Purification and characterization of vitellin from the estuarine mysid Neomysis integer (Crustacea; Mysidacea)

Invertebrates account for roughly 95% of all animals, yet surprisingly, little effort has been invested to understand their value in signaling potential environmental endocrine disruption. There has been, however, much recent attention on vitellogenin induction in egg-laying invertebrates and vertebrates as indicators of exposure to estrogenic xenobiotics. Mysid shrimp (Crustacea: Mysidacea) have been put forward by several researchers and regulatory bodies (e.g., US-EPA) as suitable test organisms for the evaluation of environmental endocrine disruption. In view of developing sensitive assays to study endocrine disruption in the estuarine mysid Neomysis integer, we isolated and characterized vitellin, the major yolk protein in eggs. Vitellin was purified using gel filtration and characterized by electrophoresis using different staining procedures. Specific (as shown by Western blotting) polyclonal antibodies were produced in rabbit against the purified vitellin of N. integer. These antisera will be used to develop immunoassays to study vitellogenesis in mysids and to detect potential stimulatory or inhibitory effects of endocrine disruptors on the production of vitellin.     

Phylogeography of the common goby, Pomatoschistus microps, with particular emphasis on the colonization of the Mediterranean and the North Sea

The phylogeographical patterns of a small marine fish, the common goby, Pomatoschistus microps, were assessed at 12 sites along the northeastern Atlantic coasts and the western Mediterranean Sea. A combination of two genetic markers was employed: cellulose acetate allozyme electrophoresis (CAGE) and sequence analysis of a 289 bp fragment of the mitochondrial locus cytochrome b. Both markers were congruent in revealing significant differences between samples (global FST = 0.247 for the allozymes and PhiST = 0.437 for the mitochondrial DNA data) and a pattern of isolation-by-distance. Phylogeographical analyses yielded a shallow branching structure with four groups. Three of those were confined to the Atlantic basin and showed a star-like pattern. The fourth group contained a central haplotype occurring at the edges of the species' distribution, accompanied by a few more rare variants, which were restricted to the Mediterranean Sea. A genetic break was observed around the British Isles, with distinct haplotypes dominating at either side of the English Channel. A significantly negative correlation between the degree of genetic diversity and latitude was recorded both for mitochondrial DNA (mtDNA) and allozymes in the Atlantic basin. Gene flow analysis suggested that recolonization of the North Sea and the coasts of western Scotland and Ireland may have taken place from a glacial refugium in the Southern Bight of the North Sea. These results are discussed in the perspective of possible postglacial migration routes of marine fish along the northeastern Atlantic coasts.     

Phylogeography of North Atlantic intertidal tardigrades: refugia,cryptic speciation and the history of the Mid‐Atlantic Islands

Aim To analyse the phylogeographical history of intertidal tardigrades in the North Atlantic in order to improve our understanding of geographical differentiation in microscopic organisms, and to understand the potential importance of the Mid‐Atlantic Islands as stepping stones between the American and European coasts of the Atlantic Ocean. Location Twenty‐four localities from the Mid‐Atlantic Islands (Greenland, Iceland and the Faroe Islands) and both sides of the North Atlantic Ocean. Methods A mitochondrial marker (cytochrome c oxidase subunit I) was sequenced from individual tardigrades belonging to the genus Echiniscoides. The existence of cryptic species was detected using generalized mixed Yule coalescence analysis; lineage ages were estimated with relaxed clock methods; and the degree of geographical differentiation was analysed with samova analyses, haplotype networks and Mantel tests. Results Echiniscoides hoepneri, previously known only from Greenland, was recovered throughout the Mid‐Atlantic Islands. The Faroe Islands population was isolated from Greenland and Iceland, but overall genetic variation was low. The morphospecies Echiniscoides sigismundi had high genetic variation and consisted of at least two cryptic species. A northern and a southern species were both recovered on both sides of the Atlantic, but only the northern species was found on the Mid‐Atlantic Islands. The northern species showed signs of long‐term isolation between the Western and Eastern Atlantic, despite the potential of the Mid‐Atlantic islands to act as stepping‐stones. There was no sign of long‐term isolation in the southern species. The Mid‐Atlantic individuals of the northern species were of Eastern Atlantic origin, but Greenland and Iceland showed signs of long‐term isolation. The genetic pattern found in the southern species is not clearly geographical, and can probably be best explained by secondary contact between former isolated populations. Main conclusions North Atlantic intertidal tardigrades from the genus Echiniscoides showed strong geographical differentiation, and the Mid‐Atlantic Islands seemed unimportant as stepping stones across the Atlantic. The geographical variation of the northern species of E. sigismundi suggests post‐glacial recolonization from several refugia.     

