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     

Geographic clines and stepping-stone patterns detected along the East Pacific Rise in the vetigastropod Lepetodrilus elevatus reflect species crypticism

Three different molecular markers (i.e. seven allozyme loci, two nuclear gene loci and, mtCOI DNA sequences) were used to assess the genetic structure of the vent gastropod Lepetodrilus elevatus collected from three vent fields along the East Pacific Rise (13°N, 9°50′N and 17°S). While allozymes and nuclear loci suggested a strong stepping-stone pattern, a multivariate analysis performed on allozymic frequencies showed the presence of two distinct evolutionary lineages: the first situated in the north from 13°N to 9°50′N and the second in the south from 9°50′N to 17°S. The analysis of mitochondrial DNA sequences confirmed the separation of L. elevatus into two distinct clades with a divergence of 6.5%, which is consistent with the interspecific level of sequence variation in other vent species. A divergence time of 6–14 Mya was estimated between the two clades from previous clock calibrations. Our results suggest that these taxa followed an allopatric speciation between the northern and southern parts of the EPR with a recent demographic expansion of the southern clade to the north and a subsequent secondary contact (clade hybridisation). This speciation was probably reinforced by a habitat specialisation of the two cryptic species because the southern clade was mainly found associated with mussel-dominated communities and the northern clade with tubeworm-dominated communities. However, the analysis of shell morphology failed to separate the two cryptic species based on this sole criterion although they differed from Lepetodrilus elevatus galriftensis (Galapagos population) by a higher shell elevation. Within each clade, genetic differentiation was not related to the distance across populations and could be within vent field as important as between fields. While both clades appear to be in expansion since their speciation, significant excesses in heterozygotes suggest a very recent and local bottleneck at 17°S, probably due to massive site extinction in this region.     

Molecular population structure of the marine benthic copepod Microarthridion littorale along the southeastern and Gulf coasts of the USA

Relationships among populations (southeast Atlantic and northern Gulf coast, USA) of a ubiquitous, estuarine, harpacticoid copepod (Microarthridion littorale Poppe) were estimated using sequence data from two loci: one mitochondrial [cytochrome b (Cyt b)] and one nuclear [first internal transcribed spacer of ribosomal DNA (ITS-1)]. Copepods were collected from seven estuaries in 1997/98. Allelic phylogenies based on both genes were generally concordant, and suggested that M. littorale populations are structured over large geographic scales (hundreds of kilometers). Three well-supported groups were found in both gene trees comprising clades of alleles sampled from South Carolina, Florida, and Louisiana. Alleles from the Savannah, Georgia sample formed a monophyletic group using the Cyt b data, but this clade was not distinguishable with comparable ITS-1 data. A single specimen from Louisiana was classified in different clades depending on the locus assayed. Received: 11 June 1998 / Accepted: 29 April 1999     

Sympatry of distinct mitochondrial DNA lineages in a copepod inhabiting estuarine creeks in the southeastern USA

The population genetic structure of the meiobenthic harpacticoid copepod Microarthridion littorale (Poppe) was examined with a geographic survey of a 348 bp fragment of the mitochondrial cytochrome b gene. Copepods were collected from ten locations on the coast from North Carolina to Georgia, USA, from January 1997 to November 1998. Sequence divergence among 198 individuals was as much as 4.3%, and three divergent mitochondrial clades were uncovered that differed by six to nine nucleotide changes. A rapid assay was developed to distinguish among mitochondrial clades, and an additional 333 specimens were surveyed. The three lineages co-occurred in seven of ten sampling locations. Data analyses were carried out separately for individuals assayed by DNA sequencing as well as for a combined data set that included individuals typed by restriction endonuclease digestion. An analysis of molecular variance indicated that a significant proportion of the total genetic variance could be partitioned among populations, although no significant correlation between geographical and genetic distance was detected.     

