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We analyzed two morpho-species, Astyanax aeneus and Astyanax caballeroi, coexisting in Lake Catemaco, with ecological differences previously associated with different trophic niches. While A. aeneus is a widely distributed species, A. caballeroi is endemic to Lake Catemaco. Due to the contrasting morphology between these two sympatric species, they were originally assigned to different genera, but little is known about their genetic differentiation. We hypothesized that these two species, which present differentiation in morphology and in trophic niches, co-occur in concomitance with assortative mating and form distinct genetic clusters. We tested this prediction by typing a set of 12 microsatellites in 348 individuals of the two morpho-species, comparing their genetic structure patterns with that of an allopatric population of A. aeneus (i.e., the Maquinas population). Genetic structure analysis, assignment analyses, and estimation of gene flow between the sympatric morpho-species were carried out. Contrary to expectations, there is a lack of genetic differentiation between the A. aeneus and A. caballeroi morpho-species. However, there was a genetic differentiation between Lake Catemaco and Río Maquinas populations of A. aeneus. In addition, we found an asymmetric gene flow pattern, with a larger migration rate from A. aeneus to A. caballeroi than in the other direction. Finally, the allopatric population of A. aeneus showed lower levels of genetic diversity than those reported for both morpho-species in the lacustrine system. Consequently, our results support the notion that these morpho-species are either in an early phase of speciation or represent a single polymorphic species. This model provides relevant information to understanding the presence of polymorphisms under gene flow and their potential impact on the speciation process. [ABSTRACT FROM AUTHOR]

Background: The Spanish toothcarp (Aphanius iberus Valenciennes, 1846) is a small fish endemic to the eastern coastline of the Iberian Peninsula and is currently listed as "Endangered" (category IUCN: EN). It mainly inhabits brackish waters which can exhibit large fluctuations in temperature and salinity throughout the year. The genetics of A. iberus are not well-known since most studies have only evaluated the genetic structure of the species under a conservation framework in order to identify its potential conservation units. Different phylogenetic relationships of Aphanius have been published based on some particular genes. In the present study, the entire mitochondrial genome of A. iberus was obtained for the first time in the context of an A. iberus reference genome and a hypothesis regarding its phylogenetic position was considered. Methods and results: The mitogenome (a circular doble-stranded DNA sequence of 16,708 bp) was reconstructed and aligned against 83 Cyprinodontiformes and two outgroup taxa to identify the phylogenetic position of A. iberus. PartitionFinder was first used to test for the best evolutionary model and the phylogenetic analyses were performed using two methods: Maximun-Likelihood Approximation (IQ-Tree) and Bayesian inference (MrBayes). Our results show that A. iberus forms a sister group with Orestias ascotanensis, a cyprinodontiform species native to South America. Conclusions: The results were congruent with the traditional morphometric reconstructed trees and with a geological vicariant hypothesis involving Cyprinodontiformes where Aphaniidae is shown as a monophyletic family separated from the family Cyprinodontidae. The information gathered from this study is not only valuable for improving our understanding of the evolutionary history of A. iberus, but for future genomic studies involving the species. [ABSTRACT FROM AUTHOR]

Conservation of biodiversity entails not just the preservation of specific taxa, but also genetic diversity. Despite the fact that molecular data appear to be crucial for freshwater fish conservation management, the information about genetic diversity of the genus Luciobarbus Heckel, 1843 populations in Morocco is still very rare. Accordingly, this study's main purpose was to present genetic and biogeographic information of nine Luciobarbus species out of 15 Luciobarbus species native to Morocco. Sequencing of the complete cytochrome b gene (1140 bp) confirmed the presence of two evolutionary lineages of Luciobarbus in Morocco and displayed low to high level of genetic diversity within Luciobarbus populations. Populations with the largest size had the highest level of genetic diversity, according to our findings. Moreover, the "star-like" shape of the haplotype networks along with low and insignificant value of the Harpending raggedness index supported the hypothesis of population expansion in Luciobarbus populations. Nevertheless, additional investigations should ideally be undertaken separately for each species in order to untangle genetic diversity trends. [ABSTRACT FROM AUTHOR]

