Morphometric and genetic analysis as proof of the existence of two sturgeon species in the Guadalquivir river

Morphometric and genetic methods were used to identify two sturgeon species, Acipenser naccarii Bonaparte, 1836, and A. sturio Linnaeus, 1758, captured in some of the principal rivers of the Iberian Peninsula, including the Guadalquivir. After measuring 25 Iberian specimens from a fishery and several Spanish and Portuguese museums and applying stepwise discriminant analysis (SDA), four specimens preserved in different museums [two specimens from the Guadalquivir river (EBD-8173 and EBD-8174), one specimen from the Tagus river (MUC1) and one specimen from the Mondego river (MUC46B)], as well as five specimens captured in the Guadalquivir river in the 1940s but not preserved (CM1, CM2, CM3, CM4 and CM5), were identified as A. naccarii. After cloning and characterisation of a satellite-DNA family, HindIII, from A.␣naccarii genome, its absence from the genome of A.␣sturio was determined. Using this satellite-DNA as a genetic marker and by means of dot-blotting, we demonstrate that the DNA of the two specimens captured during the mid-1970s in the Guadalquivir river cross-hybridised with HindIII satellite-DNA sequences of A.␣naccarii. We conclude that A. naccarii is autochthonous to the Iberian Peninsula and is not, as was previously believed, endemic to the Adriatic Sea. Received: 28 November 1996 / Accepted: 10 March 1997     

Chemistry of the postpharyngeal gland secretion and its implication for the phylogeny of IberianCataglyphis species (Hymenoptera: Formicidae)

Summary A comparative morphological and chemical study of six endemic species ofCataglyphis from the Iberian Peninsula:C. ibericus, C. rosenhaueri, C. hispanicus, C. humeya, C. velox andC. floricola and the Moroccan speciesC. bombycinus is described. The morphological study relied primarily on genitalia characteristics, whereas the chemical study concentrated on the postpharyngeal gland constituents. Cladograms based on the morphological and chemical data were performed using Ward's method. The dendrogram based on morphological features revealed that the IberianCataglyphis can be classified into three species groupsalbicans, altisquamis andemmae. The same pattern occurred when the dimethylalkanes constituents of the postpharyngeal gland were utilised as character states, with a slight displacement of species within thealtisquamis group. However, when the complete hydrocarbon blend was utilised major discrepancies in the dendrograms occurred.Cataglyphis velox proved to be very similar toC. bombycinus, whereasC. floricola clustered with the other two species of thealtisquamis group. Based on the geographical distribution and paleontological data (Tinaut 1993) it is assumed thatC. floricola recently invaded the Iberian Peninsula. Based on the chemical findings we postulate that chemical character displacement occurred inC. floricola as a result of its sympatry withC. velox after the former colonized the Iberian Peninsula. We further discuss the possible reason for the different dendrograms obtained when only the dimethylalkanes are considered and its implication for the communicative role of the postpharyngeal gland secretion in these ants.     

Failure to confirm previous identification of two putative museum specimens of the Atlantic sturgeon, Acipenser sturio, as the Adriatic sturgeon, A. naccarii

MtDNA analyses were employed to examine the species identification of three preserved museum sturgeon specimens from the River Guadalquivir, two of which were originally described as Acipenser sturio L., 1758 by Hernando (1975; Doñana, Acta Vertd 2: 263–264). Later the same two specimens were identified as A. naccarii Bonaparte, 1836 by Garrido-Ramos et?al. (1997; Mar Biol 129: 33–39) on the basis of morphometric and nuclear DNA analyses. Three different laboratories using independent techniques were unsuccessful in extracting authentic DNA from tissue samples from the three specimens and in obtaining any verifiable PCR product. We cannot confirm the previous molecular identification of two of these specimens by Garrido-Ramos et?al. as A. naccarii. We suggest that these specimens are indeed A. sturio.     

