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Background: Polyploidy, although still poorly explored, represents an important evolutionary event in several cyprinid clades. Herein, Catlocarpio siamensis and Probarbus jullieni - representatives of the paleotetraploid tribe Probarbini, were characterized both by conventional and molecular cytogenetic methods., Results: Alike most other paleotetraploid cyprinids (with 2n = 100), both species studied here shared 2n = 98 but differed in karyotypes: C. siamensis displayed 18m + 34sm + 46st/a; NF = 150, while P. jullieni exhibited 26m + 14sm + 58st/a; NF = 138. Fluorescence in situ hybridization (FISH) with rDNA probes revealed two (5S) and eight (18S) signals in C. siamensis , respectively, and six signals for both probes in P. jullieni . FISH with microsatellite motifs evidenced substantial genomic divergence between both species. The almost doubled size of the chromosome pairs #1 in C. siamensis and #14 in P. jullieni compared to the rest of corresponding karyotypes indicated chromosomal fusions., Conclusion: Based on our findings, together with likely the same reduced 2n = 98 karyotypes in the remainder Probarbini species, we hypothesize that the karyotype 2n = 98 might represent a derived character, shared by all members of the Probarbini clade. Besides, we also witnessed considerable changes in the amount and distribution of certain repetitive DNA classes, suggesting complex post-polyploidization processes in this small paleotetraploid tribe., Competing Interests: Experiments were performed in accordance with ethical protocols, and anesthesia using clove oil was administered prior to sacrificing the animals, as approved by the Ethics Committee of Khon Kaen University and by the RGJ committee under no. PHD/K0081/2556.Not applicableThe authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Mammalian carnivores have been extensively studied by cross-species chromosome painting, which indicated a high degree of karyotypic conservatism in the cat-like suborder Feliformia relative to the ancestral carnivore karyotype (ACK). The first exception to this high degree of karyotypic conservation in feliforms was recently confirmed in genets, mesocarnivores belonging to the basal family Viverridae. Here, we present a comparative analysis of the chromosome rearrangements among 2 subspecies of the small-spotted genet Genetta genetta (the Iberian nominate and the Arabian grantii) and the panther genet G. maculata, the 2 most common and widespread genets, using whole-chromosome paints from the domestic cat (Felis catus). The chromosome homology maps and the presence of numerous interstitial telomeric sites in both genet species strengthen the hypothesis that a highly rearranged karyotype compared to the ACK may occur throughout Genetta. The karyotype of G. maculata appears to have undergone more rearrangements than that of G. genetta, which is an older lineage. Notably, we identified a tandem fusion distinguishing G. g. genetta and G. g.grantii. As G. g. grantii is morphologically and genetically distinctive, and tandem fusions have been associated with substantial postzygotic isolation in mammals, this cytogenetic finding flags the subspecies for future taxonomic investigations., (© 2018 S. Karger AG, Basel.)

Androgenesis among vertebrates is considered a rare phenomenon, with some cases reported so far, but linked to experiments involving gamete manipulation (artificial androgenesis). Herein, we report the first empirical evidence of the natural occurrence of spontaneous androgenesis in a vertebrate, the Squalius alburnoides allopolyploid complex. A genetically screened random sample of a natural population was allowed to reproduce in an isolated pond without any human interference, and the viable offspring obtained was later analysed for paternity. Both nuclear and mitochondrial markers showed that the only allodiploid fish found among all the allotriploid offspring was androgenetically produced by an allodiploid male. This specimen had no female nuclear genomic input, and the sequence of the mitochondrial fragment examined differed from that of the male progenitor, matching one of the parental females available in the pond, probably the mother. The possible role of androgenesis in the reproductive dynamics of this highly successful vertebrate complex is discussed., Competing Interests: We have no competing interests.

