Origin and history of Phoxinus (Cyprinidae) introductions in the Douro basin (Iberian Peninsula): an update inferred from genetic data

The number of non-native freshwater fishes in the Iberian Peninsula has been greatly increasing. In this study, individuals of the genus Phoxinus were detected in 18 out of 138 stream sites sampled across the Douro Basin in 2017 and 2018. A total of 26 individuals were barcoded using partial cytochrome c oxidase subunit I (COI) and cytochrome b (cytb) genes for species identification and determination of geographical origin. Molecular data provided the first record of a second Phoxinus species in western Douro (Portugal, Iberian Peninsula), with haplotypes closely matching those found in the Charente River (southern France). This species is suspected to be a recent introduction associated with the use of minnows as live bait by freshwater anglers, which was facilitated by human movements between France and Portugal. Individuals from watercourses in eastern Douro (Spain) were genetically assigned to Phoxinus bigerri, an introduced species previously known for that region, which confirms reports of introduction events from Ebro to Douro Basin probably also related to freshwater angling and facilitated by geographic proximity. The potential ecological impacts of this genus in the region are unknown and need further investigation.We acknowledge Fernando Teixeira, Fernando Miranda, Mario Ferreira, Sara Carona, Jose Pedro RamiAo and Francisco Carvalho for the valuable assistance during fieldwork. We specially thank Maria Filomena MagalhAes for previous fruitful discussions and logistic support. We are grateful to Matthias F. Geiger and Andrea Corral Lou for facilitating genetic data and coordinates of sampling sites. Finally, we appreciate the comments of the three anonymous reviewers that improved the quality of the manuscript. AFF and AGR were supported by the project FRESHING founded by the Portuguese Foundation for Science and Technology (FCT) and COMPETE (PTDC/AAGMAA/2261/2014 - POCI-01-0145-FEDER-356016824). FMSM was supported by the FCT PhD grant SFRH/BD/104703/2014. This study was conducted as part of the projects FRESHING and FRESHCO. The latter is also supported by FCT and COMPETE (PTDC/AGR-FOR/1627/2014 - 04/SAICT/2015) and UID/AGR/04033/2019. Logistic support was also facilitated by the ENVMETAGEN - Capacity Building at InBIO for Research and Innovation Using Environmental Metagenomics project at CIBIO laboratories (668981; EUH2020-WIDESPREAD-2014-2)

Distribution of Eurasian minnows ( Phoxinus

The Eurasian minnows of the genus Phoxinus are small cyprinid species, widely distributed across Europe and Asia. Currently, there are at least 15 species in Phoxinus, with preliminary data suggesting more to be described. Despite the discernible increase in research integrating molecular phylogenetic approaches with traditional taxonomy and systematics, inter- and intraspecific relationships in the genus Phoxinus are still poorly known. The aims of this research were to: (i) compile data on the distribution of Phoxinus species in Croatia and Bosnia and Herzegovina from the sampling conducted by authors from 2006 to 2016 and from literature sources, and (ii) provide a detailed insight on distribution ranges of genetic clades and species of Phoxinus in the western Balkans. Additional 118 localities in Croatia and 8 locations in Bosnia and Herzegovina were added to already known 160 localities from the literature data. Molecular analyses of mitochondrial DNA indicate: (i) the presence of Phoxinus marsilii in Croatia (Drava drainage, the Papuk Mountain), around 260 km south of its known distribution range in Hungary, and (ii) an exceptional genetic variability of P. lumaireul in the Western Balkans

Identification of rare genetic variation of NLRP1 gene in familial multiple sclerosis

The genetic etiology and the contribution of rare genetic variation in multiple sclerosis (MS) has not yet been elucidated. Although familial forms of MS have been described, no convincing rare and penetrant variants have been reported to date. We aimed to characterize the contribution of rare genetic variation in familial and sporadic MS and have identified a family with two sibs affected by concomitant MS and malignant melanoma (MM). We performed whole exome sequencing in this primary family and 38 multiplex MS families and 44 sporadic MS cases and performed transcriptional and immunologic assessment of the identified variants. We identified a potentially causative homozygous missense variant in NLRP1 gene (Gly587Ser) in the primary family. Further possibly pathogenic NLRP1 variants were identified in the expanded cohort of patients. Stimulation of peripheral blood mononuclear cells from MS patients with putatively pathogenic NLRP1 variants showed an increase in IL-1B gene expression and active cytokine IL-1β production, as well as global activation of NLRP1-driven immunologic pathways. We report a novel familial association of MS and MM, and propose a possible underlying genetic basis in NLRP1 gene. Furthermore, we provide initial evidence of the broader implications of NLRP1-related pathway dysfunction in MS

From historical expedition diaries to whole genome sequencing

Torpedo rays (Torpedinidae, Torpediniformes) are small to moderately large batoids that produce an electric discharge. They are distributed worldwide in temperate and tropical seas and are, as a result of their bottom‐dwelling behaviour, susceptible to trawl fishing and often end up as victims of bycatch. The distribution ranges of most recognized species seem to be restrictedhowever, their species‐level systematics is not adequately resolved. In the genus Torpedo, in which many species require revision, there are possibly several undescribed species, while numerous misidentifications add to the complexity of the issue. In the latest lists of living rays, 13 species are accepted in the genus Torpedo, including three of doubtful validity and several recently discovered undescribed species. Among the valid species is the critically endangered, possibly extinct, Torpedo suessii Steindachner, 1898, the Red Sea torpedo, of which only four specimens have been recorded in the literature until now, three of which still exist in the fish collection of the Natural History Museum of Vienna. Museum collections are the most important archive of biodiversity on Earth, and are increasingly being used for various studies, including phylogenetics, population genomics, and biogeography. Nevertheless, molecular analysis of old museum material remains challenging because the genetic material has degraded, is fragmented, and of low quantity. In molecular taxonomy, the necessity of including type specimens as name‐bearing specimens is increasingly recognized. Here, the extended specimen approach was applied to re‐describe the lectotype of T. suessii. The approach included research of historical information and whole genome sequencing, followed by genome assembly and phylogenetic analysis