Phylogeography of the Alpine salamander, Salamandra atra (Salamandridae) and the influence of the Pleistocene climatic oscillations on population divergence

Fifty individuals of the endemic Alpine salamander, Salamandra atra, representing 13 populations throughout the range of the two currently recognized subspecies, atra and aurorae, were examined for sequence variation in a large portion (1050 bp) of the mitochondrial cytochrome b gene. We revealed a large number of mitochondrial DNA (mtDNA) haplotypes (10). Interpopulation sequence divergence was very low, ranging from 0 to 3.1%. The relationships among haplotypes were poorly resolved. The divergence time estimate between several mtDNA haplotypes suggested a pre-Pleistocene differentiation approximately 3 million years ago. Moreover, the impact of the Pleistocene glaciations on the phylogeographical patterns appears to have been secondary, although a somewhat reduced genetic variability was found in populations living in areas that were directly affected by the glaciation.     

Phylogeography of Emerita analoga (Crustacea,Decapoda, Hippidae), an eastern Pacific Ocean sand crab with long‐lived pelagic larvae

Aim Phylogeographic analyses have confirmed high dispersal in many marine taxa but have also revealed many cryptic lineages and species, raising the question of how population and regional genetic diversity arise and persist in dynamic oceanographic settings. Here we explore the geographic evolution of Emerita analoga, an inter‐tidal sandy beach crab with an exceptionally long pelagic larval phase and wide latitudinal, amphitropical, distribution. We test the hypothesis that eastern Pacific E. analoga constitute a single panmictic population and examine the location(s), timing and cause(s) of phylogeographic differentiation. Location Principally the eastern Pacific Ocean. Methods We sequenced cytochrome c oxidase subunit I (COI) from 742 E. analoga specimens collected between 1997 and 2000 and downloaded homologous sequences of congeners from GenBank. We reconstructed a phylogeny for Emerita species using maximum likelihood and Bayesian methods and estimated times to most recent common ancestors (TMRCAs), using a COI divergence rate of 1% Myr^?1 and timing of closure of the Central American Seaway. We constructed the COI haplotype network of E. analoga using statistical parsimony, calculated population genetic and spatial structure statistics in Arlequin , and estimated the demographic history of E. analoga using Bayesian skyline analysis. Results Population subdivision and allele frequency differences were insignificant among north‐eastern Pacific locations over 2000 km apart (Φ_ST = 0.00, P = 0.70), yet two distinct phylogroups were recovered from the north‐eastern and south‐eastern Pacific (Φ_CT = 0.87, P < 0.001). Amphitropical differentiation of these temperate clades occurred after TMRCA 1.9 ± 0.02 (mean ± SE) Ma and E. analoga has expanded into its present‐day north‐eastern Pacific range since c. 250 ka. Main conclusions Emerita analoga is not panmictic but is very widely dispersed and approaching genetic homogeneity, i.e. ‘eurymixis’, in the north‐eastern Pacific. North‐eastern and south‐eastern Pacific populations of E. analoga probably became isolated c. 1.5 Ma as the tropical eastern Pacific Ocean warmed and expanded, intensifying barriers to gene flow. The fragmentation of a widespread ancestral species previously connected by long‐distance gene flow (‘soft vicariance’) coincident with changing oceanographic conditions may be a common theme in the evolution of Emerita species and in other highly dispersive taxa. Highly dispersive species may differentiate because of, not despite, the dynamic oceanographic setting.     

