criterion), observed and expectedheterozygosities (Ho and He), per sample and overallFIS estimators after 10 000 replicates were calculatedfor each sample using FSTAT, version 2.9.3 (Goudet,1995), whereas the genetic differentiation betweensamples was quantified as the Nei’s (1978) unbiasedmeasure of the genetic distances between pairwisepopulations, as calculated using POPULATION,version 1.2.28 (http://www.cnrs-gif.fr/pge). PairwiseFST were further calculated after 5000 repli-cates (ARLEQUIN, version 3.1; http://lgb.unige.ch/arlequin).

ISSR analysisAn overview study was conducted on ten individualsof P. agilis from HE assaying ten primers. Theanalysis showed five informative and reliable pri-mers containing differently anchored dinucleotide andtrinucleotide repeats, capable of screening differentparts of the genome (Table 2), which were hence usedto amplify the genomes of 18 (PP, GI, CH, MA) and 20(HE, OM) individuals, respectively. The 25-mL volumePCR reaction mixture contained 0.5 U of Taq DNAPolymerase, 1¥ reaction buffer, 2.5 mM of MgCl2,0.2 mM of primer, 200 mM of each dNTP, and up to30 ng of genomic DNA. The PCR profile was the sameas that used for SSU analysis, except for the anneal-ing temperature. For all primers, negative controlsand replicates were included in the amplifications tocheck for the occurrence of PCR artefacts and toverify the repeatability of results. The PCR productswere analysed by electrophoresis using a 2% agarosegel in 1¥ TAE buffer. Gels were run, with standard100-bp ladders as reference, at 4 V cm-1 for approxi-mately 3 h and subsequently stained by soaking the

Table 1. Enzyme systems screened

Code Enzyme E.C. NL NA

APK Arginine kinase 2.7.3.3 1 4GAPDH Glyceraldheyde-3-P dehydrogenase 1.2.1.12 3 2,2,2GPI Glucose-6-P isomerase 5.3.1.9 1 5G6PDH Glucose-6-P dehydrogenase 1.1.1.49 2 1,2IDH Isocitrate dehydrogenase (NADP+) 1.1.1.42 1 3LDH L-lactate dehydrogenase 1.1.1.27 2 1,2MDH Malate dehydrogenase 1.1.1.37 2 3,2ME Malic enzyme (NADP+) 1.1.1.40 1 5PEP-A Peptidase A (substrate Leu-Gly) 3.4.13 1 5PGDH Posphogluconate dehydrogenase 1.1.1.44 1 4PGM Phosphoglucomutase 5.4.2.2 1 8

NA, number of alleles detected; NL, number of loci detected.

Table 2. Primer names and sequences used in the inter-simple sequence repeats (ISSR) analysis, number of polymorphicbands per primer, and range of molecular weight in base pairs (bp) amplified by polymerase chain reaction-ISSR

Primer Sequence (5′- to 3′) Ta (°C) No. of bandsSize range ofbands (bp)

SAS1 (GTG)4GC 55 26 480–2640SAS3 (GAG)4GC 51 26 350–1800UBC809 (AG)8G 55 18 400–1500UBC811 (GA)8C 55 21 400–2200UBC827 (AC)8G 55 23 350–3000

Ta, annealing temperature.

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gel in a 1 mL 10 mL-1 ethidium bromide solution forultraviolet visualization of the banding patterns.

To estimate the allelic frequencies from thedatasets of dominant markers, the method proposedby Zhivotovsky (1999) was chosen and a Bayesianframework was used (Shoemaker, Painter & Weir,1999). This method allows the application of theinbreeding coefficient to earlier information, avoid-ing the assumption that populations are in Hardy–Weinberg equilibrium. To set this value, the averageFIS value for the P. agilis samples from the Mediter-ranean allozymic dataset was calculated after 10 000replicates. The percentage of polymorphic loci (PLP)and the heterozygosity for each sample, and theF-statistics values were obtained using AFLP-SURV,version 1.0 (Vekemans, 2002), applying the methodoutlined by Lynch & Milligan (1994).

The genetic structure was inferred by the Bayesianmodel-based clustering algorithm implemented inthe software STRUCTURE, version 2.2.3 (Pritchard,Stephens & Donnelly, 2000), applying the methoddescribed in Evanno, Regnaut & Goudet (2005). Thismethod allows the identification of clusters of indi-viduals according to their genetic information (i.e.the multilocus individual genotype). We tested boththe no-admixture and admixture model (the latterbecause it is more powerful in detecting shared recentancestry) (Pritchard et al., 2000). For each value of Kranging from 1 to the maximum number of popula-tions plus 3 (where K is the number of assumedgenetic clusters), five independent runs were con-ducted (100 000 iterations after a burn-in period of50 000).

MORPHOLOGICAL AND KARYOLOGICAL DATA

Specimens were studied alive on semisquashedmounts and on sections. For microscopic analyses,fully mature specimens, after relaxation in an isotonicMgCl2 solution, were fixed in Bouin’s fluid and em-bedded in Paraplast at 56 °C; serial sections wereobtained at 4-mm thickness, stained with Mayer’shaematoxylin and eosin, and mounted in Depex. Forthe analyses of the intra- and interpopulation vari-ability of P. agilis, 30 fully mature specimens ofpopulations from the western (PP, GI) and easternMediterranean (MA, CH) regions and 11 specimensof the Adriatic (OM) and Baltic (HE) populations, ofwhich only fewer mature specimens were available,were sectioned. Biometric analyses took into accountany morphological parameter that could be confi-dently measured with the aid of an ocular microme-ter: (1) total body length; (2) distance between maleand female pores; (3) length of the copulatory organ;(4) width of the copulatory organ; (5) length of thepenis papilla; (6) thickness of the proximal muscle

sheath of the copulatory bulb; thickness of the (7)muscular and (8) prostatic components of the penispapilla; length of the (9) dorsal and (10) ventral cilia;and (11) length of the dorsal rhabdoids. Karyologicaltechniques were conducted sensu Curini-Galletti,Puccinelli & Martens (1989). For each population, 20specimens were studied. Relative lengths (r.l. = lengthof chromosome ¥ 100/total length of haploid genome)and centromeric indices (c.i. = length of short arm ¥100/length of entire chromosome) were obtainedfrom measurements of the camera lucida drawingsof plates showing average contraction. Chromo-some classification was made sensu Levan, Fredga &Sandberg (1964).