Freshwater ecosystems are under multiple threats in modern times such as water extraction for human consumption, industries and agricultural activities, water contamination and habitat destruction for example. At the same time the biodiversity of these ecosystems are often poorly studied, especially in arid countries such as Iran. In this work, we study one of the ecologically important members of Iranian freshwater fauna, freshwater crab species of the genus Potamon. Here, we barcoded the different populations occurring in the country and delimited the species to allow for a better understanding of their distribution and taxonomy. In this study, we evaluated the taxonomical statues of Potamon species in Iran using genetic data. In addition, we created the first barcoding reference for Iranian freshwater crabs, which is an important resource for future environmental and conservation studies. [ABSTRACT FROM AUTHOR]

Achondrostoma salmantinum is a small, endangered freshwater fish endemic to Spain. Although the species is a member of the widely distributed family Leuciscidae, its geographic range is restricted to a few rivers in the southwestern Duero Basin. Its populations are in decline, with some now extirpated from rivers within its historical distribution. A captive breeding program has already been implemented for the species; however, there are no population genetic studies on A. salmantinum, despite the importance of information on genetic variability and variation as fundamentals tools for management and conservation efforts. Here, we assessed the genetic diversity of A. salmantinum and defined its Operational Conservation Units (OCUs). We sampled throughout the entire known distribution area of A. salmantinum, and analysed both nuclear and mitochondrial genes and 4123 single nucleotide polymorphisms (SNPs). Contrary to expectations, due to its small distribution area within the same basin, A. salmantinum showed signs of marked genetic erosion and a strong population structure that is not associated with the current hydrogeographic configuration of the region, but rather with historical geomorphological processes. On the basis of our genetic analyses, we identified four OCUs for the species: (I) Águeda Sub-basin; (II) Uces Sub-basin, Huebra River and the right-bank tributaries of Huebra Sub-basin; (III) left-bank tributaries of Huebra Sub-basin and (IV) Camaces River, a small left-bank tributary of the Huebra River. Overall, our findings provide insight on the population genetic structure of A. salmantinum and on some intrinsic and extrinsic factors that threaten the viability of the species. [ABSTRACT FROM AUTHOR]

The use of 3D scaffolds based on mesoporous bioactive glasses (MBG) enhanced with therapeutic ions, biomolecules and cells is emerging as a strategy to improve bone healing. In this paper, the osteogenic capability of ZnO-enriched MBG scaffolds loaded or not with osteostatin (OST) and human mesenchymal stem cells (MSC) was evaluated after implantation in New Zealand rabbits. Cylindrical meso-macroporous scaffolds with composition (mol %) 82.2SiO2–10.3CaO–3.3P2O5–4.2ZnO (4ZN) were obtained by rapid prototyping and then, coated with gelatin for easy handling and potentiating the release of inorganic ions and OST. Bone defects (7.5 mm diameter, 12 mm depth) were drilled in the distal femoral epiphysis and filled with 4ZN, 4ZN + MSC, 4ZN + OST or 4ZN + MSC + OST materials to evaluate and compare their osteogenic features. Rabbits were sacrificed at 3 months extracting the distal third of bone specimens for necropsy, histological, and microtomography (µCT) evaluations. Systems investigated exhibited bone regeneration capability. Thus, trabecular bone volume density (BV/TV) values obtained from µCT showed that the good bone healing capability of 4ZN was significantly improved by the scaffolds coated with OST and MSC. Our findings in vivo suggest the interest of these MBG complete systems to improve bone repair in the clinical practice. [ABSTRACT FROM AUTHOR]

The genus Herichthys is widely considered to be the monophyletic representative of Cichlidae in northeastern Mexico and southern Texas. It is also the northernmost distributed genus of Neotropical Cichlids. Its distribution stretches over an area that is characterized by an intricate geologic and climatic history that affected its temporal and spatial diversification north of the Trans-Mexican Volcanic-Belt. We access the evolutionary history of the genus Herichthys based on a phylogenetic reconstruction using a mitochondrial fragment of gene Cox1. We evaluate its morphological variation, its correspondence with molecular differentiation and suggest a biogeographical scenario based on a molecular clock and demographic history. Furthermore, we describe Nosferatu new genus, composed of Nosferatu pame (assigned as type species), N. molango, N. pratinus, N. bartoni, N. labridens, N. pantostictus, and N. steindachneri. Genus is characterized by a transition to prolongation in the size of the symphysial pair of teeth relative to that of the other teeth in the outer row of the upper jaw; breeding pigmentation that consists of darkening of ventral area extending over nostrils, opercular series, or pectoral fins; depressed dorsal fin rarely expands beyond anterior third of caudal fin; and an elongated, elastic, smooth caecum adhered to a saccular stomach. We also describe Herichthys tepehua n. sp. found in the Pantepec, Cazones, Tenixtepec, Tecolutla, and Solteros rivers, in Veracruz, Mexico. Moreover, we provide re-descriptions for some of the species in Herichthys and propose a biogeographic hypothesis for both genera, based on available information on the geological and climate history of the area of study, associated to dating retrieved in our phylogenetic analysis. [ABSTRACT FROM AUTHOR]

