Abstract

Pseudomonocelis ophiocephala (Schmidt, 1861), a mesopsammic proseriate, is common in a variety of shallow water habitats in the Mediterranean. The genetic relationships between morphologically indistinguishable populations across the Mediterranean were surveyed by means of combined allozyme and Random Amplified Polymorphic DNA (RAPD) analysis. Genetic distances, UPGMA cluster analysis and F-statistics based on 27 allozyme genetic loci and 68 RAPD primer fragments were consistent in showing that the taxon Pseudomonocelis ophiocephala is a complex of sibling species, consisting of four taxonomic units. The four species differ in distribution and habitat: sibling A is widespread in lower intertidal habitats of the Mediterranean, in well-sorted, medium- to coarse-grained sand; siblings B, C and D show a restricted distribution (Corsican–Sardinian region and Elba Island, west and east coast of Greece, respectively) in low-energy marine habitats. Given that the species of the complex are morphologically indistinguishable, the type material is absent, and that there are two siblings in the type locality (Corfu Island, Greece), a neotype is designated for P. ophiocephala. The three further siblings are named; species descriptions are based on non-morphological characters (karyotype, allozymic and RAPD patterns). Distributions and reconstruction of phylogenetic relationships based on allozyme data suggest that both allopatric and ecological speciation have played a role in the evolutionary history of the species complex.

INTRODUCTION

The actual extent of marine biodiversity is presently under debate (reviewed by, e.g., Gray, 1997). The number of described species is deemed under-representative even for charismatic macrofaunal groups (Bouchet et al., 2002). The problem is particularly acute in groups pertaining to the meiofauna (Lambshead, 1993; Godfray & Lawton, 2001; Blaxter, 2003), and especially for ‘soft-bodied’ taxa, such as Rhabditophora (‘Platyhelminthes’partim) (cf. Jondelius et al., 2002), which are routinely excluded from ecological and biodiversity studies owing to problems in identification of fixed material (Cannon & Faubel, 1988).

However, the Rhabditophora are important components of meiobenthic communities worldwide, and their diversity and biomass, particularly in intertidal areas, may even exceed those of the Nematoda (Martens & Schockaert, 1986). The relative abundance of Rhabditophora peaks in coarse, high-energy sediments, where they may represent up to 95% of total meiofauna (Remane, 1933; Radziejewska & Stankowska-Radziun, 1979).

The particular difficulty in the classification of most rhabditophoran species, based on observations on both living and sectioned specimens (Cannon & Faubel, 1988), constitutes a major hindrance to their study. Furthermore, the relative abundance of sibling species in the group makes species attribution critical (Curini-Galletti, 2001). In response to the difficulties inherent to the study of Rhabditophora, a molecular protocol for the identification of meiofaunal ‘turbellarians’ has even been proposed (Litvaitis et al., 1994). Indeed, classification of rhabditophoran taxa such as the Monocelidinae (Proseriata: Monocelididae) is particularly problematical as most members of the subfamily lack sclerotized structures in the male copulatory organ, a crucial problem because this is the basis for species identification in Proseriata (see, e.g. Ax, 1956). Not surprisingly, the geographical ranges of members of the Monocelidinae are amongst the widest known in Proseriata, raising suspicions that these distributions may result from the lumping together of undetected sibling species. Recently, the widespread amphiatlantic species Monocelis lineata (O.F. Muller, 1774) was shown, on the basis of genetic evidence, to consist of at least six sibling species, distinguished by range and/or ecological requirements (Casu & Curini-Galletti et al., 2004).

Pseudomonocelis ophiocephala (Schmidt, 1861), an abundant Mediterranean shallow water species (Murina, 1981), is among the Monocelidinae with wide distribution and plastic ecology. Previous assays on 13 populations showed the existence of two karyotypes (‘a’ and ‘b’). Karyotype ‘a’, with chromosome 1 acrocentric and chromosome 2 metacentric, was widely distributed in populations from Corsica to Israel. Karyotype ‘b’, with chromosome 1 at the border between subtelo- and submetacentric, and chromosome 2 submetacentric, was found in populations from the Corsican–Sardinian complex, including Elba Island, and on the west and east coasts of Greece. In some cases, populations with different karyotypes were found in sites only a few kilometres apart. A morphological analysis, based on 21 characters, revealed a marked heterogeneity among populations for all characters considered, but failed to reproduce the two clusters revealed by karyotyping (Curini-Galletti & Casu, 2005). Habitat heterogeneity, ranging from high-energy coarse to low-energy, silty sediments, and, in the Corsican–Sardinian complex, to reduced sediments under a ‘banquette’ (a thick layer of stranded dead leaves) of the seagrass Posidonia oceanica (L.) Delile, suggested that a biochemical and genetic analysis of populations was needed, in order to assess their taxonomic status.

Allozyme electrophoresis is a well-established technique for the study of the genetic structure of natural populations, and it has been applied to a wide range of organisms (reviewed by, e.g. Thorpe, 1982). The method has been extensively used to investigate cryptic speciation and interspecific relationships in many species, especially in taxa occurring in sympatry (reviewed by Thorpe & Solè-Cava, 1994). Furthermore, RAPD markers have been applied to identify species (Crossland et al., 1993; Dinesh et al., 1993), to separate species complexes, and to confirm close relations between populations of marine invertebrate species (e.g., Manchenko & Radashevsky, 1994, 1998; Sato & Masuda, 1997). In many respects, RAPDs have replaced or complemented enzyme electrophoresis studies (e.g., De Wolf, Backeljau & Verhagen, 1998; Mamuris, Stamatis & Triantaphyllidis, 1999; Huang, Peakall & Hanna, 2000). The distinct advantage of the RAPD technique is that it requires such small amounts of DNA that even tiny, single specimens can be used for a number of analyses. However, not all variation at RAPD loci can be detected and not all primers produce polymorphisms. Given that the RAPD technique is less laborious than other fingerprinting methods, producing results with low statistical error (Naish et al., 1995), and that it does not require prior knowledge of DNA sequences (Hadrys, Balick & Schierwater, 1992), it could be introduced as a complementary tool to treat questions related to population genetics (Mendel, Nestel & Gafny, 1994;Damato & Corach, 1997; Tassanakajon et al., 1997). The advantages and disadvantages of each technique in the field of population genetics have been reported in a number of review articles (e.g., Hadrys et al., 1992; Carvalho & Hauser, 1994; Ward & Grewe, 1994), and many authors have underlined the importance of simultaneous applications of different method in population genetics (Carvalho & Hauser, 1994; Ward & Grewe, 1994; Ward, Woodwark & Skibinski, 1994). The results of a combined allozyme and RAPD analysis are presented here.

MATERIAL AND METHODS

Samples

This study was conducted on 18 samples of Pseudomonocelis ophiocephala. Specimens were collected in the following localities (see Fig. 1 and Table 1 for specific details on stations): France: Occelluccia (OCC), reduced medium sand beneath a Posidonia oceanica‘banquette’ (May 1996); Calvi (CVI), harbour, medium-coarse sand (May 1996); Ventilegne (VEN), reduced medium sand beneath a ‘banquette’ of P. oceanica (April 2000). Italy: Porto Torres (PTO), harbour, at the mouth of the river Mannu, medium-coarse sand (September 2000); Cala Lunga (CLU), reduced medium sand beneath a ‘banquette’ of P. oceanica (March 1999); Livorno (LIV), harbour, medium-coarse sand (July 1996); Portoferraio (PFE), at the mouth of the river Madonnina, medium-coarse sand (April 1999); Biodola (BIO), reduced medium-coarse sand beneath a ‘banquette’ of P. oceanica (February 1999); La Strea (STR), medium sand (March 2000). Croatia: Omiš (OMI), town beach, medium-coarse sand (June 2001). Greece: Gouviá (GOU), very sheltered, silty medium-fine sand (December 1997); Corfu (COR), Yacht Club harbour, coarse sand (December 1997); Patrás (PAT), about 5 km south of town, at the sheltered outlet of an artificial channel, silty medium sand (September 1998); Maliakós (MLK), at the mouth of a stream, silty coarse sand (September 1998); Nea Mihanióna (NMI), harbour, silty coarse sand (September 1998); Kámena Voúrla (KVO), town beach, silty very coarse sand (September 1998); Piraeus (PIR), Glyfada, medium-coarse sand (September 1998); Israel: Haifa (HAI), small harbour north of Tel Shiqmona, medium-coarse sand (October 1997). As outgroup, we used a sample of the congeneric species Pseudomonocelis cetinae Meixner, 1943, collected at Porto Pozzo (PPc) (Italy) (April 1999).

Figure 1.

Location of sampling sites of Pseudomonocelis ophiocephala and P. cetinae. For population codes see Table 1.

Figure 1.

Location of sampling sites of Pseudomonocelis ophiocephala and P. cetinae. For population codes see Table 1.

Table 1.

Location of samples of Pseudomonocelis ophiocephala and P. cetinae collected in the Mediterranean

Sampling station Codea,b Latitude; Longitude Karyotype Genetic analysisc 
Porto Torres PTOa 40°50′N; 08°24′E ‘a’ A, R 
Cala Lunga CLUa 38°58′N; 08°24′E ‘b’ A, R 
Occelluccia OCCa 42°33′N; 08°43′E ‘b’ 
Calvi CVIa 42°34′N; 08°45′E ‘a’ 
Ventilegne VENa 41°26′N; 09°07′E ‘b’ A, R 
Livorno LIVa 43°33′N; 10°18′E ‘a’ 
Portoferraio PFEa 42°48′N; 10°19′E ‘a’ A, R 
Biodola BIOa 42°48′N; 10°19′E ‘b’ 
Omiš OMIa 43°26′N; 16°41′E ‘a’ A, R 
La Strea STRa 40°15′N; 17°53′E ‘a’ A, R 
Gouviá GOUa 39°39′N; 19°50′E ‘b’ 
Corfu CORa 39°37′N; 19°55′E ‘a’ 
Patrás PATa 38°14′N; 21°44′E ‘b’ A, R 
Maliakós MLKa 38°52′N; 22°33′E ‘b’ A, R 
Nea Mihanióna NMIa 40°27′N; 22°51′E ‘b’ 
Kámena Voúrla KVOa 38°45′N; 22°51′E ‘b’ A, R 
Piraeus PIRa 37°56′N; 23°35′E ‘a’ A, R 
Haifa HAIa 32°55′N; 34°55′E ‘a’ 
Porto Pozzo PPcb 41°12′N; 09°16′E – 
Sampling station Codea,b Latitude; Longitude Karyotype Genetic analysisc 
Porto Torres PTOa 40°50′N; 08°24′E ‘a’ A, R 
Cala Lunga CLUa 38°58′N; 08°24′E ‘b’ A, R 
Occelluccia OCCa 42°33′N; 08°43′E ‘b’ 
Calvi CVIa 42°34′N; 08°45′E ‘a’ 
Ventilegne VENa 41°26′N; 09°07′E ‘b’ A, R 
Livorno LIVa 43°33′N; 10°18′E ‘a’ 
Portoferraio PFEa 42°48′N; 10°19′E ‘a’ A, R 
Biodola BIOa 42°48′N; 10°19′E ‘b’ 
Omiš OMIa 43°26′N; 16°41′E ‘a’ A, R 
La Strea STRa 40°15′N; 17°53′E ‘a’ A, R 
Gouviá GOUa 39°39′N; 19°50′E ‘b’ 
Corfu CORa 39°37′N; 19°55′E ‘a’ 
Patrás PATa 38°14′N; 21°44′E ‘b’ A, R 
Maliakós MLKa 38°52′N; 22°33′E ‘b’ A, R 
Nea Mihanióna NMIa 40°27′N; 22°51′E ‘b’ 
Kámena Voúrla KVOa 38°45′N; 22°51′E ‘b’ A, R 
Piraeus PIRa 37°56′N; 23°35′E ‘a’ A, R 
Haifa HAIa 32°55′N; 34°55′E ‘a’ 
Porto Pozzo PPcb 41°12′N; 09°16′E – 
a

Sample of P. ophiocephala;

b

Sample of P. cetinae;

c

A, allozymic analysis; R, RAPD analysis

Samples were collected by scooping up the superficial layer of sandy sediments (c. 0.5 L) from the lower intertidal level to a depth of 20–30 cm. The animals were extracted from sand in the laboratory using the MgCl2 decantation technique, as described by Martens (1984). For each population, a sample (30 specimens) was studied karyologically, according to the techniques reported by Curini-Galletti, Puccinelli & Martens (1989) (see introduction, Curini-Galletti & Casu, 2005) (Table 1). Specimens were then kept alive in small boxes containing sea water at 18 °C, prior to genetic analysis.