Mitochondrial gene variation in Mercenaria clam sibling species reveals a relict secondary contact zone in the western Gulf of Mexico

We investigated phylogeographic relationships among American Mercenaria taxa by assessing variation in a 444 nucleotide fragment of the mitochondrial 16S ribosomal gene in clams sampled from four representative sites in January to November 1994. Three of these sites were in the Gulf of Mexico, one was on the Atlantic coast in South Carolina. Direct sequencing of this amplified gene fragment in 85 individuals revealed 21 haplotypes. Phylogenetic analyses consistently resolved this variation into three well supported clades, and within-clade genetic divergence levels were markedly lower than among-clade values. One of the clades, A, was taxon-specific, in that it solely and exclusively contained specimens of M. mercenaria (Linnaeus, 1758) sampled in South Carolina. The other two clades, B and C, were the most divergent and both encompassed specimens of M. campechiensis (Gmelin, 1791) and of M. campechiensis texana (Dall, 1902), sampled from the three Gulf of Mexico sites. Clade B was found at high frequencies at all three Gulf sites, whereas Clade C occurred at low frequencies at two western Gulf sites. We interpret this pattern as resulting from the secondary contact and introgression of two allopatrically differentiated Mercenaria taxa in the western Gulf of Mexico. Clade C haplotypes may represent relict mitochondrial lineages from original Gulf Mercenaria spp. populations that predate massive mitochondrial introgression by M. campechiensis. We further propose that the M. campechiensis texana nuclear genome is a mosaic, heavily weighted toward M. campechiensis, but containing some relict alleles inherited from the precontact population, especially those governing shell characteristics, which may be adaptive in cohesive sediments of bays and estuaries in the northwestern Gulf of Mexico.     

Mitochondrial cytochrome b variation in sleeper sharks (Squaliformes: Somniosidae)

Sleeper sharks are a poorly studied group of deep-sea sharks. The subgenus, Somniosus, contains three morphologically similar species: S. microcephalus found in the Arctic and North Atlantic; S. pacificus in the North Pacific; and S. antarcticus in the Southern Ocean. These sharks have been reported mainly in temperate to polar waters and occasionally in subtropical locations. They have not been recorded in tropical waters. This study investigates the relationships among the accepted species of Somniosus through analysis of mitochondrial cytochrome b sequence variation. Seventy-five samples were examined from four sampling locations in the North Pacific, Southern Ocean and North Atlantic. Twenty-one haplotypes were found. A minimum spanning parsimony network separated these haplotypes into two divergent clades, an S. microcephalus and an S. pacificus/antarcticus clade, strongly supporting the distinction of S. microcephalus as a separate species from the Pacific sleeper shark species. Analysis of genetic structure within the S. pacificus/antarcticus clade (analysis of molecular variance, allele frequency comparisons, and a nested clade analysis) showed limited or no differences amongst three populations. Further examination of genetic variation at more variable mtDNA and nuclear markers is needed to examine the species status of S. pacificus and S. antarcticus.     

Phylogeography of two species of <Emphasis Type="Italic">Lysidice</Emphasis> (Polychaeta,Eunicidae) associated to the seagrass <Emphasis Type="Italic">Posidonia oceanica</Emphasis> in the Mediterranean Sea