Hybrid complexes are composed of organisms with multiple combinations of parental genomes (genomotypes) that interconnect through nets of crosses. Although several such complexes are well established without speciation or extinction, mechanisms shaping their dynamics remain poorly understood. In this study, we quantified the reproductive success of the allopolyploid Iberian fish Squalius alburnoides in experimental free-access and directional crosses involving the most common genomotypes. Specifically, we analysed the paternity of the offspring produced when females had free access to male genomotypes and quantified variations in egg allocation, fertilization rate, and offspring survival among crosses involving each male genomotype. The composition of the offspring produced from free-access crosses varied significantly from that expected from random mating, suggesting that offspring production and viability are not independent of parental male genomotype. Moreover, directional crosses producing the genomotype most commonly found in wild populations appeared to be the most successful, with females laying more eggs, and fertilization rate and offspring survival being the highest. These results suggest that reproductive dynamics plays a relevant role in structuring the genomotype composition of populations and opens a path to future research on the ecology and evolutionary biology of allopolyploids and their multiplicity of possible evolutionary pathways., (© 2016 The Author(s).)

Background: Iberian Leuciscinae are greatly diverse comprising taxa of hybrid origin. With highly conservative karyotypes, Iberian Chondrostoma s.l. have recently demonstrated sub-chromosomal differentiation and rapid genome restructuring in natural hybrids, which was confirmed by ribosomal DNA (rDNA) transposition and/or multiplication. To understand the role of repetitive DNAs in the differentiation of their genomes, a genetic and molecular cytogenetic survey was conducted in Achondrostoma oligolepis, Anaecypris hispanica, Iberochondrostoma lemmingii, I. lusitanicum, Pseudochondrostoma duriense, P. polylepis, Squalius pyrenaicus and hybrids between A. oligolepis x (P. duriense/P. polylepis), representing 'alburnine', chondrostomine and Squalius lineages., Results: Partial Rex3 sequences evidenced high sequence homology among Leuciscinae (≥98 %) and different fish families (80-95 %) proposing a relatively recent activity of these elements in the species inspected. Low nucleotide substitution rates (<20 %) and intact ORFs suggests that Rex3 may in fact be active in these genomes. The chromosomal distribution of Rex3 retroelement was found highly concentrated at pericentromeric and moderately at subtelomeric blocks, co-localizing with 5S rDNA loci, and correlating with blocks of heterochromatin and C0t-1 DNA. This accumulation was evident in at least 10 chromosome pairs, a pattern that seemed to be shared among the different species, likely pre-dating their divergence. Nevertheless, species-specific clusters were detected in I. lusitanicum, P. duriense, P. polylepis and S. pyrenaicus demonstrating rapid and independent differentiation. Natural hybrids followed the same patterns of accumulation and association with repetitive sequences. An increased number of Rex3 clusters now associating also with translocated 45S rDNA clusters vouched for other genomic rearrangements in hybrids. Rex3 sequence phylogeny did not agree with its hosts' phylogeny but the observed distribution pattern is congruent with an evolutionary tendency to protect its activity, a robust regulatory system and/or events of horizontal transfer., Conclusions: This is the first report directed at retroelement physical mapping in Cyprinidae. It helped outlining conceivable ancestral homologies and recognizing retrotransposon activation in hybrids, being possibly associated with genome diversification within the subfamily. The extensive diversity of Iberian Leuciscinae makes them excellent candidates to explore the processes and mechanisms behind the great plasticity distinguishing vertebrate genomes.

Previous studies have shown that assortative mating acts as a driver of speciation by countering hybridization between two populations of the same species (pre-zygotic isolation) or through mate choice among the hybrids (hybrid speciation). In both speciation types, assortative mating promotes speciation over a transient hybridization stage. We studied mate choice in a hybrid vertebrate complex, the allopolyploid fish Squalius alburnoides. This complex is composed by several genomotypes connected by an intricate reproductive dynamics. We developed a model that predicts the hybrid complex can persist when females exhibit particular mate choice patterns. Our model is able to reproduce the diversity of population dynamic outcomes found in nature, namely the dominance of the triploids and the dominance of the tetraploids, depending on female mate choice patterns and frequency of the parental species. Experimental mate choice trials showed that females exhibit the preferences predicted by the model. Thus, despite the known role of assortative mating in driving speciation, our findings suggest that certain mate choice patterns can instead hinder speciation and support the persistence of hybrids over time without speciation or extinction.

