Maria Judite Alves

Mitochondrial DNA variation in the highly endangered cyprinid fish Anaecypris hispanica: importance for conservation

Anaecypris hispanica is a cyprinid fish which is endemic to the Guadiana River basin in the Iberian Peninsula, and whose abundance and geographical range have contracted considerably during the last 20 years. We investigated mitochondrial DNA cytochrome b and control region variation among specimens representative of nine tributaries, using direct sequencing and diagnostic restriction fragment length polymorphism. The samples from the Caia, Degebe, Ardila, and Odeleite rivers exhibited haplotypes that differed by a large number of site differences, which may be indicative of population bottlenecks that have caused stochastic extinction of haplotypes. In contrast, the populations from the Xévora, Chança, Carreiras, Vascão and Foupana rivers exhibited low levels of nucleotide diversity, which together with high haplotype diversity may also be indicative of genetic bottleneck events, with subsequent population expansion. Phylogenetic analyses, a minimum spanning network, and an analysis of molecular variance revealed geographical structuring, suggesting limited or no gene flow between populations. The populations from extreme southern rivers (Foupana and Odeleite) are monophyletic entities, suggesting that they have been isolated, probably as a consequence of brackish water upstream of their confluence with the Guadiana. The results suggest that the Foupana and the Odeleite populations, and the remaining northern populations altogether should be managed as three distinct Evolutionary Significant Units (ESUs). Within the northern ESU, four Management Units (MUs) should be considered.

A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal.

Background The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal. Methodology/Principal Findings Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A–E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E). Conclusion/Significance We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.

Diversification within glacial refugia: tempo and mode of evolution of the polytypic fish Barbus sclateri

A diversity of evolutionary processes can be responsible for generating and maintaining biodiversity. Molecular markers were used to investigate the influence of Plio‐Pleistocene climatic oscillations on the evolutionary history of taxa restricted to the freshwaters of a classical glacial refugium. Population genetic, phylogenetic and phylogeographical methods allowed the inference of temporal dynamics of cladogenesis and processes shaping present‐day genetic constitution of Barbus sclateri, a polytypic taxon found in several independent river drainages in southern Iberian Peninsula. Results from different analyses consistently indicate several range expansions, high levels of allopatric fragmentation, and admixture following secondary contacts throughout its evolutionary history. Using a Bayesian demographical coalescent model on mitochondrial DNA sequences calibrated with fossil evidence, all cladogenetic events within B. sclateri are inferred to have occurred during the Pleistocene and were probably driven by environmental factors. Our results suggest that glaciation cycles did not inhibit cladogenesis and probably interacted with regional geomorphology to promote diversification. We conclude that this polytypic taxon is a species complex that recently diversified in allopatry, and that Pleistocene glaciation–deglaciation cycles probably contributed to the generation of biological diversity in a classical glacial refugium with high endemicity.

Paralog-Specific Primers for the Amplification of Nuclear Loci in Tetraploid Barbels (Barbus: Cypriniformes)

Thirty paralog-specific primers were developed, following an intron-primed exon-crossing strategy, for S7 and growth hormone genes in Barbus (subgenera Barbus and Luciobarbus). We found that paralog-specific amplification requires the use of only one paralog-specific primer, allowing their simultaneous use with universal exon-primed intron-crossing primers of broad taxonomic applicability. This hybrid annealing strategy guarantees both specificity and generality of amplification reactions and represents a step forward in the amplification of duplicated nuclear loci in polyploid organisms and members of multigene families. Assays of several representative taxa identified high levels of segregating single nucleotide polymorphisms (SNPs) and nucleotide diversity within each of these subgenera. Additionally, several insertions-deletions (indels) that are diagnostic across species are found in intronic regions. Therefore, these primers provide a reliable source of valuable nuclear SNP and indel data for population and species level studies of barbels, such as applied conservation and basic evolutionary studies.

Allocation of nutrients during the reproductive cycle of Ophidiaster ophidianus (Echinodermata: Asteroidea)

The reproductive cycle of Ophidiaster ophidianus (strictly protected status) from São Miguel Island, in the Azorean Archipelago was studied. The reproductive strategy; the energy allocation of each sex during the reproductive cycle and the nutritional condition of the population were analyzed. Gonadal index (GI) showed a clear seasonal pattern with spawning between August and October but histological examination revealed that gamete release can occur throughout the entire year. The pyloric caeca index (PCI) showed little annual variation but with an inverse relationship with the GI. Allocation of energy to the gonads and to the pyloric caeca reflected the seasonal reproductive strategy of this species. Individuals were able to simultaneously develop gonads, pyloric caeca, and quickly regenerate lost arms. There was a major expenditure of energy by females compared to males but, sexual size dimorphism was not observed. The reproductive pattern observed in O. ophidianus combining rich food availability and seawater temperatures characteristic of a temperate zone may be the key to the success of this species in the Azorean oceanic Island.