The aim of this work was to define the phylogeographic patterns of the two species of polychaete Eunicidae, Lysidice ninetta Audouin and Milne Edwards and Lysidice collaris Grube, both associated as sheath borers to the Mediterranean seagrass Posidonia oceanica, and with reference to their different origin and their actual geographic distribution. L. ninetta is distributed in the Atlantic and in the Mediterranean Sea while L. collaris is a tropical species, whose introduction into the Mediterranean Sea through the Suez Canal (lessepsian migrant) has been hypothesized in recent years. The two species have been often confused in the past, although they appear morphologically distinct. They share the same microhabitat (Posidonia sheaths) and they co-exist along a broad bathymetric range (1–30 m). Several populations for both taxa were sampled all along the coast of the Mediterranean basin. A variable no coding region of nuclear DNA (rDNA, ITS1) and a portion of a more conservative coding region of mitochondrial DNA (sub-unit one of citochrome oxidase, COI) were used as molecular markers. Both markers confirmed the separation between the two species. Low intraspecific polymorphism was present in L. collaris, together with absence of phylogeographic structure. In L. ninetta, instead, the presence of intraspecific cryptic lineages, sympatric in some sites, was recorded. Clustering of single populations in the two main clades was not always consistent between markers. The mitochondrial COI region showed more resolution at the given spatial scale. Our results suggest that Lysidice collaris could be recently introduced into the Mediterranean Sea from one or more separate events. On the other hand, for L. ninetta one could presume a re-colonization of the Mediterranean basin from the Atlantics, after the Messinian crisis (dry-out of the Mediterranean Sea, 5.5 my) with the subsequent separation of intraspecific lineages. The phylogeographic patterns of both Lysidice spp. are disjoined with respect to that of the host plant, P. oceanica. The obtained results suggest that environmental constraints and evolutionary history of these polychaetes and their host plant act in different ways to determine their actual genetic spatial structure.     

Phylogeography of the Southwestern Atlantic menhaden genus <Emphasis Type="Italic">Brevoortia</Emphasis> (Clupeidae,Alosinae)

Among pelagic fish, the Southwestern Atlantic menhaden genus Brevoortia (Clupeidae, Alosinae) constitutes an important species model to investigate the patterns of genetic differentiation. It is abundant in the Río de la Plata estuary and in the Atlantic coastal lagoons system from Uruguay and Southern Brazil. To access in the taxa discrimination and population structure in Brevoortia we perform a phylogeographic approach based on mitochondrial cytochrome b (cyt-b) sequences including 240 individuals from 16 collecting sites. Among the 720 bp cyt-b sequenced, 199 correspond to variables and 88 to phylogenetically informative sites. High values of haplotype diversity (h = 1.000) and nucleotide diversity (π = 0.061), as well as an average of 0.084 polymorphic segregating sites and 46 different haplotypes were found. Maximum likelihood analysis based on the GTR + I + G model and Bayesian inference strongly support the idea that B. aurea is the only species of the genus inhabiting the Southwestern Atlantic region. Our analyses revealed a complex population pattern characterized by the existence of long-term highly structured genetic assemblages of mixed stocks. Each monophyletic entity included individuals from different collecting sites, different age groups and collected in different years. Our data also suggest that the recruitment of unrelated mtDNA haplotypes carried out by individuals within schools could be occurring in the same nursery areas revealing the existence of many different maternal lineages. A scenario where different simultaneously and successively mixed mtDNA lineages remain historically connected through basal haplotypes among different clades could explains more accurately the complex and ordered metapopulation dynamic found in this pelagic fish.     

The utility of the 16S gene in investigating cryptic speciation within the Brachionus plicatilis species complex

Recent reports indicate an extensive amount of molecular evolution separating cryptic taxa as well as significant population structure at a microgeographical scale. Appropriate molecular markers are particularly suitable for distinguishing cryptic biological species. In this study, we examine the phylogenetic utility of 16S rRNA in elucidating the evolutionary relationships within the recently described euryhaline Brachionus plicatilis species complex. In addition, we assess the applicability of this marker in the genetic identification and monitoring of rotifer populations. We have sequenced a 378-bp fragment of the mitochondrial 16S rRNA gene in laboratory reference strains, hatchery clones as well as collections from a wild population of the subsaline Lake Koroneia (Northern Greece). Also, restriction fragment length polymorphism (RFLP) analysis was performed with eight restriction endonucleases. Rotifer samples are distinguished into six genetically divergent lineages. Average sequence divergence between lineages is 0.1038. The evolutionary relationships and divergence time-scales revealed with the 16S sequence data are in agreement with previous analyses using different mitochondrial and nuclear markers. The 16S region appears to have several advantages over other regions of the genome regarding use of species-specific primers, ease of amplification from single specimens and undiluted informational content over both recent and more ancient separations. It has also exhibited maximum discriminatory power (100% success) between lineages during RFLP analysis. The 16S assayed region has proven especially informative and consistent in detecting, supporting and establishing the lineage status within the B. plicatilis species complex both from a phylogenetic perspective and as an identification tool.     

