Species Phylogeny versus Gene Trees: A Case Study of an Incongruent Data Matrix Based on Paphiopedilum Pfitz. (Orchidaceae).

The phylogeny of the genus Paphiopedilum based on the plastome is consistent with morphological analysis. However, to date, none of the analyzed nuclear markers has confirmed this. Topology incongruence among the trees of different nuclear markers concerns entire sections of the subgenus Paphiopedilum. The low-copy nuclear protein-coding gene PHYC was obtained for 22 species representing all sections and subgenera of Paphiopedilum. The nuclear-based phylogeny is supported by morphological characteristics and plastid data analysis. We assumed that an incongruence in nuclear gene trees is caused by ancestral homoploid hybridization. We present a model for inferring the phylogeny of the species despite the incongruence of the different tree topologies. Our analysis, based on six low-copy nuclear genes, is congruent with plastome phylogeny and has been confirmed by phylogenetic network analysis.

Genetic History of the Remnant Population of the Rare Orchid Cypripedium calceolus Based on Plastid and Nuclear rDNA.

The lady's slipper orchid (Cypripedium calceolus), which inhabits shady deciduous and mixed forests and meadows, is now threatened with extinction in many European countries, and its natural populations have been dramatically declining in recent years. Knowledge of its evolutionary history, genetic variability, and processes in small populations are therefore crucial for the species' protection. Nowadays, in south-west Poland, it is only distributed in seven small remnant and isolated populations, which we examined. One nuclear (ITS rDNA) and two plastid (accD-psa1, trnL-F) markers were analyzed and compared globally in this study. Based on the nuclear marker, the most common ancestor of C. calceolus and Cypripedium shanxiense existed about 2 million years ago (95% HPD: 5.33-0.44) in Asia. The division of the C. calceolus population into the European and Asian lineages indicated by C/T polymorphism started about 0.5 million years ago (95% HPD: 1.8-0.01). The observed variation of plastid DNA, which arose during the Pleistocene glacial-interglacial cycles, is still diffuse in Poland. Its distribution is explained by the result of fragmentation or habitat loss due to human impact on the environment.

Revision of Gyrodactylus salaris phylogeny inspired by new evidence for Eemian crossing between lineages living on grayling in Baltic and White sea basins.

In this research, grayling-specific Gyrodactylus salaris Malmberg, 1957 isolates from Baltic Sea basin were collected in Sweden for the first time. Samples were obtained in three drainage systems: Kalixälven (River Kaitum), Ljungan (River Sölvbacka strömmar), and Umeälven (River Juktån). Three molecular markers were analysed: nuclear ITS rDNA (Internal Transcribed Spacer) and ADNAM1 (Anonymous DNA Marker 1), and mitochondrial cox1 gene. As a result, four new mitochondrial haplotypes were identified (III-C1tt, III-C1ttht, IX-A1tt and X-A1tt). The ADNAM1 analyses resulted in revealing two new alleles (WS4 and BS9) and two new genotypes (T6 and T7). T7 seems to be an indicator of ancient crossing between Baltic and White Sea lineages of the parasite which happened during a first 3000-year period of Eemian interglacial about 130,000 years ago in the connection between Baltic and White Sea. Molecular clock estimates were adjusted, revealing the mean substitution rate and the divergence rate among branches of 3.6% (95% HPD: 2.2%-5.2%) and 7.2% per million years, respectively. As a result, cox1 phylogeny rooted with the introgressed haplotypes has been revised and altered in accordance to new data, revealing fourteen equidistant lineages five of which have been excluded from the study. Based on the new phylogenetic approach, including the molecular clock, this work suggests an overall revision of G. salaris phylogeny and attempts at precisely drawing the division of lineages within this polytypic species as well as proposes unification in nomenclature for its strains.

Horizontal transmission of the ectoparasite Gyrodactylus arcuatus (Monogenea: Gyrodactylidae) to the next generation of the three-spined stickleback Gasterosteus aculeatus.