Molecular data as a possible tool for tracing groundwater flow in karst environment

Karst areas have some of the most complex aquifers in the world. Because of extreme groundwater oscillations, different hydrological connections are active depending on the season. Therefore, the complex water network remains poorly investigated, despite many geological, hydrological and geomorphological studies performed. The methodology of water tracing was developed especially to determine water interconnections in karst. In additionto tracing with salt, dye or temperature, the use of rich endemic fauna, their dispersion and population structure is lately considered. In this paper, we used population structure of endemic fish species Delminichthys adspersus to confirm hydrological data as well as suggest new underground water connections in part of central Dinaric karst. Moreover, we propose a holistic approach of karst area studies, combining hydrology with biology to develop a better strategy for the protection of vulnerable karst ecosystems

Revised classification of former genus Phoxinellus using nuclear DNA sequences

Molecular data inferred from three nuclear DNA regions were used to re-examine the phylogenetic position and taxonomic status of former Phoxinellus taxa. Using either distance method, maximum likelihood or MCMC, phylogenetic tree revealed statistically well supported clade of Delminichthys adspersus, Delminichthys ghetaldii, Delminichthys jadovensis, and Delminichthys krbavensis occupying a sister position to Pelasgus prespensisfurthermore, Phoxinellus pseudalepidotus emerged as a sister taxon to Delminichthys-Pelasgus group. There was a moderate support for sister relationship between Telestes croaticus and Telestes fontinalis, while the position of Telestes metohiensis varied depending on the region and method used. The topology of taxa within Delminichthys was weakly supported and remained pretty much unresoved. Our results confirm a previous notion that the former genus Phoxinellus is not a monophyletic group but rather a grouping of independent lineages

Contrasting morphology with molecular data: an approach to revision of species complexes based on the example of European Phoxinus (Cyprinidae)

Abstract Background Molecular taxonomy studies and barcoding projects can provide rapid means of detecting cryptic diversity. Nevertheless, the use of molecular data for species delimitation should be undertaken with caution. Especially the single-gene approaches are linked with certain pitfalls for taxonomical inference. In the present study, recent and historical species descriptions based upon morphology were used as primary species hypotheses, which were then evaluated with molecular data (including in type and historical museum material) to form secondary species hypotheses. As an example of cryptic diversity and taxonomic controversy, the European Phoxinus phoxinus species complex was used. Results The results of the revision showed that of the fourteen primary species hypotheses, three were rejected, namely P. ketmaieri, P. likai, and P. apollonicus. For three species (P. strandjae, P. strymonicus, P. morella), further investigation with increased data sampling was suggested, while two primary hypotheses, P. bigerri and P. colchicus, were supported as secondary species hypotheses. Finally, six of the primary species hypotheses (P. phoxinus, P. lumaireul, P. karsticus, P. septimanae, P. marsilii and P. csikii) were well supported by mitochondrial but only limitedly corroborated by nuclear data analysis. Conclusion The approach has proven useful for revision of species complexes, and the study can serve as an overview of the Phoxinus genus in Europe, as well as a solid basis for further work

Fish migrate underground

Complex aquatic systems of karst harbour a rich but little-investigated biodiversity. In Croatia and Bosnia-Herzegovina karst, temporal springs are inhibited by a group of minnow-like fishes that retreat to the associated ground water during dry seasons and spend several months underground. The most abundant species in this group is Delminichthys adspersus (Heckel 1843), which also has the most fragmented distribution range. To determine the population composition and dispersal patterns, and to detect potential underground migration, a large genetic data set comprising 544 specimens of D. adspersus covering most of its distribution area was analysed. Analysis of mitochondrial cytochrome b sequence (-1000 bp) and eight microsatellite loci showed that D. adspersus comprises at least three subpopulations with gene flow occuring among them. Coalescent-based analysis revealed a complex migration pattern, with several unidirectional dispersal paths, including between temporal springs that share no surface connection. The results of this study suggest the existence of recurrent underground migration of fish in a karst environment and demonstrate the complexity of its hydrological network. The findings are relevant to conservation strategies for endemic karst organisms and karst ecosystems as a whole

Novel data support validity of <I>Phoxinus chrysoprasius</I> (Pallas, 1814) (Actinopterygii, Leuciscidae)

The common minnow species Cyprinus chrysoprasius, previously synonymised to Phoxinus phoxinus, was originally described from the Crimean Peninsula (Black Sea – Sea of Azov basin). A genetic analysis of the mitochondrial cytochrome oxydase 1 in the context of a phylogenetic study of European Phoxinus showed that it represents a distinct genetic clade and potentially a valid species. In the present study, we approach the issue following a broader, both genetic and morphological, study in order to check and support the validity of native Crimean Phoxinus under the earliest available name of the species: P. chrysoprasius. Our data demonstrate a reliable genetic distance of this minnow from geographically neighbouring clades and species, and a certain morphological distinctiveness. In order to determine the taxonomic concept of P. chrysoprasius, as a species involved in a genetically well-differentiated, but phenotypically poorly structured complex of east-European Phoxinus, a neotype for the species, based on topotypical material, is herein described and designated. The original type locality of the species is also clarified