Fish diversification at the pace of geomorphological changes: evolutionary history of western Iberian Leuciscinae (Teleostei: Leuciscidae) inferred from multilocus sequence data.

The evolutionary history of western Iberian Leuciscinae, obligatory freshwater fish, is directly linked to the evolution of the hydrographic network of the Iberian Peninsula after its isolation from the rest of Europe, which involved dramatic rearrangements such as the transition from endorheic lakes to open basins draining to the Atlantic. Previous phylogenetic research on western Iberian leuciscines, using mainly mitochondrial DNA and more recently one or two nuclear genes, has found contradictory results and there remain many unresolved issues regarding species relationships, taxonomy, and evolutionary history. Moreover, there is a lack of integration between phylogenetic and divergence time estimates and information on the timing of geomorphological changes and paleobasin rearrangements in the Iberian Peninsula. This study presents the first comprehensive fossil-calibrated multilocus coalescent species tree of western Iberian Leuciscinae (including 14 species of Achondrostoma, Iberochondrostoma, Pseudochondrostoma and Squalius endemic to the Iberian Peninsula, seven of which endemic to Portugal) based on seven nuclear genes, and from which we infer their biogeographic history by comparing divergence time estimates to known dated geological events. The phylogenetic pattern suggests slow-paced evolution of leuciscines during the Early-Middle Miocene endorheic phase of the main Iberian river basins, with the shift to exorheism in the late Neogene-Quaternary allowing westward dispersals that resulted in many cladogenetic events and a high rate of endemism in western Iberia. The results of this study also: (i) confirm the paraphyly of S. pyrenaicus with respect to S. carolitertii, and thus the possible presence of a new taxon in the Portuguese Tagus currently assigned to S. pyrenaicus; (ii) support the taxonomic separation of the Guadiana and Sado populations of S. pyrenaicus; (iii) show the need for further population sampling and taxonomic research to clarify the phylogenetic status of A. arcasii from the Minho basin and of the I. lusitanicum populations in the Sado and Tagus basins; and (iv) indicate that A. occidentale, I. olisiponensis and P. duriensis are the most ancient lineages within their respective genera.

Establishment of a coastal fish in the Azores: recent colonisation or sudden expansion of an ancient relict population?

The processes and timescales associated with ocean-wide changes in the distribution of marine species have intrigued biologists since Darwin's earliest insights into biogeography. The Azores, a mid-Atlantic volcanic archipelago located >1000 km off the European continental shelf, offers ideal opportunities to investigate phylogeographic colonisation scenarios. The benthopelagic sparid fish known as the common two-banded seabream (Diplodus vulgaris) is now relatively common along the coastline of the Azores archipelago, but was virtually absent before the 1990 s. We employed a multiple genetic marker approach to test whether the successful establishment of the Azorean population derives from a recent colonisation from western continental/island populations or from the demographic explosion of an ancient relict population. Results from nuclear and mtDNA sequences show that all Atlantic and Mediterranean populations belong to the same phylogroup, though microsatellite data indicate significant genetic divergence between the Azorean sample and all other locations, as well as among Macaronesian, western Iberian and Mediterranean regions. The results from Approximate Bayesian Computation indicate that D. vulgaris has likely inhabited the Azores for ∼ 40 (95% confidence interval (CI): 5.5-83.6) to 52 (95% CI: 6.32-89.0) generations, corresponding to roughly 80-150 years, suggesting near-contemporary colonisation, followed by a more recent demographic expansion that could have been facilitated by changing climate conditions. Moreover, the lack of previous records of this species over the past century, together with the absence of lineage separation and the presence of relatively few private alleles, do not exclude the possibility of an even more recent colonisation event.

Different stocks of brook lamprey in Spain and their origin from Lampetra fluviatilis at two distinct times and places.

Using the mitochondrial non-coding region I, it was shown that the two Spanish Lampetra planeri populations (Cares-Deva and Olabidea-Ugarana) correspond to different genetic units. The Cares-Deva population is probably a recent offshoot of Lampetra fluviatilis, being the Olabidea-Ugarana population less diverse and of older origin.

Gaidropsarus (Gadidae, Teleostei) of the North Atlantic Ocean: a brief phylogenetic review.