In the parthenogenetic monogeneans of the genus Gyrodactylus Nordmann, 1832, the genetic diversity within or between hosts is determined by the relative roles of lateral transmission and clonal propagation. Clonality and limited transmission lead to high-amplitude metapopulation dynamics and strong genetic drift. In Baltic populations of the three-spined stickleback Gasterosteus aculeatus Linnaeus, the local mitochondrial diversity of Gyrodactylus arcuatus Bychowsky, 1933 is very high, and spatial differentiation weak. To understand the transmission dynamics in a single location, the transmission of the parasite from adults to next generation sticklebacks was investigated in a northern Baltic brackish water location. By sequencing 777 nt of cox1, as many as 38 separate mitochondrial haplotypes were identified. In August, the intensity of gyrodactylid infection on adult hosts was high, the haplotype diversity (h) was extreme and differentiation between fish was negligible (total h = 0.926, mean h = 0.938). In October, only 46% of the juvenile sticklebacks carried G. arcuatus. The number of parasites per young fish followed a Poisson distribution 0.92 ± 1.04 (mean ± SD) on October 2, and was clearly overdispersed 2.38 ± 5.00 on October 25. The total haplotype diversity of parasites on juveniles was nearly as high as in adults (h = 0.916), but the mean per fish was only h = 0.364 (FST = 0.60), due to low intensity of infection and rapid clonal propagation of early arrivals. The initial first come first served advantage of the first gyrodactylid colonisers will be lost during the host adulthood via continuous transmission. Nesting and polygamy are suggested as factors maintaining the high genetic diversity of the parasite population. The transmission dynamics and, consequently, the population structure of Baltic G. arcuatus is fundamentally different from that of G. salaris Malmberg, 1957, on the Baltic salmon Salmo salar Linnaeus.

Ancient and modern genome shuffling: Reticulate mito-nuclear phylogeny of four related allopatric species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae), ectoparasites on the Eurasian minnow Phoxinus phoxinus (L.) (Cyprinidae).

Phylogenetic analyses including four allopatric species of Gyrodactylus von Nordmann, 1832 on the Eurasian minnow Phoxinus phoxinus (L.) (Cyprinidae) revealed incongruence between the nuclear ITS1-5.8S-ITS2 and mitochondrial cox1 phylogenies due to ancient hybridisation. Gyrodactylus pannonicus Molnár, 1968 was sampled close to its type-locality, the upper reaches of River Tisza, tributary of Danube in the Black Sea Basin. Faunistic search detected three new related species with maximum composite likelihood distances in cox1 between 16.8-23.2% (tentatively 1.3 to 1.8 My of divergence). Gyrodactylus albolacustris n. sp. recorded in the White Sea Basin, eastern Baltic Basin and Mongolia was close to G. pannonicus in the nuclear ITS (divergence of 0.9%), but diverged in cox1 by 19.8%. The Mongolian isolate of G. albolacustris n. sp. diverged from the European isolates in cox1 by 8.9%, suggesting 0.7 My of isolation. The two other new species differed from G. pannonicus by >4% in ITS and some large indels in ITS1, and by >20% in cox1. Gyrodactylus danastriae n. sp. was found in River Strwiaz, a tributary of the River Dniester (Black Sea Basin) and was characterised by smaller size of anchors and by 29-41 bp dimorphic insertion in ITS1. Gyrodactylus botnicus n. sp. is considered endemic in the Baltic Basin, but was also found in the White Sea Basin as a postglacial immigrant, where it had hybridised with G. albolacustris n. sp. in spite of the high divergence in ITS (3.9%) and cox1 (22%). The discordant nuclear and mitochondrial phylogenies revealed an ancient mitochondrial introgression: G. albolacustris n. sp. was derived from a hybridisation combining proto-pannonicus ITS with proto-danastriae mitochondria, perhaps 1.3 My ago. The postglacial hybridisation of G. albolacustris n. sp. (as the donor of mtDNAalb and ITSalb) and G. botnicus n. sp. (donor of the ITSbot) offered a model of shuffling of the genomic components: the process of the homogenisation and stabilisation of nuclear ITS (concerted evolution) and the lineage sorting has hardly begun.