Allozyme electrophoresis

On average, 100 individuals from each sample were scored by horizontal cellulose acetate electrophoresis. We analysed entire individuals of P. ophiocephala and P. cetinae due to the small size of these species (1–3 mm). Each specimen was put in 5 µL of grinding buffer [Tris (0.05 m), EDTA (0.01 m), Mercaptoethanol (0.2%), Triton X100 (0.1%), corrected to pH 8.0 with HCl] in a 25 µL microwell where it was manually homogenized using a glass pestle. Supernatant fractions were applied to cellulose acetate membranes for electrophoresis (30 min at 300 V). A preliminary screening was performed on specimens from the reference population (Porto Torres, PTO) in order to optimize analytical conditions for the activity and resolution of enzymes. A total of 22 enzyme systems was assayed using the Tris EDTA maleate electrode buffer [Tris (0.1 m), EDTA (0.01 m), MgCl2 (0.001 m), corrected to pH 7.8 with maleic acid] and Tris citrate electrode buffer [Tris (0.1 m), MgCl2 (0.001 m), corrected to pH 7.2 with citric acid]. Enzyme staining was carried out according to procedures described by Pasteur et al. (1987) with slight modifications. Enzymes with scarce activity and resolution were discarded. We concentrated our efforts on 17 enzymes: aldolase ALD (E.C. 4.1.2.13), alkaline phosphatase AKP (E.C. 3.1.3.1); arginine kinase, APK (E.C. 2.7.3.3); fructokinase, FK (E.C. 2.7.1.4); glyceraldheide-3-P dehydrogenase, GAPDH (E.C. 1.2.1.12); glycerol-3-phosphate dehydrogenase, αGPD (E.C. 1.1.1.8); glucose-6-phosphate isomerase, GPI (E.C. 5.3.1.9); glucose-6-phosphate dehydrogenase, G6PDH (E.C. 1.1.1.49); hexokinase, HK (E.C. 2.7.1.1); isocitrate dehydrogenase (NADP+), IDH (E.C. 1.1.1.42); l-lactate dehydrogenase, LDH (E.C. 1.1.1.27); malate dehydrogenase, MDH (E.C. 1.1.1.37); malic enzyme (NADP+), ME (E.C. 1.1.1.40); mannose-6-phosphate isomerase, MPI (E.C. 5.3.1.8); peptidase A, PEP A (E.C. 3.4.11, substrate Leu-Gly); phosphogluconate dehydrogenase, PGDH (E.C. 1.1.1.44); phosphoglucomutase, PGM (E.C. 5.4.2.2). Nomenclature for protein-coding loci followed Shaklee et al.'s (1990) recommendations. For each locus the arbitrary value 100 was assigned to the commonest allele. Slower and faster electromorphs, representing other alleles, were given lower or higher numbers corresponding to their relative mobilities, respectively. Alleles were calibrated by running individuals from different populations on the same electrophoretic membrane.

RAPD analysis

Ten samples of P. ophiocephala (30 individuals for each sample) were also analysed using the RAPD technique (Table 1). A set of 26 RAPD 10-mer primers designed by the authors were obtained from Operon Technologies and Genset SA. Initially they were tested on five individuals of P. ophiocephala from each locality, in order to find out repeats producing a suitable number of variable bands.

Genomic DNA was extracted from the entire individuals using the QIAGEN DNeasy Tissue kit (QIAGEN Inc.) according to the manufacturer's instructions. Once extracted, DNA was stored in solution at 4 °C until RAPD-PCR amplifications.

Samples were amplified in 25 µL PCR reaction mixture containing 10 × PCR reaction buffer (Pharmacia), 3 mm MgCl2, 0.2 µm primer, 200 µm of each dNTP (Roche), 1 unit of Taq DNA Polymerase (Pharmacia), and up to 30 ng of genomic DNA. Amplification was performed in a MJ PTC-100 Thermal Cycler (MJ research) programmed for 45 cycles of 1 min denaturation at 94 °C, 1 min low stringency annealing at 36 °C, and 1 min primer extension at 72 °C to complete partial amplification. At the end a post-treatment for 5 min at 72 °C and a final cooling at 4 °C were performed. For each primer, negative controls and replicates were included in the amplifications in order to verify the occurrence of PCR antifacts and the repeatability of banding patterns.

The PCR products were analysed by electrophoresis using a 1.5% agarose gel in 1× TAE buffer (0.04 m Tris-acetate, and 0.001 m EDTA). Gels were run at 50 V for 3 h and stained by soaking gel in a 1 µL/10 mL ethidium bromide solution for 15 min. RAPD banding patterns on gels were visualized using a photo-UV transilluminator system and recorded by digital photography. One hundred base pair ladders (DNA Molecular Weight Marker XIV, Roche) were run for reference with each primer. We took a conservative approach to scoring by choosing only distinct, intense and reproducible bands.

Because RAPD markers are interpreted as dominant diallelic markers, the dominant allele determines the presence of the band, namely AA and Aa individuals have the (1) phenotype, whereas aa individuals have the (0) phenotype. Thus, each variable band was scored as present (1) or absent (0), and was considered to represent the phenotype of a distinct locus. Assuming that each RAPD band represents a single diallelic locus in Hardy–Weinberg equilibrium, the presence/absence dataset can be converted into allele frequencies (see, e.g. Apostol et al., 1996).

Statistical analysis of allozyme data

Allele frequencies (Appendix 1), mean number of alleles, percentage of polymorphic loci (P99), observed and expected heterozygosities were determined separately for each sample of P. ophiocephala and for the sample of the outgroup P. cetinae (Table 2), using BIOSYS software (Swofford & Selander, 1981). Deviations from Hardy–Weinberg equilibrium were assessed for each sample by means of an exact test using the Markov chain algorithm in the GENEPOP package (Raymond & Rousset, 1995). Differences in heterozygosity were tested for significance between all possible pairs of samples by paired t-tests of arcsine square-root transformed values of single locus Ho (Archie, 1985). Multiple tests were adjusted using the sequential Bonferroni correction with an initial α = 0.05 to correct for Type I error (Hochberg, 1988).

Table 2.

Within-sample estimates of genetic variability (±SE) based on allozymic dataset. MSL, mean sample size per locus; MAL, mean number of alleles per locus; P99, percentage of polymorphic loci (99% cut-off); Ho, observed heterozygosity; He, expected heterozygosity. For population codes see Table 1

 MSL MAL P99 Ho He 
PTO 63.0 ± 6.2 1.1 ± 0.1 11..1 0.046 ± 0.026 0.050 ± 0.028 
CVI 59.5 ± 6.7 1.1 ± 0.1 11..1 0.035 ± 0.021 0.038 ± 0.022 
PFE 39.5 (5.4) 1.1 ± 0.1  7.4 0.030 ± 0.022 0.033 ± 0.023 
LIV 58.2 (6.5) 1.1 ± 0.1 11..1 0.032 ± 0.018 0.042 ± 0.025 
STR 55.2 (6.3) 1.1 ± 0.1 11..1 0.025 ± 0.015 0.029 ± 0.018 
OMI 31.1 (4.6) 1.1 ± 0.1 11..1 0.047 ± 0.027 0.051 ± 0.028 
COR 57.6 (6.3) 1.1 ± 0.1 11..1 0.044 ± 0.024 0.054 ± 0.030 
PIR 84.3 (4.1) 1.2 ± 0.1 14..8 0.058 ± 0.027 0.062 ± 0.029 
HAI 58.7 (6.6) 1.2 ± 0.1 18..5 0.041 ± 0.020 0.052 ± 0.025 
CLU 26.5 ± 1.9 1.1 ± 0.1 11..1 0.041 ± 0.031 0.036 ± 0.024 
VEN 28.0 ± 1.8 1.1 ± 0.1 7..4 0.032 ± 0.023 0.031 ± 0.022 
OCC 63.1 ± 6.8 1.1 ± 0.1 14..8 0.018 ± 0.012 0.024 ± 0.017 
BIO 67.4 (5.5) 1.1 ± 0.1 14..8 0.052 ± 0.027 0.054 ± 0.028 
GOU 51.7 (6.9) 1.1 ± 0.1 14..8 0.028 ± 0.019 0.035 ± 0.024 
PAT 75.6 (4.8) 1.1 ± 0.1 14..8 0.033 ± 0.021 0.034 ± 0.022 
NMI 66.0 (6.7) 1.3 ± 0.1 22..2 0.061 ± 0.024 0.082 ± 0.032 
MLK 62.9 (6.2) 1.1 ± 0.1 14..8 0.035 ± 0.020 0.037 ± 0.021 
KVO 57.0 (6.3) 1.0 ± 0.0  3.7 0.006 ± 0.006 0.007 ± 0.007 
PPc 81.1 (5.2) 1.1 ± 0.1  7.4 0.033 ± 0.023 0.036 ± 0.025 
 MSL MAL P99 Ho He 
PTO 63.0 ± 6.2 1.1 ± 0.1 11..1 0.046 ± 0.026 0.050 ± 0.028 
CVI 59.5 ± 6.7 1.1 ± 0.1 11..1 0.035 ± 0.021 0.038 ± 0.022 
PFE 39.5 (5.4) 1.1 ± 0.1  7.4 0.030 ± 0.022 0.033 ± 0.023 
LIV 58.2 (6.5) 1.1 ± 0.1 11..1 0.032 ± 0.018 0.042 ± 0.025 
STR 55.2 (6.3) 1.1 ± 0.1 11..1 0.025 ± 0.015 0.029 ± 0.018 
OMI 31.1 (4.6) 1.1 ± 0.1 11..1 0.047 ± 0.027 0.051 ± 0.028 
COR 57.6 (6.3) 1.1 ± 0.1 11..1 0.044 ± 0.024 0.054 ± 0.030 
PIR 84.3 (4.1) 1.2 ± 0.1 14..8 0.058 ± 0.027 0.062 ± 0.029 
HAI 58.7 (6.6) 1.2 ± 0.1 18..5 0.041 ± 0.020 0.052 ± 0.025 
CLU 26.5 ± 1.9 1.1 ± 0.1 11..1 0.041 ± 0.031 0.036 ± 0.024 
VEN 28.0 ± 1.8 1.1 ± 0.1 7..4 0.032 ± 0.023 0.031 ± 0.022 
OCC 63.1 ± 6.8 1.1 ± 0.1 14..8 0.018 ± 0.012 0.024 ± 0.017 
BIO 67.4 (5.5) 1.1 ± 0.1 14..8 0.052 ± 0.027 0.054 ± 0.028 
GOU 51.7 (6.9) 1.1 ± 0.1 14..8 0.028 ± 0.019 0.035 ± 0.024 
PAT 75.6 (4.8) 1.1 ± 0.1 14..8 0.033 ± 0.021 0.034 ± 0.022 
NMI 66.0 (6.7) 1.3 ± 0.1 22..2 0.061 ± 0.024 0.082 ± 0.032 
MLK 62.9 (6.2) 1.1 ± 0.1 14..8 0.035 ± 0.020 0.037 ± 0.021 
KVO 57.0 (6.3) 1.0 ± 0.0  3.7 0.006 ± 0.006 0.007 ± 0.007 
PPc 81.1 (5.2) 1.1 ± 0.1  7.4 0.033 ± 0.023 0.036 ± 0.025 