Phylogeography of an east Australian wet-forest bird, the satin bowerbird (Ptilonorhynchus violaceus), derived from mtDNA, and its relationship to morphology

Australian wet forests have undergone a contraction in range since the mid-Tertiary, resulting in a fragmented distribution along the east Australian coast incorporating several biogeographical barriers. Variation in mitochondrial DNA and morphology within the satin bowerbird was used to examine biogeographical structure throughout almost the entire geographical extent of these wet forest fragments. We used several genetic analysis techniques, nested clade and barrier analyses, that use patterns inherent in the data to describe the spatial structuring. We also examined the validity of the two previously described satin bowerbird subspecies that are separated by well-defined biogeographical barriers and tested existing hypotheses that propose divergence occurs within each subspecies across two other barriers, the Black Mountain corridor and the Hunter Valley. Our data showed that the two subspecies were genetically and morphologically divergent. The northern subspecies, found in the Wet Tropics region of Queensland, showed little divergence across the Black Mountain corridor, a barrier found to be significant in other Wet Tropics species. Biogeographical structure was found through southeastern Australia; three geographically isolated populations showed genetic differentiation, although minimal divergence was found across the proposed Hunter Valley barrier. A novel barrier was found separating inland and coastal populations in southern New South Wales. Little morphological divergence was observed within subspecies, bar a trend for birds to be larger in the more southerly parts of the species' range. The results from both novel and well-established genetic analyses were similar, providing greater confidence in the conclusions about spatial divergence and supporting the validity of these new techniques.     

Erodium maritimum (Geraniaceae), a species with an uneven and fragmented distribution along the Western Mediterranean and European Atlantic coasts,has a weak genetic structure

Erodium maritimum L. is an annual species presenting heterogeneous, sometimes very small, and distant populations, distributed along a discontinuous coastal strip of the European Atlantic and the central and western Mediterranean basin. The aim of this study is to investigate genetic variation and geographic structure changes across its large distribution. Fourteen populations of E. maritimum were studied using AFLP fingerprints, together with their population sizes, reproductive systems and flower visitors. AFLP markers revealed the genetic structure of the species to be weak. Many individuals from one population clustered together with those of other populations, showing a high degree of genetic admixture. Despite having a self‐compatible reproductive system, populations (especially the largest ones) showed high levels of genetic polymorphism, and the majority of genetic variation was contained within populations. The low genetic structure suggests high levels of gene flow, which might be explained through the dispersability of the species’ fruits. Finally, recommendations are provided for management strategies to facilitate the conservation of this endangered species.     

Comparative phylogeography of sympatric sister species,Clevelandia ios and Eucyclogobius newberryi (Teleostei,Gobiidae), across the California Transition Zone

It is paradigmatic in marine species that greater dispersal ability often, but not always, results in greater gene flow and less population structure. Some of the exceptions may be attributable to studies confounded by comparison of species with dissimilar evolutionary histories, i.e. co-occurring species that are not closely related or species that are closely related but allopatric. Investigation of sympatric sister species, in contrast, should allow differences in phylogeographic structure to be attributed reliably to recently derived differences in dispersal ability. Here, using mitochondrial DNA control region sequence, we first confirm that Clevelandia ios and Eucyclogobius newberryi are sympatric sister taxa, then demonstrate considerably shallower phylogeographic structure in C. ios than in E. newberryi. This shallower phylogeographic structure is consistent with the higher dispersal ability of C. ios, which most likely results from the interaction of habitat and life-history differences between the species. We suggest that the paradigm will be investigated most rigorously by similar studies of other sympatric sister species, appended by thorough ecological studies, and by extending this sister-taxon approach to comparative phylogeographic studies of monophyletic clades of sympatric species.     

Historical biogeography of Iberobathynella (Crustacea, Syncarida, Bathynellacea), an aquatic subterranean genus of Parabathynellids, endemic to the Iberian Peninsula

• 1 The origin and distribution of the species of freshwater interstitial (stygobiont) syncarids of the Iberobathynella genus (Crustacea, Parabathynellidae), endemic to the Iberian Peninsula, are considered in the context of evolutionary biogeography.
• 2 We established the most likely phylogenetic relationships between the 14 known species of the Iberobathynella genus on the basis of morphological characters. We assumed a common marine ancestor and estimated the possible phylogenetic ages of the 14 inland species from the number of cladogram nodes.
• 3 The land ages at the sites where the species are found help to tell us when the landmasses emerged definitively: most species are found in areas that have undergone one or more marine transgressions/regressions since the Triassic and which could therefore have been colonized by the common ancestors.
• 4 We assessed the Boutin–Coineau two‐step model of colonization and evolution in an attempt to elucidate the evolutionary history of Iberobathynella and to offer a plausible palaeogeographical scenario. We looked for concordance between the possible chronology of Iberobathynella taxa colonization and their phylogenetic relationships, but found none of significance. Hence, we cannot reject the possibility that evolutionary clade divergence within the genus was independent of sea‐level changes in the Senonian (88.5–65 mya) and Eocene (56.5–35.4 mya) epochs. We can therefore neither confirm nor rule out a correlation.