Displaced phylogeographic signals from Gyrodactylus arcuatus, a parasite of the three-spined stickleback Gasterosteus aculeatus, suggest freshwater glacial refugia in Europe.

We examined the global mitochondrial phylogeography of Gyrodactylus arcuatus, a flatworm ectoparasite of three-spined stickleback Gasterosteus aculeatus. In accordance with the suggested high divergence rate of 13%/million years, the genetic variation of the parasite was high: haplotype diversity h=0.985 and nucleotide diversity π=0.0161. The differentiation among the parasite populations was substantial (Φst=0.759), with two main allopatric clades (here termed Euro and North) accounting for 54% of the total genetic variation. The diversity center of the Euro clade was in the Baltic Sea, while the North clade was spread across the Barents and White Seas. A single haplotype within the North clade was found in the western and eastern Pacific Ocean. Divergence of main clades was estimated to be circa 200 thousand years ago. Each main clade was further divided into six distinct subclades, estimated to have diverged in isolation since 135 thousand years ago. This second division corresponds approximately to the Eemian interglacial predating the last glacial maximum. A demographic expansion of the subclades is associated with colonisation of northern Europe since the last glacial maximum, circa 15-40 thousand years ago. The parasite phylogeny is most likely explained by sequential isolated bottlenecks and expansions in numerous allopatric refugia. The postglacial intermingling and high variation in the marine parasite populations, separately in the Baltic and Barents Seas, suggest low competition of divergent parasite matrilines, coupled with a large population size and high rate of dispersal of hosts. The genetic contribution of the assumed refugial fish populations maintaining the parasite during the last glacial maximum was not detected among the marine sticklebacks, which perhaps were infected after range expansion.

Morphological and molecular characterization of Ceratomyxa gurnardi sp. n. (Myxozoa: Ceratomyxidae) infecting the gallbladder of the grey gurnard Eutrigla gurnardus (L.) (Scorpaeniformes, Triglidae).

The myxosporean specimens were noted in grey gurnard Eutrigla gurnardus (L.) from the area near the Shetland Islands. The structure and dimensions of its vegetative stage differ from earlier descriptions. A sequence of small subunit ribosomal RNA gene obtained during the current study differs from other Ceratomyxa spp. available in GenBank. A phylogenetic position of parasite based on the 18S rDNA fragment was estimated. The proposed name for this myxosporean is Ceratomyxa gurnardi sp. n.

A monogenean fish parasite, Gyrodactylus chileani n. sp., belonging to a novel marine species lineage found in the South-Eastern Pacific and the Mediterranean and North Seas.

Gyrodactylus chileani n. sp. is the first Gyrodactylus species reported from Chile. It is an ectoparasite living on fins and skin of a small fish, the Chilean tidal pond dweller Helcogrammoides chilensis (Cancino) (Perciformes: Tripterygiidae). A phylogenetic analysis based on 5.8S+ITS2 of rDNA placed the new species close to marine Gyrodactylus species found in Europe: G. orecchiae Paladini, Cable, Fioravanti, Faria, Cave & Shinn, 2009 on gilthead seabream Sparus aurata L. from the Adriatic and Tyrrhenian Sea fish farms (Perciformes: Sparidae), and an undescribed species on the black goby Gobius niger L. from the North Sea (Perciformes: Gobiidae). A morphological description of the latter species is unavailable. These geographically distant parasite samples on different host families form a new well supported Gyrodactylus orecchiae lineage. Using molecular phylogenetics, it is shown that the marine species groups of Gyrodactylus may have a worldwide distribution.

Two new Antarctic Gyrodactylus species (Monogenoidea): description and phylogenetic characterization.