UPGMA cluster analysis on pairwise Nei's (1978) unbiased genetic distances between populations was performed to construct an unrooted majority rule consensus tree using the NEIGHBOR and DRAWGRAM programs (PHYLIP package, Felsenstein, 1995). Nodes of the tree were tested using bootstrapping with 5000 replicates. In addition, populations with karyotype ‘a’ (Table 1) were ordinated in a bi-dimensional space with non-metric multidimensional scaling (MDS) (Guiller, Bellido & Madec, 1998) to examine the relationships depicted in the original distance matrix. Because divergence time among populations may be estimated from genetic distance data if a constant evolutionary rate of allozymes is assumed (see Thorpe, 1982, and Chao & Carr, 1993 for reviews), we assumed a divergence rate of 0.2 DN/Myr (Nei, 1987). This approach, even if subject to large standard errors (Avise, 1994), has proven to be useful in dating cladogenetic allopatric events (Sbordoni et al., 1990), especially for genetic distance values smaller than 1.

Levels of genetic heterogeneity among samples were quantified by FST using Weir & Cockerham's (1984) jack-knifed unbiased estimators. FST was tested for difference from zero permuting (5000 replicates) alleles within and between samples, respectively, over all loci with the software FSTAT (Goudet, 1995). Furthermore, we used the software ARLEQUIN (available on the web at http://lgb.unige.ch/arlequin) to perform an analysis of molecular variance (amova, Excoffier, Smouse & Quattro, 1992) on allozyme data.

Indirect jack-knifed estimates of gene flow (Nm, the effective number of migrants per generation) were obtained using Wright's (1943) island model, [FST = 1/(4Nm + 1)]. The relation between genetic divergence and geographical distance was determined using the Mantel test. We applied the test to the log-transformed matrix of pairwise nautical geographical distance and the matrix of pairwise estimates of gene flow [FST/(1 − FST)] (Rousset, 1997) using the program GENETIX (Belkhir et al., 1996).

Statistical analysis of RAPD data

Level of polymorphism (P99), heterozygosity (H) and jack-knifed F-statistics (Weir & Cockerham, 1984) values were obtained using the software TFPGA (Miller, 1997). We used t-tests (Archie, 1985) to assess the significance of differences in heterozygosity between all possible pairs of samples. An UPGMA dendrogram was constructed using the programs NEIGHBOR and DRAWGRAM (Felsenstein, 1995) on the basis of a matrix of Nei's (1978) genetic distance.

Hierarchical relationships were estimated by analysis of molecular variance (AMOVA) using amova PREP (Miller, 1998) and WINamova (Excoffier et al., 1992). We calculated P-values from a random permutation test using 10 000 replicates, and Φ-statistics representing the probability of obtaining by chance alone a more extreme variance than the observed values (Excoffier et al., 1992). All statistical analyses were performed applying the method outlined by Lynch & Milligan (1994).

Cladistic analysis

The analysis was based on the allozyme dataset; only polymorphic loci were considered. Coding was achieved by treating loci as characters, alleles as character states, and heterozygotes as polymorphisms, following Pleijel & Eide (1996). Clusters of populations revealed by allozyme and RAPD analysis were considered terminal taxa. Allozyme configurations for these terminal taxa were obtained by pooling respective populations. Pseudomonocelis cetinae was used as outgroup. The data matrix had five taxa, and 21 characters (Table 3): (1) AKP-1* (three alleles); (2) APK* (five alleles); (3) ALD* (two alleles); (4) FK* (two alleles); (5) GAPDH-1* (two alleles); (6) GAPDH-2* (two alleles); (7) GAPDH-3* (two alleles); (8) áGPD-2* (two alleles); (9) GPI* (six alleles); (10) HK-1* (two alleles); (11) HK-3* (two alleles); (12) IDH* (three alleles); (13) LDH-2* (four alleles); (14) MDH-1* (four alleles); (15) MDH-2* (two alleles); (16) ME* (three alleles); (17) MPI-1* (four alleles); (18) MPI-2* (three alleles); (19) PEP* (six alleles); (20) PGDH* (two alleles); (21) PGM* (six alleles) (Table 3). The matrix was edited in MACCLADE (Maddison & Maddison, 1992) and the parsimony analysis performed in paup (Swofford, 1993). Exhaustive search (with collapse option in effect) was applied, and all minimal trees were kept. Clade support was assessed by bootstrap and jack-knife (1000 replicates).

Table 3.

Character matrix of the four clusters of populations of Pseudomonocelis ophiocephala based on allozyme and RAPD analysis; P. cetinae is defined as outgroup

Characters 10 11 12 13 14 15 16 17 18 19 20 21 
Cluster A 0 & 1 0 & 1 & 3 & 4 0 & 3 0 & 2 0 & 2 0 & 2 0 & 2 & 3 & 4 0 & 2 
Cluster B 1 & 2 0 & 3 0 & 1 0 & 1 1 & 2 4 & 5 
Cluster C 1 & 3 0 & 2 0 & 1 1 & 2 1 & 2 
Cluster D 0 & 2 0 & 2 0 & 1 & 2 0 & 1 0 & 1 2 & 3 0 & 3 
P. cetinae 1 & 2 4 & 5 
Characters 10 11 12 13 14 15 16 17 18 19 20 21 
Cluster A 0 & 1 0 & 1 & 3 & 4 0 & 3 0 & 2 0 & 2 0 & 2 0 & 2 & 3 & 4 0 & 2 
Cluster B 1 & 2 0 & 3 0 & 1 0 & 1 1 & 2 4 & 5 
Cluster C 1 & 3 0 & 2 0 & 1 1 & 2 1 & 2 
Cluster D 0 & 2 0 & 2 0 & 1 & 2 0 & 1 0 & 1 2 & 3 0 & 3 
P. cetinae 1 & 2 4 & 5 

RESULTS

Allozymes

Twenty-seven allozyme loci were resolved in the 18 populations of Pseudomonocelis ophiocephala and in the population of P. cetinae. Allele frequencies are given in Appendix 1. Eleven alleles were locality-private in P. ophiocephala: namely GPI*106 in Calvi (CVI); MDH-1*102 in Omiš (OMI); LDH-2*98, ME*110, MPI-2*102 and PEP*102 in Haifa (HAI); PEP*94 in Occelluccia (OCC); MPI-1*96 in Patrás (PAT), IDH*102 and LDH-2*96 in Nea Mihanióna; and PGM*102 in Maliakós (MLK). Eleven alleles were species-private of P. cetinae, namely APK*104, ALD*102, Gapdh-1*105, Gapdh-2*105, Gapdh-3*110, ΑGPD-2*102, GPI*108, MDH-1*106, MPI-1*105, MPI-2*98 and PEP*110.

The levels of within-population genetic variation in P. ophiocephala were very low (Table 2). The mean number of alleles per locus ranged from 1.0 ± 0.0 to 1.3 ± 0.1, and the percentage of polymorphic loci ranged from 3.7% to 22.2% under the 0.99 criterion. The values of Ho ranged from 0.006 ± 0.006 to 0.061 ± 0.024 (mean Ho = 0.039), and He from 0.007 ± 0.007 to 0.082 ± 0.032 (mean He = 0.044) (Table 2). One out of 58 probability tests of genotypic fit with Hardy–Weinberg predictions showed significant departures from Hardy–Weinberg proportions after the Bonferroni correction. This deviation was caused by heterozygote deficiencies and occurred at the MDH-1* locus in the Cala Lunga (CLU) sample (P < 0.05). Probability values obtained by pairwise t-tests on arcsine square-root transformed values of observed heterozygosities gave no significant cases after the sequential Bonferroni adjustment.

The UPGMA tree of Nei's (1978) genetic distances grouped samples of P. ophiocephala in four distinct monophyletic clusters (A–D in Fig. 2), provided with bootstrap support higher than 50. The range of Nei's (1978) genetic distances between pairs of populations of the four groups varied from D = 0.142–0.683, whereas those within each group were substantially lower (D = 0.001–0.076, D = 0.012–0.049, D = 0.013 and D = 0.015–0.078 for group A, B, C and D, respectively). Genetic distances between samples of P. ophiocephala and P. cetinae were high, ranging from 0.543 to 1.369. A summary of the pairwise values of genetic distance is given in Table 4.

Figure 2.

UPGMA dendrogram of Nei's (1978) genetic distances among samples based on allozyme data. Bootstrap support > 50 is indicated by an asterisk. For population codes see Table 1.

Figure 2.

UPGMA dendrogram of Nei's (1978) genetic distances among samples based on allozyme data. Bootstrap support > 50 is indicated by an asterisk. For population codes see Table 1.

Table 4.

