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.

Morphological differences between two ecologically similar sympatric fishes.

Morphological differentiation and microhabitat segregation of two ecologically similar populations of pelagic planktivorous fishes, Coregonus albula and the smaller Coregonus fontanae, were studied in Lake Stechlin (northern Germany). Both populations performed diel vertical migrations, although C. fontanae was always situated in deeper pelagic water than C. albula both during day and night. Landmark-based geometric morphometrics revealed that sympatric C. albula and C. fontanae differ in external morphology, with main differences found in head length and eye position, as well as in length and width of the caudal peduncle. Moreover, while C. albula has a similar morphology over all sizes, the shape of C. fontanae changes with size. Accordingly, the morphology of the two is most different at smaller size. Although the morphological differences may reflect adaptations to the slightly differing microhabitats of the two populations, there is no conclusive evidence that this correspondence between ecology and morphology is the main mechanism behind the coexistence of the closely related coregonids in Lake Stechlin.

Matrilinear phylogeography and demographical patterns of Rutilus rutilus: implications for taxonomy and conservation.

A phylogeographic analysis of mitochondrial DNA sequence variation was carried out to infer the geographical distribution of the genealogical lineages and the historical demography of roach Rutilus rutilus (L.). A total of 265 individuals from 52 sites covering most of the Eurasian distribution range were sequenced for a 475 bp fragment of the mitochondrial cytochrome b gene. The monophyletic roach contained two deep clades that dated back to the Pliocene. The Ponto-Caspian clade comprised populations from Greece to Siberia with a likely palaeorefugium at the west coast of the Caspian Sea. This clade largely corresponds to individuals with morphological features described as Rutilus heckelii. The west European clade included individuals from central and western Europe with the Danube and Dniester basins as possible palaeorefugia. This clade largely corresponds to individuals with morphological features described as R. rutilus. A suture-zone of the two main lineages was observed along the coastal region of the Black Sea. The neutrality tests and the mismatch distributions indicated a demographic expansion during the Middle-Pleistocene for both clades.

When invaders meet locally adapted types: rapid moulding of hybrid zones between sculpins (Cottus, Pisces) in the Rhine system.

Hybrid zones are commonly studied to dissect the processes that drive divergence among lineages, which have incomplete barriers of reproduction. Most hybrid zones have existed for an extended time making inferences on the initial mode of formation difficult. It is a priori unclear how fast a nascent hybrid zone would form as a response to endogenous and exogenous factors. We have studied several hybrid zones between two lineages of sculpins (Cottus spp.), which emerged due to a recent range expansion of one of the lineages along the river Rhine in the early 1990s. Applying a dense sampling across two contact areas and using a highly informative set of 45 microsatellite markers we found pronounced genetic structure. Steep genetic clines suggest that strong selective forces have shaped the respective hybrid zones from the beginning. We find that the zones are coupled to ecological transitions from small streams to larger rivers. The width of these zones is much smaller than estimates of annual individual dispersal distances, as estimated outside of the hybrid zones. The pattern is apparently not strongly affected by pre- or postzygotic reproductive isolation because numerous backcross hybrids occur within the zones. This suggests that strong natural selection acts against immigrant genotypes. The study exemplifies how local adaptation can play a key role in preventing admixture in dependence of the ecological context.

Phylogeography of the bullhead Cottus gobio (Pisces: Teleostei: Cottidae) suggests a pre-pleistocene origin of the major central European populations.

