Historical dispersal and host-switching formed the evolutionary history of a globally distributed multi-host parasite - The Ligula intestinalis species complex.

Studies on parasite biogeography and host spectrum provide insights into the processes driving parasite diversification. Global geographical distribution and a multi-host spectrum make the tapeworm Ligula intestinalis a promising model for studying both the vicariant and ecological modes of speciation in parasites. To understand the relative importance of host association and biogeography in the evolutionary history of this tapeworm, we analysed mtDNA and reduced-represented genomic SNP data for a total of 139 specimens collected from 18 fish-host genera across a distribution range representing 21 countries. Our results strongly supported the existence of at least 10 evolutionary lineages and estimated the deepest divergence at approximately 4.99-5.05 Mya, which is much younger than the diversification of the fish host genera and orders. Historical biogeography analyses revealed that the ancestor of the parasite diversified following multiple vicariance events and was widespread throughout the Palearctic, Afrotropical, and Nearctic between the late Miocene and early Pliocene. Cyprinoids were inferred as the ancestral hosts for the parasite. Later, from the late Pliocene to Pleistocene, new lineages emerged following a series of biogeographic dispersal and host-switching events. Although only a few of the current Ligula lineages show narrow host-specificity (to a single host genus), almost no host genera, even those that live in sympatry, overlapped between different Ligula lineages. Our analyses uncovered the impact of historical distribution shifts on host switching and the evolution of host specificity without parallel host-parasite co-speciation. Historical biogeography reconstructions also found that the parasite colonized several areas (Afrotropical and Australasian) much earlier than was suggested by only recent faunistic data.

Testing the Applicability of MALDI-TOF MS as an Alternative Stock Identification Method in a Cryptic Species Complex.

Knowledge of intraspecific variability of a certain species is essential for their long-term survival and for the development of conservation plans. Nowadays, molecular/genetic methods are the most frequently used for this purpose. Although, the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has become a promising alternative tool to specify intraspecific variability, there is a lack of information about the limitations of this method, and some methodological issues need to be resolved. Towards this goal, we tested the sensitivity of this method on an intraspecific level, using genetically identified individuals of a cryptic fish species complex collected from five distinct populations. Additionally, some methodologic issues, such as the effect of (1) delayed sample preparation, (2) clove oil anaesthetization, and (3) different tissue types (muscle, and brain) were investigated using the MS analysis results. Our results show that the delayed sample preparation has a fundamental effect on the result of MS analysis, while at the same time the clove oil did not affect the results considerably. Both the brain and muscle samples were usable for cryptic species identification, but in our opinion this method has limited applicability for population-level segregation. The application of MALDI-TOF MS to the exploitable toolkit of phylogenetic and taxonomic researches could be used to broaden conclusions.

New record of metacercariae of the North American Posthodiplostomum centrarchi (Digenea, Diplostomidae) in pumpkinseed (Lepomis gibbosus) in Hungary.

Two species of the genus Posthodiplostomum (Digenea: Diplostomatidae) (Posthodiplostomum brevicaudatum Nordmann, 1832 and Posthodiplostomum cuticola Nordmann, 1832) are known as parasites of Hungarian native fishes. Metacercariae of P. cuticola are widespread in Europe and cause black spot disease. Several species of Posthodiplostomum were described also from North America but none of them has been isolated in Hungary up to now. Posthodiplostomum centrarchi Hoffman, 1958 has been detected recently in pumpkinseeds (Lepomis gibbosus L., 1758) in several European countries. Posthodiplostomum centrarchi was isolated for the first time in Hungary from pumpkinseeds caught in the Maconka water reservoir in 2015. Thereafter, several natural waters (e.g. the River Danube, Lake Balaton and the Sió channel) were sampled in order to determine its presence and distribution. Only the native species P. cuticola was detected in Lake Balaton on cyprinids but a relatively high infection rate of P. centrarchi was observed in the Sió channel close to the lake. Pathological changes were absent, and metacercariae were mostly attached to the surface of the liver, kidney and heart. The phylogenetic analysis of the ITS and COI sequences of P. centrarchi and P. cuticola clustered into two distinct branches, which was in agreement with the morphological results.

The mixed phylogenetic origin of northern pike (Esox lucius Linnaeus 1758) populations in the Middle Danubian drainage.