Two new species of Gyrodactylus Nordmann, 1832 (Gyrodactylidae, Monogenoidea) are described, i.e., Gyrodactylus coriicepsi n. sp. from gills of black rockcod, Notothenia coriiceps Richardson, 1844, and Gyrodactylus nudifronsi n. sp. from gills of gaudy notothen, Lepidonotothen nudifrons Lönnberg, 1905, collected in waters surrounding the South Shetland Islands, Antarctica. Gyrodactylus coriicepsi n. sp. is characterized by a long, narrowing membrane of the ventral bar and a marginal hook sickle with beveled heel and toe. Gyrodactylus nudifronsi n. sp. is characterized by a conspicuous ventral bar process and barely curved marginal hook sickle with curved heel and toe. Definitive species identification and phylogenetic position were based on sequences of the rDNA region (ITS1-5.8S-ITS2). Both new species belong to a monophyletic group (bootstrap support 99%) that includes the European marine species Gyrodactylus perlucidus Bykhovskiy and Polyanskiy, 1953 (on Zoarces viviparus), Gyrodactylus flesi Malmberg, 1957 and G. robustus Malmberg, 1957 (both on Platichthys flesus), and the freshwater species Gyrodactylus hrabei Ergens, 1957 and Gyrodactylus mariannae (both on Cottus poecilopus). The observations suggest an evolutionary continuum of the marine Gyrodactylus spp. fauna in the northern and southern hemispheres, which indicates the marine origin of some freshwater Gyrodactylus species.

Molecular faunistics of accidental infections of Gyrodactylus Nordmann, 1832 (Monogenea) parasitic on salmon Salmo salar L. and brown trout Salmo trutta L. in NW Russia.

Salmon Salmo salar L. and brown trout S. trutta L. juveniles were examined for the presence of accidental monogenean ectoparasitic species of Gyrodactylus Nordmann, 1832 in the Baltic and White Sea basins of Russian Karelia in order to estimate the frequency of host-switching attempts on an ecological timescale. To collect phylogeographical information and for exact species identification, the parasites were characterised by nuclear internal transcribed spacer sequences of rDNA (ITS) and, for some species, also by their mitochondrial DNA (CO1 gene) sequences. Four accidental Gyrodactylus species were observed on salmon and brown trout. A few specimens of G. aphyae Malmberg, 1957, the normal host of which is the Eurasian minnow Phoxinus phoxinus (L.), were observed on lake salmon from the Rivers Kurzhma (Lake Kuito, White Sea basin) and Vidlitsa (Lake Ladoga, Baltic basin). G. lucii Kulakovskaya, 1952, a parasite of the northern pike Esox lucius L., was observed on salmon in the Kurzhma. In the River Vidlitsa, two specimens of G. papernai Ergens & Bychowsky, 1967, normally on stone loach Barbatula barbatula (L.), were found on salmon. On anadromous White Sea salmon in the River Pulonga in Chupa Bay, a few salmon parr carried small colonies of G. arcuatus Bychowsky, 1933, which were shown to have originated from the local three-spined stickleback Gasterosteus aculeatus L. consumed as prey. No specimens of Gyrodactylus salaris Malmberg, 1957 were observed, although the Pulonga is the nearest salmon spawning river to the River Keret', which is heavily infected with introduced G. salaris. In the River Satulinoja, Lake Ladoga, three specimens of G. lotae Gusev, 1953, from burbot Lota lota (L.), were collected from a single brown trout S. trutta. All nonspecific gyrodactylid infections on salmonids were judged to be temporary, because only a few specimens were observed on each of the small number of infected fishes. The prevalence of endemic G. salaris was also low, only 1% (Nfish = 296) in Lake Onega and 0.7% (Nfish = 255) in Lake Ladoga, while brown trout specific Gyrodactylus species were not observed on any of the 429 trout examined from the Ladoga basin. The host-specific and unspecific burden of Gyrodactylus spp. on these 'glacial relict' populations of salmon and brown trout was very low, suggesting a generalised resistance against the co-evolved freshwater parasite community, or some kind of 'vaccination' effect. These hypotheses deserve further testing.

Hybrid origin of Baltic salmon-specific parasite Gyrodactylus salaris: a model for speciation by host switch for hemiclonal organisms.

