Jörg Bohlen

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.

Synthesis of clonality and polyploidy in vertebrate animals by hybridization between two sexual species.

Because most clonal vertebrates have hybrid genomic constitutions, tight linkages are assumed among hybridization, clonality, and polyploidy. However, predictions about how these processes mechanistically relate during the switch from sexual to clonal reproduction have not been validated. Therefore, we performed a crossing experiment to test the hypothesis that interspecific hybridization per se initiated clonal diploid and triploid spined loaches (Cobitis) and their gynogenetic reproduction. We reared two F1 families resulting from the crossing of 14 pairs of two sexual species, and found their diploid hybrid constitution and a 1:1 sex ratio. While males were infertile, females produced unreduced nonrecombinant eggs (100%). Synthetic triploid females and males (96.3%) resulted in each of nine backcrossed families from eggs of synthesized diploid F1s fertilized by haploid sperm from sexual males. Five individuals (3.7%) from one backcross family were genetically identical to the somatic cells of the mother and originated via gynogenesis; the sperm of the sexual male only triggered clonal development of the egg. Our reconstruction of the evolutionary route from sexuality to clonality and polyploidy in these fish shows that clonality and gynogenesis may have been directly triggered by interspecific hybridization and that polyploidy is a consequence, not a cause, of clonality.

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.

Genome differentiation in a species pair of coregonine fishes: an extremely rapid speciation driven by stress-activated retrotransposons mediating extensive ribosomal DNA multiplications

BackgroundSympatric species pairs are particularly common in freshwater fishes associated with postglacial lakes in northern temperate environments. The nature of divergences between co-occurring sympatric species, factors contributing to reproductive isolation and modes of genome evolution is a much debated topic in evolutionary biology addressed by various experimental tools. To the best of our knowledge, nobody approached this field using molecular cytogenetics. We examined chromosomes and genomes of one postglacial species pair, sympatric European winter-spawning Coregonus albula and the local endemic dwarf-sized spring-spawning C. fontanae, both originating in Lake Stechlin. We have employed molecular cytogenetic tools to identify the genomic differences between the two species of the sympatric pair on the sub-chromosomal level of resolution.ResultsFluorescence in situ hybridization (FISH) experiments consistently revealed a distinct variation in the copy number of loci of the major ribosomal DNA (the 45S unit) between C. albula and C. fontanae genomes. In C. fontanae, up to 40 chromosomes were identified to bear a part of the major ribosomal DNA, while in C. albula only 8–10 chromosomes possessed these genes. To determine mechanisms how such extensive genome alternation might have arisen, a PCR screening for retrotransposons from genomic DNA of both species was performed. The amplified retrotransposon Rex1 was used as a probe for FISH mapping onto chromosomes of both species. These experiments showed a clear co-localization of the ribosomal DNA and the retrotransposon Rex1 in a pericentromeric region of one or two acrocentric chromosomes in both species.ConclusionWe demonstrated genomic consequences of a rapid ecological speciation on the level undetectable by neither sequence nor karyotype analysis. We provide indirect evidence that ribosomal DNA probably utilized the spreading mechanism of retrotransposons subsequently affecting recombination rates in both genomes, thus, leading to a rapid genome divergence. We attribute these extensive genome re-arrangements associated with speciation event to stress-induced retrotransposons (re)activation. Such causal interplay between genome differentiation, retrotransposons (re)activation and environmental conditions may become a topic to be explored in a broader genomic context in future evolutionary studies.

Spawning habitat in the spined loach, Cobitis taenia (Cypriniformes: Cobitidae)

Abstract Spawning habitat of the endangered spined loach Cobitis taenia was studied in northwestern Germany. The distribution of eggs of the loach in the field was best correlated with the density of vegetation but had little correlation with current velocity, water depth, or bottom substrate. In the aquarium, the loach chose dense vegetation for an oviposition site, as inferred by the direct observations of spawning and the location of spawned eggs. It is concluded that such habitat specialization is an important element in the autecology of the endangered fish and a critical stage in the habitat requirements of its populations.

New mtDNA data reveal a wide distribution of the Japanese ginbuna Carassius langsdorfii in Europe.

In this study, records on the occurrence of the Japanese ginbuna Carassius langsdorfii from northern Germany, north-western Italy and southern Bosnia and Herzegovina are presented. The new findings, in addition to former studies reported in the Czech Republic and Greece, show that C. langsdorfii is much more widespread in Europe than was previously believed.

Molecular phylogeographic analyses of the loach Oxynoemacheilus bureschi reveal post-glacial range extensions across the Balkans

Rivers on the Balkan Peninsula can be separated into ichthyofaunistic areas with different endemic fish species. The Vardar River contains a particularly large number of endemics, indicating its complete and long-term isolation from neighbouring river systems. One of the few species shared with other rivers is the loach species Oxynoemacheilus bureschi. In this study, the genetic analyses of 175 individuals of O. bureschi from 17 sites, covering the entire distribution of the species, including the Rivers Vardar (= Axios), Struma (= Strymon), Mesta (= Nestos) and Danube, were performed using one mitochondrial and one nuclear marker. Genetic differentiation among populations was in general low. Shared haplotypes were common and occurred even between distant localities and different river systems. This points to a high degree of gene flow among populations and rejects the hypothesis that the population in the Vardar River represents a relict from an early colonization of the Balkan Peninsula. In contrast, the results suggest that populations in the Vardar River, as well as those in the Danube River, are of recent origin, and a human-mediated introduction cannot be excluded. On the other hand, the populations in the Aggitis River, a left tributary of the lower Struma River, were clearly separated from the rest of the species and represent a long-term isolated lineage. Demographic analyses suggest a recent population expansion for O. bureschi, in which the population in the Aggitis River was not involved.