Key Role of European Rabbits in the Conservation of the Western Mediterranean Basin Hotspot

Abstract: The Mediterranean Basin is a global hotspot of biodiversity. Hotspots are said to be experiencing a major loss of habitat, but an added risk could be the decline of some species having a special role in ecological relationships of the system. We reviewed the role of European rabbits (Oryctolagus cuniculus) as a keystone species in the Iberian Peninsula portion of the Mediterranean hotspot. Rabbits conspicuously alter plant species composition and vegetation structure through grazing and seed dispersal, which creates open areas and preserves plant species diversity. Moreover, rabbit latrines have a demonstrable effect on soil fertility and plant growth and provide new feeding resources for many invertebrate species. Rabbit burrows provide nest sites and shelter for vertebrates and invertebrates. In addition, rabbits serve as prey for a number of predators, including the critically endangered Iberian lynx (Lynx pardinus) and Spanish Imperial Eagle (Aquila adalberti). Thus, the Mediterranean ecosystem of the Iberian Peninsula should be termed “the rabbit's ecosystem.” To our knowledge, this is the first empirical support for existence of a multifunctional keystone species in a global hotspot of biodiversity. Rabbit populations have declined drastically on the Iberian Peninsula, with potential cascading effects and serious ecological and economic consequences. From this perspective, rabbit recovery is one of the biggest challenges for conservation of the Mediterranean Basin hotspot.     

Settlement of the wreckfish (<Emphasis Type="Italic">Polyprion</Emphasis><Emphasis Type="Italic">americanus</Emphasis>)

The wreckfish (Polyprion americanus) is a panoceanic, long-lived, deep-water demersal species, characterized by an extended pelagic juvenile phase associated with floating objects. In the present study morphometric data from 435 specimens collected from Greece (Crete), Italy (Ionian Sea) and the Atlantic coast of France from September 1999 to March 2001 were analyzed to estimate the settlement size of the species and associated changes in morphology. The fishery sample included specimens from both the pelagic and the demersal stage. Length-at-settlement (TL₅₀) and 95% confidence intervals were estimated by fitting a logistic function. Eleven morphometric characters were analyzed, and the existing inflection points, reflecting alterations in body shape, were defined: (1) iteratively, as the transition point, splitting the data set into two groups, for which the reduced major axis functions, between a character and total length, of the successive groups best fit (minimum sum of squares of residuals) the combined data set and (2) where the second derivative of the fitted third-order polynomial functions to morphometric ratios equaled zero. The main size range within which wreckfish settled was 56-65 cm, and the means of the inflection points defined by the two methods were 61 and 64 cm. These results indicate changes in functional morphology associated with settlement. The monitoring of wreckfish juveniles caught in the wild and kept individually in captivity showed changes in growth and food intake at approximately the estimated settlement length and suggested that temperature was the most likely factor triggering settlement.     

Phylogeography of the European stalked barnacle (Pollicipes pollicipes): identification of glacial refugia

The alternation of glacial and interglacial events during the Pleistocene has produced changes in species distribution ranges leading to bottlenecks and alterations of patterns of gene flow. The European stalked barnacle, Pollicipes pollicipes, is a sessile pedunculate cirripede that inhabits the rocky intertidal frame, from Senegal to the northwestern coast of France. In this work, we have analyzed a fragment of the mitochondrial gene cytochrome c oxidase subunit I for 569 individuals of P. pollicipes in order to investigate whether the shifts in climatic conditions that occurred during the Pleistocene influenced the current pattern of distribution of genetic variation of P. pollicipes. A pre-last glacial maximum pattern of demographic expansion was found, in concordance with many other North Atlantic marine species. On the other hand, three potential glacial refugia were identified: North African coasts, northwestern Iberian Peninsula and English Channel/Brittany.     

Recent strandings of the giant jellyfish Rhizostoma luteum Quoy and Gaimard, 1827 (Cnidaria: Scyphozoa: Rhizostomeae) on the Atlantic and Mediterranean coasts

We present reports of sightings of living and stranded specimens of Rhizostoma luteum on the Atlantic coast of Morocco and along the south shore of the Iberian Peninsula in June–July 2012 and in January–February 2013. During summer 2012 and following the dominant currents, the jellyfish first appeared in the Gulf of Cadiz west of the Strait of Gibraltar. Subsequently, seven additional sightings were reported east of the Strait, in the Alboran Sea. In winter 2013, another event of stranded individuals of this species occurred in the Gulf of Cadiz. A phylogenetic analysis performed on the mitochondrial cytochrome c oxidase I (COI) gene sequence in specimens from both stranding events confirmed the morphological classification, ratifying that R. luteum differs from Rhizostoma octopus and Rhizostoma pulmo. This study records the presence of this species for the first time in 60 years.     