Biodiversity is currently declining in the Mediterranean, particularly in freshwater ecosystems. The Iberian Peninsula is not an exception with many fish species threatened or endangered. This is the case of Parachondrostoma arrigonis, which has experienced a marked reduction of more than 80 % in both number and area of occupancy in the last 20 years. Species historical records showed a widespread distribution in the Júcar Basin, but according to recent records, P. arrigonis was completely absent in the main river course, being restricted to only some tributaries. Current fish distribution was grouped into only seven fish population nuclei geographically isolated among them. Only two population nuclei showed with well-developed fish populations. Factors affecting P. arrigonis distribution are associated to human activities, such as river channelization, flow regulation, pollution and habitat degradation, being concentrated in the middle and lower river reaches where the Júcar nase is in marked regression. Another key factor was the presence of introduced species, reporting a strong spatial segregation with the translocated Pseudochondrostoma polylepis, an ecologically similar species, and with the exotic piscivorous Micropterus salmoides and Esox lucius, which are currently occupying most of the P. arrigonis historical range. Because its dramatic situation, conservation measures must be urgently taken, for instance, by establishing conservation areas, an exotic fish removal programme, and by developing a flow regulation program to minimize dam effects. However it is necessary to increase our knowledge regarding the habitat requirements of the Júcar nase and its interactions with invasive species. If measures are not implemented its extinction seems inevitable. [ABSTRACT FROM AUTHOR]

We studied the genetic structure of the Mediterranean killifish Aphanius fasciatus. Analysis of the sequence variation in a 372-bp portion of the mitochondrial control region in 623 fish from 27 sampling sites along the species’ distributional range (Tyrrhenian coast, Sardinia, Sicily, Adriatic coast, Malta, Tunisia, and Greece) yielded 120 distinct haplotypes. Most of the haplotypes are unique, and only 15 % are shared among different populations. The high FST value (=0.80) suggests a strong population genetic structuring. The phylogenetic analysis based on Bayesian inference, maximum likelihood and maximum parsimony, and the median-joining network show a sharp separation of the Southeastern Sicilian populations (belonging to the Hyblean region) and of the fluvial Tunisian population of Rio Melah from the others. The Adriatic, the Eastern Sicilian, and the Greek populations are well differentiated, while the group of populations from the Central-Western Mediterranean does not show a clear pattern of differentiation. Our findings indicate that the current genetic structuring of A. fasciatus reflects historical geographical patterns occurring within the Mediterranean basin from the Late Miocene to the Pleistocene. The presence of divergent evolutionary entities in the Hyblean region and the Tunisian Rio Melah supports their inclusion as target areas for the conservation of A. fasciatus. [ABSTRACT FROM AUTHOR]

The genetic relationships of a population of brook lamprey Lampetra planeri from Spain were analysed using a fragment of the mitochondrial non-coding region in order to describe its relationships with other European Lampetra populations. DNA sequences were obtained from ten larvae, all corresponding to a single private haplotype. Its closest haplotype differed by five mutations and was found in several samples of Lampetra fluviatlis, both from the North Sea and the Baltic. These results argue for the great conservation value of this brook lamprey population, likely originated from L. fluviatilis ancestors. [ABSTRACT FROM AUTHOR]