To test whether the characters above may offer aclue to differentiate populations, unifactorial analysisof variance (ANOVA) was performed on morphologicaland karyological variables. Following significanteffects in the ANOVA (P < 0.01), a posteriori analyseswere performed [Scheffé test on unbalanced morpho-logical data; Student–Newman–Keuls (SNK) test onbalanced karyological data], in order to detect homo-geneous groupings of populations at a = 0.05 (Under-wood, 1997). Principal component analysis (PCA) wasused to test the hypothesis that different populationsdid not differ for morphometric and karyological char-acters. Because body length was significantly corre-lated with most morphological measurements, a PCAbased on morphological data was performed on datatransformed, to achieve normal distribution and tofully eliminate size effect, by converting original mea-surements into ln arcsine square-root proportionvalues (Suchentrunk et al., 2007). The normality ofeach variable was then proved by a Kolmogorov–Smirnov test. Variables that were significantlycorrelated with each other (P < 0.05) even after trans-formation were not considered for further analyses.PCAs were performed on correlations, using SPSS,version 13 (SPSS Inc.). Analyses of similarities(ANOSIM) (Clarke, 1993), performed on similaritymatrices based on Euclidean distances, were carriedout to confirm significant differences among (1) popu-lations and (2) population clusters, using PRIMER 5(Plymouth Marine Laboratory).

CROSSBREEDING EXPERIMENTS

Cultures of specimens were maintained in the labo-ratory at 18 ± 0.5 °C and 36‰ salinity and fed weeklywith crushed Sphaeroma sp. (Crustacea: Isopoda).The Baltic population (HE) proved impossible tomaintain in cultures because the offspring were short-lived and could not be grown to sexual maturity.Therefore, crossbreeding experiments could only becarried out on the five Mediterranean populations,which showed good survival and remarkable longe-

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vity in the laboratory, with individuals attaining 25months of age. Experiments were carried out by iso-lating pairs of newborns in 20-mL containers filledwith seawater, under the culture conditions describedabove. The offspring produced weekly by pairs werecounted and relocated. To obtain a balanced design, incase one or both members of the pair died before orwithin 5 weeks after the onset of sexual maturity (asindicated by the formation of the copulatory organs),the replicate was considered as lost, and a new pairwas set as replacement. ANOVAs were used toanalyse differences in reproductive outputs (i.e thenumber of offspring produced) among pairs of speci-mens derived from: (1) the same population; (2)different populations; and (3) different species. Forintrapopulation crossings, 20 replicates (i.e. pairs)were considered for each of the five populations.For interpopulation crossings, all possible combina-tions (i.e. CH ¥ GI; CH ¥ MA; CH ¥ PP; CH ¥ OM;MA ¥ PP; MA ¥ GI; MA ¥ OM; PP ¥ OM; PP ¥ GI; andGI ¥ OM) were considered, with 20 replicates percombination. For interspecific crossings, newborns ofeach of the five populations were placed in containersalong with newborns of the congeneric P. ophio-cephala (population from Cala Dragunara, North-West Sardinia) (ten replicates per combination). Toexclude biases caused by the differential survival ofpairs, statistical analyses were based on the meannumber of offspring produced by a given pair perweek. Cochran’s C-test was used to test the assump-tions of homogeneity of variance, and log-normaltransformation was used to remove heteroscedastic-ity. The SNK test was used a posteriori to comparemeans.

RESULTSSSU ANALYSIS

A total of 26 SSU sequences were obtained (GenBankAccession nos. FJ422480–FJ422505), 19 of whichwere 828 bp in length, whereas seven individualsshowed a deletion of 1 bp. The 48 polymorphic siteswere all phylogenetically informative in parsimony;whereas there were 52 nucleotide changes. Theaverage number of transitionsal and transversionalpairs, and mutations among populations, was 12, 6,and 18, respectively (Table 3). The estimate of haplo-type diversity among the populations yielded a valueof Hd = 0.723. Only four different haplotypes weredetected: one of them was site specific for HE and onefor OM, whereas one haplotype was basin specific forpopulations from the Western Mediterranean (GI andPP) and one again for the Eastern Mediterraneancounterpart (MA and CH). MP analysis yielded aconsensus tree (Fig. 2 shows the tree obtained

without the Goloboff criterion because such correctiondid not indicate differences in tree topology), whichplaced P. cf. cavernicola from the Indian Ocean as theoutgroup of the Atlanto-Mediterranean populationsattributed to P. agilis, whereas M. lineata, Minonaileanae, and Peraclistus sp. nov. were more distantlyrelated to the ingroup. Remarkably, individuals of theunpigmented species P. ophiocephala and P. caputdra-conis grouped within the cluster that included thetwo western Mediterranean populations of P. agilis(Fig. 2). Within the P. agilis group, the MP treeshowed four main clusters (bootstrap value > 70%)(A, B, C, and D; Fig. 2), corresponding, respectively, toindividuals from the: (1) Eastern Mediterranean (MAand CH); (2) Western Mediterranean (GI and PP); (3)Adriatic (OM); and (4) Baltic (HE) regions. Networkanalysis, carried out on the dataset comprising onlythe samples of the genus Pseudomonocelis, sets P. cf.cavernicola as the basal branch (Fig. 3) and revealedthe early separation between populations from theBaltic (HE) and Mediterranean (OM, GI, PP, MA, CH)regions. In the Mediterranean branch, individualsfrom OM sharply split from the individuals from thewestern (GI and PP) and eastern Mediterranean (MAand CH) regions, in addition to splitting from thoseof P. ophiocephala and P. caputdraconis (Fig. 3). Con-sistent with the MP dendrogram, individuals of theunpigmented Pseudomonocelis species were closer tothe western Mediterranean samples of P. agilis, fromwhich they were separated by few mutations only,whereas 14 mutations occurred between the westernand eastern Mediterranean populations of P. agilis(Fig. 3).