Homogenization dynamics and introduction routes of invasive freshwater fish in the Iberian Peninsula.

Nonnative invasive species are one of the main global threats to biodiversity. The understanding of the traits characterizing successful invaders and invasion-prone ecosystems is increasing, but our predictive ability is still limited. Quantitative information on biotic homogenization and particularly its temporal dynamics is even scarcer. We used freshwater fish distribution data in the Iberian Peninsula in four periods (before human intervention, 1991, 1995, and 2001) to assess the temporal dynamics of biotic homogenization among river basins. The percentage of introduced species among fish faunas has increased in recent times (from 41.8% in 1991 to 52.5% in 2001), leading to a clear increase in the similarity of community composition among basins. The mean Jaccard's index increase (a measure of biotic homogenization) from the pristine situation to the present (17.1%) was similar to that for Californian fish but higher than for other studies. However, biotic homogenization was found to be a temporally dynamic process, with finer temporal grain analyses detecting transient stages of biotic differentiation. Introduced species assemblages were spatially structured along a latitudinal gradient in the Iberian Peninsula, with species related to sport fishing being characteristic of northern basins. Although the comparison of fish distributions in the Iberian Peninsula and France showed significant and generalized biotic homogenization, nonnative assemblages of northeastern Iberian basins were more similar to those of France than to those of the rest of the Iberian Peninsula, indicating a main introduction route. Species introduced to the Iberian Peninsula tended to be mainly piscivores or widely introduced species that previously had been introduced to France. Our results indicate that the simultaneous analysis of the spatial distribution of introduced assemblages (excluding native species that reflect other biogeographical patterns) and their specific traits can be an effective tool to detect introduction and invasion routes and to predict future invaders from donor regions.     

Reproduction and sex reversal of the solitary vermetid gastropod<Emphasis Type="Italic"> Serpulorbis arenarius</Emphasis>

The reproductive biology of the Mediterranean vermetid gastropod Serpulorbis arenarius, a subtidal sessile solitary species that lives in shady rocky communities, was studied at two localities south-east of the Iberian Peninsula. Its population density is usually low, with specimens sometimes sparsely distributed. Its shell often appears to be covered by epibiotic organisms. The sex ratio is biased toward males (63%) in the populations of SE Spain, and the results point towards hermaphroditism with alternating sexuality. The results obtained suggest that all specimens are first males, change to females under some as yet unknown condition and, after the ovogenesis cycle has been completed, revert to the original sexual state (male). The possible adaptive forces that favour hermaphroditism in this species are discussed, and we hypothesise that the seizure of a spermatophore by males may stimulate sex change to female. Brooding females (51% of females studied) were found from March to December. In males, histological study of testes revealed that spermatogenesis seems to occur throughout the year. Females brooded between 5 and 28 (=15) egg capsules, which were attached by stalks to the inner surface of the shell. It is noteworthy that the gonads of two of the specimens brooding egg capsules were developing testes. The newly laid egg capsules were filled with an organic intracapsular matrix, in which 40–153 (=94) eggs were embedded. The mean diameter of the uncleaved ova was 352.5 µm. The organic intracapsular matrix and some nurse eggs were used as an extra-embryonic food source. Hatching occurred at an advanced veliger stage, immediately followed by metamorphosis. Therefore, the larval development of S. arenarius is of a lecithotrophic intracapsular type, with a short, free-swimming/crawling phase.Communicated by O. Kinne, Oldendorf/Luhe     

Genetic differentiation in three Sepia species (Mollusca: Cephalopoda) from Galician waters (north-west Iberian Peninsula)

