Dispersal ability, habitat characteristics, and sea-surface circulation shape population structure of Cingula trifasciata (Gastropoda: Rissoidae) in the remote Azores Archipelago.

BACKGROUND: In the marine realm, dispersal ability is among the major factors shaping the distribution of species. In the Northeast Atlantic Ocean, the Azores Archipelago is home to a multitude of marine invertebrates which, despite their dispersal limitations, maintain gene flow among distant populations, with complex evolutionary and biogeographic implications. The mechanisms and factors underlying the population dynamics and genetic structure of non-planktotrophic gastropods within the Azores Archipelago and related mainland populations are still poorly understood. The rissoid Cingula trifasciata is herewith studied to clarify its population structure in the Northeast Atlantic Ocean and factors shaping it, with a special focus in intra-archipelagic dynamics. RESULTS: Coupling microsatellite genotyping by amplicon sequencing (SSR-GBAS) and mitochondrial datasets, our results suggest the differentiation between insular and continental populations of Cingula trifasciata, supporting previously raised classification issues and detecting potential cryptic diversity. The finding of connectivity between widely separated populations was startling. In unique ways, dispersal ability, habitat type, and small-scale oceanographic currents appear to be the key drivers of C. trifasciata's population structure in the remote Azores Archipelago. Dispersal as non-planktotrophic larvae is unlikely, but its small-size adults easily engage in rafting. Although the typical habitat of C. trifasciata, with low hydrodynamics, reduces the likelihood of rafting, individuals inhabiting algal mats are more prone to dispersal. Sea-surface circulation might create dispersal pathways for rafts, even between widely separated populations/islands. CONCLUSIONS: Our results show that gene flow of a marine non-planktotrophic gastropod within a remote archipelago can reveal unanticipated patterns, such that the understanding of life in such areas is far from well-understood. We expect this work to be the starting of the application of SSR-GBAS in other non-model marine invertebrates, providing insights on their population dynamics at distinct geographical scales and on hidden diversity. How transversal is the role played by the complex interaction between functional traits, ecological features, and sea-surface circulation in the population structure of marine invertebrates can be further addressed by expanding this approach to more taxa.

Genetic diversity and differentiation patterns in Micromeria from the Canary Islands are congruent with multiple colonization dynamics and the establishment of species syngameons.

BACKGROUND: Especially on islands closer to the mainland, such as the Canary Islands, different lineages that originated by multiple colonization events could have merged by hybridization, which then could have promoted radiation events (Herben et al., J Ecol 93: 572-575, 2005; Saunders and Gibson, J Ecol 93: 649-652, 2005; Caujapé-Castells, Jesters, red queens, boomerangs and surfers: a molecular outlook on the diversity of the Canarian endemic flora, 2011). This is an alternative to the scenario where evolution is mostly driven by drift (Silvertown, J Ecol 92: 168-173, 2004; Silvertown et al., J Ecol 93: 653-657, 2005). In the former case hybridization should be reflected in the genetic structure and diversity patterns of island species. In the present work we investigate Micromeria from the Canary Islands by extensively studying their phylogeographic pattern based on 15 microsatellite loci and 945 samples. These results are interpreted according to the hypotheses outlined above. RESULTS: Genetic structure assessment allowed us to genetically differentiate most Micromeria species and supported their current classification. We found that populations on younger islands were significantly more genetically diverse and less differentiated than those on older islands. Moreover, we found that genetic distance on younger islands was in accordance with an isolation-by-distance pattern, while on the older islands this was not the case. We also found evidence of introgression among species and islands. CONCLUSIONS: These results are congruent with a scenario of multiple colonizations during the expansion onto new islands. Hybridization contributes to the grouping of multiple lineages into highly diverse populations. Thus, in our case, islands receive several colonization events from different sources, which are combined into sink populations. This mechanism is in accordance with the surfing syngameon hypothesis. Contrary to the surfing syngameon current form, our results may reflect a slightly different effect: hybridization might always be related to colonization within the archipelago as well, making initial genetic diversity to be high to begin with. Thus the emergence of new islands promotes multiple colonization events, contributing to the establishment of hybrid swarms that may enhance adaptive ability and radiation events. With time, population sizes grow and niches start to fill. Consequently, gene-flow is not as effective at maintaining the species syngameon, which allows genetic differentiation and reproductive isolation to be established between species. This process contributes to an even further decrease in gene-flow between species.

Molecular phylogeny and character evolution of carnivorous plant families in Caryophyllales--revisited.