GENETIC STRUCTURE OF POPULATIONS

AllozymesThe 11 enzymes analysed yielded a total of 16 loci, 14of which were polyallelic (Table 1). A summary of themean estimates of intrapopulation genetic variability

Table 3. Number of transitions (below) and transversions(above) between populations referred to Pseudomonocelisagilis (small subunit dataset)

PP GI MA CH OM HE

PP – 0 2 2 5 6GI 0 – 2 2 4 5MA 6 6 – 0 6 8CH 6 6 0 – 6 7OM 8 7 11 10 – 5HE 12 11 14 12 11 –

CH, Charaki; GI, Giglio Island; HE, Helsingør; MA:Maliakós; OM, Omiš; PP, Porto Pozzo.

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is provided in Table 4. The lowest levels of within-sample genetic variability were observed in thesample from GI, whereas the highest estimatesoccurred in MA. The mean value of FIS (0.147 ± 0.087)was statistically significant (P < 0.01), and confirmedthe overall heterozygote deficit in the Mediterraneanpopulations of P. agilis, as further demonstratedby the statistically significant values of FIS obtained

for MA (P < 0.001) and OM (P < 0.01). The analysisof interpopulation Nei’s genetic distance conductedamong the samples of P. agilis varied from 0.009 (PPversus GI) to 1.107 (OM versus MA) (Table 5). Valuesof pairwise FST, ranging from 0.250 (PP versus GI) to0.936 (GI versus OM), were all statistically significant(P < 0.001).

ISSRsThe number of fragments and sizes of bands for eachISSR primer ranged from 18 (UBC809) to 26 (SAS1and SAS3) and from 350 bp (SAS3 and UBC827) to3000 bp (UBC827), respectively (Table 2). In particu-lar, in P. agilis from HE, 16 unique bands (bandspresent in only one population) were found, whereasMediterranean samples showed numerous uniquebands ranging from 1 (GI) to 13 (OM). Setting thevalue of FIS corresponding to a moderate deficitof heterozygotes for the Bayesian analyses, as hademerged from the allozyme survey (FIS ~ 0.15), thenumber of polymorphic loci and the values ofheterozygosity ranged from PLP = 12.3% (HE) toPLP = 25.5% (CH), and to H = 0.050 (MA) to H = 0.080(CH), respectively (Table 6). The output of model-based clustering analysis based upon admixturemodel with correlated allelic frequencies demon-strated that the highest hierarchical structurepresent in the data fitted the model with K = 4(Fig. 4). In addition to the cluster comprising HEspecimens, Mediterranean samples were splitted intothree clusters that matched the western (PP, GI) andeastern (CH, MA) basins of Mediterranean sea, andthe Adriatic (OM). Moreover, the high membershipcoefficient (q > 0.9), with which all but one individualwere assigned to the corresponding cluster, suggestedthat their genotype belonged exclusively to one group,thus reinforcing the hypothesis of a sharp separa-tion between these clusters. Finally, a STRUCTUREsurvey demonstrated the absence of gene flow amongthe sample analysed. The results obtained by theno-admixture model are not reported because they donot add any further useful information.

MORPHOLOGY

Both P. agilis and P. cetinae were originally describedas lacking an external vagina. However, duringthe study of sections, a distinct, bifurcated externalvagina, resulting from the distal splitting of thevaginal duct, and similar to that present in P. ophio-cephala (Schockaert & Martens, 1987), was unexpect-edly observed in all specimens belonging to westernand eastern Mediterranean populations The charac-ter has relevant phylogenetical implications and isdiscussed in detail below. Morphometric analysiswas based on characters shared by all populations,

Figure 2. Maximum parsinomy consensus dendrogrambased on a small subunit dataset. Bootstrap values > 50%are reported at each node. Individuals with body pigmen-tation are indicated in red; individuals with bifid vaginaare underlined. PP, Porto Pozzo; GI, Giglio Island; MA,Maliakós; CH, Charaki; OM, Omiš; HE, Helsingør.

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and thus excluded the presence of the vagina fromthe matrix. Based on unifactorial ANOVAs, popula-tions nonetheless were significantly differentiated foreach character. Homogeneous groupings detected byScheffé tests were not consistent among variables,

and showed overlaps among populations included inthe groupings (Table 7). PCA analysis was performedon the transformed variables, accounting for correla-tions of characters with body length. The main twocomponents extracted by PCA explained 79.1% of

Figure 3. Median-joining network among individuals belonging to the genus Pseudomonocelis, based on SSU dataset.Clusters A, B, C, D are as in Figure 2. HE, PP, Porto Pozzo; GI, Giglio Island; MA, Maliakós; CH, Charaki; OM, Omiš;HE, Helsingør.

Table 4. Measures of intrapopulation genetic variability (± standard error) calculated for the Mediterranean populationsreferred to Pseudomonocelis agilis (allozimic dataset)

PP GI MA CH OM

P95 6.1% 6.1% 18.8% 25.0% 6.1%Ho 0.026 ± 0.017 0.015 ± 0.009 0.056 ± 0.026 0.106 ± 0.036 0.020 ± 0.016He 0.031 ± 0.017 0.019 ± 0.011 0.080 ± 0.043 0.098 ± 0.049 0.029 ± 0.016FIS 0.161NS 0.187NS 0.296*** -0.087NS 0.315**

P95, level of polymorphism; Ho, observed heterozygosity; He, expected heterozygosity. NS, not significant.**P < 0.01; ***P < 0.001.CH, Charaki; GI, Giglio Island; MA, Maliakós; OM, Omiš; PP, Porto Pozzo.