The systematics of the genus Sepia is not yet clear. Morphological evidence has led to S. officinalis Linnaeus, 1758 being considered as belonging to the subgenus Sepia sensu stricto, and S. orbignyana Férussac, 1826 and S. elegans Blainville, 1827 as belonging to the subgenus Rhombosepion. Samples of 30 individuals of S. officinalis and S. orbignyana from both sides of an oceanographic boundary off the north-west Iberian Peninsula, and a sample of S. elegans from the northern side, were collected in 1993–1994. Allozyme electrophoresis for 32 presumptive loci revealed low levels of genetic variability for the three Sepia species (mean expected heterozygosity estimates were <0.052). No significant differences in allozyme frequencies were detected among populations of either S. officinalis or S. orbignyana. The genetic identities (I) of S. officinalis and S. orbignyana (I=0.12) and of S. elegans (I=0.13) were significantly different from that of S. orbignyana and S. elegans (I=0.49). The former are typical of values for confamilial genera, and a new generic status is proposed for the latter two species, which become Rhombosepion orbignyana (Férussac, 1826) and R. elegans (Blainville, 1827).     

Allozyme heterozygosity and escape response performance of the scallops, <Emphasis Type="Italic">Argopecten purpuratus</Emphasis> and <Emphasis Type="Italic">Placopecten magellanicus</Emphasis>

Multilocus allozyme heterozygosity (MLH) has been positively correlated with growth in some marine bivalves and was suggested to facilitate swimming activity in pectinids. Using two highly mobile scallops, Placopecten magellanicus and Argopecten purpuratus, we examined escape response performance and morphometric characteristics as a function of allelic variability at metabolic loci. Ten allozyme systems were used for A. purpuratus and 7 for P. magellanicus. In each species, the morphometric characteristics and escape response parameters were analyzed separately using principal components analysis (PCA) and the scores of the major principal components were related to allozyme heterozygosity. In both P. magellanicus and A. purpuratus, positive correlations were found between MLH and morphometric parameters, but escape response parameters were only positively linked to MLH in P. magellanicus, and then weakly. The hypothesis that MLH improves fitness of pectinids by increasing the capacity to escape predators is not supported.     

Phylogeography of the sea star Marthasterias glacialis (Asteroidea, Echinodermata): deep genetic divergence between mitochondrial lineages in the north-western mediterranean

We explore the phylogeography of the broadcast spawner Marthasterias glacialis along south Europe and Azores. Sequences of the cytochrome c oxidase gene from 225 specimens, belonging to 10 localities, were analysed. We found 73 haplotypes grouped within two lineages (divergence 2.9%). One lineage was Atlanto–Mediterranean, whereas another one was exclusively Mediterranean. Estimation of lineages split goes back to 830,000–580,000 (±120,000) years ago. This suggests that sea-level oscillations during the Pleistocene glaciations promoted gene flow interruption, lineage divergence between basins and cryptic speciation. Secondary contact between populations allowed a recolonization of the Mediterranean by the Atlantic lineage. When animals of the Atlanto–Mediterranean lineage were considered separately, F _st index and AMOVA did not show significant differences between populations along either the Iberian Peninsula or basins. Isolation by distance between populations was not detected, and only populations of Plymouth and Azores showed significant differences to all the others. The remoteness of Azores islands might explain the structure of this population. Haphazard arrival of larvae and local extinctions rather than contemporary restricted gene flow might be responsible for the distinctive population structure of Plymouth.     

Comparison of larval fish assemblages in three large estuarine systems, KwaZulu-Natal, South Africa