Understanding the mechanisms underlying diversification and speciation by introgressive hybridization is currently one of the major challenges in evolutionary biology. Here, the analysis of hybridization between two pairs of Iberian Leuciscinae provided new data on independent hybrid zones involving Achondrostoma oligolepis (AOL) and Pseudochondrostoma duriense (PDU), and confirmed the occurrence of hybrids between AOL and Pseudochondrostoma polylepis (PPO). A multilevel survey combining morphological, genetic and cytogenomic markers on a vast population screening successfully sorted the selected fishes as admixed. Results were similar in both AOL × PDU and AOL × PPO systems. Overall, hybrid morphotypes, cytogenomic data and genetic profiling indicated preferential backcrossing and suggested AOL as a major genomic contributor. Moreover, results implied AOL as more permissive to introgression than PDU or PPO. Although PDU- and PPO-like individuals appeared more resilient to genome modifications, AOL appeared to be more involved and affected by the ongoing hybridization events, as chromosomal translocations were only found in AOL-like individuals. All hybrids analysed evidenced extensive ribosomal DNA (rDNA) polymorphism that was not found in parental species, but usually seen falling within the range of possible parental combinations. Yet, transgressive phenotypes that cannot be explained by normal recombination, including more rDNA clusters than expected or the occurrence of syntenic rDNAs, were also detected. Present results proved rapid genomic evolution providing the genetic novelty for species to persist. In addition, although the ultimate consequences of such apparently extensive and recurrent events remain unknown, modern genome-wide methodologies are of great promise towards answering questions concerning the causes, dynamics and impacts of hybridization.

The glochidium larvae of freshwater mussels of the family Unionidae need to find suitable hosts to attach themselves and metamorphose into free-living juveniles. The specificity of the host-parasite relationship was investigated for the Iberian Unio tumidiformis Castro, 1885 by means of experimental infections and also by analyzing naturally infected fish. The process of encapsulation of glochidia was studied using scanning electron microscopy. Unio tumidiformis has proven to be an unusual host-specific unionid mussel, apparently parasitizing only fish of the genus Squalius Bonaparte, 1837. Successful encapsulation or complete metamorphosis was observed in five fish taxa: S. aradensis (Coelho, Bogutskaya, Rodrigues et Collares-Pereira), S. caroliterti (Doadrio), S. pyrenaicus (Günther), S. torgalensis (Coelho, Bogutskaya, Rodrigues et Collares-Pereira) and S. alburnoides (Steindachner) complex (only for the nuclear hybrids with at least one copy of the S. pyrenaicus genome). Complete metamorphose was achieved in 6 to 14 days at mean temperatures ranging from 21.8 to 26.1 degrees C. The current study provides support for cell migration being the main force of cyst formation and shows the influence of potential host's genome in response to the infection process to determine the success of the metamorphosis.

In this study, the description of the karyotypes of the endangered chubs Squalius aradensis (Coelho, Bogutskaya, Rodrigues and Collares-Pereira, 1998) and Squalius torgalensis (Coelho, Bogutskaya, Rodrigues and Collares-Pereira, 1998) is presented by means of conventional (Giemsa-staining, Chromomycin A3 (CMA3)-fluorescence, Silver-impregnation (Ag-NORs)) and molecular (fluorescence in situ hybridization (FISH) with 18S rDNA probe) protocols. These endemic sister-species have an allopatric but adjacent distribution in the most southwestern part of the Iberian Peninsula. Diploid chromosome number was invariably 2n = 50 and karyotypes of both species were grossly similar, composed of metacentric and submetacentric elements with a reduced number of acrocentric pairs. Sequential staining using FISH with an 18S rDNA probe, CMA3 and Ag-NORs treatments revealed consistent positive signals located at the end of the short arms of a submetacentric chromosome pair, likely homologous in both species. While providing useful cytogenetic comparative data against other members of the genus Squalius Bonaparte, 1837, the work aimed to draw attention towards the conservation of two narrow-range and highly confined fish species.