Matrix of pairwise Nei's (1978) genetic distances based on allozymic dataset. For population codes see Table 1

 PTO CVI PFE LIV STR OMI COR PIR HAI CLU VEN OCC BIO GOU PAT NMI MLK KVO 
CVI 0.008                  
PFE 0.012 0.036                 
LIV 0.007 0.028 0.001                
STR 0.008 0.013 0.015 0.013               
OMI 0.006 0.015 0.023 0.018 0.013              
COR 0.015 0.023 0.020 0.017 0.019 0.027             
PIR 0.038 0.043 0.042 0.039 0.041 0.052 0.016            
HAI 0.063 0.062 0.076 0.071 0.068 0.075 0.032 0.035           
CLU 0.217 0.230 0.222 0.221 0.206 0.209 0.237 0.257 0.301          
VEN 0.166 0.174 0.180 0.177 0.164 0.168 0.188 0.206 0.244 0.048         
OCC 0.234 0.247 0.238 0.238 0.224 0.226 0.254 0.273 0.316 0.020 0.035        
BIO 0.216 0.228 0.223 0.222 0.206 0.207 0.237 0.258 0.301 0.016 0.049 0.012       
GOU 0.456 0.468 0.481 0.474 0.470 0.456 0.474 0.487 0.520 0.636 0.558 0.660 0.604      
PAT 0.468 0.485 0.491 0.485 0.485 0.467 0.488 0.503 0.537 0.659 0.579 0.683 0.627 0.013     
NMI 0.248 0.273 0.253 0.249 0.273 0.257 0.224 0.210 0.236 0.490 0.424 0.508 0.466 0.232 0.233    
MLK 0.166 0.187 0.175 0.170 0.188 0.171 0.146 0.142 0.167 0.375 0.315 0.393 0.353 0.306 0.335 0.058   
KVO 0.166 0.184 0.183 0.178 0.185 0.164 0.152 0.179 0.184 0.363 0.304 0.383 0.337 0.280 0.308 0.078 0.015  
PPc 0.767 0.773 0.776 0.771 0.778 0.767 0.765 0.761 0.788 0.560 0.609 0.569 0.543 1.323 1.369 0.993 0.814 0.830 
 PTO CVI PFE LIV STR OMI COR PIR HAI CLU VEN OCC BIO GOU PAT NMI MLK KVO 
CVI 0.008                  
PFE 0.012 0.036                 
LIV 0.007 0.028 0.001                
STR 0.008 0.013 0.015 0.013               
OMI 0.006 0.015 0.023 0.018 0.013              
COR 0.015 0.023 0.020 0.017 0.019 0.027             
PIR 0.038 0.043 0.042 0.039 0.041 0.052 0.016            
HAI 0.063 0.062 0.076 0.071 0.068 0.075 0.032 0.035           
CLU 0.217 0.230 0.222 0.221 0.206 0.209 0.237 0.257 0.301          
VEN 0.166 0.174 0.180 0.177 0.164 0.168 0.188 0.206 0.244 0.048         
OCC 0.234 0.247 0.238 0.238 0.224 0.226 0.254 0.273 0.316 0.020 0.035        
BIO 0.216 0.228 0.223 0.222 0.206 0.207 0.237 0.258 0.301 0.016 0.049 0.012       
GOU 0.456 0.468 0.481 0.474 0.470 0.456 0.474 0.487 0.520 0.636 0.558 0.660 0.604      
PAT 0.468 0.485 0.491 0.485 0.485 0.467 0.488 0.503 0.537 0.659 0.579 0.683 0.627 0.013     
NMI 0.248 0.273 0.253 0.249 0.273 0.257 0.224 0.210 0.236 0.490 0.424 0.508 0.466 0.232 0.233    
MLK 0.166 0.187 0.175 0.170 0.188 0.171 0.146 0.142 0.167 0.375 0.315 0.393 0.353 0.306 0.335 0.058   
KVO 0.166 0.184 0.183 0.178 0.185 0.164 0.152 0.179 0.184 0.363 0.304 0.383 0.337 0.280 0.308 0.078 0.015  
PPc 0.767 0.773 0.776 0.771 0.778 0.767 0.765 0.761 0.788 0.560 0.609 0.569 0.543 1.323 1.369 0.993 0.814 0.830 

The FST value for the whole P. ophiocephala sample was very high (FST = 0.802 ± 0.045, P < 0.001), showing that most of the genetic variation was among populations. Remarkably, FST values were substantially lower when calculated among populations of each group [FST(A) = 0.393 ± 0.114, FST(B) = 0.440 ± 0.051, FST(C) = 0.230 ± 0.091, FST(D) = 0.527 ± 0.093, P < 0.001].

On the basis of the presence of four groups distinguished by the UPGMA tree (Fig. 2), we estimated the hierarchical relationships by multilocus analysis of molecular variance (AMOVA). The total genetic variation was partitioned (i) among groups, (ii) among populations within groups, and (iii) within groups (Table 5). The AMOVA indicated that the greatest portion of the genetic variation was found among groups (80.1%), whereas the amount of variance found within groups and among populations within groups was substantially lower (14.8% and 5.1%, respectively). However, all three Φ-values were significant by permutation tests (Table 5).

Table 5.

Results of hierarchical analysis of molecular variance (AMOVA) based on allozymic dataset.

Source of variation d.f. Variance component Percentage of variance Φ-statistics P 
Among groups (siblings)    3 1.37630 80.09 ΦSC = 0.254 < 0.0002 
Among populations within groups (siblings)   14 0.08706  5.07 ΦST = 0.852 < 0.0002 
Within groups (siblings) 3374 0.25500 14.84 ΦCT = 0.801 < 0.0002 
Source of variation d.f. Variance component Percentage of variance Φ-statistics P 
Among groups (siblings)    3 1.37630 80.09 ΦSC = 0.254 < 0.0002 
Among populations within groups (siblings)   14 0.08706  5.07 ΦST = 0.852 < 0.0002 
Within groups (siblings) 3374 0.25500 14.84 ΦCT = 0.801 < 0.0002 

A non-metric multidimensional scaling analysis was performed on populations belonging to cluster A. The MDS plot clearly showed a sharp distinction between western and eastern Mediterranean populations, separated along the horizontal axis (Fig. 3). For these populations, we obtained a low estimate of the effective number of migrants per generation (Nm = 0.084). Furthermore, the Mantel test, performed to assess isolation-by-distance within group A, indicated a significant relationship between geographical separation and genetic distance (G = 2.426, P = 0.016, after 5000 permutations).

Figure 3.

Multidimensional scaling of Nei's (1978) genetic distances between populations of group A based on allozyme data. The relatively low value of stress indicates reliability of graphic representation. For population codes see Table 1.

Figure 3.

Multidimensional scaling of Nei's (1978) genetic distances between populations of group A based on allozyme data. The relatively low value of stress indicates reliability of graphic representation. For population codes see Table 1.

RAPDs

The RAPD survey was performed on a subsample of ten populations of P. ophiocephala (Table 1). Seven of the 26 primers tested produced clear DNA fragments and the same profiles in repeat tests, and could be scored with confidence (Table 6). Furthermore, we excluded from the analysis any inadequate pattern or unsatisfactory amplification generated by these primers. Omitting the DNA template from the PCR reaction (i.e. negative control) failed to produce a banding pattern. No primer was monomorphic. The size of the fragment varied approximately from 200 bp to 3600 bp, and the number of the reproducible and well-resolved bands from 6 to 13 (Table 6). On the whole, we found eight private bands: five in the sample from Patrás (PAT) and one in the samples from Omiš (OMI), Cala Lunga (CLU) and Kámena Voúrla (KVO), respectively. The percentage of polymorphic loci (0.99 cut-off level) ranged from 19.1% to 51.5%, and the values of heterozygosities from 0.051 ± 0.012 to 0.159 ± 0.040 (mean H = 0.137). These values did not differ significantly by t-tests of arcsine square-root transformed values. Within-sample estimates of genetic variability based on RAPD dataset are given in Table 7.

Table 6.

Primer names and sequences, number of total (polymorphic) bands per primer and range of molecular weight in base pairs (bp) amplified PCR-RAPD

Primer Sequence (5′-3′) No. of bands Range of molecular weight (bp) 
C-02 GTGAGGCGTC 11 (11)  300–3000 
C-05 GATGACCGCC 12 (12)  300–3300 
C-08 TGGACCGGTG  6 (6) 1200−2700 
C-11 AAAGCTGCGG 13 (13)  400–3500 
C-16 CACACTCCAG 11 (11)  400–3600 
C-18 TGAGTGGGTG  7 (7)  200–2800 
R2 CGGCAAGCTC  8 (8)  300–2800 
Primer Sequence (5′-3′) No. of bands Range of molecular weight (bp) 
C-02 GTGAGGCGTC 11 (11)  300–3000 
C-05 GATGACCGCC 12 (12)  300–3300 
C-08 TGGACCGGTG  6 (6) 1200−2700 
C-11 AAAGCTGCGG 13 (13)  400–3500 
C-16 CACACTCCAG 11 (11)  400–3600 
C-18 TGAGTGGGTG  7 (7)  200–2800 
R2 CGGCAAGCTC  8 (8)  300–2800 
Table 7.

Within-sample estimates of genetic variability (±SE) based on RAPD dataset. P99, percentage of polymorphic bands (99% cutoff); H, heterozygosity. For population codes see Table 1

Sample No. of bands Private bands P99 H 
PTO 37 – 51..5 0.145 ± 0.036 
PFE 36 – 39..7 0.113 ± 0.029 
STR 37 – 47..1 0.151 ± 0.039 
OMI 38 48..5 0.159 ± 0.040 
PIR 37 – 47..1 0.134 ± 0.034 
CLU 30 45..6 0.127 ± 0.032 
VEN 26 – 38..2 0.104 ± 0.027 
PAT 32 47..1 0.159 ± 0.040 
MLK 19 – 19..1 0.051 ± 0.012 
KVO 26 35..3 0.100 ± 0.022 
Sample No. of bands Private bands P99 H 
PTO 37 – 51..5 0.145 ± 0.036 
PFE 36 – 39..7 0.113 ± 0.029 
STR 37 – 47..1 0.151 ± 0.039 
OMI 38 48..5 0.159 ± 0.040 
PIR 37 – 47..1 0.134 ± 0.034 
CLU 30 45..6 0.127 ± 0.032 
VEN 26 – 38..2 0.104 ± 0.027 
PAT 32 47..1 0.159 ± 0.040 
MLK 19 – 19..1 0.051 ± 0.012 
KVO 26 35..3 0.100 ± 0.022 

The UPGMA tree (Fig. 4), based on the matrix of pairwise Nei's (1978) genetic distances (Table 8), was consistent with the tree based on the allozyme dataset in showing four distinct group of populations (A–D in Fig. 2). Yet, the arrangement of the populations within group A in the allozyme and RAPD trees was different. Furthermore, distance matrices were not significantly correlated (Mantel test: G = 1.396, P = 0.101, after 5000 permutations). Estimates of FST using the expected heterozygosity values for the ten samples of P. ophiocephala, confirmed the presence of high levels of genetic divergence among populations (FST = 0.515 ± 0.029).

Figure 4.

UPGMA dendrogram of Nei's (1978) genetic distance among samples based on RAPD data. Bootstrap support > 50 is indicated by an asterisk. For population codes see Table 1.

Figure 4.

UPGMA dendrogram of Nei's (1978) genetic distance among samples based on RAPD data. Bootstrap support > 50 is indicated by an asterisk. For population codes see Table 1.

Table 8.