Understanding the population structure, population dynamics and processes that give rise to polyploidy and helps to maintain it is central to our knowledge of the evolution of asexual vertebrates. Previous studies revealed high genetic diversity and several reproductive pathways in the southern populations of the Squalius alburnoides hybrid complex. In contrast, lower genetic variability and the associated limited chance of introducing new genetic combinations may threaten the survival of the northern Mondego populations. We analysed the genetic diversity and structure of nine populations of S. alburnoides in the Iberian Peninsula using microsatellite loci to provide further insights on the evolutionary history of this complex. Special attention was given to the less-studied northern populations (Mondego and Douro basins). Marked population structure, a high frequency of private alleles and a high diversity of some biotypes in the Douro basin indicate that some northern populations may not be at high risk of extinction, contrary to what was expected. The genetic diversity found in the northern Douro populations contradicts the general trend of remarkable genetic impoverishment northwards that occurs in other species and regions. The results indicate the possible existence of a glacial refugium in the Rabaçal River, corroborating findings in other species of this region. Historical events seem to have affected the geographical patterns of genetic variability found among and within the northern and southern populations of this complex and contributed to different patterns of genome composition. Therefore, historical events might have a major role in the long-term persistence of some polyploid hybrid taxa. [ABSTRACT FROM AUTHOR]

Myxoma virus and the European rabbit are one of the best-studied examples of coevolution of pathogen virulence and host resistance. Since the introduction of the virus in Spain in 1953, a decrease in its virulence has been observed; however, most strains are still considered highly virulent. To determine whether this attenuation is due to molecular differences, and to characterise the field strains in Spain and the genetic changes that have occurred since the introduction of the virus, we analysed 7,741 bp in 97 virus samples from 12 localities. We found an extremely low genetic variability and an absence of a geographic structure. We defined 35 haplotypes, none of which were identical to the original Lausanne strain. Three genetic groups were determined and were found to occur at different frequencies in different locations. Overall, virus evolution deviated from neutrality and did not conform to a strict molecular clock, probably due to the existence of a strong selective pressure that acts differently across the viral genome. [ABSTRACT FROM AUTHOR]

In this paper, we analysed the phylogeography of the Critically Endangered fish Chondrostoma lusitanicum (Cyprinidae) endemic to Portugal. A fragment of the mitochondrial cyt b gene and one from the nuclear beta-actin gene were sequenced. The results support recognition of 3 ESU's distributed as follows: 1) Sado River, 2) the Tagus River basin and 3 independent nearby coastal rivers (collectively named ‘West’) and 3) Lagoa de Albufeira, a small coastal lagoon between Tagus and Sado basins. [ABSTRACT FROM AUTHOR]

The molecular divergence and phylogenetic relationships of the Iberian populations of Aphanius iberus were established using allozymes and the complete cytochrome b gene sequence. Congruent results were found with both nuclear and mitochondrial molecular markers. The Mediterranean and Atlantic populations are clearly differentiated into two independent lineages. Their high molecular divergence suggests an early isolation, and the absence of gene flow among the populations indicates their independent evolution. The nuclear and mitochondrial data reveal monophyletic clustering of the two geographical lineages, but provide weak support for the population relationships. However, the mitochondrial results differentiated the Villena population as a distinct mitochondrial unit within the Mediterranean group. Geographically broad studies across the distribution range of A. iberus have helped to elucidate the patterns of diversification of this species. The genetic divergence found between the Atlantic and the Mediterranean populations is of the same order as those found among recognized species of cyprinodontids. The identification of two discrete evolutionary lineages has important implications for the conservation of this species, since its recovery requires the recognition and preservation of natural diversity. The Mediterranean and Atlantic lineages should be managed separately to avoid loss of their genetic identity, and the genetic uniqueness of the populations should be preserved by using wild stocks as the source of genetic diversity in captive breeding programmes. [ABSTRACT FROM AUTHOR]

To understand further the fragmentation of the hydrographical basins and the processes of divergence and speciation of freshwater fishes of the Iberian Peninsula, 10 populations of the Iberian endemic cyprinid Chondrostoma lemmingii were studied using 26 loci encoding 19 enzymes and the complete nucleotide sequence of the mitochondrial cytochrome b gene. Nuclear and mitochondrial DNA markers showed complete congruence in assessing the genetic differentiation among the samples analysed. This congruence was supported by a Mantel test in which a significant correlation (r=0.89) between Nei’s genetic distances and sequence divergence (uncorrected p distances) was obtained. Diagnostic loci, Nei’s genetic distances, and FST values, as well as the percentage of sequence divergence indicate that the Duero basin population accumulates the highest level of genetic differentiation. A moderate divergence was also observed among populations of the rest of the basins. Phenetic and phylogenetic relationships support the hypothesis that the differentiation process was not only due to hydrographical basin isolation but also due to an ancient endorrheism event, previous to hydrographical configuration, that could explain the marked differentiation of the Duero basin population. [ABSTRACT FROM AUTHOR]