 The structure of the larval fish assemblages in three large estuarine systems on the KwaZulu-Natal coast of South Africa was examined using a combination of univariate, distributional and multivariate techniques. The database was comprised of a full annual set of larval fish samples taken from each estuarine system: Durban Harbour, Richards Bay Harbour and St Lucia Estuary. The mean monthly densities of each species in each system were used in the species matrix, and the mean monthly values of salinity, temperature and turbidity were used in the environmental matrix. The mean species diversity and evenness index were significantly higher in Durban Harbour (H′ = 1.03, J′ = 0.65) than in the other two systems. The cumulative dominance curve showed that St Lucia Estuary has a high dominance of a few species, with Richards Bay Harbour intermediate and Durban Harbour being the most diverse. Classification and MDS (multiple-dimensional scaling) analyses of larval fish densities in all three systems grouped together into three main clusters on the basis of system. The species similarity matrix (inverse analysis) clustered into five groups at the 25% similarity level. The MDS analysis of the same matrix showed that the groups separated out according to the degree of estuarine association of a species and hence habitat type. The species most responsible for system groupings were: Glossogobius callidus, Gilchristella aestuaria, Stolephorus holodon, Croilia mossambica and Gobiid 12. The “best fitting” of the environmental variables to explain the larval fish community patterns in each system was turbidity on its own (weighted Spearman's rank correlation, ρ_w = 0.55). The relationship of larval densities to environmental conditions was shown to be species-specific with estuarine species (e.g. G. callidus and G. aestuaria), having a strong positive correlation with temperature and turbidity but negative correlations with salinity. In summary, much longer term studies with more sites within each system are needed to assess whether the larval fish assemblages are stable or at an equilibrium (both spatially and temporally) and whether these assemblages are indicative of the relative “health” and nursery function of the system. Received: 28 April 1999 / Accepted: 30 May 2000     

Heterospecific call recognition and phonotaxis in the orientation behavior of the marbled newt,Triturus marmoratus

The role of acoustic cues as reference cues for orientation by amphibians has been demonstrated in anurans, the only amphibian group that engages in acoustic communication, but not in urodeles. Orientation responses of marbled newts, Triturus marmoratus, were studied to determine whether heterospecific calls elicited positive phonotaxis. The orientation tests consisted in presenting either a familiar acoustic stimulus, the advertisement calls of natterjack toads (Bufo calamita), or a control stimulus, the advertisement calls of European green toads (Bufo viridis) that the newts would not be expected to recognize. Marbled newts and natterjack toads occur in simpatry, but T. marmoratus and B. viridis are allopatric species. Thus, T. marmoratus is distributed over the northern half of the Iberian Peninsula, whereas B. viridis occurs in the Balearic Islands, but not over the Iberian Peninsula. Newts were released in a circular arena while a recorded chorus of natterjack toads or European green toads played outside the arena to determine whether they displayed positive phonotactic orientation. Our results show that marbled newts performed positive phonotaxis when exposed to the breeding calls of natterjack toads, but not to those of European green toads. Newts chose a compass course in the direction of the advertisement calls of B. calamita. Acoustic information might improve orientation accuracy. This study is the first to provide evidence of heterospecific call recognition and positive phonotactic response in urodeles.Communicated by W. Wiltschko     

Cryptic species and population structuring of the Atlantic and Pacific seabob shrimp species, Xiphopenaeus kroyeri and Xiphopenaeus riveti

Seabob shrimps of the genus Xiphopenaeus are important fishery resources along the Atlantic and Pacific coasts of Central and South America. The genus was considered to comprise two species: the Atlantic Xiphopenaeus kroyeri (Heller, Sitzungsber Math Naturwiss cl kaiserliche Akad Wiss Wien 45:389–426, 1862), and the Pacific Xiphopenaeus riveti (Bouvier, Bull Mus Hist Nat Paris 13:113–116, 1907). In a recent review, Xiphopenaeus was regarded as a monotypic genus, on the basis that no clear morphological differences could be found between Pacific and Atlantic specimens (Pérez Farfante and Kensley, Mem Mus Nat Hist Nat Paris 175:1–79, 1997). In the present work, nuclear (allozymes), and mitochondrial (Cytochrome Oxidase I) genes were used to demonstrate the validity of X. riveti and reveal the presence of two cryptic species of Xiphopenaeus within X. kroyeri in the Atlantic Ocean. The high levels of molecular divergence among these species contrast with their high morphological resemblance. Interspecific sequence divergences (Kimura 2-parameter distance) varied from 0.106 to 0.151, whereas intraspecific distances ranged from 0 to 0.008 in Xiphopenaeus sp. 1, from 0 to 0.003 in Xiphopenaeus sp. 2, and from 0.002 to 0.005 in X. riveti. In addition, five diagnostic allozyme loci were found between sympatric samples of Xiphopenaeus sp. 1 and 2 along the Brazilian coast. The results suggest that Xiphopenaeus sp. 2 from the Atlantic is more closely related to the Pacific X. riveti than to the Atlantic Xiphopenaeus sp. 1. Furthermore, a high level of genetic structuring (Xiphopenaeus sp. 1: F _ST =0.026; P<0.05; Xiphopenaeus sp. 2: F _ST =0.055; P<0.01) was found in the Brazilian Xiphopenaeus populations, indicating the presence of different genetic stocks in both Atlantic species. These findings have important commercial implications as they show that the fisheries of the two Atlantic species must be managed separately, and that each one is comprised of different populations.Communicated by O. Kinne, Oldendorf/Luhe     