Matrix of pairwise Nei's (1978) genetic distances based on RAPD dataset. For population codes see Table 1

 PTO PFE STR OMI PIR CLU VEN PAT MLK 
PFE 0.134         
STR 0.118 0.127        
OMI 0.140 0.160 0.104       
PIR 0.061 0.133 0.078 0.166      
CLU 0.113 0.147 0.127 0.199 0.115     
VEN 0.123 0.179 0.133 0.208 0.127 0.034    
PAT 0.169 0.205 0.242 0.229 0.208 0.131 0.166   
MLK 0.184 0.254 0.202 0.273 0.180 0.132 0.159 0.201  
KVO 0.116 0.169 0.145 0.174 0.135 0.054 0.070 0.127 0.086 
 PTO PFE STR OMI PIR CLU VEN PAT MLK 
PFE 0.134         
STR 0.118 0.127        
OMI 0.140 0.160 0.104       
PIR 0.061 0.133 0.078 0.166      
CLU 0.113 0.147 0.127 0.199 0.115     
VEN 0.123 0.179 0.133 0.208 0.127 0.034    
PAT 0.169 0.205 0.242 0.229 0.208 0.131 0.166   
MLK 0.184 0.254 0.202 0.273 0.180 0.132 0.159 0.201  
KVO 0.116 0.169 0.145 0.174 0.135 0.054 0.070 0.127 0.086 

In contrast with results obtained from the analysis of molecular variance performed on the allozyme dataset (Table 5), the genetic variation revealed by RAPD survey was equally divided among groups (30.3%), among populations within groups (32.3%) and within groups (37.4%) of P. ophiocephala, with significant molecular differentiation revealed by the Φ-statistics (ΦSC = 0.463, ΦST = 0.625 and ΦCT = 0.303, respectively) (Table 9).

Table 10.

Estimates of the fixation index FST (Weir & Cockerham, 1984) between pairs of clusters (see Figure 2) calculated on the allozymic dataset

FST Cluster A Cluster B Cluster C 
Cluster B 0.696 ± 0.078   
Cluster C 0.755 ± 0.086 0.860 ± 0.029  
Cluster D 0.667 ± 0.093 0.831 ± 0.047 0.760 ± 0.070 
FST Cluster A Cluster B Cluster C 
Cluster B 0.696 ± 0.078   
Cluster C 0.755 ± 0.086 0.860 ± 0.029  
Cluster D 0.667 ± 0.093 0.831 ± 0.047 0.760 ± 0.070 
Table 9.

Results of hierarchical analysis of molecular variance (AMOVA) based on RAPD dataset

Source of variation d.f. Variance component Percentage of variance Φ-statistics P 
Among groups (siblings)   3 3.38429 30.26 ΦSC = 0.463 < 0.0002 
Among populations within groups (siblings)   6 3.61017 32.28 ΦST = 0.625 < 0.0002 
Within groups (siblings) 240 4.18800 37.45 ΦCT = 0.303 < 0.0002 
Source of variation d.f. Variance component Percentage of variance Φ-statistics P 
Among groups (siblings)   3 3.38429 30.26 ΦSC = 0.463 < 0.0002 
Among populations within groups (siblings)   6 3.61017 32.28 ΦST = 0.625 < 0.0002 
Within groups (siblings) 240 4.18800 37.45 ΦCT = 0.303 < 0.0002 

Cladistic analysis

The analysis yielded one single most parsimonious tree (tree length = 24 steps; consistency index = 1.000; retention index = 1.000). Support values are generally high (Fig. 5). Cluster B result as the outgroup of a clade consisting of clusters A, D, and C. Clusters C and D, including populations of western and eastern Greece, respectively, result as sister taxa.

Figure 5.

Cladogram depicting the phylogenetic relationships among clusters of population referred to Pseudomonocelis ophiocephala s.l. Bootstrap support (above) and jack-knife values (below) are indicated at each node. For population codes see Table 1.

Figure 5.

Cladogram depicting the phylogenetic relationships among clusters of population referred to Pseudomonocelis ophiocephala s.l. Bootstrap support (above) and jack-knife values (below) are indicated at each node. For population codes see Table 1.

DISCUSSION

We employed two molecular techniques, enzyme electrophoresis and RAPD analysis, for the assessment of the genetic divergence in populations of Pseudomonocelis ophiocephala. It is well established that allozyme analysis is less expensive but often yields less information than the RAPD technique (Liu & Furnier, 1993). Yet, there are also some problems with RAPD analysis, linked to the number of assumptions that have to be made to analyse the data (Lynch & Milligan, 1994). Because of the dominance property of RAPD markers, estimates of gene frequencies calculated from RAPD datasets are less accurate than those obtained from codominant markers, such as allozymes. Therefore, in studies using RAPDs, two to ten times more individuals should be sampled per locus, and more loci should be scored per individual, in comparison with allozyme analysis (Lynch & Milligan, 1994). In the present study, although smaller numbers of individuals per locus were screened compared with the allozyme analysis (≤ 30 vs. ≤ 100 individuals per population, respectively), about 2.5 times more polymorphic loci (68 vs. 27 loci per individual, respectively) were sampled in order to estimate genetic variability between populations. Moreover, after sequential Bonferroni adjustment, no significant deviations from Hardy–Weinberg equilibrium were found, with the exception of the data for MDH-1* in Cala Lunga). This fact implies that the assumption of Hardy–Weinberg equilibrium may also be acceptable for the RAPD dataset.

Within -population genetic variability

The allozyme analysis of P. ophiocephala revealed very low levels of within-population genetic variability. The polymorphism percentage and observed heterozygosities (P99 = 1.0–1.3%, Ho = 0.006–0.061, respectively) were much lower than those detected in other marine invertebrates (Selander, 1977; Nevo, Beiles & Ben-Shlomo, 1984), whereas they were comparable to values found in other Monocelididae (Casu & Curini-Galletti, 2004). Furthermore, the occurrence of a number of fixed alleles (Appendix 1) indicates that populations of P. ophiocephala are demographically unstable, possibly as a result of periodic bottlenecks or recent founder-effect events. The habitat of P. ophiocephala is limited to the lower intertidal zone of sheltered to moderately exposed areas, such as coves and small harbours, often with a freshwater outlet. This subjects the populations to considerable variations in chemical and physical parameters, such as temperature, O2 content and salinity, and, to some extent, hydrodynamic conditions and sediment grain size. Such variations may be the basis for the marked fluctuation of population size (including temporary disappearance) that we have frequently witnessed during successive samplings throughout the year (authors’ unpubl. data), and lead to the reduction or loss of genetic variability (see, e.g., Ayala & Valentine, 1978).

The allozyme survey revealed 12 polymorphic loci (about 44% of all loci) in the 18 samples investigated. For the RAPD technique, we used seven primer-pairs to identify 68 reliable polymorphisms (100% of all DNA fragments) in the ten samples analysed. According to the allozyme data, the genotypic proportions differed significantly from Hardy–Weinberg proportions in only one of 58 tests. This is less than a 2% occurrence and one which would be expected by chance alone, suggesting that breeding within the 18 populations sampled was substantially panmictic. We can conclude from these data that it is unlikely that populations of P. ophiocephala are involved in phenomena such as non-random mating, inbreeding or the Wahlund effect. The very short generation times of P. ophiocephala (specimens, in laboratory, attain sexual maturity at 4–5 weeks of age) (authors’ unpubl. data) may also rapidly undo the effect of recurrent demographic breakdowns on the Hardy–Weinberg equilibrium (e.g. Hanski & Gilpin, 1991; Lessios, Weinberg & Starczac, 1994; Lewis & Thorpe, 1994; Piertney & Carvalho, 1995; Burton, 1997).

As expected, the amount of within-population genetic variability found by means of RAPD markers was considerably higher than that detected by allozyme electrophoresis, with the exception of the heterozygosity values which were substantially comparable (H = 0.051–0.159 vs. He = 0.007–0.082, respectively). The selective pressure acting on the products of genes codifying for proteins may explain the fact that the allozyme data has shown lower levels of within-population genetic variation than that using RAPD markers, where random DNA is assayed.

Among -population genetic variability

A previous study of the morphology and karyology of P. ophiocephala revealed two main results. The first is that populations of P. ophiocephala can be grouped into two clusters, distinguished on the basis of karyotype and which occupy a largely overlapping range across the Mediterranean. Second, no consistent groupings among populations could be identified on the basis of morphological characters. The coexistence in sympatry of morphologically undistinguishable populations with distinct karyotypes, indicates that at least two sibling species were present within the taxon (Curini-Galletti & Casu, 2005).

The present genetic survey further confirms the existence of a complex of sibling species within the morphospecies P. ophiocephala, albeit at a more complex scale than inferred by karyology alone. In fact, four distinct genetic clusters can be recognized, whose significant genetic heterogeneity is confirmed by FST analysis. The extremely high FST value from the allozyme data (0.803) indicates that most of the total variance was explained by the between-sample component (Hedrick, 1999). The overall FST value (0.515) calculated from RAPD data, although lower, was in agreement with that calculated from the allozyme data. Analysis of the data using the amova test indicated a large amount of genetic variance between groups (80.1%, for the allozyme data vs. 30.3%, for the RAPD data), further demonstrating the genetic divergence among the four clusters of P. ophiocephala.

Analysis of Nei's (1978) genetic distance between populations using the allozyme data confirms that the populations could be grouped into four different clusters, with distance values ranging from D = 0.142–0.683. It is assumed that values of Nei's (1978) genetic distance below D ≈ 0.11 indicate con-specificity, and those above D ≈ 0.22 correspond with interspecific differentiation, with a ‘grey zone’ between the above values (Thorpe & Solé-Cava, 1994). Notably, values of genetic distances between some populations of clusters A and B and between all populations of clusters A and D are not clearly above the species differentiation threshold. However, the karyotype of populations belonging to cluster A distinctly differs from that of populations of clusters B and D, without any intermediates known (Curini-Galletti & Casu, 2005), and values of FST calculated between each pair of clusters were significantly high (Table 10). Furthermore, populations of clusters A and B maintain their distinction in sympatry (see below). In addition to the karyotype data, the presence of unique molecular configurations in each of four clusters (see Appendix 2), allows their recognition as distinct species, according with, e.g. the diagnosable phylogenetic species concept, defining species as ‘the smallest aggregation of (sexual) populations’…‘diagnosable by a unique combination of character states’ (Wheeler & Platnick, 1997). The lack of morphological differentiation among populations (Curini-Galletti & Casu, 2005) otherwise fits the definition of sibling species [genetically divergent populations indistinguishable on morphological basis (Mayr & Ashlock, 1991; Knowlton, 1993)].

Estimates of the time of divergence based on allozyme genetic distance (Nei, 1978) indicate that populations of the P. ophiocephala species complex diverged approximately 2.3–2.1 Mya, with an early emergence of siblings C and B, in the eastern and western Mediterranean basins, respectively, whereas divergence between the younger species pair (siblings D and A) can be dated about 1.5 Mya. These dates are consistent with a Plio-Pleistocene intrabasin radiation of the four siblings, after the Messinian ‘crisis’ (dated about 5.5 Mya) (Hsu, Ryan & Cita, 1973). Results of cladistic analysis are not consistent with the above scenario, in that sibling B appears as the most basal taxon, while siblings C and D are sister taxa. This discrepancy appears mainly to result from the number of private alleles of sibling C, uninformative in a cladistic approach, but determining higher genetic distance, and hence age of divergence, according to the formula proposed by Nei (1987).