Genetic evidence for the existence of cryptic species in an endangered clam <Emphasis Type="Italic">Coelomactra antiquata</Emphasis>

Coelomactra antiquata is a commercially important bivalve species, but has been suffering from severe population decline due to over-exploitation and the deterioration of environmental conditions. Previous genetic survey of C. antiquata conducted with allozymes combined with morphology revealed high levels of genetic differentiation between northern and southern populations which suggests a cryptic species might exist in C. antiquata. To test this hypothesis, amplified fragment length polymorphisms (AFLPs) and 16S rRNA gene sequence were used to re-evaluate the spatial genetic structure of six populations of C. antiquata along the coast of China. Both genetic markers display a sharp genetic break between the four northern populations (northern lineage) and two southern population (southern lineage). Large numbers of private alleles (AFLP) were found within the northern or southern populations and a deep divergence of about 6.5% in 16S rRNA gene sequence between the northern and southern lineages suggests the occurrence of potential cryptic or sibling species of C. antiquata. Applying previously published rates of mutation, divergence between the two lineages is estimated to have occurred approximately 3 million years ago and may be due to allopatric isolation during the middle Pliocene times. While no genetic differentiation was found within the northern or southern populations in both AFLP and 16S mtDNA markers, the results indicate that the northern and southern lineage should be managed separately and any translocation between the two areas should be avoided.     

Genetic structure of round scad mackerel <Emphasis Type="Italic">Decapterus macrosoma</Emphasis> (Carangidae) in the Indo-Malay archipelago

Round scad mackerel sampled in 1995-1998 were analysed for genetic variation using mitochondrial-DNA and nuclear-DNA markers. Sequence variation for a fragment of the cytochrome b gene (mitochondrial), amplified by polymerase chain reaction, was screened across individuals using single strand conformation polymorphism (SSCP). Sequence analysis of all SSCP haplotypes indicated two mitochondrial clades separated by, on average, 2.3% nucleotide divergence. The geographic distribution of haplotypes was homogeneous (Weir and Cockerham's [^(q)] \hat \theta =-0.002). Also, no geographic heterogeneity was detected for length polymorphism of Intron 1 of the gene encoding aldolase B ([^(q)] \hat \theta =0.005). Although homogeneity in allele frequencies throughout the Indo-Malay archipelago conformed to the expectations for a widely distributed pelagic fish in a highly connected habitat, this was at variance with the sharp geographic structure previously uncovered in Indian scad mackerel, Decapterus russelli, a fish with life-history characteristics similar to D. macrosoma. A remarkable similarity, however, was the occurrence of two similarly distinct clades within each species, suggesting a common history of geographic isolation. Low sea levels in the Pleistocene might have caused the separation and vicariance of populations within both D. macrosoma and D. russelli. Subsequent genetic exchange between populations would then have erased allele-frequency differences at the cytochrome b and aldolase B loci in D. macrosoma while some barrier to gene flow was maintained in D. russelli.     

Multi-marker estimate of genetic connectivity of sole (Solea solea) in the North-East Atlantic Ocean

A thorough knowledge on the genetic connectivity of marine populations is important for fisheries management and conservation. Using a dense population sampling design and two types of neutral molecular markers (10 nuclear microsatellite loci and a mtDNA cytochrome b fragment), we inferred the genetic connectivity among the main known spawning grounds of sole (Solea solea L.) in the North-East Atlantic Ocean. The results revealed a clear genetic structure for sole in the North-East Atlantic Ocean with at least three different populations, namely the Kattegat/Skagerrak region, the North Sea and the Bay of Biscay, and with indications for a fourth population, namely the Irish/Celtic Sea. The lack of genetically meaningful differences between biological populations within the southern North Sea is likely due to a large effective population size and sufficient connection (gene flow) between populations. Nevertheless, an isolation-by-distance pattern was found based on microsatellite genotyping, while no such pattern was observed with the cytochrome b marker, indicating an historical pattern prevailing in the latter marker. Our results demonstrate the importance of a combined multi-marker approach to understand the connectivity among marine populations at region scales.     