When comparing the known picture of polyploidy in animals and in plants, it is possible to recognize some similarities, namely: (i) multiple and recurrent origins in several well-established taxonomic groups; (ii) a strong and regular association with hybridization events; (iii) the production of genotypic diversity; (iv) a rapid genomic reshuffling; (v) a very active role of transposable elements in allopolyploids; (vi) a comparatively privileged occurrence in harsher environments when compared with their diploid relatives, and (vii) gene silencing and divergence of duplicated genes without disruption of duplicated loci. Research on polyploidy was highly biased towards plants during the last century because polyploidy in animals was for long time considered rare, occasional and irrelevant from an evolutionary perspective. However, as empirically observed in plants, genome rediploidization starts in polyploid organisms immediately after the polyploid shock. Given the speed and dynamicity of this process, evidence of genome multiplication is completely erased over time, and hence, only the most recent events are likely to be acknowledged. Although varying in expression between and within taxonomic groups, polyploidy and hybridization are ubiquitous in animals and may be recurrent, fostering evolution. Since evolutionary allopolyploid genomes behave as biologically diploid, zoologists have to challenge the old paradigm of an irrelevant evolutionary role in animals using current genomic and cytogenomic tools. These methods are most likely to reveal the role of polyploid mechanisms in producing evolutionary novelties. Nonsexual complexes are the perfect models to bridge the gap between empirical and theoretical research, while the evolutionary process is in action. Such animal complexes represent a transient stage that, in general, moves towards a polyploid stage, where bisexuality might be recovered, ultimately giving rise to a new gonochoric species. These pathways are herein illustrated by the Iberian allopolyploid Squalius alburnoides. Some general aspects on this fish's complex are updated and reviewed, namely the reproductive modes of the distinct genomotypes, since variable ploidies and genomic combinations occur in natural populations. Most recent data on the mechanisms of gene expression regulation and the importance of the genomic context in driving allelic expression are also included. It was first demonstrated that a regulatory mechanism involving dosage compensation by gene-copy silencing exists in allotriploid females and that allelic expression patterns differed either between genomically equivalent individuals or within the same individual (between tissues and genes). Thus, instead of a whole haplome inactivation, a biased silencing towards repression of a specific allele was observed as well as a reduction of the transcript levels to the diploid state. See also sister article focusing on plants by Tayalé and Parisod in this themed issue., (Copyright © 2013 S. Karger AG, Basel.)

Iberian Leuciscinae have been used in many studies as models to explore topics such as hybridization, allopolyploidy, modes of reproduction, and evolution. This article focuses on the contribution of cytogenomics to foster research in this group of cyprinid fish. Conventional and molecular banding results were reviewed, facilitating comparative analysis between nase and chub taxa inhabiting Portuguese freshwaters. Hybridization is known to occur within both Chondrostoma s.l. and Squalius genera although polyploidy has only been reported in the latter; the reasons behind such differential genome flexibility remain unidentified. FISH tools allowed recognizing additional chromosome markers, confirming NOR polymorphism and distinguishing species and their hybrids. Recombination and genome instability were detected in homoploid and polyploid hybrid genomes supporting active NOR transposition. However, the multiplication of rDNAs in these species does not seem to be associated with Rex3 retroelement, though hybrids were not surveyed. CGH and GISH allowed reaffirming the hybrid origin of S. alburnoides and confirming that the conservative karyotype patterns within Iberian leuciscines are restricted to the macrostructure. Current data also support the usefulness of mapping repetitive DNAs, especially for nonmodel compact genomes with less variable karyotypes and sequence data resources unavailable, like in many cyprinid lineages.

Karyotypes and chromosomal features of both minor and major ribosomal RNA genes (rDNA) were investigated in nine Iberian chondrostomine species by fluorescent in situ hybridization (FISH) with 5S and 45S rDNA probes. All species presented invariably diploid values of 2n = 50 and the characteristic leuciscin karyotype pattern with 6-7 metacentric (m), 15-16 submetacentric (sm) and 3-4 subtelo- to acrocentric (st/a) chromosome pairs. The largest chromosome pair of the set was st/a as typical of Leuciscinae and no heteromorphic chromosomes could be unequivocally associated to sex determination. Achondrostoma occidentale and Pseudochondrostoma willkommii were cytogenetically characterized for the first time while Achondrostoma arcasii and Iberochondrostoma lemmingii were revisited regarding previous karyotype descriptions. Remarkable variability in number and location was observed for both molecular chromosome markers, especially within Achondrostoma and Iberochondrostoma genera. Clusters of 5S rDNA were mostly terminally associated to st/a chromosomes varying from four to eight positive signals, whilst NOR sites directly detected by the 45S rDNA probe were identified in sm chromosomes varying from three to six independent clusters. Frequent population bottlenecks in Mediterranean-type semiarid habitats were hypothesized to explain not only such extensive polymorphism which seems unique among leuciscin cyprinids but also the increased probability of fixation of rDNA translocation events.