Table 5. Matrices of pairwise Nei’s (1978) genetic distances (below), and pairwise FST (above) (allozymic dataset)

PP GI MA CH OM

PP – 0.250*** 0.898*** 0.878*** 0.919***GI 0.009 – 0.909*** 0.889*** 0.936***MA 0.723 0.730 – 0.686*** 0.921***CH 0.683 0.682 0.200 – 0.907***OM 0.439 0.446 1.107 1.077 –

CH, Charaki; GI, Giglio Island; MA, Maliakós; OM, Omiš; PP, Porto Pozzo.***P < 0.001.

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total variance (PC1 = 66.9%; PC2 = 12.2%). In par-ticular, PC-1 significantly discriminated populations(ANOVA: d.f = 5,141, F = 730.08; P < 0.001).

The bidimensional plot (Fig. 5A) clearly separatesspecimens from the Adriatic (OM) and the Baltic(HE); specimens from the western (PP, GI) andeastern (MA, CH) Mediterranean showed a partialoverlap. ANOSIM among populations yielded a globalvalue of R = 0.734 (P = 0.001). The highest separationamong groups (R = 0.952; P = 0.001) was attained byclustering populations into three units: OM; HE;CH + MA + PP + GI. The alternative solution withfour units (OM; HE; CH + MA; PP + GI), compatiblewith phylogroups, received less support (R = 0.762,P = 0.001). In any case, pairwise comparisons pro-duced R-values markedly lower than the global Ronly for pairs consisting of western Mediterranean(GI–PP; R = 0.286; P = 0.001) and eastern Mediterra-nean populations (MA–CH; R = 0. 435; P = 0.001),indicating poor separation among the specimens ofthese pairs of populations.

KARYOLOGY

All populations showed karyotypes with the haploidnumber n = 3. A unifactorial ANOVA based on therelative length and centromeric index of each chro-mosome was significant for each variable at P < 0.01.Homogeneous groupings detected by the SNK testwere not consistent among variables (Table 8). Themain two components extracted by PCA explained92% of total variance (PC1 = 77.7%; PC2 = 14.3%)(Fig. 5B). ANOSIM carried out on separate popula-tions yielded a value of global R = 0.842 (P = 0.001).The highest separation among groups (R = 0.951,

P = 0.001) was obtained clustering populations intofour units (OM; HE; CH + MA; PP + GI), correspond-ing to phylogroups. The three group hypothesis(OM; HE; CH + MA + PP + GI) received far lesssupport (R = 0.543; P = 0.001). Pairwise comparisonsproduced markedly lower R-values only for the pairsconsisting of the western Mediterranean (GI–PP;R = 0.092; P = 0.007) and eastern Mediterraneanpopulations (MA–CH; R = 0.085; P = 0.007), indicat-ing very poor separation among specimens of thesepairs of populations.

REPRODUCTIVE BIOLOGY

Intrapopulational crossing experiments showedobvious differences in the reproductive outputs, withwestern Mediterranean populations (GI; PP) produc-ing a significantly higher number of offspring per pairper week (15.45 ± 0.99 and 22.63 ± 2.07, respectively)than OM (11.13 ± 0.88) and the eastern Mediter-ranean populations, particularly the latter (MA:3.8 ± 0.35; CH: 5.96 ± 1.04) (F = 57.61; P << 0.001;SNK test: MA = CH < OM < GI < PP). Crossings be-tween specimens from different populations andspecies demonstrated:

1. No offspring were produced in any interspecificcrossing experiments, showing total reproductiveincompatibility among the specimens of P. agilisand P. ophiocephala;

2. Similarly, no offspring were produced in the inter-populational crossings involving the Mediterra-nean populations of P. agilis, with the exceptionof pairs derived from the same geographical area[i.e. western (GI ¥ PP) and eastern (CH ¥ MA)

Table 6. Measures of intrapopulation genetic variability (± SE) calculated for the populations referred to Pseudomono-celis agilis (inter-simple sequence repeats dataset)

PP GI MA CH OM HE

PLP 22.8% 15.8% 20.2% 25.4% 20.2% 12.3%H 0.063 ± 0.012 0.071 ± 0.015 0.050 ± 0.011 0.080 ± 0.014 0.059 ± 0.012 0.054 ± 0.011

CH, Charaki; GI, Giglio Island; H, heterozygosity; HE, Helsingør; MA, Maliakós; OM, Omiš; PLP, level of polymorphism;PP, Porto Pozzo.

Figure 4. Bayesian model-based clustering of individuals referred to Pseudomonocelis agilis. Individuals are representedby vertical bars.

INTEGRATIVE TAXONOMIC APPROACH TO PSEUDOMONOCELIS 915

© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 907–922

Tab

le7.

Bio

met

rica

lda

ta(m

ean

s±

SD

)an

dth

ere

sult

sof

anan

alys

isof

vari

ance

and

Sch

effè

test

ofth

esi

xpo

pula

tion

sre

ferr

edto

Pse

ud

omon

ocel

isag

ilis

Ch

arac

ter

PP

GI

MA

CH

OM

HE

F5,

141

PS

chef

fète

st

114

40±

47.4

1671

.4±

54.6

1544

±61

1984

.6±

72.2

1885

.4±

6316

76.4

±56

25.9

5<

0.00

1(P

P-M

A)(

MA

-GI-

HE

)(G

I-H

E-O

M)

(OM

-CH

)2

45±

2.9

51±

2.3

44±

1.4

52.6

±2.

860

.8±

2.2

20.8

±1.

666

2.6

<0.

001

(HE

)(O

M)(

CH

-MA

-GI-

PP

330

.63

±0.

7333

.07

±0.