Meiofaunal cryptic species revealed by confocal microscopy: the case of Xenotrichula intermedia (Gastrotricha)

The phylum Gastrotricha includes about 750 species of meiobenthic marine and freshwater species that are often widely distributed. The microscopic size, short life cycle, low motility of adults, and the absence of larval stages normally required for dispersal raise doubts about the putative cosmopolitan distribution of many of gastrotrich species. The phenomenon of cosmopolitanism is acknowledged for all major meiobenthic taxa (“the meiofauna paradox”) and can be explained, at least in part, with the existence of sibling species, so far identified primarily by molecular analysis. In this paper, we report the discovery of sibling species in the marine chaetonotidan Xenotrichula intermedia using confocal laser scanning microscopy (CLSM). A total of 40 specimens collected from two geographically separate populations, the Mediterranean (Adriatic Sea) and the Arabian Gulf (Kuwait), were investigated. Fifteen specimens of each population were studied in vivo with a contrast interference microscope (DIC) in order to obtain the main morphometric parameters; ten other animals (five in each population) were fixed and marked with fluorescent phalloidin for the observation of their muscular systems under CLSM. The metrics and meristic data of the two populations fall within the range of measures recognized for the species. Pairwise comparisons (t-test) in general did not reveal statistically significant differences between the traits of specimens belonging to the two populations; moreover, multivariate analyses (cluster- and MDS analysis) were unable to separate clearly the Mediterranean from the Arabian specimens. In contrast, an examination of their muscular systems revealed clear dissimilarities between the two geographic groups. In particular, while the Italian specimens possessed incomplete circular bands and dorsoventrally orientated muscles that are partially inserted into the basal lamina of the cuticle, in both splanchnic- and somatic positions, their Arabian counterparts showed partial dorsoventral bands and complete circular muscles that surround the entire body of the animal in the same corresponding positions. Additional differences in the position of helicoidal bands (present in a more anterior location in the Italian specimens) are noted between Mediterranean and Arabian specimens. Since the investigated animals share a similar interstitial habitat, a working hypothesis connects the differences in the musculature to possible differences in the reproductive modality of specimens between the two populations.     

Use of Coarse‐Resolution Models of Species’ Distributions to Guide Local Conservation Inferences

Abstract: Distribution models are used increasingly for species conservation assessments over extensive areas, but the spatial resolution of the modeled data and, consequently, of the predictions generated directly from these models are usually too coarse for local conservation applications. Comprehensive distribution data at finer spatial resolution, however, require a level of sampling that is impractical for most species and regions. Models can be downscaled to predict distribution at finer resolutions, but this increases uncertainty because the predictive ability of models is not necessarily consistent beyond their original scale. We analyzed the performance of downscaled, previously published models of environmental favorability (a generalized linear modeling technique) for a restricted endemic insectivore, the Iberian desman (Galemys pyrenaicus), and a more widespread carnivore, the Eurasian otter (Lutra lutra), in the Iberian Peninsula. The models, built from presence–absence data at 10 × 10 km resolution, were extrapolated to a resolution 100 times finer (1 × 1 km). We compared downscaled predictions of environmental quality for the two species with published data on local observations and on important conservation sites proposed by experts. Predictions were significantly related to observed presence or absence of species and to expert selection of sampling sites and important conservation sites. Our results suggest the potential usefulness of downscaled projections of environmental quality as a proxy for expensive and time‐consuming field studies when the field studies are not feasible. This method may be valid for other similar species if coarse‐resolution distribution data are available to define high‐quality areas at a scale that is practical for the application of concrete conservation measures.     