The siblings of the P. ophiocephala species complex are characterized by different geographical range and/or habitat. Sibling A occurs in moderately exposed sediments with coarse to medium granulometry across the whole Mediterranean. Sibling B is found exclusively in the presence of the ‘banquette’ of the seagrass Posidonia oceanica in the Corsican–Sardinian region and Elba Island (western Mediterranean). Siblings C and D occur in low-energy, silty sediments on the west and east coast of Greece (eastern Mediterranean), respectively. Sister species relationships of the latter species, as established by cladistic analysis, are highly suggestive of speciation by means of allopatric divergence. Remarkably, the range of siblings A, B and C partially overlaps, and sibling pairs A and B, and A and C, have been found in locations only a few kilometres apart. However, the allozyme FST (FST A + B = 0.696, FST A + C = 0.755, respectively) data indicate the absence of gene flow, and the occurrence of discriminating loci in sympatric populations (see Appendix 1) represents strong evidence of differentiation at the species level (Thorpe & Solé-Cava, 1994).

Most sibling species in recent marine environments are sympatric (Knowlton, 1993), suggesting that within-province speciation (either allopatric or sympatric) is relatively common in nearshore marine environments (Marko, 1998). Both estimates of time of divergence and cladistic analysis revealed that sibling species A, with distinct karyotype and habitat, is not a basal taxon (Fig. 5). Based on a wider karyotypic study of the genus Pseudomonocelis, there are indications that karyotype ‘a’ is apomorphic (M. Curini-Galletti & M. Casu, unpubl. data). Furthermore, because P. cetinae, as well as sibling species B, C and D, occurs in low energy, confined environments (Schockaert & Martens, 1987; pers. observ.), the higher energy habitat of sibling species A may be similarly derived. It may thus be hypothesized that sibling A is the result of speciation through microhabitat specialization (high-energy vs. low-energy sediments), as often seen in coexisting sister species (Hellberg, 1998, and references therein). ‘Ecological inviability’ of hybrids could have then been further strengthened by chromosome rearrangements, potentially deleterious in hybrids. It is worth noting that small, isolated populations of interstitial flatworms, subject to repeated oscillations in numbers of individuals, offer ideal conditions for the fixation of chromosomal rearrangements (cf. Rieseberg, 2001). In this scenario, the present wide distribution of sibling A could be the result of dispersal, presumably easier for a species colonizing a less fragmented, and more energetic habitat. As for most meiofaunal organisms, P. ophiocephala s.l. lacks a larval stage. This, together with the reduced mobility of adults, provides limited potential for active dispersal. Passive recruitment in the water column is the main way of colonizing or re-colonizing new areas, and for the exchange of individuals and thus genes between geographically separated populations (Palmer, 1988).

There are indirect indications that this may be the case. Values of Nm for sibling species A and B in a comparable area (Corsican–Sardinian complex and adjacent Ligurian–Tyrrhenian sea area), based on the same number of populations (four), yielded markedly different results (Nm = 6.802 and Nm = 0.103, respectively). Estimates for siblings C (two populations) and sibling D (three populations) were similarly very low (Nm = 0.045 and Nm = 0.104, respectively). It is worth noting that estimates of gene flow with a Nm value lower than 1.0 indicate that gene exchanges are not sufficient to prevent genetic divergence as a result of genetic drift (see Slatkin, 1987 for a review).

On the other hand, the higher dispersal potential of sibling A is not altogether sufficient to prevent genetic divergence across the whole Mediterranean (average effective migration number of Nm = 0.084 for all populations). In particular, the enzyme survey indicated a sharp genetic differentiation between western and eastern populations of sibling A, as highlighted in the multidimensional scaling plot given in Figure 3, with a genetic hiatus, originating from a conspicuous decreasing of the rate of gene exchange between western and eastern populations, corresponding to the Straits of Otranto (Adriatic sea). However, it remains an open question whether the genetic discontinuity occurred during the glacial periods, when the physical connection between basins was considerably reduced (Por, 1989), a situation presently maintained by the limited dispersal capability of the species, or as a consequence of different selective regimes related to differences in the abiotic parameters (especially salinity and temperature) of western and eastern Mediterranean basins (Sarà, 1985; Stocker, 1992). Notably, the former hypothesis is corroborated by the estimate of the divergence time between western and eastern populations of siblings A (about 0.3 Mya), corresponding to the late Pleistocene. Nonetheless, the distribution of genetic variability detected by means of RAPD markers did not show the geographical pattern revealed by the allozymes. The higher level of genetic differentiation exhibited by allozymes compared to RAPDs (allozyme FST = 0.803 vs. RAPD FST = 0.515), usually considered neutral markers, and the occurrence of private alleles and alleles alternatively fixed in western and eastern populations suggests that some loci may be under selection.

Conservation and the naming of siblings

As a whole, the P. ophiocephala species complex shows traits, such as limited distribution, fragmented habitat, restricted bathymetrical range and poor dispersal capability, which may potentially threaten its conservation.

The preferred habitat for all the species of this complex (typically consisting of small coves) is particularly prone to human-induced disturbance across the Mediterranean (Bourcier, 1996). During this study, we have observed the local extinction of at least one population (Calvi), caused by the destruction of the residual beach within the harbour, and there are serious concerns for a further number of populations. The demise of the genetically distinct Calvi population is particularly regrettable, because the level of its genetic differentiation was sufficient to allow its recognition as an important unit of evolutionary divergence (see, e.g. Mayr, 1963; Bush, 1975; Mayr & Ashlock, 1991).

Sibling B as a whole appears particularly endangered, as it is restricted in range and only found in the presence of a ‘banquette’ of Posidonia oceanica seagrass. Seasonal removal of the ‘banquette’ may in fact result in colonization of the beach by sibling A, as we have witnessed at the sites of Ventilegne (Corsica) and Cala Lunga (Sardinia) (pers. observ.). This may be part of a natural cycle, allowing coexistence of siblings A and B, with relative peaks of abundance related to presence/absence of the ‘banquette’. In fact, degradation of the mass of seagrass leaves produces highly anoxic sediments (Holmer, Duarte & Marba, 2003), to which sibling B appears adapted, as distinct from the congenerics. At Ventilegne and Cala Lunga, hydrodynamic conditions allow for natural removal of the ‘banquette’ during the summer months. It is worth noting that in small, deep coves, the ‘banquette’, although varying in thickness, may persist from year to year, and allow year-round occupancy of the site by sibling B. Such areas may act as a source of potential colonizers for neighbouring, more open beaches, where the presence of a ‘banquette’ is seasonal. Unfortunately, the ‘banquette’ is increasingly seen as a hindrance to summer tourism because it impacts visually and on bathing, and is removed from many sites on a regular basis. The spread of this practice may seriously endanger the survival of sibling B.

In this worrying scenario, given that names may be helpful for specifying conservation efforts, both for the insertion in lists of endangered species and easiness of reference, we purposefully decided to give each of the sibling species a formal classification (see Appendix 2).

CONCLUSION

The results of this study parallel similar research showing that many cosmopolitan marine invertebrate species, which lack accessible characters allowing unequivocal species discrimination, are in reality complexes of sibling species (Grassle & Grassle, 1976; Manchenko & Radashevsky, 1994,, 1998; Klautau et al., 1999; Maltagliati et al., 2000). This finding in an abundant and comparatively well studied ‘species’ further questions the extent of our appreciation of marine biodiversity, particularly in groups such as interstitial Rhabditophora where molecular research is not commonplace in taxonomy.

Furthermore, our data reveal that marine interstitial flatworm species may have restricted distribution and specialized habitat requirements. These facts, coupled with demographic instability and limited potential for dispersal, raise concerns for their conservation. It thus seems reasonable to affirm that the lack of resolution of complexes of sibling species in meiofaunal organisms may contribute to the underestimation of the rate of marine extinctions, so far exclusively known for macrofaunal organisms (Roberts & Hawkins, 1999).

ACKNOWLEDGEMENTS

The following people and institutions offered hospitality during field collections: Eviatar Nevo (Institute of Evolution, University of Haifa, Israel), Ferdinando Boero and Adriana Giangrande (Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, University of Lecce, Italy); STARESO s.a. (Pointe Revellata, Calvi, France). We thank Ferruccio Maltagliati, Giorgio Binelli, Marilena Meloni, Tiziana Lai and Piero Cossu for technical assistance and for providing valuable comments and discussions. This research was partly supported by the European Community INTERREG III project, and by the ‘Centro di Eccellenza’ of the University of Sassari.

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APPENDIX 1

Allele frequencies at 27 allozyme loci in 16 samples of Pseudomonocelis ophiocephala and one of P. cetinae. For population codes see Table 1. Private alleles are in bold. N, sample size.

Locus Population
 
PTO CVI PFE LIV STR OMI COR PIR HAI 
Akp-1* 
(N60 20 58 27 25 14 25 65 20 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Akp-2* 
(N25 20 58 27 10 14 25 45 20 
1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Apk
(N100 89 56 100 32 80 49 76 47 
100 0.430 0.798 0.286 0.295 0.719 0.350 0.592 0.684 0.723 
91 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
97 0.570 0.202 0.714 0.705 0.281 0.650 0.408 0.316 0.277 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Ald
(N22 100 56 100 79 98 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Fk
(N22 100  56 100 80 30 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gapdh-1* 
(N30 30 20 30 14 20 30 74 30 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gapdh-2* 
(N30 30 20 30 14 20 30 74 30 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gapdh-3* 
(N56 100 92 100 57 20 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
αGPD-1
(N29 30 20 30 16 15 80 15 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
αGPD-2
(N29 15 20 30 16 15 80 15 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gpi* 
(N100 99 16 90 78 37 100 100 100 
100 0.580 0.328 1.000 0.878 0.923 0.608 0.350 0.290 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.650 0.710 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
104 0.420 0.657 0.000 0.122 0.077 0.392 0.000 0.000 0.000 
106 0.000 0.015 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
108 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
G6pdh-1*          
(N60 30 16 40 58 28 38 100 41 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
G6pdh-2* 
(N60 15 29 28 18 97 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-1* 
(N27 99 70 100 100 18 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Hk-2* 
(N27 99 94 100 100 18 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-3* 
(N27 99 58 100 100 18 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Idh
(N100 31 15 61 26 28 33 92 51 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Ldh-1* 
(N100 34 74 57 70 21 52 100 82 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Ldh-2* 
(N100 55 26 55 70 21 50 100 80 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.994 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.006 
Mdh-1* 
(N100 100 13 26 76 39 68 78 52 
100 1.000 1.000 1.000 1.000 1.000 0.692 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.308 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mdh-2* 
(N100 63 26 79 18 68 74 60 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Me* 
(N99 42 16 58 77 39 74 36 30 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.750 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 
Mpi-1* 
(N58 30 16 30 25 30 31 91 20 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mpi-2* 
(N58 30 16 30 16 30 31 91 48 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.760 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.240 
Pep
(N82 66 50 26 44 100 24 24 35 
100 0.250 0.129 0.590 0.558 0.136 0.000 0.479 0.229 0.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.688 0.000 
96 0.750 0.871 0.410 0.442 0.864 1.000 0.521 0.083 0.129 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.871 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Pgdh* 
(N100 100 90 99 100 30 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Pgm
(N100 88 16 98 100 24 80 98 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.781 1.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.219 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Locus Population
 