The phylogenetic relationships of 106 European cyprinid taxa were determined based on the complete nucleotide sequence (1140 bp) of the mitochondrial cytochrome b gene. The molecular phylogeny was used (1) to revise the current systematics of European cyprinids, (2) to establish the phylogenetic utility of traditional morphological characters that are widely used in Cyprinidae systematics, and (3) to discuss alternative hypotheses on the biogeography of the family in Europe. The age of the major lineages within European cyprinids was tentatively estimated with a molecular clock and showed full agreement with the fossil record of the group. Moreover, the results provided unambiguous evidence for a close phylogenetic affinity of some Caucasian and Greek endemic cyprinid taxa (e.g., B. capito and B. brachycephalus and Leuciscus keadicus, Barbus graecus, and B. albanicus, respectively) to Iberian and North African, but not Central European, cyprinids. The existence of such unexpected phylogenetic relationships refutes the classical hypothesis on the biogeography of European cyprinids, which assumes a dispersal of the cyprinid fauna from central Europe to southern Europe and northern Africa during the Miocene (and, hence, predicts a close phylogenetic relationship of all Caucasian, Greek, Iberian, and North African cyprinids to central European taxa). Instead, the existence of a Mediterranean realm independent of the central European route seems plausible based on the molecular evidence. It is likely that the new biogeographical scenario proposed here might apply to other primary freshwater European animals with low dispersal abilities, including fish, amphibians, and invertebrates. [ABSTRACT FROM AUTHOR]

Classification and ordination methods used to examine the internal complexity of the Mediterranean Tagus River catchment based on fish distribution revealed that it is not a homogeneous biogeographical unit. The indigenous fishes analyzed in this study are distributed through the basin forming geographical communities (chorotypes), some of which are associated with environmental factors like river morphology, water quality or geographical location. Nevertheless, 40% of the variation in species occurrence remains unexplained by either environmental or geographical variables, suggesting that historical factors may influence the freshwater fish distribution patterns. Three main biogeographical areas, delimited by significant boundaries, were identified. Two of them are identified as the upper and the middle-lower basins of the Tagus River catchment; the third corresponds to the Alagón River and seems to be linked to historical factors of the catchment. [ABSTRACT FROM AUTHOR]

We report the synthesis and study of new Mo(V) dimeric complexes with oxosulphido bridges and n-pentyl, n-hexyl, n-heptyl, iso-octyl, cyclohexyl and phenyl carboxylates as coligands. The complexes, of general formula MoOS(RCO), are compared to the Mo carboxylates with monoxo and dioxo bridges. Their i.r. and electronic spectra are sensitive to substitution of sulphur atoms in the bridge system. The results are discussed in the light of the structural information available for oxomolybdenum(V) complexes. [ABSTRACT FROM AUTHOR]

Six new organometallic derivatives of Rh(I), belonging to the general structure [Rh(CO)(L)(Cl)], were synthesized and characterized by chemical analysis and IR determinations. The following ligands (L) were employed: 2-aminothiazole, thiazole, 2-amino-6-bromobenzothiazole, 5-chloro-2-methylthiobenzothiazole, 2-bromo-thiazole, and 2-isopropylthiazole. These new complexes were assayed in vitro with KB cells and in vivo with mice bearing established P388 and L1210 leukemias. Assays against S180 and Ehrlich ascitic tumors were also performed. Two complexes displayed antitumor activity against ascitic tumors. [ABSTRACT FROM AUTHOR]