The phylogenetic relationships among the North Atlantic Gaidropsarus and between the three Gaidropsarinae genera Gaidropsarus, Ciliata and Enchelyopus are reviewed with the hitherto most comprehensive taxonomic sampling of this group. Phylogenetic results (maximum parsimony, maximum likelihood and Bayesian inference) based on nuclear (rhodopsin) and concatenated mitochondrial (12s, 16s and cytb) markers clearly support this subfamily. For the north-eastern Atlantic species of Gaidropsarus, two previously unreported clades were strongly supported, clarifying the relationships within the genus, and revealing fewer distinct taxa in the north Atlantic Gaidropsarus than previously stipulated. The data challenge the specific status of Gaidropsarus mediterraneus and Gaidropsarus guttatus and raise doubts concerning the distinctiveness of other species. A taxonomic revision of the genus is suggested.

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes.

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

Phylogeographical analysis reveals multiple conservation units in brook lampreys Lampetra planeri of Portuguese streams.

The populations of brook lamprey Lampetra planeri of Portuguese Rivers were analysed phylogeographically using a fragment of 644 bp of the mitochondrial control region of 158 individuals from six populations. Samples representing L. planeri and migratory lampreys Lampetra fluviatilis of rivers draining to the North Sea and the Baltic Sea were also included to assess the relationships of Portuguese samples. The data support a clear differentiation of all the populations studied. Several populations, which are isolated among themselves and also from the migratory lampreys, proved to be entirely composed of private haplotypes, a finding that supports some time of independent evolutionary history for these populations. This, combined with the geographic confinement to small water bodies, justifies the recognition of at least four conservation units in the Portuguese rivers Sado, São Pedro, Nabão and Inha.

Phylogenetic analysis of peri-Mediterranean blennies of the genus Salaria: molecular insights on the colonization of freshwaters.

In this paper, the phylogenetic relationships of the marine blenny Salaria pavo and the freshwater S. fluviatilis and S. economidisi were analyzed using four molecular markers: the mitochondrial 12S rRNA, 16S rRNA, and the control region and the nuclear first intron of the S7 ribosomal protein. The monophyly of Salaria is supported, as well as that of S. pavo and that of all the freshwater members of Salaria. Thus, the present results support a single origin for all freshwater Mediterranean blenniids. Our results reject the placement of the species of Salaria in the genus Lipophrys as proposed in previous studies. Using a molecular clock calibrated with trans-Isthmian geminate blenniid species, the split between the ancestor of the freshwater lineage and the ancestor of S. pavo is tentatively placed in the Middle Miocene (well before the Messinian). The marine S. pavo displays a very low level of intraspecific sequence divergence consistent with a Pleistocene bottleneck. S. fluviatilis is a paraphyletic entity with S. economidisi nested within it. A Moroccan population of S. fluviatilis is more divergent than S. economidisi, both in nuclear and mitochondrial genes. Fish from Israel together with some Turkish samples represent the second oldest split. It is argued that these populations may represent cryptic species. Thus, further studies on the taxonomy of these freshwater blennies are urgently needed.

Insights on speciation patterns in the genus Iberochondrostoma (Cyprinidae): evidence from mitochondrial and nuclear data.

In this paper, the patterns of cladogenesis in the cyprinid fish genus Iberochondrostoma were analysed using a mitochondrial (cytochrome b) and a nuclear (beta-actin) gene fragment. The two genes yielded discordant results. While the cytochrome b gene yielded a fully dichotomous tree, where all species of the genus are monophyletic, the much slower beta-actin gene yielded star-like relationships. However, when information from both genes was considered together, the data suggested the persistence of a very large central unit from which at least two peripheral clades arose at different times. This pattern which is akin to peripatric speciation was shown to be compatible with the paleogeographical information available. It is suggested that combining the techniques of phylogeny and phylogeography and the use of multiple markers varying in their rate of evolution may enrich our understanding of speciation and evolution of clades beyond species level.

Nitrite reductase silencing as a tool for selecting spontaneous haploid plants.

Using tobacco as a model species, we have developed a simple procedure for the selection of spontaneous haploid plants under horticultural conditions, which does not require the use of any selective agent. One transgenic tobacco plant, homozygous for an antisense transgene able to silence the expression of nitrite reductase host genes, and encoding the second enzyme of the nitrate assimilation pathway, was used to pollinate two different cultivars of wild type tobacco plants. Seeds were sown at high density in the greenhouse and watered with a nutrient solution containing nitrate. Green plants able to develop normally emerged at a frequency of 5.10(-4) in a mass of chlorotic retarded plants. Phenotypic and genetic analysis, chloroplast counting in stomatal guard cells and molecular hybridizations revealed that 22% of these plants were gynogenetic haploid plants exhibiting the maternal phenotype whereas the remaining 78% were true diploid plants that have lost the antisense transgene. These results demonstrate that a transgene able to silence the expression of a housekeeping gene can be utilized as a counter-selectionable marker for the rapid and easy selection of spontaneous haploid plants in transformable species.