Host switching explains the high species number of ectoparasitic, viviparous, mainly parthenogenetic but potentially hermaphroditic flatworms of the genus Gyrodactylus. The starlike mitochondrial phylogeny of Gyrodactylus salaris suggested parallel divergence of several clades on grayling (also named as Gyrodactylus thymalli) and an embedded sister clade on Baltic salmon. The hypothesis that the parasite switched from grayling to salmon during the glacial diaspora was tested using a 493-bp nuclear DNA marker ADNAM1. The parasites on salmon in lakes Onega and Ladoga were heterozygous for divergent ADNAM1 alleles WS1 and BS1, found as nearly fixed in grayling parasites in the White Sea and Baltic Sea basins, respectively. In the Baltic salmon-specific mtDNA clade, the WS/BS heterozygosity was maintained in 23 out of the 24 local clones. The permanently heterozygous clade was endemic in the Baltic Sea basin, and it had accumulated variation in mtDNA (31 variable sites on 1600 bp) and in the alleles of the nuclear locus (two point mutations and three nucleotide conversions along 493 bp). Mendelian shuffling of the nuclear alleles between the local clones indicated rare sex within the clade, but the WS/BS heterozygosity was lost in only one salmon hatchery clone, which was heterozygous WS1/WS3. The Baltic salmon-specific G. salaris lineage was monophyletic, descending from a single historical hybridization and consequential host switch, frozen by permanent heterozygosity. A possible time for the hybridization of grayling parasite strains from the White Sea and Baltic Sea basins was during the Eemian interglacial 132 000 years bp. Strains having a separate divergent mtDNA observed on farmed rainbow trout, and on salmon in Russian lake Kuito were suggested to be clones derived from secondary and tertiary recombination events.

Escape from an evolutionary dead end: a triploid clone of Gyrodactylus salaris is able to revert to sex and switch host (Platyhelminthes, Monogenea, Gyrodactylidae).

Diploid parthenogenesis, with rare sex, is considered as the basic mode of reproduction among the hermaphroditic and viviparous Gyrodactylus. A particular strain of the monogenean parasite Gyrodactylus salaris (RBT clone) was recognized by an invariable, unique mitochondrial DNA haplotype in rainbow trout (Oncorhynchus mykiss) farms. The RBT clone was shown to be triploid and asexual by analyzing a 493 bp sequence of a nuclear DNA marker. Three alleles were present as heterozygous in all 237 individuals sampled in years 2001-2005 from five isolated Finnish farms. The triploid clone probably originated from a diploid oocyte fertilized by a non-self hermaphrodite, most probably in a fish farm. Identical mitochondrial COI gene (1606 bp) was also found in G. salaris parasites on landlocked salmon (Salmo salar) in two rivers draining to the lake Kuitozero, Russian Karelia. In the river Pisto, the clone was triploid, but the diagnostic "short" nuclear allele of the RBT clone was replaced by an allele typical for salmon specific parasites in the Lake Onega. The clone in the river Kurzhma was diploid, having lost the "short" allele, but still heterozygous for the other two alleles of the RBT clone. Evidently, the triploid parthenogenetic RBT clone had produced diploid oocytes, when (as a female) stimulated by a non-self mate in the new environment. The genetic reorganization coincided with a switch to the salmon host. Participation of triploids into the gene pool of the species is rarely reported in animals, and the triploidy is generally considered as an irreversible dead-end of the evolution. Liberalism in ploidy level may significantly add to the evolutionary options available for a parasite in ever-changing environments.

In vitro adenine nucleotide catabolism in African catfish spermatozoa.

It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 degrees C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 degrees C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP-->ADP-->AMP (adenosine/IMP)-->inosine-->hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.

Inhibition by tributyltin of herring skeletal muscle lactate dehydrogenase activity.