Genomic In Situ Hybridization (GISH) is a powerful tool to identify and to quantify genomic constituents in allopolyploids, and is mainly based on hybridization of highly and moderate repetitive sequences. In animals, as opposed to plants, GISH has not been widely used in part because there are technical problems in obtaining informative results. Using the allopolyploid Squalius alburnoides Steindachner, 1866 fish complex as a model system, we succeeded in overcoming methodological constraints when dealing with parental species with a small genome size. This hybridogenetic complex has biotypes with different genome compositions and ploidy levels, but parental chromosomes are small, morphologically very similar and therefore cannot be distinguished by conventional cytogenetic approaches. Specimens have a small genome (C-value1.2 pg) with a low level of highly and moderate repetitive sequences, mainly located at pericentromeric chromosome regions. Since it is well known that probe annealing depends on probe concentration and hybridization time to obtain uniform hybridization signals along the chromosome arms, we progressively increased the amount of labeled probes from 100ng up to 1µg and the incubation time from overnight up to 5 days. We also made other smaller improvements. Results showed a clear enhancement of signals with respect to previous data, allowing an accurate and reproducible assignment of the parental genomes in both diploid and triploid fish.It was thus evidenced that high probes' concentrations and long incubation time are the key to obtain, without extra image editing, uniform and reliable hybridization signals in metaphase chromosomes of animal hybrids from species with small genome size.

In the Squalius alburnoides fish complex, allotriploid females (3n = 75) reproduce mostly by meiotic hybridogenesis, producing haploid gametes by means of the elimination of the heterospecific chromosome set and recombination between the 2 homospecific genomes. A synaptonemal complexes (SCs) analysis was performed in specimens from a confined southern population (Quarteira, Portugal) to understand chromosome dynamics during gametogenesis. The comparative study between hybrid females with QAA genome composition and the parental bisexual species Squalius aradensis (2n = 50, QQ genome) evidenced: (i) that allotriploid meiocytes comprise the complete chromosome set (75 chromosomes) in prophase I, proving the heterospecific genome (Q) is only excluded after pachytene stage, and (ii) a 2-phase synaptic process where initially, exclusively homologous SCs form and the unmatched univalents remain in a bouquet conformation, followed by the establishment of extensive non-homologous SCs with multivalent associations among the later. These findings disagree with most literature concerning the meiotic process in allotriploid vertebrates, since the most accountable mechanisms (premeiotic exclusion of the unmatched chromosome set and whole genome endoduplication) were not observed., (Copyright © 2012 S. Karger AG, Basel.)

A non-intrusive method for individual identification of Iberian cyprinid complex Squalius alburnoides is presented, with possible application to other fish complexes and species. The proposed methodology is based on scale patterns that vary greatly between individuals.

The recent allocation of the cyprinid complex Squalius alburnoides to the genus Iberocypris in the Handbook of European Freshwater Fishes is refuted by confirming evidence of a close relationship with the genus Squalius. Squalius alburnoides is here defined as a complex based on the existence of a hybrid form with distinct genome combinations and ploidies and of a reconstituted nuclear non-hybrid male form also with hybrid origin.