PTO CVI PFE LIV STR OMI COR PIR HAI 
Akp-1* 
(N60 20 58 27 25 14 25 65 20 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Akp-2* 
(N25 20 58 27 10 14 25 45 20 
1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Apk
(N100 89 56 100 32 80 49 76 47 
100 0.430 0.798 0.286 0.295 0.719 0.350 0.592 0.684 0.723 
91 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
97 0.570 0.202 0.714 0.705 0.281 0.650 0.408 0.316 0.277 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Ald
(N22 100 56 100 79 98 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Fk
(N22 100  56 100 80 30 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gapdh-1* 
(N30 30 20 30 14 20 30 74 30 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gapdh-2* 
(N30 30 20 30 14 20 30 74 30 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gapdh-3* 
(N56 100 92 100 57 20 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
αGPD-1
(N29 30 20 30 16 15 80 15 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
αGPD-2
(N29 15 20 30 16 15 80 15 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Gpi* 
(N100 99 16 90 78 37 100 100 100 
100 0.580 0.328 1.000 0.878 0.923 0.608 0.350 0.290 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.650 0.710 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
104 0.420 0.657 0.000 0.122 0.077 0.392 0.000 0.000 0.000 
106 0.000 0.015 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
108 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
G6pdh-1*          
(N60 30 16 40 58 28 38 100 41 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
G6pdh-2* 
(N60 15 29 28 18 97 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-1* 
(N27 99 70 100 100 18 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Hk-2* 
(N27 99 94 100 100 18 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-3* 
(N27 99 58 100 100 18 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Idh
(N100 31 15 61 26 28 33 92 51 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Ldh-1* 
(N100 34 74 57 70 21 52 100 82 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Ldh-2* 
(N100 55 26 55 70 21 50 100 80 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.994 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.006 
Mdh-1* 
(N100 100 13 26 76 39 68 78 52 
100 1.000 1.000 1.000 1.000 1.000 0.692 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.308 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mdh-2* 
(N100 63 26 79 18 68 74 60 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Me* 
(N99 42 16 58 77 39 74 36 30 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.750 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 
Mpi-1* 
(N58 30 16 30 25 30 31 91 20 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mpi-2* 
(N58 30 16 30 16 30 31 91 48 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.760 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.240 
Pep
(N82 66 50 26 44 100 24 24 35 
100 0.250 0.129 0.590 0.558 0.136 0.000 0.479 0.229 0.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.688 0.000 
96 0.750 0.871 0.410 0.442 0.864 1.000 0.521 0.083 0.129 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.871 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Pgdh* 
(N100 100 90 99 100 30 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Pgm
(N100 88 16 98 100 24 80 98 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.781 1.000 
94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.219 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Locus Population
 
CLU VEN OCC BIO GOU PAT NMI MLK KVO PPc 
Akp-1 
(N30 30 18 98 20 90 18 100 40 94 
100 0.000 0.000 0.000 0.000 0.000 0.000 0.167 0.140 0.000 0.000 
102 0.000 0.000 0.000 0.485 1.000 1.000 0.833 0.860 1.000 0.628 
104 1.000 1.000 1.000 0.515 0.000 0.000 0.000 0.000 0.000 0.372 
Akp-2* 
(N30 30 15 80 20 60 20 81 20 40 
1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Apk
(N42 27 100 45 31 49 81 48 29 45 
100 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
91 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 
95 0.000 0.000 0.000 0.000 0.435 0.224 0.000 0.000 0.000 0.000 
97 0.000 0.000 0.000 0.000 0.565 0.776 1.000 1.000 1.000 0.000 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Ald
(N30 30 100 60 100 100 100 70 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Fk
(N30 30 100 60 100 100 100 70 100 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 1.000 1.000 1.000 1.000 
98  0.000  0.000   0.000   0.000   1.000   1.000   0.000   0.000   0.000   0.000 
Gapdh-1* 
(N44 30 30 30 30 90 29 30 60 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Gapdh-2* 
(N47 30 30 60 30 90 29 30 60 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Gapdh-3* 
(N35 30 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
αGPD-1
(N20 15 60 15 90 15 30 100 25 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
αGPD-2
(N11 15 60 15 90 15 30 100 25 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Gpi* 
(N12 55 100 73 100 75 100 49 19 100 
100 1.000 0.773 0.980 0.952 0.005 0.007 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 
96 0.000 0.000 0.000 0.000 0.995 0.993 0.000 0.000 0.000 0.000 
104 0.000 0.227 0.020 0.048 0.000 0.000 0.000 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
108 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
G6pdh-1*           
(N20 30 65 100 22 100 61 100 50 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
G6pdh-2* 
(N20 30 36 98 10 100 20 100 40 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-1* 
(N30 30 100 100 100 100 100 100 100 100 
100 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 1.000 0.000 
96 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 
Hk-2* 
(N30 30 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-3* 
(N30 30 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 1.000 1.000 1.000 1.000 
94 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 
Idh
(N32 11 79 24 36 46 73 31 17 71 
100 0.031 0.636 0.000 0.000 1.000 1.000 0.890 1.000 1.000 0.000 
94 0.969 0.364 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.110 0.000 0.000 0.000 
Ldh-1* 
(N10 21 67 43 47 88 24 32 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Ldh-2* 
(N27 21 81 44 57 28 74 24 24 86 
100 1.000 1.000 1.000 1.000 0.307 0.000 0.041 0.979 1.000 1.000 
94 0.000 0.000 0.000 0.000 0.693 1.000 0.730 0.021 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.230 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mdh-1* 
(N26 43 26 30 79 46 92 39 33 74 
100 0.442 1.000 0.308 0.300 1.000 1.000 1.000 1.000 1.000 0.000 
96 0.558 0.000 0.692 0.700 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Mdh-2* 
(N22 43 80 44 24 50 75 39 38 40 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.400 1.000 0.895 1.000 
96 0.000 0.000 0.000 0.000 1.000 1.000 0.600 0.000 0.105 0.000 
Me* 
(N30 22 63 54 42 33 92 30 37 93 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.451 1.000 1.000 1.000 
98 0.000 0.000 0.000 0.000 1.000 1.000 0.549 0.000 0.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mpi-1* 
(N30 30 15 100 20 83 20 100 41 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.446 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 1.000 0.554 1.000 1.000 1.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Mpi-2* 
(N30 30 15 100 20 74 20 100 50 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Pep
(N13 30 53 23 50 36 59 23 30 44 
100 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
94 0.000 0.000 0.075 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 1.000 1.000 0.925 1.000 1.000 1.000 0.203 0.413 1.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.797 0.587 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Pgdh* 
(N27 14 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 
98 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 1.000 0.000 
Pgm
(N20 18 100 35 66 64 100 49 19 54 
100 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.867 1.000 0.000 
94 0.000 0.000 0.000 0.000 0.008 0.031 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.992 0.969 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.133 0.000 0.000 
104 0.275 1.000 0.985 0.686 0.000 0.000 0.000 0.000 0.000 0.454 
106 0.725 0.000 0.015 0.314 0.000 0.000 0.000 0.000 0.000 0.546 
Locus Population
 
CLU VEN OCC BIO GOU PAT NMI MLK KVO PPc 
Akp-1 
(N30 30 18 98 20 90 18 100 40 94 
100 0.000 0.000 0.000 0.000 0.000 0.000 0.167 0.140 0.000 0.000 
102 0.000 0.000 0.000 0.485 1.000 1.000 0.833 0.860 1.000 0.628 
104 1.000 1.000 1.000 0.515 0.000 0.000 0.000 0.000 0.000 0.372 
Akp-2* 
(N30 30 15 80 20 60 20 81 20 40 
1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Apk
(N42 27 100 45 31 49 81 48 29 45 
100 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
91 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 
95 0.000 0.000 0.000 0.000 0.435 0.224 0.000 0.000 0.000 0.000 
97 0.000 0.000 0.000 0.000 0.565 0.776 1.000 1.000 1.000 0.000 
104 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Ald
(N30 30 100 60 100 100 100 70 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Fk
(N30 30 100 60 100 100 100 70 100 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 1.000 1.000 1.000 1.000 
98  0.000  0.000   0.000   0.000   1.000   1.000   0.000   0.000   0.000   0.000 
Gapdh-1* 
(N44 30 30 30 30 90 29 30 60 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Gapdh-2* 
(N47 30 30 60 30 90 29 30 60 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Gapdh-3* 
(N35 30 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
αGPD-1
(N20 15 60 15 90 15 30 100 25 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
αGPD-2
(N11 15 60 15 90 15 30 100 25 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Gpi* 
(N12 55 100 73 100 75 100 49 19 100 
100 1.000 0.773 0.980 0.952 0.005 0.007 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 
96 0.000 0.000 0.000 0.000 0.995 0.993 0.000 0.000 0.000 0.000 
104 0.000 0.227 0.020 0.048 0.000 0.000 0.000 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
108 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
G6pdh-1*           
(N20 30 65 100 22 100 61 100 50 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
G6pdh-2* 
(N20 30 36 98 10 100 20 100 40 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-1* 
(N30 30 100 100 100 100 100 100 100 100 
100 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 1.000 0.000 
96 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 
Hk-2* 
(N30 30 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Hk-3* 
(N30 30 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 1.000 1.000 1.000 1.000 
94 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 
Idh
(N32 11 79 24 36 46 73 31 17 71 
100 0.031 0.636 0.000 0.000 1.000 1.000 0.890 1.000 1.000 0.000 
94 0.969 0.364 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.110 0.000 0.000 0.000 
Ldh-1* 
(N10 21 67 43 47 88 24 32 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 
Ldh-2* 
(N27 21 81 44 57 28 74 24 24 86 
100 1.000 1.000 1.000 1.000 0.307 0.000 0.041 0.979 1.000 1.000 
94 0.000 0.000 0.000 0.000 0.693 1.000 0.730 0.021 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.000 0.230 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mdh-1* 
(N26 43 26 30 79 46 92 39 33 74 
100 0.442 1.000 0.308 0.300 1.000 1.000 1.000 1.000 1.000 0.000 
96 0.558 0.000 0.692 0.700 0.000 0.000 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
106 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Mdh-2* 
(N22 43 80 44 24 50 75 39 38 40 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.400 1.000 0.895 1.000 
96 0.000 0.000 0.000 0.000 1.000 1.000 0.600 0.000 0.105 0.000 
Me* 
(N30 22 63 54 42 33 92 30 37 93 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.451 1.000 1.000 1.000 
98 0.000 0.000 0.000 0.000 1.000 1.000 0.549 0.000 0.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Mpi-1* 
(N30 30 15 100 20 83 20 100 41 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.000 0.446 0.000 0.000 0.000 0.000 
98 0.000 0.000 0.000 0.000 1.000 0.554 1.000 1.000 1.000 0.000 
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Mpi-2* 
(N30 30 15 100 20 74 20 100 50 100 
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
Pep
(N13 30 53 23 50 36 59 23 30 44 
100 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
94 0.000 0.000 0.075 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
96 1.000 1.000 0.925 1.000 1.000 1.000 0.203 0.413 1.000 0.000 
98 0.000 0.000 0.000 0.000 0.000 0.000 0.797 0.587 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 
Pgdh* 
(N27 14 100 100 100 100 100 100 100 100 
100 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 
98 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 1.000 0.000 
Pgm
(N20 18 100 35 66 64 100 49 19 54 
100 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.867 1.000 0.000 
94 0.000 0.000 0.000 0.000 0.008 0.031 0.000 0.000 0.000 0.000 
96 0.000 0.000 0.000 0.000 0.992 0.969 0.000 0.000 0.000 0.000 
102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.133 0.000 0.000 
104 0.275 1.000 0.985 0.686 0.000 0.000 0.000 0.000 0.000 0.454 
106 0.725 0.000 0.015 0.314 0.000 0.000 0.000 0.000 0.000 0.546 