A Ploidy Difference Represents an Impassable Barrier for Hybridisation in Animals. Is There an Exception among Botiid Loaches (Teleostei: Botiidae)?

One of the most efficient mechanisms to keep animal lineages separate is a difference in ploidy level (number of whole genome copies), since hybrid offspring from parents with different ploidy level are functionally sterile. In the freshwater fish family Botiidae, ploidy difference has been held responsible for the separation of its two subfamilies, the evolutionary tetraploid Botiinae and the diploid Leptobotiinae. Diploid and tetraploid species coexist in the upper Yangtze, the Pearl River and the Red River basins in China. Interestingly, the species ‘Botia’ zebra from the Pearl River basin combines a number of morphological characters that otherwise are found in the diploid genus Leptobotia with morphological characters of the tetraploid genus Sinibotia, therefore the aim of the present study is to test weather ‘B.’ zebra is the result of a hybridisation event between species from different subfamilies with different ploidy level. A closer morphological examination indeed demonstrates a high similarity of ‘B.’ zebra to two co-occurring species, the diploid Leptobotia guilinensis and the tetraploid Sinibotia pulchra. These two species thus could have been the potential parental species in case of a hybrid origin of ‘B.’ zebra. The morphologic analysis further reveals that ‘B.’ zebra bears even the diagnostic characters of the genera Leptobotia (Leptobotiinae) and Sinibotia (Botiinae). In contrast, a comparison of six allozyme loci between ‘B.’ zebra, L. guilinensis and S. pulchra showed only similarities between ‘B.’ zebra and S. pulchra, not between ‘B.’ zebra and L. guilinensis. Six specimens of ‘B.’ zebra that were cytogenetically analysed were tetraploid with 4n = 100. The composition of the karyotype (18% metacentric, 18% submetacentric, 36% subtelocentric and 28% acrocentric chromosomes) differs from those of L. guilinensis (12%, 24%, 20% and 44%) and S. pulchra (20%, 26%, 28% and 26%), and cannot be obtained by any combination of genomes from L. guilinensis and S. pulchra. Phylogenetic reconstructions based on sequence data of the mitochondrial cytochrome b gene and the nuclear RAG-1 gene invariably places ‘Botia’ zebra as sister species to S. pulchra, while L. guilinensis is only distantly related. The presented combination of genetic data demonstrates that ‘B.’ zebra is not the result of a hybridisation, but a species of tetraploid genus Sinibotia with a striking morphological evolution towards an enormous similarity with a co-occurring, but not directly related species. The complete lack of knowledge of the ecology of these species, their main predators or their ecological interactions hampers any conclusion regarding the evolutionary advantage of such adaptation.

Loaches and the environment in two provinces in Northern Vietnam

Abstract. During the last decades, agriculture activities in the mountainous northern provinces of Vietnam intensified drastically, and today rice fields occupy the complete valleys of local streams and rivers. Upstream of the fields, many dams were built mainly for irrigation purposes; sometimes stopping the flow through the lower courses of the creek completely. Illegal fishing with electro-fishing gears is a common way to improve food supply for local villagers. Larger rivers are impacted by non-selective fishing, gravel mining and pollution. These factors represent threats to the local fish communities, which typically include one or several species of loaches. With usage of electro-fishing gear and hand net we surveyed 16 localities of the river basins of the River Ky Cung and the River Bang Giang. Both rivers belong to the drainage area of the River Pearl in the provinces Lang Son and Cao Bang in Northern Vietnam. We focused on getting maximum number of fish species with special attention to loaches at each locality. Ten species of loaches were found; at least two of them represent undescribed species. The main conclusion is that the ichthyofauna of Northern Vietnam still holds a hidden diversity, but at the same time is under strong anthropogenic pressure, so that parts of the diversity may be lost soon.

Dynamics of tandemly repeated DNA sequences during evolution of diploid and tetraploid botiid loaches (Teleostei: Cobitoidea: Botiidae)

Polyploidization has played an important role in the evolution of vertebrates, particularly at the base of Teleostei–an enormously successful ray-finned fish group with additional genome doublings on lower taxonomic levels. The investigation of post-polyploid genome dynamics might provide important clues about the evolution and ecology of respective species and can help to decipher the role of polyploidy per se on speciation. Few studies have attempted to investigate the dynamics of repetitive DNA sequences in the post-polyploid genome using molecular cytogenetic tools in fishes, though recent efforts demonstrated their usefulness. The demonstrably monophyletic freshwater loach family Botiidae, branching to evolutionary diploid and tetraploid lineages separated >25 Mya, offers a suited model group for comparing the long-term repetitive DNA evolution. For this, we integrated phylogenetic analyses with cytogenetical survey involving Giemsa- and Chromomycin A3 (CMA3)/DAPI stainings and fluorescence in situ hybridization with 5S/45S rDNA, U2 snDNA and telomeric probes in representative sample of 12 botiid species. The karyotypes of all diploids were composed of 2n = 50 chromosomes, while majority of tetraploids had 2n = 4x = 100, with only subtle interspecific karyotype differences. The exceptional karyotype of Botia dario (2n = 4x = 96) suggested centric fusions behind the 2n reduction. Variable patterns of FISH signals revealed cases of intraspecific polymorphisms, rDNA amplification, variable degree of correspondence with CMA3+ sites and almost no phylogenetic signal. In tetraploids, either additivity or loci gain/loss was recorded. Despite absence of classical interstitial telomeric sites, large blocks of interspersed rDNA/telomeric regions were found in diploids only. We uncovered different molecular drives of studied repetitive DNA classes within botiid genomes as well as the advanced stage of the re-diploidization process in tetraploids. Our results may contribute to link genomic approach with molecular cytogenetic analyses in addressing the origin and mechanism of this polyploidization event.