European Lampreys: New Insights on Postglacial Colonization, Gene Flow and Speciation

Ice ages are known to be the most dominant palaeoclimatic feature occurring on Earth, producing severe climatic oscillations and consequently shaping the distribution and the population structure of several species. Lampreys constitute excellent models to study the colonization of freshwater systems, as they commonly appear in pairs of closely related species of anadromous versus freshwater resident adults, thus having the ability to colonize new habitats, through the anadromous species, and establish freshwater resident derivates. We used 10 microsatellite loci to investigate the spatial structure, patterns of gene flow and migration routes of Lampetra populations in Europe. We sampled 11 populations including the migratory L. fluviatilis and four resident species, L. planeri, L. alavariensis, L. auremensis and L. lusitanica, the last three endemic to the Iberian Peninsula. In this southern glacial refugium almost all sampled populations represent a distinct genetic cluster, showing high levels of allopatric differentiation, reflecting long periods of isolation. As result of their more recent common ancestor, populations from northern Europe are less divergent among them, they are represented by fewer genetic clusters, and there is evidence of strong recent gene flow among populations. These previously glaciated areas from northern Europe may have been colonized from lampreys expanding out of the Iberian refugia. The pair L. fluviatilis/L. planeri is apparently at different stages of speciation in different locations, showing evidences of high reproductive isolation in the southern refugium, and low differentiation in the north.

Diversity of shallow-water asteroids (Echinodermata) in the Azorean Archipelago

A comprehensive review of the literature on shallow-water asteroids (Echinodermata) recorded in Azores was carried out to establish a definitive list for the Archipelago. A total of 49 echinoderm species was compiled, comprising members of all extant classes; Crinoidea (1), Asteroidea (12), Ophiuroidea (11), Echinoidea (10) and Holothuroidea (7). References of asteroids occurence in the Azores, including historical records and distribution of asteroids from the Azores are given. Seven asteroid species recorded found in the Azores are found also in the Mediterranean and adjoining Atlantic coastlines, three are recorded from the Mediterranean and both sides of the Atlantic, one is limited to the Eastern Atlantic and one is circumtropical. Differences between the littoral hydrological conditions found on the North American coast and those in Azores makes the colonization from American coasts particularly difficult and probably are related to chance events like the episodic anomalies found in the general pattern of oceanic circulation. Asteroids are important in determining habitat structure for other species and can represent a substantial portion of the ecosystem biomass.

Genetics of lineage diversification and the evolution of host usage in the economically important wheat curl mite, Aceria tosichella Keifer, 1969

BackgroundUnderstanding the mechanisms that underlie the diversification of herbivores through interactions with their hosts is important for their diversity assessment and identification of expansion events, particularly in a human-altered world where evolutionary processes can be exacerbated. We studied patterns of host usage and genetic structure in the wheat curl mite complex (WCM), Aceria tosichella, a major pest of the world’s grain industry, to identify the factors behind its extensive diversification.ResultsWe expanded on previous phylogenetic research, demonstrating deep lineage diversification within the taxon, a complex of distinctive host specialist and generalist lineages more diverse than previously assumed. Time-calibrated phylogenetic reconstruction inferred from mitochondrial DNA sequence data suggests that lineage diversification pre-dates the influence of agricultural practices, and lineages started to radiate in the mid Miocene when major radiations of C4 grasses is known to have occurred. Furthermore, we demonstrated that host specificity is not phylogenetically constrained, while host generalization appears to be a more derived trait coinciding with the expansion of the world’s grasslands. Demographic history of specialist lineages have been more stable when compared to generalists, and their expansion pre-dated all generalist lineages. The lack of host-associated genetic structure of generalists indicates gene flow between mite populations from different hosts.ConclusionsOur analyses demonstrated that WCM is an unexpectedly diverse complex of genetic lineages and its differentiation is likely associated with the time of diversification and expansion of its hosts. Signatures of demographic histories and expansion of generalists are consistent with the observed proliferation of the globally most common lineages. The apparent lack of constrains on host use, coupled with a high colonization potential, hinders mite management, which may be further compromised by host range expansion. This study provides a significant contribution to the growing literature on host-association and diversification in herbivorous invertebrates.