     

Blastodinium galatheanum sp. nov. (Dinophyceae) a parasite of the planktonic copepod Acartia negligens (Crustacea,Calanoida) in the central Atlantic Ocean

A new dinoflagellate species, Blastodinium galatheanum sp. nov., was found parasitizing the planktonic copepods Acartia negligens and Acartia sp. in the Atlantic Ocean between the Azores and the Cape Verde Islands. These copepods have not previously been reported hosting a Blastodinium species. Characters that distinguish the new species are the shape and size of the trophic stage, its host species, and its predominantly solitary existence. Dinospores of Blastodinium galatheanum sp. nov. are peridinioid in nature and morphologically indistinguishable from dinospores of two other previously investigated Blastodinium species. SSU rRNA gene sequences from two isolates of this new species were almost identical and showed similarities to SSU rRNA sequences of other species of Blastodinium. A phylogenetic analysis based on SSU rRNA gene sequences suggested monophyly for all existing sequences of Blastodinium spp., including a sequence from the type species B. pruvoti, presented here for the first time.     

Biogeographical distribution of Hartaetosiga strains (Choanoflagellatea,Craspedida, Salpingoecidae) including morphological and transcriptomic data from a transect across the Atlantic Ocean

The genus Hartaetosiga Carr, Richter and Nitsche, 2017 comprised up to now only three species, H. gracilis (Kent) Carr, Richter, Nitsche, 2017, H. balthica (Wylezich and Karpov) Carr, Richter and Nitsche, 2017 and H. minima (Wylezich and Karpov) Carr, Richter and Nitsche, 2017. Based on distinct molecular data these species were relocated from the strictly freshwater genus Codosiga (Ehrenberg) Bütschli, 1878 to a new genus comprising brackish and marine species. During the cruise MSM82/2 across the Atlantic Ocean in 2019, surface water samples were taken from 15 stations along a transect ranging from 35°S to 23°N. We were able to isolate and cultivate 14 strains of the genus Hartaetosiga. Morphometric data showed no distinct morphological traits allowing for a species delineation, indicating a cryptic species complex within the genus. Based on cultivation, morphological data, and molecular analyses, we recorded H. gracilis for the first time from off-shelf waters of the Atlantic Ocean and could describe a new species, H. australis n. sp. This new species was recorded from sampling stations in the Southern Hemisphere only, which may indicate a potential biogeographic restriction likely caused by the Equatorial Counter Current (ECC), dividing the northern and southern surface waters.     

Population structure of the dusky pipefish (Syngnathus floridae) from the Atlantic and Gulf of Mexico,as revealed by mitochondrial DNA and microsatellite analyses

Aim To elucidate the historical phylogeography of the dusky pipefish (Syngnathus floridae) in the North American Atlantic and Gulf of Mexico ocean basins. Location Southern Atlantic Ocean and northern Gulf of Mexico within the continental United States. Methods A 394‐bp fragment of the mitochondrial cytochrome b gene and a 235‐bp fragment of the mitochondrial control region were analysed from individuals from 10 locations. Phylogenetic reconstruction, haplotype network, mismatch distributions and analysis of molecular variance were used to infer population structure between ocean basins and time from population expansion within ocean basins. Six microsatellite loci were also analysed to estimate population structure and gene flow among five populations using genetic distance methods (F_ST, Nei’s genetic distance), isolation by distance (Mantel’s test), coalescent‐based estimates of genetic diversity and migration patterns, Bayesian cluster analysis and bottleneck simulations. Results Mitochondrial analyses revealed significant structuring between ocean basins in both cytochrome b (Φ_ST = 0.361, P < 0.0001; Φ_CT = 0.312, P < 0.02) and control region (Φ_ST = 0.166, P < 0.0001; Φ_CT = 0.128, P < 0.03) sequences. However, phylogenetic reconstructions failed to show reciprocal monophyly in populations between ocean basins. Microsatellite analyses revealed significant population substructuring between all locations sampled except for the two locations that were in closest proximity to each other (global F_ST value = 0.026). Bayesian analysis of microsatellite data also revealed significant population structuring between ocean basins. Coalescent‐based analyses of microsatellite data revealed low migration rates among all sites. Mismatch distribution analysis of mitochondrial loci supports a sudden population expansion in both ocean basins in the late Pleistocene, with the expansion of Atlantic populations occurring more recently. Main conclusions Present‐day populations of S. floridae do not bear the mitochondrial DNA signature of the strong phylogenetic discontinuity between the Atlantic and Gulf coasts of North America commonly observed in other species. Rather, our results suggest that Atlantic and Gulf of Mexico populations of S. floridae are closely related but nevertheless exhibit local and regional population structure. We conclude that the present‐day phylogeographic pattern is the result of a recent population expansion into the Atlantic in the late Pleistocene, and that life‐history traits and ecology may play a pivotal role in shaping the realized geographical distribution pattern of this species.     