BACKGROUND: Pikes, members of genus Esox, are widespread freshwater predators of the northern hemisphere, and important sport fish also. From the Carpathian basin only one species, the northern pike (E. lucius) is noted. At the same time the pike stocks living in this area show high level of phenotypic variance (e.g. various body pattern) and its growth varies highly both among and within populations. These features usually explained by the environmental diversity of the area. Whereas we think that genetic reasons -e.g. the appearance of other/new pike species in the area- may also be responsible for these observed features. Since as no detailed information have been published from the pike populations of this area, so far; we conducted phylogenetic and morphological assay on 88 pike specimens, collected from 49 Middle Danubian sampling sites. RESULTS: Our phylogenetic surveys showed that the northern pike appear in the study area solely, but all the three of its major lineages (Northern, Circumpolar, Southern) were indicated. Only six specimens represent the Northern lineage, collected from the western part of the study area. The Circumpolar and Southern lineages were common in the Carpathian basin, but the Southern lineage showed higher levels of haplotype diversity than the Circumpolar clade. Which indicates that only the Southern lineage is native in the area, while the other two groups could have appeared in the Middle Danubian system either spontaneously or by human introduction. Moreover, the different clades appeared in the same populations, suggesting the opportunity of inter-lineage hybridisation. From the studied morphologicalal features, the number of scales on the lateral line and the head length showed significant differences among the lineages. At the same time the body pattern of the studied individuals seems to be rather influenced by the ontogenic changes than phylogeny. CONCLUSIONS: The high phenotypic variability of Middle Danubian northern pike populations may be due that all of its three major clades appeared and came in secondary contact in the area. In the within watershed spread of the non-native lineages the human stocking/transfer may play a considerable role.

Increased spatial resolution of sampling in the Carpathian basin helps to understand the phylogeny of central European stream-dwelling gudgeons.

BACKGROUND: Phylogenetic studies of widespread European fish species often do not completely cover their entire distribution area, and some areas are often excluded from analyses than others. For example, Carpathian stocks are often omitted from these surveys or are under-represented in the samples. However, this area served as an extra-Mediterranean refugia for many species; therefore, it is assumed that fish stocks here may show special phylogenetic features. For this reason, increased spatial resolution of sampling, namely revealing genetic information from unexamined Carpathian areas within the range of doubtful taxa, may help us better understand their phylogenetic features. To test this hypothesis, a phylogenetic investigation using a partial mtCR sequence data was conducted on 56 stream-dwelling freshwater fish (Gobio spp.) individuals collected from 11 rivers of the data-deficient Southeastern Carpathian area. Moreover, we revieved the available phylogenetic data of Middle-Danubian stream-dwelling gudgeon lineages to delineate their distribution in the area. RESULTS: Seven out of the nine detected haplotypes were newly described, suggesting the studied area hosts distinct and diverse Gobio stocks. Two valid species (G. obtusirostris, G. gobio), and a haplogroup with doubtful phylogenetic position" G. sp. 1" were detected in the area, showing a specific spatial distribution pattern. The distribution of the detected lineages in the Middle-Danubian area correspond with recent and paleo hydrogeographic features, at the same time mainly on their bordering areas show considerable overlap. CONCLUSIONS: Despite the relatively limited geographic range of the study, our results provide important information which contributes to a better understanding of the phylogenetic, taxonomic and distribution features of Central European gudgeons. The genetically confirmed distribution data of the indicated lineages corresponds well with the recent and near-recent hydrogeographic features of the area, and assumes several hybrid zones in the Carpathian Basin. Additionally, the results show that the middle and lower Danubian watershed cannot be excluded from the range of G. gobio. Moreover, the" G. sp. 1", is slightly differentiated but phylogenetically distinct entity, and is the only Gobio taxa thus far detected in the middle and lower Tisza-basin. However, further investigations are necessary to clarify the taxonomic position of this group.

Comparison of the Genetic Structure of Invasive Bigheaded Carp (Hypophthalmichthys spp.) Populations in Central-European Lacustrine and Riverine Habitats.

Bigheaded carps (bighead carp, Hypophthalmichthys molitrix, and silver carp, Hypophthalmichthys nobilis) and their hybrids play an important ecological and economic role in their original habitat, while their introduced stocks may pose serious ecological risks. To address questions about the persistence and invasiveness of these fish, we need to better understand their population structures. The genetic structures of bigheaded carp populations inhabiting Lake Balaton and the Tisza River were examined with ten microsatellite markers and a mitochondrial DNA marker (COI). The Lake Balaton stock showed higher genetic diversity compared with the Tisza River stock. Based on hierarchical clustering, the Tisza population was characterized only by only silver carps, while the Balaton stock included hybrid and silver carp individuals. All COI haplotypes originated from the Yangtze River. Based on the high genomic and mitochondrial diversity, along with the significant deviation from H-W equilibrium and the lack of evidence of bottleneck effect, it can be assumed that bigheaded carps do not reproduce in Lake Balaton. The present stock in Balaton may have originated from repeated introductions and escapes from the surrounding fishponds. The Tisza stock consists solely of silver carp individuals. This stock appears to have significant reproductive potential and may become invasive if environmental factors change due to climate change.