Life-history variability of two non-native centrarchids, pumpkinseed Lepomis gibbosus and largemouth bass Micropterus salmoides, was evaluated in three stream stretches of the lower River Guadiana drainage (south-west Iberian Peninsula) with different degrees of regulated flows. Abundance, condition and population structure differed among populations for both species, but invasion success was lower in the least regulated river. Lepomis gibbosus were abundant and had multiple age classes in the three river sites, whereas M. salmoides were less abundant and mainly represented by young-of-the-year fish. Juvenile growth in L. gibbosus was similar in all three populations, though longevity was slightly greater in the population from the River Guadiana mainstream. Lepomis gibbosus exhibited a long reproductive season, but the duration of season, size at maturity and reproductive effort varied among populations. The life-history differences found demonstrate the importance of species adaptation to local conditions which might favour their invasion success. Lepomis gibbosus were more adaptable and resilient to local conditions, whereas M. salmoides seemed dependent on reservoirs and large rivers for maintenance of riverine populations.

Fish show an amazing variety of mechanisms of sex determination and modes of reproduction. Of these, simultaneous hermaphroditism has rarely been described. Here, we report a novel case of this phenomenon in adults of the Squalius alburnoides allopolyploid cyprinid complex. We found evidence for the simultaneous presence of mature male and female gonads on the basis of gonad gross morphology and histological analyses. Different stages of male and female germ cell maturation were identified, including motile spermatocytes. We hypothesize on the genetic and/or environmental causes of this phenomenon., (Copyright 2010 S. Karger AG, Basel.)

Pseudochondrostoma duriense and Pseudochondrostoma polylepis hybridize extensively with Achondrostoma oligolepis in natural populations. In this first survey, karyotypes were comparatively analyzed by C-, AgNOR- and CMA(3)-banding procedures in pure (non-introgressed) fish specimens. Leuciscinae pattern was evidenced in the three species: metacentrics and submetacentrics dominance, a big subtelo/acrocentric (marker) chromosome pair and a 2n = 50; small macro-structural differences were observed. Heterochromatin was centromere-associated. Exceptions were found at sm1 and st/a1 long arms and at m1, sm3 and sm6 short arms. The st/a1 band was telomeric in the straight-mouth nases and sub-terminal in A. oligolepis. Multiple NORs of heterochromatic nature were found in sm pairs of the three species. Signals were telomeric except for one pair in A. oligolepis. Two to four structural and two functional NORs were found in P. duriense and P. polylepis, and four to six structural and four functional NORs in A. oligolepis. Species-specific markers will prove useful in hybrid zones' cytogenetic characterization and for in-depth studies of genome compatibility-related issues in future studies.

This study aimed to define the karyotype of the recently described Iberian endemic Iberochondrostoma almacai, to revisit the previously documented chromosome polymorphisms of its sister species I.lusitanicum using C-, Ag-/CMA(3) and RE-banding, and to compare the two species genome sizes. A 2n = 50 karyotype (with the exception of a triploid I.lusitanicum specimen) and a corresponding haploid chromosome formula of 7M:15SM:3A (FN = 94) were found. Multiple NORs were observed in both species (in two submetacentric chromosome pairs, one of them clearly homologous) and a higher intra and interpopulational variability was evidenced in I.lusitanicum. Flow cytometry measurements of nuclear DNA content showed some significant differences in genome size both between and within species: the genome of I. almacai was smaller than that of I.lusitanicum (mean values 2.61 and 2.93 pg, respectively), which presented a clear interpopulational variability (mean values ranging from 2.72 to 3.00 pg). These data allowed the distinction of both taxa and confirmed the existence of two well differentiated groups within I. lusitanicum: one that includes the populations from the right bank of the Tejo and Samarra drainages, and another that reunites the southern populations. The peculiar differences between the two species, presently listed as "Critically Endangered", reinforced the importance of this study for future conservation plans.