APPENDIX 2

The finding of four sibling species within the taxon Pseudomonocelis ophiocephala raises problems of nomenclature, as to which of the siblings the nominal taxon should pertain. The type-locality for Monocelis ophiocephala Schmidt (1861) is given as ‘Corfu’. Samplings on the island of Corfu revealed the presence of two species of the complex, reported here as siblings A and C, which raises a potentially worrisome taxonomic problem. However, in the introduction to his paper, Schmidt (1861) gave a brief description of the locations from where most of his ‘sand-dwelling’ species were found. Among these, the first locality listed is the area beneath the Corfu castle. A search around the castle revealed one accessible stretch of sand, at present within the premises of the local ‘Yacht Club’. Sorting of the sediment from this beach yielded one species only, namely sibling A, to which we deem the nominal taxon should apply. It is an added bonus for nomenclatural stability that the nominal taxon remains attached to the most common and widespread of the siblings.

We consider that the special conditions for neotype designation apply in this case, as there is the need to clarify the taxonomic status and the type-locality of a nominal taxon (ICZN, 1999: Art. 75). In fact, the deposition of type-specimens by Schmidt (1861) is not declared, and types are considered unavailable (cf. Sluys, 1989). Furthermore, the process of neotype designation has already been performed for Schmidt's species (as in the case of Dugesia sagitta Schmidt, 1861) (De Vries, 1984).

Besides the (very limited) original diagnosis, the morphology of P. ophiocephala has been described at length by Meixner (1943) and Schockaert & Martens (1987). The morphospecies is characterized by the presence of a bifurcated vagina and a complex vagina-bursa system (see Meixner, 1943), allowing immediate discrimination among congenerics. Analysis of 21 morphological characters of ten populations, belonging to siblings A (CVI, STR, COR, PIR, HAI), B (OCC), C (GOU, PAT), and D (MLK, NMI), did not reveal the existence of any feature allowing unequivocal discrimination among siblings. In the following diagnoses, species are therefore distinguished on the basis of the unique characters known at present: karyotype (when suitable), allelic configuration, and RAPDs profiles. Given the resolution power of the latter, the number and length of each fragment in base pairs (bp) should be considered in a ±30-bp range. The type series is implemented with standard, morphological slides, which may allow further studies. Localities given for each sibling are based on karyotypical and/or allozyme data. Type material (slides with sectioned specimens and diskettes with allozyme and RAPDs profiles) are stored in the collections of the Swedish Museum of Natural History (Stochkolm, Sweden) (SMNH).

Pseudomonocelis ophiocephala (Schmidt, 1861)

Synonymy: ‘Sibling A’ in present paper.

Neotype: a mature specimen fixed in 100° Alcohol (xii.1997) (SMNH 6032).

Type locality: Corfu Island (Greece), beneath Corfu Castle, beach in the premises of the Yacht Club (v.1987, iv.1993, xii.1997, ix.1998).

Paratypes: one specimen from the type locality, sagittally sectioned) (SMNH 6033); cellulose acetate membrane with discriminating allozymic patterns of specimens from the type locality (SMNH 6033); 3.5″ floppy disk with pictures of RAPD 2% agarose gel of specimens from the type population showing discriminating bands (SMNH 6033).

Distribution: besides the type locality, the species is known from France: Calvi (v.1984, ix.1984, i.1987, v.1994, v.1996, x.1996, not found from onwards and presumably extinct, see above); Ventilegne (iv.2000, ix.2001). Italy: Dragunara, Capo Caccia (ix.1996, viii.1998), Porto Torres (iii,x.1997, iv.1998, ix.2000), Alghero (vii.1996, vi.1997, ii.1999), S. Antioco Island, Cala Lunga (vii.1996, iii,ix.1999), Livorno (iv.1987, ii.1991, vii.1992, x.1993, vii.1996), Quercianella (iv.1991), Elba Island, Porto Ferraio (i.1992, iv.1999), Santa Marinella (iii.1992, ii.1994, ii.2004), Porto Cesareo, La Strea (v.1987, iv.1993, v.1996, iii.2000), Acquatina (v.1996, ix.2003). Croatia: Omiš (vi.2001). Greece: Piraeus, Glyfada (iv.1993, xii.1997, ix.1998). Israel: Haifa (iii.1987, ii.1988, x.1997).

Description: a member of the Pseudomonocelis ophiocephala sibling complex (see Meixner, 1943 and Schockaert & Martens, 1987 for a detailed description of the morphospecies), characterized by: karyotype of type ‘a’ (see Curini-Galletti & Casu, 2005: figs 2D, E; tables 1,3); presence of unique allele APK*100; presence of unique RAPD bands: primer C-16 (700 bp); primer R-2 (1000 bp).

Remarks: see above for the justification of neotype designation. A very common and widespread species in lower intertidal habitats of the Mediterranean, in well-sorted, medium-to-coarse sand.

Pseudomonocelis caputserpentissp. nov.

Synonymy: ‘Sibling B’ in present paper.

Holotype: a mature specimen fixed in 100° Alcohol (v.1996) (SMNH 6034).

Type locality: Occelluccia Cove, Point Revellata (Corsica, France) (v.1984, ix.1984, i.1987, v.1994, v.1996, x.1996, v.2000, viii.2001).

Additional material: paratype: one specimen from the type locality, sagittally sectioned (Iv.1992) (SMNH 6035); cellulose acetate membrane with discriminating allozymic patterns of specimens from the type locality (SMNH 6035); 3.5″ floppy disk with picture of RAPD 2% agarose gel of specimens from the type population showing discriminating bands (SMNH 6035).

Etymology: from Latin caput (head) and serpens (snake). The name is coined after the nominal taxon P. ophiocephala[from greek ′οφις (snake) and κɛφαλη′ (head)].

Distribution: besides the type locality, the species is known from France, Corsica: St. Fleurent (ix.1984); Sagone (v.1994); Ventilegne (i.1997, iv.2000); Italy, Sardinia: S. Antioco Island, Cala Lunga (xii.1989, ii.1994, iii.1999) Cala Sapone (ii.1994, iii.1999). Elba Island, La Biodola (xii.1995, ii.1999).

Description: a member of the Pseudomonocelis ophiocephala sibling complex (see Meixner, 1943 and Schockaert & Martens, 1987), characterized by:

  • presence of unique alleles APK*91 and HK-1*96.

  • presence of unique RAPD bands: primer C-02 (2800 bp); primer C-05 (3000 bp; 1600 bp); primer C-08 (1700 bp); primer C-11 (400 bp); and primer C-18 (2800 bp).

The species has karyotype of type ‘b’ (see Curini-Galletti & Casu, 2005: fig. 2G; tables 1,3).

Remarks: species with distribution limited to the Corsican–Sardinian complex, and to the adjacent Elba Island. Exclusively found in reduced sediments beneath the ‘banquette’ of Posidonia oceanica.

Pseudomonocelis caputdraconissp. nov.

Synonymy: ‘Sibling C’ in present paper.

Holotype: a mature specimen fixed in 100° alcohol (xii.1997) (SMNH 6036).

Type locality: Gouviá (Corfu Island, Greece) (v.1987, iv.1993, xii.1997, ix.1998).

Additional material: paratype: one specimen from the type locality, sagittally sectioned (ix.1998) (SMNH 6037); cellulose acetate membrane with discriminating allozymic patterns of specimens from the type locality (SMNH 6037); 3.5″ floppy disk with picture of RAPD 2% agarose gel of specimens from the type population showing discriminating bands (SMNH 6037).

Distribution: besides the type locality, the species is known from Kanoni (Corfu Island) (v.1987, iv.1993, not found in subsequent samplings, after refurbishment of the beach with clean, coarse sand); Patrás (Greece) (iv.1993, ix.1998).

Etymology: from latin caput (head) and draco (drake, snake).

Description: a member of the Pseudomonocelis ophiocephala sibling complex (see Meixner, 1943 and Schockaert & Martens, 1987), characterized by:

  • presence of unique alleles APK*95, FK*98, GPI*96, HK-3*94 and PGM*94.

  • presence of unique RAPD bands: primer C-02 (500 bp); primer C-11 (1600 bp); primer C-16 (1600 bp; 1500 bp); primer C-18 (1700 bp).

The species has karyotype of type ‘b’ (see Curini-Galletti & Casu, 2005:Tables 1,3)

Remarks: the species appears exclusive of mixed, silty sediments. It is only known from Corfu Island and Patras. Albeit a wider distribution along western Greece appears reasonable, samplings in apparently suitable areas, from Igoumenitza to Kyparissia, failed to yield the species.

Pseudomonocelis caputanguissp. nov.

Synonymy: ‘Sibling D’ in present paper.

Holotype: a mature specimen fixed in 100° alcohol (ix.1998) (SMNH 6038).

Type locality: Nea Mihanióna (Gulf of Termaikós, Greece) (v.1987, xii.1997, ix.1998).

Additional material: paratype: one specimen from the type locality, sagittally sectioned (ix.1998) (SMNH 6039); cellulose acetate membrane with discriminating allozymic patterns of specimens from the type locality (SMNH 6039); 3.5″ floppy disk with picture of RAPD 2% agarose gel of specimens from the type population showing discriminating bands (SMNH 6039).

Distribution: besides the type locality, the species is known from other localities in Aegean Sea: Peraia (xii.1997), Maliakós (v.1987, xii.1997, ix.1998), Kámena Voúrla (ix.1998), Skiátos Island, town beach (ix.2000).

Etymology: from Latin caput (head) and anguis (snake).

Description: a member of the Pseudomonocelis ophiocephala sibling complex (see Meixner, 1943 and Schockaert & Martens, 1987), characterized by:

  • APK, GPI exclusively found in the allelic forms APK*97 and GPI*98.

  • presence of unique RAPD bands: primer C-02 (600 bp); primer C-05 (2500 bp, 400 bp); primer C-08 (1200 bp); primer C-11 (2500 bp); and primer C-16 (400 bp).

The species has karyotype of type ‘b’ (see Curini-Galletti & Casu, 2005: fig. 2F, tables 1,3).

Remarks: the species is found in mixed, silty sediments. At the type locality, congregating in enormous numbers at the mouth of a sewer, within the harbour. Probably widespread in northern Aegean Sea, where it appears to be the only species of the complex.