Microcystis Chemotype Diversity in the Alimentary Tract of Bigheaded Carp.

Most cyanobacterial organisms included in the genus Microcystis can produce a wide repertoire of secondary metabolites. In the mid-2010s, summer cyanobacterial blooms of Microcystis sp. occurred regularly in Lake Balaton. During this period, we investigated how the alimentary tract of filter-feeding bigheaded carps could deliver different chemotypes of viable cyanobacteria with specific peptide patterns. Twenty-five Microcystis strains were isolated from pelagic plankton samples (14 samples) and the hindguts of bigheaded carp (11 samples), and three bloom samples were collected from the scums of cyanobacterial blooms. An LC-MS/MS-based untargeted approach was used to analyze peptide patterns, which identified 36 anabaenopeptin, 17 microginin, and 13 microcystin variants. Heat map clustering visualization was used to compare the identified chemotypes. A lack of separation was observed in peptide patterns of Microcystis that originated from hindguts, water samples, and bloom-samples. Except for 13 peptides, all other congeners were detected from the viable and cultivated chemotypes of bigheaded carp. This finding suggests that the alimentary tract of bigheaded carps is not simply an extreme habitat, but may also supply the cyanobacterial strains that represent the pelagic chemotypes.

Repeatability, Reproducibility, Separative Power and Subjectivity of Different Fish Morphometric Analysis Methods.

We compared the repeatability, reproducibility (intra- and inter-measurer similarity), separative power and subjectivity (measurer effect on results) of four morphometric methods frequently used in ichthyological research, the "traditional" caliper-based (TRA) and truss-network (TRU) distance methods and two geometric methods that compare landmark coordinates on the body (GMB) and scales (GMS). In each case, measurements were performed three times by three measurers on the same specimen of three common cyprinid species (roach Rutilus rutilus (Linnaeus, 1758), bleak Alburnus alburnus (Linnaeus, 1758) and Prussian carp Carassius gibelio (Bloch, 1782)) collected from three closely-situated sites in the Lake Balaton catchment (Hungary) in 2014. TRA measurements were made on conserved specimens using a digital caliper, while TRU, GMB and GMS measurements were undertaken on digital images of the bodies and scales. In most cases, intra-measurer repeatability was similar. While all four methods were able to differentiate the source populations, significant differences were observed in their repeatability, reproducibility and subjectivity. GMB displayed highest overall repeatability and reproducibility and was least burdened by measurer effect. While GMS showed similar repeatability to GMB when fish scales had a characteristic shape, it showed significantly lower reproducability (compared with its repeatability) for each species than the other methods. TRU showed similar repeatability as the GMS. TRA was the least applicable method as measurements were obtained from the fish itself, resulting in poor repeatability and reproducibility. Although all four methods showed some degree of subjectivity, TRA was the only method where population-level detachment was entirely overwritten by measurer effect. Based on these results, we recommend a) avoidance of aggregating different measurer's datasets when using TRA and GMS methods; and b) use of image-based methods for morphometric surveys. Automation of the morphometric workflow would also reduce any measurer effect and eliminate measurement and data-input errors.

Population Genetic Patterns of Threatened European Mudminnow (Umbra krameri Walbaum, 1792) in a Fragmented Landscape: Implications for Conservation Management.

The European mudminnow (Umbra krameri) is a Middle Danubian endemic fish species, which is characterised by isolated populations living mainly in artificial habitats in the centre of its range, in the Carpathian Basin. For their long term preservation, reliable information is needed about the structure of stocks and the level of isolation. The recent distribution pattern, and the population genetic structure within and among regions were investigated to designate the Evolutionary Significant, Conservation and Management Units (ESUs, CUs, MUs) and to explore the conservation biological value of the shrinking populations. In total, eight microsatellite loci were studied in 404 specimens originating from eight regions. The results revealed a pronounced population structure, where strictly limited gene flow was detected among regions, as well as various strengths of connections within regions. Following the results of hierarchical structure analyses, two ESUs were supposed in the Carpathian Basin, corresponding to the Danube and Tisza catchments. Our results recommend designating the borders of CUs in an 80-90km range and 16 clusters should be set up as MUs for the 33 investigated populations. How these genetic findings can be used to better allocate conservation resources for the long term maintenance of the metapopulation structure of this threathened endemic fish is discussed.