Leuciscine cyprinids possess a nearly invariant diploid number (2n = 50) with an extremely uniform karyotype comprising of 8 pairs of metacentric, 13-15 pairs of submetacentric and 2-4 pairs of subtelocentric (st) to acrocentric (a) chromosomes. The largest pair is characteristically an st/a element-the 'leuciscine' cytotaxonomic marker. Previously, the interspecific homology of this chromosome pair could not be assessed owing to the inability to produce euchromatic or serial banding patterns. In the present study, we used laser-microdissection (15-20 copies of the marker chromosome) to construct a whole chromosome probe (WCP) from the marker chromosome of the roach Rutilus rutilus to ascertain the interspecific homology of marker chromosomes by cross-species in-situ hybridization. WCP was hybridized to chromosomes of widely distributed (Abramis brama, Alburnoides bipunctatus, Alburnus alburnus, Aspius aspius, Ballerus ballerus, B. sapa, Blicca bjoerkna, Chondrostoma nasus, Leucaspius delineatus, Leuciscus leuciscus, L. idus, R. rutilus, Scardinius erythrophthalmus, Squalius cephalus, and Vimba vimba) and Iberian endemic species (Achondrostoma oligolepis, Iberochondrostoma almacai, I. lusitanicum, Pseudochondrostoma duriense, S. alburnoides and S. pyrenaicus). Cross-species in-situ hybridization to chromosomes of Phoxinus phoxinus, a representative of leuciscine sister lineage, showed the same pattern as in all of the leuciscins. The probe consistently hybridized to the distal part of the short arm of the marker chromosome, indicating sequence homology.

Squalius alburnoides is a widely distributed intergeneric hybrid complex with fish of both sexes, varying ploidy levels and proportions of the parental genomes. Its dispersal routes were here delineated and framed by the reconstruction of the phylogeny and phylogeography of other Squalius with which it hybridizes, based on the available data on the paleohydrographical history of the Iberian Peninsula. Results based on sequences of cytochrome b and beta-actin genes showed that: proto-Squalius pyrenaicus originated at least five species as it dispersed throughout the Iberian Peninsula in the Mio-Pliocene; the S. alburnoides complex likely had a single origin in the bulk of Iberia, in the Upper Tagus/Guadiana area, when hydrographical rearrangements allowed the contact between its ancestors (around 700,000 years ago); interspecific crosses allowed the introgression of mitochondrial and nuclear genes of S. alburnoides in allopatric species/populations of other Squalius and vice-versa; and reconstituted S. alburnoides non-hybrid males may contribute to the replacement of the typical mtDNA of the complex (in the populations where they occur, crosses with females of other Squalius seem to have been especially frequent). A number of dispersal events and colonization routes are proposed.

The endemic Iberian minnow Squalius alburnoides is a complex of fishes of hybrid origin including both males and females with distinct ploidy levels and varying proportions of the parental genomes. In this paper we demonstrated that in contrast to many vertebrate hybrid lineages the sperm of triploid hybrid males of S. alburnoides is viable and fully functional. Flow cytometry and analysis of sequences of a fragment of the beta-actin nuclear gene applied to progenitors and offspring evidenced that these males produced their sperm clonally, as already described for diploid hybrids. The presence of different types of fertile males (nonhybrid diploids with normal meiosis and both diploid and triploid hybrids) coupled with hybridogenetic meiosis in females endows this vertebrate complex with a high level of independence from other species and contributes to maintain its genetic variability.

The karyotype of the endangered fish Anaecypris hispanica was revisited using advanced cytogenetic techniques to elucidate its putative relationship with the paternal ancestor of the hybrid complex Squalius alburnoides and to clarify some of the recently described cytogenetic patterns of the complex. The results of chromomycin A3 and Ag staining, as well as fluorescent in situ hybridization with 28S and 5S rDNA and the (TTAGGG)n telomeric probes, were compared with the patterns observed in specimens of the all-male nonhybrid lineage of S. alburnoides complex, which is considered to reconstitute the nuclear genome of the probably extinct paternal ancestor. Several cytogenetic features observed in A. hispanica specimens were indeed shared by S. alburnoides nuclear nonhybrid males, supporting the hypothesis of a close evolutionary link between A. hispanica and the paternal ancestor of the complex. The genomic rearrangements involving 28S rDNA sites previously described in the S. alburnoides complex and in its maternal ancestor (S. pyrenaicus) were not detected in A. hispanica; they are, therefore, probably due to mechanisms related to hybridization and polyploidy.