8442

.7±

1.37

38.1

3±

0.68

76.0

9±

2.22

55.8

2±

1.21

79.5

9<

0.00

1(C

H-G

I-P

P)(

MA

)(H

E)(

OM

)4

30±

0.59

34.1

7±

0.75

37.7

±0.

9735

.9±

0.83

52.2

7±

2.53

43.3

6±

2.24

26.6

4<

0.00

1C

H-G

I-P

P)(

PP

-MA

)(M

A-H

E-O

M)

58.

73±

0.18

10.1

7±

0.38

14.8

±0.

4313

.87

±0.

3929

±1.

4122

.09

±1.

1284

.02

<0.

001

(GI-

PP

-CH

)(M

A)(

HE

-OM

)6

14.3

3±

0.67

12.3

±0.

6824

.17

±1.

5722

.83

±1.

534.

5±

0.23

2.09

±0.

2214

5.38

<0.

001

(HE

)(O

M)(

GI-

PP

)(P

P-C

H-M

A)

71.

4±

0.07

1.18

±0.

062.

74±

0.07

2.42

±0.

094.

09±

0.31

13.3

6±

0.54

149.

91<

0.00

1(O

M)(

GI-

HE

)(H

E-P

P)

(PP

-CH

)(M

A)

87.

83±

0.22

8.7

±0.

179.

43±

0.16

9.2

±0.

2413

.91

±0.

592

±0.

1613

7.28

<0.

001

(HE

)(C

H-G

I-P

P-M

A)(

OM

)9

3.83

±0.

123.

87±

0.08

3.17

±0.

083.

33±

0.09

3.09

±0.

164.

09±

0.09

23.9

5<

0.00

1(O

M-C

H)(

CH

-MA

)(M

A-G

I-H

E)

(GI-

HE

-PP

)10

4.37

±0.

094.

83±

0.1

3.8

±0.

113.

9±

0.11

3.45

±0.

164.

64±

0.15

25.8

9<

0.00

1(O

M-C

H)(

CH

-MA

)(M

A-H

E-G

I)(H

E-G

I-P

P)

1110

.17

±0.

2310

.4±

0.26

9.6

±0.

289.

43±

0.28

12.1

8±

0.25

10.4

5±

0.35

25.1

3<

0.00

1(C

H)(

MA

-HE

-GI-

OM

-PP

)

Ch

arac

ters

:(1

)bo

dyle

ngt

h;

(2)

dist

ance

betw

een

�an

d�

pore

s;(3

)le

ngt

han

d(4

)w

idth

ofco

pula

tory

orga

n;

(5)

len

gth

ofpe

nis

papi

lla;

(6)

wid

thof

prox

imal

mu

scle

shea

th;t

hic

knes

sof

(7)

mu

scu

lar;

and

(8)

pros

tati

cti

ssu

esof

pen

ispa

pill

a;(9

)do

rsal

and

(10)

ven

tral

cili

ale

ngt

h;(

11)

rhab

doid

len

gth

(for

mor

ede

tail

s,se

eM

ater

ial

and

Met

hod

s).

Val

ues

are

inmm

.C

H,

Ch

arak

i;G

I,G

igli

oIs

lan

d;H

E,

Hel

sin

gør;

MA

,M

alia

kós;

OM

,O

miš

;P

P,P

orto

Poz

zo.

Tab

le8.

Kar

yom

etri

cal

data

(mea

ns

±S

D)

and

the

resu

lts

ofan

anal

ysis

ofva

rian

cean

dS

tude

nt–

New

man

–Keu

lste

st(r

igh

t)of

the

six

popu

lati

ons

refe

rred

toP

seu

dom

onoc

elis

agil

is

PP

GI

MA

CH

OM

HE

F5,

119

PS

NK

test

Ch

rom

osom

e1

r.l.

36.0

5±

0.21

36.1

7±

0.22

37.0

9±

0.24

36.4

5±

0.14

38.7

9±

0.29

38.9

±0.

3118

.79

<0.

001

PP

=G

I=

MA

=C

H<<

OM

=H

Ec.

i.9.

84±

0.47

9.45

±0.

8625

.31

±1.

0227

.87

±0.

8227

.97

±0.

4840

.06

±0.

6326

9.78

<0.

001

GI

=P

P<<

CH

<M

A=

OM

<<H

EC

hro

mos

ome

2r.

l.33

.17

±0.

2333

.75

±0.

3333

.35

±0.

2134

.11

±0.

1932

.46

±0.

3332

.27

±0.

316.

85<

0.00

1H

E=

OM

=P

P=

CH

=G

I=

MA

c.i.

40.7

8±

0.36

41.3

8±

0.5

44.7

8±

0.4

45.1

7±

0.32

35.0

4±

0.38

31.2

1±

0.52

184.

81<

0.00

1H

E<<

OM

<<P

P=

GI

<<C

H<

MA

Ch

rom

osom

e3

r.l.

30.6

0±

0.2

30.0

4±

0.32

29.6

7±

0.27

29.9

4±

0.5

28.8

4±

0.31

28.7

3±

0.43

4.18

0.00

16H

E=

OM

=C

H=

GI

=M

A=

PP

c.i.

11.9

5±

0.53

11.2

2±

1.01

16.0

4±

0.6

17.8

4±

0.62

15.1

3±

0.57

16.8

9±

0.63

17.4

5<

0.00

1G

I=

PP

<<O

M=

CH

=H

E=

MA

CH

,C

har

aki;

GI,

Gig

lio

Isla

nd;

HE

,H

elsi

ngø

r;M

A,

Mal

iakó

s;O

M,

Om

iš;

PP,

Por

toP

ozzo

.r.

l.=

len

gth

ofch

rom

osom

e¥

100/

tota

lle

ngt

hof

hap

loid

gen

ome;

c.i.

=le

ngt

hof

shor

tar

m¥

100/

len

gth

ofen

tire

chro

mos

ome.

