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.

Evolutionary history of the synbranchid eels (Teleostei: Synbranchidae) in Central America and the Caribbean islands inferred from their molecular phylogeny.

Swamp eels of the genera Synbranchus and Ophisternon are secondary freshwater fishes whose biogeography provides evidence of their long residence in Mesoamerica, while their impoverished species-level taxonomy might suggest a more recent diversification or a conservative morphology. We have inferred the phylogenetic relationships of Synbranchus marmoratus and Ophisternon aenigmaticum from 45 drainages throughout South, Central America, and Cuba based on mitochondrial genes (cytochrome b and ATPase 8/6). Phylogeographic analysis supported the monophyly of Mesoamerican O. aenigmaticum although our results suggest that S. marmoratus is not a monophyletic group. We found a evolutionary differentiated Synbranchus mtDNA lineage inhabiting Las Perlas islands (Pacific Panama) that appeared to be taxonomically distinct and separated for a long period of time from the main Synbranchus clade. Major synbranchid clades were also corroborated with the nuclear RAG-1 gene (1171-bp). Application of two fish-based mtDNA clocks (1.05-1.3% pairwise divergence/million year (Ma)), is in accordance with the Gondwanian origin suggested for the Synbranchidae. The mtDNA lineages exhibited a remarkable geographic structure in Central America suggesting that vicariance has most likely promoted the Synbranchus and Ophisternon mtDNA diversification. Although our data indicate the importance of the Pacific area in Synbranchus differentiation, the mtDNA divergence between South and Central American Synbranchus is too small to support Cretaceous colonization via the proto-Antillean bridge suggested by Rosen [Syst. Zool. 24 (1976) 431]. Instead, our phylogeographic results suggest that Ophisternon and Synbranchus mtDNA clades most likely colonized Central America during the Miocene (12.7-23Ma) prior the final closure of the Isthmus of Panama (3.3Ma).

Mitochondrial diversity of Opsariichthys bidens (Teleostei, Cyprinidae) in three Chinese drainages.

We describe the phylogeographic structure of 28 Chinese populations of the cyprinid Opsariichthys bidens across three main Chinese river drainages. Our study is based on the phylogenetic analysis of the complete mitochondrial cytochrome b gene (1140 bp). We combined this analysis with population processes inferred from nested clade analysis (NCA) and mismatch distributions. Both analyses showed that Chinese O. bidens consists of five mtDNA lineages (Opsariichthys 1-5) with high genetic divergence among them. Molecular divergences (TrN+G) higher than 20% among the Opsariichthys 1-5 mtDNA lineages suggest a taxonomic underestimation at the species level. About 92% of the genetic variance among samples was explained by differences among Opsariichthys mtDNA lineages. Drainage-restricted haplotypes with high frequencies and moderate nucleotide diversity show that Opsariichthys populations have evolved independently. NCA results were congruent with the phylogeny, and unimodal mismatch distributions with negative Tajima's D values suggest population expansions in some Opsariichthys lineages. The phylogeographic structure of the Opsariichthys 1-5 mtDNA lineages appears to be related to their long-term interruption of gene flow (theta(ST)>0.97). Our results suggested that fragmentation of ancestral ranges might have caused Opsariichthys diversification in Chinese waters. However, current distribution of common haplotypes across the Yangtze and Pearl drainages suggests a recent river connection that could have favoured gene flow across drainages. Overall, the results indicated that the richness of current Asian widespread species might have been underestimated, and that the cyprinid populations of O. bidens in the Yangtze, Pearl and Hai He drainages may correspond to five species.

Phylogenetic structure of Zacco platypus (Teleostei, Cyprinidae) populations on the upper and middle Chang Jiang (=Yangtze) drainage inferred from cytochrome b sequences.

We examined the genetic structure and phylogenetic relationships of some Chinese populations from the Chang Jiang (=Yangtze) drainage of the cyprinid Zacco platypus. We sequenced the complete mitochondrial cytochrome b gene of 64 individuals from 6 upper and middle tributaries of the Sichuan and Hunan Provinces to assess their population structure and systematics. The combined analyses of the phylogenetic information and the population structure suggested that Chinese Z. platypus consist of four distinct mtDNA lineages which exhibit high genetic variation and haplotypic diversity (Zacco A-D). The high molecular divergence observed among Zacco A-D mtDNA lineages (TrN+I (0.76) distance, mean 8.9%+/-1.7%) and their phylogeographic structure indicate that all four lineages have evolved independently. Analysis of molecular variance (AMOVA) indicates that most of the genetic variation observed is found among the four Zacco mtDNA lineages (thetaCT = 0.94) suggesting restricted gene flow among the Chang Jiang populations. Long-term interruption of gene flow was also evidenced by thetaST values higher than 0.9 that could be favoured by the discontinuous distributions of the lineages inhabiting upper (Sichuan Province) and middle (Hunan Province) Chang Jiang tributaries. The significant correlation between the geographic and genetic distances provide support for the importance of geographic discontinuity in shaping the Zacco genetic structure. Nested clade analysis (NCA) results were congruent with phylogenetic relationships recovered and confirm the genetic distinctiveness of four independent Zacco groups. These four groups correspond to the four Zacco A-D mtDNA lineages recovered in the phylogeny and were defined by nucleotide synapomorphies permitting bootstrapped and Bayesian confidence of 95% or greater. The high level of mitochondrial sequence divergence separating all Zacco mtDNA lineages suggested that the Z. platypus populations from the Chang Jiang drainage probably correspond to four different species.