Recent phylogenetic analyses based on single gene and combined data sets have substantially increased our knowledge of the phylogeny of Caryophyllales s.l., indicating that additional carnivorous families are related to this alliance. In earlier contributions towards a reassessment of inter- and infrafamilial relationships slowly evolving genes had been preferred for phylogenetic inference. The resulting tree topologies based on rbcL and 18S rDNA, however, were characterized by limited resolution, low internal support and topological incongruence. Therefore genomic regions evolving more rapidly have been used in subsequent studies. Comparative sequencing of the matK gene and the flanking trnK intron region as well as combined analyses based on plastid matK, atpB, rbcL, and nuclear 18S rDNA have effectively improved resolution and internal support. Tree topologies revealed Caryophyllales s.l. as monophyletic group and indicated a clear division into two sister clades, the "core" and the "non-core" Caryophyllales (with Rhabdodendraceae and Simmondsiaceae with unclear affinities). Contrary to the "core" group (with Asteropeiaceae and Physenaceae as successive sister groups), which corresponds largely to the previous circumscription of the order, the monophyly of "non-core" Caryophyllales comprising Polygonaceae, Plumbaginaceae, Frankeniaceae, and Tamaricaceae along with the carnivorous families Droseraceae, Nepenthaceae, Drosophyllaceae, Dioncophyllaceae, and Ancistrocladaceae are a recent discovery. Based on reliable tree topologies it is hypothesized that pitfall traps of Nepenthes and snap traps typical for Aldrovanda and Dionaea were derived from a common ancestor with adhesive flypaper traps. With exception of Triphyophyllum carnivory was secondarily lost in the remaining Dioncophyllaceae (Dioncophyllum, Habropetalum) and all taxa of Ancistrocladaceae.

Introduction of a nuclear marker for phylogenetic analysis of Nepenthaceae.

Nepenthaceae, the pitcher plants of the Old World tropics show a remarkable diversity in SE Asia, especially on the islands of Borneo and Sumatra. This region is considered as a secondary center of diversity. Sequence analysis of the cpDNA TRNK intron supports this hypothesis showing the species of the Malay Archipelago as neighbour group to the isolated species from Sri Lanka, the Seychelles, and Madagascar. Based on phylogenetic reconstructions an origin of recent Nepenthaceae in the Indian subcontinent is assumed. A recent investigation focused on a non-plastid, translocated copy of the TRNK intron has revealed an incongruence to tree topology based on the cpDNA TRNK intron. Although the translocated copy emerged as insufficient for phylogenetic reconstruction of Nepenthaceae some taxa showed, contrary to the cpDNA dataset, relatively high distances to the rest of the taxa. These results indicated that the phylogeny of the TRNK intron could not reflect true phylogenetic relationships. We investigated the peptide transferase 1 (PTR1), to develop a phylogenetic marker that is based on a nuclear low copy gene in Nepenthes. All sequences obtained were probably functional, indicated by the ratio of point mutations of the single codon positions in exon and intron regions. Comparative analysis showed that this locus is of similar variability as the cpDNA TRNK intron and, contrary to the translocated copy of TRNK, potential useful for phylogenetic reconstruction. While in parts congruent to the plastid TRNK intron phylogeny, a higher divergence of some sequences in PRT1 and in the previously reported, non cpDNA dataset indicates that remnants of an older species stock persisted east of Wallace's line and on the Sunda Shelf. This suggests that plastid haplotypes existing today in the main distribution center of the Nepenthaceae could be descendants of more recently dispersed lineages that had been transmitted to an old species stock.

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

The suitability of gastropods as biomarkers: Induction of ethoxy-, pentoxy- and benzoxyresorufin-O-dealkylase in limnetic gastropods via aroclor 1254.

The measurement and induction of mixed function oxyigenases (MFOs) of limnetic gastropods were investigated to estimate their suitability as biomarkers. A determination of MFO activities was performed through the measurement of ethoxyresorufin-O-dealkylase (EROD), pentoxy-(PROD) and benzoxyresorufin-O-dealkylase-activity (BROD). Optimal measuring conditions of these activities were investigated in preparations of the digestive gland of 4 species of limnetic gastropods. Results indicate that MFO-activity inhibiting substances are accumulating in the microsomal pellet, the fraction commonly used for the measurement of MFO-activities. Therefore, the fraction used for induction studies was the postmitochondrial supernatant (PMS). EROD, PROD, and BROD activity of Planorbis planorbis and Planorbis carinatus were measured after a treatment with Aroclor 1254 for 1-17 days. Maximal induction of EROD and PROD were 6 and 10 times the value in the control group of P. carinatus, respectively induction in P. planorbis was lower. BROD-activity could not been measured in P. planorbis. In P. carinatus, BROD-activity increased to a maximum of 2 times after treatment with Aroclor 1254. The level of induction of EROD-activity is comparable to results described in analog studies with fish.