Population genetic structure of the prosobranch Nassarius reticulatus (L.) in a ria seascape (NW Iberian Peninsula) as revealed by RAPD analysis

Randomly amplified polymorphic DNA (RAPD) banding patterns were compared between samples of the netted dogwhelk Nassarius reticulatus from 11 locations along the NW Iberian Peninsula coast. To detect if rias (estuaries formed by drowned river valleys) might promote genetic differentiation, five sampling sites were located within a ria (ria of Muros) and the remaining six were scattered along open-coast areas at increasing distances from the ria mouth. Population differentiation statistics (Φ-values) were estimated using a hierarchical analysis of molecular variance (AMOVA) with samples sorted into two groups: open-coast and ria populations. Despite a high potential to disperse, AMOVA demonstrated a modest, statistically significant genetic heterogeneity among N. reticulatus populations. Most of the genetic structure resided in differences among open-coast populations; ria populations were genetically homogeneous. No obvious geographical pattern was detected for the pairwise genetic distances (non-metric multidimensional scaling; UPGMA tree; Mantel test). Unlike previous studies with other species at a variety of estuarine systems other than rias, there was no evidence that the ria of Muros may enhance the genetic divergence of N. reticulatus populations. This discrepancy is discussed in relation to the biological features of the species (high dispersal potential and a preference for mid-low estuarine habitat) and the strong hydrographic connectivity between ria and neighbouring off-shore waters.     

Species boundaries in the starfish genus Linckia

 The genetic basis for species boundaries in the starfish genus Linckia was examined using variation observed in 613 base pairs (bp) of sequence from the cytochrome oxidase I gene of mtDNA and 16 allozyme loci. Five groups within Linckia were clearly genetically differentiated; L. columbiae, L. bouvieri, two clades within L. guildingi, and one clade with two sub-clades consisting of both L. laevigata and L. multifora. Genetic divergence among these groups is consistent with interspecific variation. The two clades within L. guildingi suggest the presence of a cryptic, partially sympatric, species. Genetic divergence between these two clades implies that they have been genetically distinct from each other for at least one million years. In contrast, genetic data suggest that L. laevigata and L. multifora are a single species, despite the fact that live individuals can be distinguished by their colour and colour pattern, number of madreporites and ratio of arm length to breadth. There are probably three closely related groups within the L. laevigata/L. multifora clade –L. multifora, and two groups in L. laevigata defined by biogeographic province. It is difficult to determine boundaries for these three entities, since genetic and morphological differences are complicated by phenotypic differences arising from both environmental variation and population genetic structure. The difficulties encountered in defining species boundaries in Linckia, particularly with respect to variation arising from the overlap of Indian and Pacific biogeographic provinces, may be a general issue for many marine organisms from this region. Received: 24 May 1999 / Accepted: 6 October 1999     

Localization of RubisCO and sulfur in endosymbiotic bacteria of the gutless marine oligochaete Inanidrilus leukodermatus (Annelida)

 The chemoautotrophic potential of the two co-occurring larger and smaller bacterial endosymbionts of the gutless marine oligochaete Inanidrilus leukodermatus was determined using immunocytochemistry. An antibody directed against the Form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key CO₂-fixing enzyme of the Calvin–Benson cycle, consistently labeled the larger symbionts. Electron microscopic spectroscopy showed that the larger symbionts contained sulfur in intracellular globules and to a lesser degree in the cytoplasm. The presence of RubisCO and sulfur indicates that the larger endosymbionts of I. leukodermatus are chemoautotrophic sulfur-oxidizing bacteria. In contrast, no RubisCO or sulfur was detected in the smaller endosymbionts of this host. Received: 28 September 1999 / Accepted: 29 May 2000