916 M. CASU ET AL.

© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 907–922

Mediterranean]. In both of these crossings, thenumber of offspring produced per week (17.12 ±1.64 and 7.49 ± 0.97, respectively) was not signifi-cantly different from the reproductive output ofthe parental populations at the P < 0.01 threshold(F = 3.28, P = 0.0449; and F = 4.80, P = 0.0118,respectively). Although the fertility of the F1 wasnot quantified, hybrids obtained from each experi-ment, pooled in separate containers, produced off-spring, further suggesting a lack of reproductivebarriers within the pairs of populations.

DISCUSSION

Recent revaluation of the definition of species astemporal segments of evolutionary lineages, divergingseparately from all such lineages (De Queiroz, 2005),has profound implications for taxonomic practice.Indeed, all properties so far considered as necessaryfor former species concepts (such as monophyly, diag-nosability, intrinsic reproductive isolation) are nowconsidered as properties attained by different lin-eages after speciation, and no single line of evidenceprovides the ‘best’ marker upon which attribution ofspecies status should be based (Padial & De la Riva,2009).

The adoption of an integrative taxonomic approach,combining all sources of evidence available, is thussuggested as ideal to provide necessary informationto base taxonomic decisions (Padial et al., 2009). Theusefulness of the approach is demonstrated by thepresent study, where patterns emerging from the dif-ferent approaches are not fully concordant:

Molecular markersThe MP dendrogram and the network analysisobtained from the SSU analysis sharply separated the

pigmented individuals into four phylogroups, corre-sponding to populations from Baltic, Adriatic, westernMediterranean, and eastern Mediterranean. Thisfinding is strongly supported by the occurrence of aconstant number of mutated sites among the indi-viduals from different basins, whereas no mutationswere found among the individuals from the samebasin.

Furthermore, the European pigmented Pseudomo-nocelis do not even appear to constitute a monophy-letic group because the unpigmented P. ophiocephalaand P. caputdraconis are nested within the vaginatedMediterranean taxa.

The distribution of genetic variability detectedby ISSRs showed the same pattern as that obtainedusing the SSU sequencing. The values of Nei’s geneticdistance between populations obtained from the allo-zyme dataset (D > 0.4) confirmed the occurrence ofa sharp genetic distinction between the taxonomicunits from the three Mediterranean basins surveyed.Indeed, the values of Nei’s distance found among eachcluster were far beyond the threshold values ofD @ 0.22, which are considered as the index of inter-specific differentiation (Thorpe & Solé-Cava, 1994).

MorphologyThe unexpected finding of external vaginae in speci-mens from the Mediterranean, absent in the popula-tions from the Adriatic and the Baltic, clearly splitsthe pigmented Pseudomonocelis into two blocks,although it does not offer clues for further sub-divisions. Although the biometrical data clearlydiscriminate the avaginate populations (OM-HE),any distinction between the vaginate populationsfrom eastern and western Mediterranean did notreceive statistical support. Typological taxonomy inflatworms relies heavily on the morphology of the

Figure 5. Bidimensional plots showing the two main components of PCA based on morphological (A) and karyological (B)data. �, Porto Pozzo; �, Giglio Island; +, Maliakós; �, Charaki; �, Omiš; �, Helsingør.

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copulatory structures, particularly of the sclerotizedpieces, which may allow identification among closely-related species (Artois, 2008). The type of copulatorybulb found in the genus Pseudomonocelis, lackingdistinctive sclerotized structures, has hardly beenconsidered to be of taxonomic value (Curini-Galletti &Casu, 2005), and groupings indicated by post-hocanalysis are indeed often conflictual. In this case,the two avaginate populations appear to be clearlydistinct because the Adriatic population (OM) had avery large copulatory bulb, with a particularly strongdevelopment of the prostatic tissue in the penispapilla, whereas specimens from the Baltic (HE) hada thin layer of prostatic tissue in the penis papilla,and a very thin muscle sheath lining the seminalvesicle.

KaryologyKaryotypes are widely used as taxonomic toolsin turbellarians (Benazzi & Benazzi-Lentati, 1976),although they are not always effectual in the discri-mination of closely-related species (Curini-Gallettiet al., 1989; Casu & Curini-Galletti, 2006). In thiscase, however, the four phylogroups are indeed clearlyseparated by their karyotypes, mostly differing inthe position of centromeres in corresponding chro-mosomes, presumably as a result of mechanisms ofpericentric inversion. Among the major differences,chromosome 1 was acrocentric in the western Medi-terranean populations (PP, GI), metacentric in HE,and subtelocentric in MA, CH, and OM. Chromosome2 was submetacentric in the HE and OM populationsand metacentric in the other populations (Table 8).

Reproductive biologyThe dataset is incomplete because the Baltic popula-tion could not be reared in the laboratory. Its habitatrequirements (it was the only population found onalgae) may have proved incompatible with the cul-tural techniques adopted, which, by contrast, wereeasily tolerated by mesopsammic populations fromthe same geographical area (M.C.G., unpubl. data).Similarly, the differences in reproductive outputsobserved in the Mediterranean populations may be aresult of laboratory bias. However, because there wereclear congruencies in the patterns of eastern Medi-terranean populations, which showed markedlyreduced fertility compared to their western Mediter-ranean counterparts, an adaptive value of the differ-ences, reflecting a differential reproductive strategyin a different environment, cannot be discounted. Bycontrast, there were unequivocal results from cross-ings involving different populations and species.Indeed, only populations from the same basin showedreproductive compatibility, whereas other combina-tions among the three basins (western and eastern

Mediterranean, and Adriatic), in addition to crossingswith the congeneric P. ophiocephala, were completelyunfertile. Therefore, at least these three differentgroups of Mediterranean populations behave as dif-ferent Biological species, providing additional infer-ence for their species status.