The bullhead Cottus gobio is a small, bottom-dwelling fish consisting of populations that have not been subject to transplantations or artificial stocking. It is therefore an ideal model species for studying the colonization history of central European freshwater systems, in particular with respect to the possible influences of the Pleistocene glaciation cycles. We sampled Cottus populations across most of its distribution range, with a special emphasis on southern Germany where the major European drainage systems are in closest contact. Mitochondrial D-loop sequencing of more than 400 specimens and phylogenetic network analysis allowed us to draw a detailed picture of the colonization of Europe by C. gobio. Moreover, the molecular distances between the haplotypes enabled us to infer an approximate time frame for the origin of the various populations. The founder population of C. gobio stems apparently from the Paratethys and invaded Europe in the Pliocene. From there, the first colonization into central Europe occurred via the ancient lower Danube, with a separate colonization of the eastern European territories. During the late Pliocene, one of the central European populations must have reached the North Sea in a second step after which it then started to colonize the Atlantic drainages via coastal lines. Accordingly, we found very distinct populations in the upper and lower Rhine, which can be explained by the fact that the lower Rhine was disconnected from the upper Rhine until approximately 1 million years ago (Ma). More closely related, but still distinct, populations were found in the Elbe, the Main and the upper Danube, all presumably of Pleistocene origin. Intriguingly, they have largely maintained their population identity, despite the strong disturbance caused by the glaciation cycles in these areas. On the other hand, a mixing of populations during postglacial recolonization could be detected in the lower Rhine and its tributaries. However, the general pattern that emerges from our analysis suggests that the glaciation cycles did not have a major impact on the general population structure of C. gobio in central Europe.

Prophylactic hemodialysis in the treatment of acute renal failure. Annals of Internal Medicine, 53:992-1016, 1960.

Prophylactic hemodialysis has been employed in the treatment of 15 patients with acute renal failure due to acute tubular necrosis (12), bilateral renal cortical necrosis (two), and poststreptococcal glomerulonephritis (one). Dialyses, usually lasting six hours each, were begun before clinical evidence of uremia developed in each patient and/or before the nonprotein nitrogen reached 200 mg.%, and were repeated daily or often enough to maintain the nonprotein nitrogen below 150 mg.%. The hypothesis underlying this technic postulates (1) that wasting, sepsis and impaired wound healing in these patients may reflect tissue injury by the same dialyzable toxic agents which produce the uremic symptoms that are readily reversible by dialysis, and (2) that repeated dialyses should therefore prevent both clinical uremia and the later, often lethal sequelae. The results contrast dramatically with our own past experience in treating patients with acute renal failure with a carefully executed medical regimen together with hemodialysis on conventional indications. Except in one instance of crush injury with progressive intracerebral damage, and one brief occasion in another individual, these patients experienced a stable, convalescent clinical course, remained free of uremic symptoms or chemical imbalances, ate at least three meals daily which were unrestricted in amount and composition, and were ambulatory between dialyses unless confined to bed by associated disease. Wounds healed well. Infection either did not occur, or subsided after appropriate therapy. Fluid restriction was liberalized by means of ultrafiltration with dialysis. Regional heparinization of only the extracorporeal circuit eliminated actual or impending bleeding as a contraindication to dialysis. Chronic vessel cannulation made the frequent dialyses possible, but may have provided the route for repeated, transient bacterial contamination of the blood stream in the first hour of many dialyses. Marked anemia, despite reticulocytosis, moderate to mild weight loss and some mental deficit persisted in spite of the general clinical improvement and well-being. Three patients with tubular necrosis died after seven, 11 and 26 days of oliguria; both patients with bilateral renal cortical necrosis also succumbed, on the seventy-third and ninety-second days of renal failure, and after 29 and 40 dialyses, respectively. At autopsy, evidence of sepsis was conspicuously absent. The remaining 10 patients survived. Thus some, but not all, clinical manifestations of acute renal failure appear to be favorably influenced by prophylactic dialysis treatment. Our initial experience in this group of 15 patients does not of course prove that freedom from complications and a significantly better outlook for survival can be assured to patients with acute renal failure by these methods. However, it seems to offer a reasonable hope of this possibility which we cannot attach to management by medical measures alone, or by dialysis on conventional indications. If this hope is realized in greatly extended, subsequent series, then it seems inevitable that some form of prophylactic dialysis, or some equally effective alternative, should be adopted in treating the majority of patients with acute renal failure.