The malaria parasite Plasmodium relictum in the endemic avifauna of eastern Cuba

Island populations are vulnerable to introduced pathogens, as evidenced by extinction or population decline of several endemic Hawaiian birds caused by the malaria parasite, Plasmodium relictum (order Haemosporida). We analyzed blood samples from 363 birds caught near Guantánamo Bay, Cuba, for the presence of haemosporidian infections. We characterized parasite lineages by determining nucleotide variation of the parasite's mitochondrial cyt b gene. Fifty‐nine individuals were infected, and we identified 7 lineages of haemosporidian parasites. Fifty individuals were infected by 6 Haemoproteus sp. lineages, including a newly characterized lineage of Haem. (Parahaemoproteus) sp. CUH01. Nine individuals carried the P. relictum lineage GRW4, including 5 endemic Cuban Grassquits (Tiaris canorus) and 1 migratory Cape May Warbler (Setophaga tigrina). A sequence of the merozoite surface protein gene from one Cuban Grassquit infected with GRW4 matched that of the Hawaiian haplotype Pr9. Our results indicate that resident and migratory Cuban birds are infected with a malaria lineage that has severely affected populations of several endemic Hawaiian birds. We suggest GRW4 may be associated with the lack of several bird species on Cuba that are ubiquitous elsewhere in the West Indies. From the standpoint of avian conservation in the Caribbean Basin, it will be important to determine the distribution of haemosporidian parasites, especially P. relictum GRW4, in Cuba as well as the pathogenicity of this lineage in species that occur and are absent from Cuba.     

Nuclear ITS region of the alga Heterosigma akashiwo (Chromophyta: Raphidophyceae) is identical in isolates from Atlantic and Pacific basins

The internal transcribed spacer (ITS) region from 19 isolates of the algal genus Heterosigma (Chromophyta: Raphidophyceae) was amplified by polymerase chain-reaction (PCR) and sequenced. Isolates were obtained from both the Atlantic and Pacific basins, including Europe, eastern North America, western North America, Japan and New Zealand. This study presents evidence that all Heterosigma isolates in this study are representatives of one species (H. akashiwo). All 19 isolates, except one (LB 2005) had identical ITS sequence (98.31% similar by pairwise comparison); Isolate LB 2005 may represent a separate subspecies. Such high degree of ITS sequence identity implies that the organism has spread between oceanic regions in geologically recent times, possibly by human means. In addition to those from Heterosigma spp., the ITS regions from other marine Raphidophyceae (Chattonella antiqua, C. marina, C. subsalsa, Fibrocapsa japonica, and Olisthodiscus luteus) were amplified and sequenced using PCR. Total ITS lengths differed among the Raphidophyceae (C. antiqua, 577 base pairs (bp); C. marina, 577 bp;. C. subsalsa, 579 bp; F. japonica, 830 bp; H. akashiwo, 561 and 563 bp; O. luteus, 829 bp), but 5.8S rDNA sequences were similar in size (13 to 142 bp). The high ITS sequence identity between C. antiqua and C. marina (>99.9% by pairwise comparison) suggests the need for a taxonomic review of these species encompassing all morphological, genetic, physiological and biochemical information. Additionally, a number of cultures of Raphidophyceae were positively identified. In general, ITS comparisons among the Raphidophyceae may be most useful at the level of species determination rather than at the population level. Received: 12 July 1999 / Accepted: 16 March 2000     

Incipient speciation within a subgenus of rockfish (<Emphasis Type="Italic">Sebastosomus</Emphasis>) provides evidence of recent radiations within an ancient species flock