The activity of herring (Clupea harengus) skeletal muscle lactate dehydrogenase (EC 1.1.1.27) LDH-A4 isoenzyme was examined in the presence of tributyltin chloride (TBT). This paper reports the in vitro inhibition of LDH activity with increasing concentration of TBT. Bovine serum albumin (BSA) added to the LDH-A4 isoenzyme prior to the addition of TBT was able to protect enzyme activity against inhibition by this toxicant. The observed protection of LDH-A4 activity increased with increasing BSA concentration in the incubation medium. The results suggest that the presence of BSA could protect LDH activity from direct binding of TBT to LDH.

Initial steps of speciation by geographic isolation and host switch in salmonid pathogen Gyrodactylus salaris (Monogenea: Gyrodactylidae).

To test the hypothesis that host-switching can be an important step in the speciation of gyrodactylid monogenean flatworms, we inferred the phylogeny within a cluster of parasites morphologically close to Gyrodactylus salaris Malmberg 1957, collected from Atlantic, Baltic and White Sea salmon (Salmo salar), farmed rainbow trout (Oncorhynchus mykiss), and grayling (Thymallus thymallus) from Northern Europe. The internal transcribed spacer region of the nuclear ribosomal gene was sequenced for taxonomic identification. Parasites on grayling from the White Sea Basin differed from the others by one nucleotide (0.08%), the remainder were identical to the sequence published earlier from Norway (G. salaris on salmon), England (Gyrodactylus thymalli on grayling), and the Czech Republic (unidentified salaris/thymalli on trout). For increased resolution, 813 nucleotides of the mitochondrial COI gene of 88 parasites were sequenced and compared with 76 published sequences using phylogenetic analysis. For all tree building algorithms (NJ, MP), the parasites formed a star-like phylogeny of six definite sister clades, indicating nearly simultaneous radiation. Average K2P distances between clades were 1.8-2.6%, and internal mean distances 0.2-1.1%. The genetic distance to the nearest known relative, Gyrodactylus lavareti Malmberg, was 24%. A variable salmon-specific mitochondrial Clade I was observed both in the Baltic Basin and in pathogenic populations introduced to the Atlantic and White Sea coasts. An invariable Clade II was common in rainbow trout farms in Sweden, Denmark and Finland; the same haplotype was also infecting salmon in a landlocked population in Russian Karelia, and in Oslo fjord and Sognefjord in Norway. Four geographically vicariant sister clades were observed on graylings: Clade III in the Baltic Sea Basin; Clade IV in Karelian rivers draining to the White Sea; Clade V in Norwegian river draining to Swedish lake Vänern; and Clade VI in rivers draining to Oslo fjord. The pattern fitted perfectly with the postglacial history of grayling distribution. Widely sampled clades from salmon and Baltic grayling had basal haplotypes in populations, which were isolated early during the postglacial recolonisation. The divergence between the six clades was clear and linked with their hosts, but not wide enough to support a species status for them. Parasites from the Slovakian type population of G. thymalli were not available, so this result does not mean that G. salaris and G. thymalli are synonyms. It is suggested that the plesiomorphic host of the parasite cluster was grayling, and the switch to salmon occurred at least once when the continental ice isolated Baltic salmon in an eastern freshwater refugium, 130,000 years ago. At the same time, parasites on grayling were split geographically and isolated into several allopatric refugia. The divergence among the parasite clades allowed a tentative calibration of the evolutionary rate, leading to an estimate of the divergence of 13.7-20.3% per million years for COI coding mtDNA. The results supported the hypothesis that parallel to the allopatric mode, host switch and instant isolation by host specificity can be operated as a speciation mechanism.

The crossroads of molecular, typological and biological species concepts: two new species of Gyrodactylus Nordmann, 1832 (Monogenea: Gyrodactylidae).