Pleistocene effects on the European freshwater fish fauna: double origin of the cobitid genus Sabanejewia in the Danube basin (Osteichthyes: Cobitidae).

Biogeographical hypotheses of European freshwater fishes were inferred using phylogeographic analysis of the complete cytochrome b and ATP synthase 8 and 6 mitochondrial genes (1982bp). To test the relative importance of drainage origin versus Pleistocene glaciations in the origin of primary freshwater fishes in Europe, we reconstructed the phylogenetic relationships of the genus Sabanejewia which is distributed in European waters. The phylogenetic relationships recovered for the genus Sabanejewia (n=75) provide support for the monophyly of six main evolutionary mtDNA lineages: Sabanejewia larvata, Sabanejewia romanica, Sabanejewia aurata/Sabanejewia caucasica, Sabanejewia kubanica, Sabanejewia baltica, and the Danubian-Balkanian complex. The Caucasian-Caspian mtDNA lineages, S. kubanica, S. aurata/S. caucasica, and the Northern European S. baltica represents the sister group of the Danubian-Balkanian complex mtDNAclade, supporting a Caucasian-Northern European origin of most of mtDNA lineages of the Central European freshwater fish fauna. The mtDNA divergence observed between the Danubian Sabanejewia species is too dissimilar to support their contemporary origin. Rather, the mtDNA data suggest that the Danubian Sabanejewia lineages most likely have a double origin, indicating that the European Sabanejewia lineages have experienced different historical processes for the following reasons. First, the origin of the S. larvata and S. romanica mtDNA clades predates the origin of the Danubian-Balkanian complex, and our results showed that the completion of the Alps and the origin of the Danube drainage seem to have promoted the speciation of the earliest Sabanejewia clades in the Miocene. Second, small genetic distances and the geographical pattern found within the Danubian-Balkanian complex clade indicate that the lineages included in this clade spread recently across the Danube and Greek river drainages. The inclusion of the S. balcanica species within all mtDNA lineages suggests that cyclical cold periods during the Pleistocene glaciations have favoured its rapid expansion and genetic homogenisation across Central European and Greek waters.

Nuclear and mitochondrial data reveal high genetic divergence among Atlantic and Mediterranean populations of the Iberian killifish Aphanius iberus (Teleostei: Cyprinodontidae).

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.

The molecular systematics and biogeography of the European cobitids based on mitochondrial DNA sequences.

Phylogenetic inference regarding the biogeography and evolution of the family Cobitidae depends in large part on the correct interpretation of transitions between the morphological states of secondary sexual characters (e.g., the scale of Canestrini or lamina circularis). Here, we use the complete mitochondrial ATP synthase 8 and 6 and cytochrome b genes to provide an independent assessment of systematics and biogeographic relationships of species in the genus Cobitis, including geographic and subgeneric sampling of species with Canestrini's scale present, duplicated, or absent. The mtDNA-based phylogeny for the genus Cobitis provides the first formal hypothesis for the group and permits a phylogenetic-based assessment of the morphological transitions demonstrated by Canestrini's scale. Our data confirm the monophyly of the genus Cobitis and indicate that European Cobitis comprise six evolutionarily independent lineages. These lineages were defined by nucleotide synapomorphies permitting bootstrapped confidence estimates of 95% or greater and mtDNA genetic distances greater than 4.5% and correspond with moderate fidelity to the Cobitis groups defined by Bacescu (1962, Rev. Roum. Biol. 4, 435-448). The Caucasian lineage, C. cf. sibirica, represents the basal sister species of the genus Cobitis, supporting an eastern Asiatic origin of the European Cobitis: Cobitis sensu stricto, Acanestrinia, Bicanestrinia, Iberocobitis, and Cobitis calderoni. Phylogenetic relationships among Cobitis subgenera and species indicate that the ancestral condition of one scale of Canestrini was duplicated once at the origin of the Bicanestrinia lineage and has been independently lost by C. calderoni and C. elongata. The absence of the scale of Canestrini is the synapomorphy defining the subgenus Acanestrinia, but the mtDNA phylogeny indicates that Acanestrinia is not a natural group and places C. calderoni as the sister lineage to the subgenus Iberocobitis, a finding that is also geographically parsimonius.

An immunoenzymatic method for improving the sensitivity of antigen measurement by electroimmunodiffusion techniques. Application to the quantification of human alpha-fetoprotein.

A double antibody technique of electroimmunodiffusion, which uses glucose oxidase-labelled sheep antibodies to rabbit immunoglobulins as second antibody, is described. Primary antigen-antibody reaction is carried out with a rabbit antiserum by electroimmundiffusion. The glucose oxidase-labelled immunoreagent, being of general application, can serve for the quantification of different antigens and is here used for measurement of low levels of human alpha-fetoprotein. Reproducible results in the range of 50-800 ng/ml were obtained with a variation coefficient of 5 to 10%.