The high frequency of speciation events associated with species flocks (i.e., radiations of closely related species) provides invaluable insight into the speciation process. Investigations of the speciation process in the marine environment are rare, and therefore, the genetic analysis of the rockfish genus Sebastes, considered an ancient marine species flock, provides an opportunity to investigate this process in the sea. Using both mitochondrial and nuclear markers, we analyzed five closely related species within the rockfish subgenus Sebastosomus. Our goal was to understand the evolutionary history and genetic relationships among species within this group and to provide evidence of recent speciation events within the subgenus. In the genetic analysis of the subgenus, we found different stages of the speciation process, with greater genetic divergences among three of the five species, evidence of recent divergence between two of the five species, Sebastes entomelas and S. mystinus, and significant genetic divergence between two lineages within S. mystinus revealing a signature of incipient speciation. We also found frequency differences of the two S. mystinus lineages among sample locations and found no evidence of introgression between the lineages at the location where both coexist. Although Sebastes is an example of an ancient species flock, this study provides evidence of ongoing speciation within the genus and reveals stages of this process from incipient to distinct species.     

Predominance of clade D <Emphasis Type="Italic">Symbiodinium</Emphasis> in shallow-water reef-building corals off Kish and Larak Islands (Persian Gulf,Iran)

Scleractinian coral species harbour communities of photosynthetic taxa of the genus Symbiodinium. As many as eight genetic clades (A, B, C, D, E, F, G and H) of Symbiodinium have been discovered using molecular biology. These clades may differ from each other in their physiology, and thus influence the ecological distribution and resilience of their host corals to environmental stresses. Corals of the Persian Gulf are normally subject to extreme environmental conditions including high salinity and seasonal variation in temperature. This study is the first to use molecular techniques to identify the Symbiodinium of the Iranian coral reefs to the level of phylogenetic clades. Samples of eight coral species were collected at two different depths from the eastern part of Kish Island in the northern Persian Gulf, and Larak Island in the Strait of Hormuz. Partial 28S nuclear ribosomal (nr) DNA of Symbiodinium (D1/D2 domains) were amplified by polymerase chain reaction (PCR). PCR products were analyzed using single stranded conformational polymorphism and phylogenetic analyses of the LSU DNA sequences from a subset of the samples. The results showed that Symbiodinium populations were generally uniform among and within the populations of eight coral species studied, and there are at least two clades of Symbiodinium from Kish and Larak islands. Clade D was detected from eight of the coral species while clade C was found in two of species only (one species hosted two clades simultaneously). The dominance of clade D might be explained by high temperatures or the extreme temperature variation, typical of the Persian Gulf. Publication of this article was held up owing to technical problems. The publisher apologizes sincerely for this lengthy delay.     

Comparative population genetic structure of marine gastropods (Littorina spp.) with and without pelagic larval dispersal

 Population genetic theory predicts that marine animal species with planktonic larvae will have less genetic structure than those with direct development. We compared the genetic structure of four species of littorinid snails – two with planktonic egg capsules that hatch as planktonic larvae and two with benthic egg masses that hatch as crawl-away juveniles. We used DNA sequencing and single stranded conformational polymorphism (SSCP) to assess sequence variation in a 480 bp fragment of the mitochondrial cytochrome b gene and then used an analysis of molecular variance (AMOVA) to estimate Φ_st for populations from the northeastern Pacific coast. One of the two direct-developing species, Littorina subrotundata, had a moderate amount of population structure (Φ_st=0.209) as expected but the other direct-developing species, L. sitkana, was nearly fixed for a single haplotype that made it impossible to precisely estimate Φ_st. One of the two planktonic-developing species, L. scutulata, did not show any significant population structure (Φ_st=0.004). In contrast to our expectations, the other planktonic-developing species, L. plena, showed some weak but statistically significant population structure (Φ_st=0.052). We discuss how differences in population genetic structure between species with the same type of development may reflect differences in their historical demography. Received: 22 December 1999 / Accepted: 24 July 2000     