Nucleotide sequences of nuclear ribosomal DNA internal transcribed spacers (ITS) were used to confirm morphological identification of Gyrodactylus species in Fennoscandia. Three pairs of morphologically similar or cryptic species are compared in this study. G. branchicus Malmberg, 1964 and G. rarus Wegener, 1910, hosted by the sticklebacks Gasterosteus aculeatus L. and Pungitius pungitius (L.), respectively, displayed a genetic divergence of 0.9-1.3% along 774 nucleotides of ITS (Jukes & Cantor distance). G. branchicus isolates from the Baltic, White Sea and Scottish North Sea were invariable, but a Belgian North Sea population with a 0.4% divergence in ITS sequence has been collected. The species status of G. branchicus and G. rarus is supported by host-specificity in sympatric habitats and consistent morphological difference in the marginal hook sickles. Among Gyrodactylus on burbot Lota lota (L.) specimens were collected with their 792 bp long ITS sequences differing by 8.5%. This has led to the splitting of G. lotae Gusev, 1953 into two species, G. lotae and G. alexgusevi n. sp. The species are morphologically separable, and a similar range of variation was found in both the anchors and the marginal hooks in other collections and in the original description. Among G. macronychus-like parasites of minnow Phoxinus phoxinus (L.), divergent ITS sequences indicate two cryptic species. By comparison with the type-specimens, one was identified as G. macronychus Malmberg, 1957; the other is here designated as G. jussii n. sp., differing by 21.8% in 950-973 bp long ITS. The consequences of these findings for the species concept and species identification of Gyrodactylus are discussed.

Enzyme activities in fish spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengus.

The activities of NAD- and NADP-dependent dehydrogenases and creatine kinase were compared in extracts of spermatozoa from herring (Clupea harengus), carp (Cyprinus carpio) and catfish (Clarias gariepinus). The activity of malic enzyme in herring spermatozoa was approximately 5 and 36 times higher than in carp and catfish spermatozoa. In contrast, lactate dehydrogenase activity in herring spermatozoa was very low. Herring spermatozoa possess two isoenzymes of lactate dehydrogenase: LDH-A(2)B(2) and LDH-B(4). Both herring spermatozoa isozymes were separated, partly purified and characterized by kinetic and physico-chemical properties. The pH optima and K(m) values for pyruvate reduction were 7.1, 7.25, 7.6 and 0.22, 0.07, 0.09 mM for LDH-A(4), LDH-A(2)B(2) and LDH-B(4), respectively. The isoenzymes also have different thermostabilities. High activity of malic enzyme in herring spermatozoa suggests adaptation to metabolism at high oxygen tension.

Speciation by host switch and adaptive radiation in a fish parasite genus Gyrodactylus (Monogenea, Gyrodactylidae).

Four hundred Gyrodactylus species have been formally described, but the estimated number of species in this fish ectoparasite genus of Monogenean Platyhelminthes is more than 20,000. The unusually high species richness has lead to the hypotheses of speciation and adaptive radiation via host switching. These hypotheses were tested by reconstructing a molecular phylogeny for the subgenus G. (Limnonephrotus) which is a group of freshwater parasites, including five species infecting wild and farmed salmonids. The highly variable ITS1 and ITS2 segments and the conservative 5.8S ribosomal gene were sequenced in 22 species plus two species representing the subgenus G. (Paranephrotus) as an outgroup. The phylogeny was compared with host systematics: the species were collected from six fish families (Cyprinidae, Salmonidae, Percidae, Esocidae, Gasterosteidae, and Gobitidae). The phylogenetic analysis demonstrated that G. (Limnonephrotus) is a monophyletic group that was originally hosted by cyprinids. The speciation has occurred in two episodes, the older one manifested in genetic distances 25-33% (4-6 Myr BP). The latter speciation burst occurred in one clade only, perhaps one million years ago. This clade has been morphologically identified as a wageneri species group. It is a monophyletic group of 18 species [studied here] and contains all five salmonid parasites, but also parasites, on cyprinids, percids, esocids, and gasterosteids. In G. (Limnonephrotus), eight host switches crossing the host family barrier were observed, and at least three of them were followed by repetitive speciation. Seven host-switch events were statistically confirmed by bootstrapping. The suggested model of speciation by host switch was accepted, and interestingly the adaptive radiation seems to be a consequence of host switch to a new family (key innovation model). The molecular and ecological evolution rate of Gyrodactylus parasites is manyfold in comparison to host species, and the phylogenies are largely independent and disconnected.