Distribution and population structure of the smooth‐hound shark,Mustelus mustelus (Linnaeus, 1758), across an oceanic archipelago: Combining several data sources to promote conservation

Sharks play a key role in the structure and functioning of marine ecosystems. More ecological information is essential to implement responsible management and conservation actions on this fauna, particularly at a regional level for threatened species. Mustelus mustelus is widely found in the eastern Atlantic Ocean and catalogued as “Vulnerable” by the IUCN European assessment. In this study, data on the distribution and population structure of this species across the islands of the Canarian archipelago, located along an east to west gradient in the north‐eastern Atlantic, were collected by taking advantage of “Local Ecological Knowledge,” in terms of sightings in coastal waters and long‐term imprints on the local gastronomic heritage, and decadal fisheries landings. Both sources of quantitative data (sightings and fisheries landings) demonstrated that adults of M. mustelus has a significantly larger presence in the eastern and central, than in the western islands of the archipelago. This is also reflected on local gastronomic legacies, with a larger number of recipes in the eastern and central islands. Adult smooth‐hound sharks were significantly more observed in sandy and sandy‐rocky bottoms, with individuals seen throughout the entire year, whereas juveniles aggregate on very shallow waters in spring and summer. Such aggregations require a special management strategy, as they play a key role in critical life stages; these sites should be protected from human perturbations. We also suggest a temporal fishing ban between April and October, when individuals tend to concentrate on nearshore waters. Because of the large differences in presence of this shark among the Canary Islands, management of the species should be adapted to the specific peculiarities of each island, rather than adopting a management policy at the entire archipelago‐scale. Overall, this study sets the basis for further investigation to promote conservation of this vulnerable shark in the study region.     

Isolation and characterization of microsatellite loci in Pitcairnia albiflos (Bromeliaceae), an endemic bromeliad from the Atlantic Rainforest, and cross-amplification in other species

Eight microsatellite markers were isolated from Pitcairnia albiflos, an endangered endemic bromeliad species restricted to 'inselberg' rock outcrops in the state of Rio de Janeiro, Brazil. The number of alleles observed for each locus ranged from two to 12. Average observed and expected heterozygosities were 0.408 and 0.663, respectively. The cross-amplification test in 16 taxa suggests that the markers will be useful in numerous related bromeliad species. The loci will be used to study genetic structure and reproductive biology in fragmented inselberg populations and the origin and maintenance of barriers to gene flow between sympatric Pitcairnia species.     

Population structure of Hypochaeris salzmanniana DC. (Asteraceae), an endemic species to the Atlantic coast on both sides of the Strait of Gibraltar, in relation to Quaternary sea level changes

To detect potential Pleistocene refugia and colonization routes along the Atlantic coast, we analysed amplified fragment length polymorphisms (AFLPs) in 140 individuals from 14 populations of Hypochaeris salzmanniana (Asteraceae), an annual species endemic to the southwestern European and northwestern African coastal areas. Samples covered the total distributional range of the species, with eight populations in southwestern Spain and six populations in northwestern Morocco. Using nine primer combinations, we obtained 546 fragments in H. salzmanniana and its sister species H. arachnoidea of which 487 (89.2%) were polymorphic. The neighbour-joining tree shows that the populations south of the Loukos river in Morocco are clearly differentiated, having more polymorphic, private, and rare fragments, and higher genetic diversity, than all the other populations. The southernmost populations in Morocco, south of the river Sebou, form a large panmictic population. They are probably the oldest populations that have been relatively unaffected by stochastic processes resulting from Pleistocene glaciations. Northward migration of populations during this period may have resulted in loss of genetic diversity in specific regions, perhaps due to bottlenecks caused by rise in sea level during interglacial periods, and, in some cases, by changes in the breeding system.