TAXONOMIC INFERENCES

A combination of the approaches adopted stronglysupports the conclusion that the wide-ranging Euro-pean pigmented species P. agilis is actually composedof four species, two of which are morphologicallyindistinguishable for the parameters essayed. Thetaxon P. agilis (Schultze, 1851), whose type-locality isGreifswald (Germany), should therefore be appliedonly to populations of the Baltic-Oresund area,whereas the taxon P. cetinae Meixner (1943) is not ajunior synonym of the former and applies to thepopulation of the type-locality (mouth of the riverCetina, Omiš, Croatia, Adriatic). The western andeastern Mediterranean species, provided with exter-nal vaginae, are named in the subsequent discussionas Pseudomonocelis sp. nov. A and Pseudomonocelissp. nov. B, respectively, and will be formally describedelsewhere. The data available at present suggest thatno single morphological character or any combinationof them may allow discrimination between the twospecies, which thus fulfil the definition for crypticspecies (Bickford et al., 2006). Cryptic species appearto be indeed pervasive in the Monocelidinae (Casu &Curini-Galletti, 2004, 2006), and may challenge theperception regarding the actual species compositionof the group.

POPULATION GENETIC INFERENCES

The allozyme analysis of populations of the complexdemonstrated very low levels of intrapopulationgenetic variability (Table 3). Similar results werefound by means of ISSRs (Table 5), whose values ofheterozygosities were comparable with those detectedby allozyme electrophoresis. In this context, it isworth noting that populations referred to P. agilis s.l.occupy discrete habitats separated by ecological andtopographical boundaries, ranging from mouths ofrivers to brackish microhabitat, around fresh-wateroutlets (Schütz & Kinne, 1955; Schütz, 2007; M.C.G.,pers. observ.). Albeit specimens of pigmentedPseudomonocelis taxa have morphological and behav-ioural traits which may aid dispersal (see below), thefragmentation of the habitat occupied by the complexmay act as an hindrance to an effective gene flowamong populations, and thus exacerbate the effect ofinbreeding, leading to the loss of genetic variabilitytill to the complete fixation of a number of alleles

918 M. CASU ET AL.

© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 907–922

(Riginos & Nachman, 2001; Billot et al., 2003). Theassumption of a scarce gene flow effectiveness is con-firmed by the highly significant value of FST observedbetween the populations of Pseudomonocelis sp. nov.A (GI and PP) and Pseudomonocelis sp. nov. B (MAand CH) (Table 6).

PHYLOGENETIC AND EVOLUTIONARY INFERENCES

The most parsimonious evolutive scenario based onSSU MP and network analyses (Figs 2, 3) suggestsa complex evolutionary history of the EuropeanPseudomonocelis species, involving (1) a basal splitbetween the Atlantic and Mediterranean lines; (2) adifferentiation of the group in the Mediterranean;and (3) a secondary evolution of the species providedwith external vaginae, which differentiated betweena western Mediterranean + unpigmented taxa cladeand the eastern Mediterranean line (Pseudomonocelissp. nov. B). The evolution of a bifurcated externalvagina in the Mediterranean species is of particularinterest. In Monocelididae, when present, a vaginais provided with a single opening in the vast majorityof the species. Apart from the MediterraneanPseudomonocelis clade, a somewhat comparablebifurcated vagina is only known in Monocelis bala-nocephala (Bohmig, 1902), from Chile. The indepen-dent evolution of the character in the MediterraneanPseudomonocelis thus appears to comprise a goodmorphological marker, supporting the molecularresults obtained. Reconstructions of the phylogeneticand phylogeographic relationships in Monocelididaehave been rarely attempted. In one of the few casesreported, concerning the M. lineata species complex,a basal split was also found between the Atlanticand Mediterranean species (Casu & Curini-Galletti,2004). Attempts at dating, based on allozymes, placedthe divergence in correspondence of the Messinianera, when the Mediterranean biota suffered massiveextinctions as a result of the extensive drying up ofthe basin (Hsü, Cita & Ryan, 1973; Bianchi & Morri,2000). Although a comparable scenario is not unlikelyfor the P. agilis complex, there is as yet no informa-tion on the basis of which a molecular clock, suitablefor the Monocelididae and the other species investi-gated in the present study, could be based. Similarly,a split between the eastern and western Mediterra-nean lines was also detected in the mesopsammicM. lineata complex (Casu & Curini-Galletti, 2004), aswell as in numerous macrofaunal taxa (Calvo et al.,2009). The two basins differ in their current andhistorical ecological parameters (in particular, salin-ity, temperature, and productivity); furthermore,during glaciations, the physical connections betweenthe two basins were considerably reduced (Bianchi,2007). It is therefore plausible that allopatry and

adaptation to local conditions may have been thedriving forces in the evolution of the Mediterraneanvaginate clade. One of the more interesting resultsof this study (i.e. the nesting of the unpigmentedP. ophiocephala complex within the pigmentedPseudomonocelis species) implies that pigmentation isa plesiomorphic feature in the genus and that, in theP. ophiocephala complex, a reversal of the characterhas occurred. Indeed, the presence of a pigmentationsimilar to that of the Mediterranean species (i.e. abrownish–yellow pigment in the parenchyma and areddish–brown girdle in front of the statocyst) is ofcommon occurrence in the genus Pseudomonocelis. Inaddition to being present in the individuals of P. cf.cavernicola used as an outgroup in the present study,this pigmentation is also found in the East AfricanP. pardii (Schockaert & Martens, 1987), the Austra-lian P. schockaerti (Curini-Galletti & Cannon, 1995),and in two further, undescribed, Australian species(Curini-Galletti & Cannon, 1995). In Platyhelm-inthes, in general, cephalic pigmentation is usuallyfound in ephiphytic or semiplanktonic species exposedto light. Therefore, an adaptive value for the pigmentas a shield against excessive amounts of light, whichcould potentially damage the optic receptors, has beenproposed (Armonies, 1989). Pigmented Pseudomono-celis species tend to occur either on algae or intertid-ally, often in brackish microhabitats, where theanimals tend to concentrate in the upper layer of thesediments as a result of the reduced thickness of theoxic layer. In cultures, newborns and juveniles ofthese species show a distinct swimming behaviour(M.C.G., pers. observ.), which is presumably relatedto their dispersal and colonization of new micro-habitats, thus leading to their exposure to light.By contrast, representatives of the unpigmented P.ophiocephala complex are typical interstitial organ-isms that occur in stable, less-fragmented habitats(sandy bays), and juveniles do not show any swim-ming behaviour in culture (M.C.G., pers. observ.). It isworth noting that network analysis shows that only afew mutations differentiate the pigmented Pseudomo-nocelis sp. nov. A from P. ophiocephala and P. caput-draconis. Consequently, the switch to a differenthabitat and life style may have determined a rela-tively quick loss of pigmentation. Differentiationamong pigmented and unpigmented species may thusbe interpreted as a case of ecological speciation as aresult of habitat shift (Johannesson, 2003). The evo-lutive scenario of the group includes further stepsbecause the four unpigmented species of the P. ophio-cephala complex are differentiated by sediment pref-erence and have complex geographical patterns, withthree species occurring in allopatry and the fourthspecies (P. ophiocephala) having a range broadly over-lapping the others (Casu & Curini-Galletti, 2006).