In 1977, Gale and associates reported the synthesis and antitumor activity of a series of Pt(II) complexes containing 1,2-diaminocyclohexane as the ligand. Certain of these complexes showed superior activity and greater water solubility compared to cis-Pt(NH)Cl complexes ('Neoplatin'). In this paper we report the synthesis and antitumor activity of some 40 new water soluble platinum(II) and platinum(IV) complexes. The following classes of the cis-Pt(L)Cl complexes were obtained, where L = 1,2-diaminocyclohexane: (a) cis-Pt(L)(X), where X is a derivative of homophthalic acid or a derivative of 1,3-benzendicarboxylic acid, (b) cis-Pt(L)(X)(OH) and cis-Pt(L)(X)(Cl) complexes, where L and X are the above-mentioned ligands, (c) cis-Pt(L)(X) complexes where X is the monoanion of an organic xanthate or dithiocarbamate and L = 1,2-diaminocyclohexane, (d) their corresponding Pt(IV) analogues, Pt(L)(X)(OH) and Pt(L)(X)(Cl). All complexes were assayed against P388 tumors and/or KB cell-bearing mice. The observed antitumor activities were correlated with the structures and spectra of the complexes as well as with the results of EHMO calculations performed on the leaving ligand molecules. A number of the most active complexes were also tested for activity against ADJ/PC6 Yoshida and S-180 tumors in vivo. [ABSTRACT FROM AUTHOR]

The article presents information on endangered fish, A. baeticus and its characteristics. The fish is commonly known as Andalusian toothcarp or Salinete. It differs from all other known species of Aphanius fishes. It is characterized as an isolated fish among its populations. It is restricted to the lower reaches of the Guadalquivir river and small streams located on the southern Atlantic coastline of Spain. It is mainly founded in small saline creeks. It has four different types of habitats: freshwater lagoons, tidal channels, small saline creeks and salt exploitation mines. It shows a very short life span with a growth period from April to September. It has fast growth and reduced longevity. The Spanish National Catalogue of Threatened Species has included it in the list of endangered species.

We report the synthesis of bis(toluene dithiocarboxylate)oxovanadium(IV) and its adducts with cyclohexylamine, piperidine, morpholine, piperazine and pyrrolidine which have been characterized by physico-chemical and spectroscopic methods, and assigned the formulae [VO(CHCS)] and [VO(CHCS)·B](B=nitrogen base) and the coordination sphere [VOSN]. The effects of adduct formation are discussed on the basis of the i.r. (V=O, V−S and V−N stretching frequencies) and electronic spectra (d−d transitions). [ABSTRACT FROM AUTHOR]

We aimed to evaluate the release of two antibiotics: gentamicin and vancomycin loaded into F-doped nanotubular anodic oxide layers, as well as their bactericide effect. F-doped nanotubular oxide layers fabricated on Ti-6Al-4V loaded with gentamicin (Gm), vancomycin (Vm) and their mixture (Gm + Vm) by a previously described loading method. Antibiotic release was studied by RP-HPLC and by a biological method. Bactericidal activity was evaluated by a bacterial adherence protocol described previously using on three clinically important bacterial species. The antibiotic release steady up to 120 and 180 min for Gm and Vm, respectively, and despite the antibiotic concentration decreased, their biological activity was maintained over time. The number of living bacteria of three species tested on NT-Gm specimens was significantly lower than on NT specimens without antibiotics (P < 0.01). There are significant differences among NT-Gm and NT-Gm + Vm specimens (P < 0.05) for S. aureus 15981, S. epidermidis ATCC 35984, and P. aeruginosa ATCC 27853 and no differences between NT-Vm and NT-Gm + Vm for staphylococci (P > 0.05). In conclusion, this Gm + Vm loading method added to the properties of F-doped nanotubular oxide layers fabricated on Ti-6Al-4V, and therefore surfaces with antibacterial, biocompatible, tissue integration stimulating and spread-spectrum bactericidal properties can be obtained. [ABSTRACT FROM AUTHOR]

The article offers information on the fish called Ruivaco do Oeste. According to the IUCN criteria B1ab this species must be considered Critically Endangered. This small cyprinid (about 100 mm TL) differs from the other members of its genus by the following combination of characters, including 40-43 canaliculate scales on the lateral line, six to seven scales above the lateral line, and two to three scales below the lateral line. It occurs in three small coastal streams in west central Portugal. The species is now restricted to small isolated pockets located upstream of the major sources of pollution in the rivers Alcabrichel and Sizandro.

The article presents information on leuciscus carolitertii fish that is an endangered species. The fish is a medium sized species. Its body is covered by big scales marginally black spotted. The fish can be found in a side variety of habitats both upstream and downstream. Its diet is omnivorous during juvenile state, feeding on arthropods and other animals when they are adults. The fish has been cited as rare in the Spanish Red Data Book.