Spatiotemporal analysis of population genetic structure in <Emphasis Type="Italic">Geomonhystera disjuncta</Emphasis> (Nematoda,Monhysteridae) reveals high levels of molecular diversity

Species identification in the phylum Nematoda is complicated due to the paucity of easily obtainable diagnostic morphological features. Furthermore, the cosmopolitan distribution of several species despite low dispersal abilities makes cryptic diversity potentially substantial within this phylum. We conducted a population genetic survey in the marine nematode Geomonhystera disjuncta in Belgium and The Netherlands in two seasons. The mitochondrial cytochrome oxidase c subunit 1 (COI) gene was screened with the single-strand conformation polymorphism method in 759 individuals. The 43 haplotypes were grouped into five lineages, with low divergences within (<3%) and high divergences between lineages (>14%). Analysis of the nuclear ITS region yielded concordant tree topologies, indicating the presence of five cryptic taxa within G. disjuncta. Analysis of Molecular Variance (AMOVA) illustrated a significant structuring in all lineages and temporal fluctuations in haplotype frequencies within and between locations. Metapopulation dynamics and/or priority effects best explained this structuring. Finally, our data indicate that the COI gene may be useful for DNA barcoding purposes.     

Phylogeographic patterns of the mysid Mesopodopsis slabberi (Crustacea, Mysida) in Western Europe: evidence for high molecular diversity and cryptic speciation

The phylogeographic patterns among populations of Mesopodopsis slabberi (Crustacea, Mysida), an ecological important mysid species of marine and estuarine habitats, were analysed by means of DNA sequencing of a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) and the 16S ribosomal RNA genes. Samples of M. slabberi collected from five Atlantic and two Western Mediterranean populations were investigated. Very high levels of within-population molecular diversity were observed in all samples (mean h=0.807 and π=0.0083), with exception of the Mediterranean Ebro population which contained only one haplotype. Differentiation among populations was high, and a clear phylogeographic break was observed between the Atlantic and Mediterranean populations. Moreover, a strong differentiation was detected between both populations in the Western Mediterranean Sea (Alicante and Ebro delta), while two divergent lineages occurred in sympatry within the Atlantic Mondego estuary. The high congruence between both the COI and 16S rRNA sequence data, the reciprocal monophyly of the different mitochondrial clades and the levels of nucleotide divergence between them suggest the presence of a complex of cryptic species within M. slabberi. Estimations of divergence time between the different mitochondrial lineages indicate that a split occurred during the late Miocene/early Pliocene. Such a divergence could be concordant with vicariant events during sea-level drops within the Mediterranean region at that time. However, within the Mediterranean Sea, the potential of divergence through ecological diversification cannot be ruled out.     

Genetic divergence between two morphologically similar varieties of the kuruma shrimp<Emphasis Type="Italic"> Penaeus japonicus</Emphasis>

The kuruma shrimp Penaeus japonicus is widely distributed throughout the Indo-West Pacific. Two morphologically similar varieties, I and II, are recognized from the South China Sea. The two varieties are characterized by different color banding patterns on the carapace, but there are no distinct differences in morphometric traits between them based on measurement of 13 characters. Sequence data and restriction profiles of the mitochondrial genes reveal that these two varieties represent distinct clades, with sequence divergences of about 1% (473 bp) in 16S rRNA, 6–7% (504 bp) in cytochrome oxidase I, and 16–19% (470 bp) in the control region. Analysis of amplified fragment length polymorphism confirms that the two varieties are genetically distinct. We also investigated the geographical distribution of the two varieties in the western Pacific by analyzing specimens collected from Japan and Singapore. Shrimps from Japan and Singapore have been found to belong to varieties I and II, respectively, suggesting that the two varieties have different geographical distribution. Phylogenetic study reveals that the two varieties are more closely related to each other than to the other phylogenetically related Penaeus species. Results from this study suggest the occurrence of two cryptic species in the kuruma shrimp P. japonicus.Communicated by M.S. Johnson, Crawley