INTEGRATIVE TAXONOMIC APPROACH TO PSEUDOMONOCELIS 919

© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 907–922

The preliminary data obtained in the present study,involving only two species (P. ophiocephala and P.caputdraconis), suggest a comparatively recent sce-nario for their divergence because only two mutationsoccur between them. However, their speciation pro-cesses need to be studied in more detail.

CONSERVATION INFERENCES

The fragmentation of a formerly wide-ranging speciesinto discrete taxonomic units, each with limited distri-bution, raises issues related to the conservation ofthese species. The habitat of the three Mediterraneanpigmented species is limited to the sediments consist-ing mostly of well sorted coarse sand, in confined areaswith fresh-water outlets. They usually avoid evenslightly silty sediments, where other Monocelididspecies occur (M.C.G., pers. observ.). The survival ofthese populations is thus linked to the availability ofthe required habitat, which is often affected by humanactivity. A search in the Omiš area for the type-population of P. cetinae proved particularly frustratingbecause the mouth of the river Cetina has been heavilymodified by urban development subsequent to Meix-ner’s times, and individuals of the species were onlyfound in literally few square meters of a relativelyunaffected area. Similarly, repeated researches con-ducted by one of us (M.C.G.) in the Baltic for P. agilis,in the stations indicated by Schütz & Kinne (1955) andAx (1959), were completely unsuccessful, and thespecies was later serendipitously found by M.C.G.during sampling for other organisms in the Helsingørharbour. In the light of the results discussed above,each known population of P. agilis, P. cetinae,Pseudomonocelis sp. nov. A and Pseudomonocelis sp.nov. B, may be thus regarded as an evolutionarysignificant unit (Ryder, 1986), namely as a geneticallyand ecologically characterized population.

CONCLUSIONS

The study of the widespread pigmented speciesP. agilis using an integrative approach showed ahitherto unsuspected taxonomic diversity: not only isthe disjunct Baltic population not conspecific with theMediterranean populations, but also within the Medi-terranean populations, three distinct taxa can be rec-ognized, two of which share previously undetectedrelationships with a complex of unpigmented, crypticspecies. It is thus evident that the choice of pigmen-tation (which appears to be a plesiomorphic featurefor the genus Pseudomonocelis and is presumably ofadaptive value in meiobenthic Platyhelminthes) as ataxonomic marker for the group is to be rejected. Thisfinding exemplifies the overall poor state of knowledgeregarding the taxonomy of meiofauna, even in com-

paratively well-studied areas as Europe, and hints tothe pitfalls connected with specific attribution basedon one or very few supposedly clearly diagnosticmarkers, which may lead to overlooking other morpho-logical differences. Furthermore, morphology alonedoes not offer any clues in the discrimination betweenPseudomonocelis sp. nov. A and Pseudomonocelis sp.nov. B, which instead is supported by molecular andkaryological data, and cross-breeding experiments.In this case at least, the resolution power, at thespecies level, of SSU points to its potential usefulnessas DNA-barcode for taxonomy of Platyhelminthesbecause the potential of the COI (Folmer et al., 1994)as a species-specific diagnostic tool has not yet beenexplored in the group. The results of this researchhighlight the following conclusions, which may have amore general scope for meiofauna:

1. The reconstruction of phylogenetic relationshipsof mesopsammic congeneric species suggests acomplex pattern of speciation involving both allo-patric processes, operating on different scales (atthe level of Atlantic/Mediterranean disjunction andat the intra-Mediterranean level), and processesconnected to speciation by habitat shift, with only afew mutations occurring among some of the termi-nal species, suggesting a comparatively recent datefor their divergence. Models of speciation in theSea are, as a rule, based on vagile macrofaunalorganisms, with much longer generation timesthan meiobenthic organisms and generally withplanctonic larval stages, allowing for wide dispersal(Palumbi, 1994). Mesopsammic organisms maythus show different scales and tempos of speciation,which are yet to be explored in detail;

2. Despite their potential for along-coast dispersal atthe very least (Committo & Tita, 2002), mesopsam-mic organisms may have limited distributions andstrict ecological requirements. Human activitiesmay thus unknowingly pose a threat to theirsurvival;

3. Finally, the combination of cryptic species andsmall-scale distribution suggests that the contribu-tion of meiofauna to marine biodiversity may beseriously underestimated at present.

ACKNOWLEDGEMENTS

We gratefully acknowledge Giulia Ceccherelli andMassimo Scandura (University of Sassari) for theirinvaluable help during the statistical analysis, andthe anonymous referees for their useful comments.The research benefited from a grant by the ItalianMinistry of Research (MIUR-PRIN 2007 ‘Approcciointegrato all’identificazione dei Proseriati’).

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