The diploid-polyploid Squalius alburnoides complex resulted from interspecific hybridization. The chromosomal mapping of 28S and 5S ribosomal genes and of (TTAGGG)n telomeric repeats was performed on specimens from the complex and from the sympatric bisexual species S. pyrenaicus (the complex maternal ancestor) as part of an investigation of the evolutionary relationships between genomic constitutions and the consequences of the ongoing polyploidization process in terms of chromosome reshaping. Contrasting results were obtained. While results with 5S rDNA and telomeric probes gave an impression of genomic stability, the variability detected with 28S rDNA probe suggested quite the opposite. The 5S rDNA probe mapped constantly to three chromosomes per haploid genome with apparently conserved locations in morphologically similar chromosomes; conversely, prominent intra- and inter-individual variations of 28S rDNA and of syntenic sites with 5S rDNA were detected with regard to number, size and location. Hypotheses for the causes of such polymorphisms are discussed. The terminal position of most 28S rDNA sites and the absence of detectable interstitial telomeric sequences suggest a mechanism that does not involve major chromosomal rearrangements. These fishes share similar patterns for the studied cytogenetic markers which may be taken as evidence of an apparent stability that may be hiding extensive and subtle genome variations that are possibly related to an ongoing evolutionary process of genome tetraploidization and speciation.

In this paper we describe a simple approach using double peaks in chromatograms generated as artefacts in the vicinity of heterozygous indels, to identify the specific sequences present in individual strands of a given DNA fragment. This method is useful to assign bases in individuals that are heterozygous at multiple sites. In addition, the relative sizes of the double peaks help to determine the ploidy level and the relative contribution of the parental genomes in hybrids. Our interpretation was confirmed with the analysis of artificial mixtures of DNA of two different species. Results were robust with varying PCR and sequencing conditions. The applicability of this method was demonstrated in hybrids of the Squalius alburnoides complex and in heterozygotes of Chondrostoma oligolepis. Far from being limited to these fish models and the gene where it was tested (beta-actin), this sequence reconstruction methodology is expected to have a broader application.

Despite the growing outcome of results that put doubt upon the reliability of silver (Ag) staining and chromomycin A3 (CMA3) fluorescent banding in the detection of major ribosomal gene sites (NORs), these methods have been widely used, especially in fishes. In order to clarify the previous patterns obtained with those techniques, we performed fluorescence in situ hybridisation (FISH) with 28S rDNA probe followed by sequential CMA3 and Ag staining in diploid non-hybrid males of the Squalius alburnoides complex and in Squalius pyrenaicus. The results from all the studied specimens revealed a lack of correlation between classical and molecular techniques. Not just some other regions besides NORs were stained with CMA3 and Ag, but also the majority of the 28S rDNA sites were not detected. Care should then be taken in considering CMA3- and Ag-stained sites as NORs since their accuracy for that purpose may not always correspond to the expectations., (Copyright 2005 S. Karger AG, Basel)

Chromosomal locations of major ribosomal sites, i.e. NOR-phenotypes, were assigned in Squalius alburnoides complex using sequential chromomycin A3 (CMA3)- and silver (Ag)-staining. This hybridogenetic Iberian minnow comprises diploid, triploid and tetraploid forms that arose by interspecific hybridisation between S. pyrenaicus and an unknown species. Inheritance of NOR patterns was studied by means of crossing experiments involving most diploid-polyploid forms of the S. alburnoides complex with identified specific genotype constitution. In all the specimens studied, the NORs were localised in the short arms of submetacentric chromosomes. Although S. pyrenaicus presented only one pair of NOR-bearing chromosomes, the data from experimental crosses evidenced that S. alburnoides complex was characterised by a multiple NOR phenotype composed of one chromosome pair with stable NORs and two chromosome pairs with NOR site polymorphism of presence/absence type. These data suggest that the karyotype of the unknown parental species of the S. alburnoides complex should have a multiple NOR pattern and emphasised the role of the all-male diploid linage in the dynamics and evolutionary potential of the S. alburnoides complex allowing the preservation of the missing ancestor genome. Cross-analyses evidenced that in spite of the high polymorphic nature of NORs in this fish complex, we have no reason to reject the hypothesis that their inheritance patterns were in accordance with Mendelian segregation.

The hybrid minnow Squalius alburnoides comprises diploid and polyploid forms with altered modes of reproduction. In the present paper, we report a cross where a triploid female generated both large, triploid and small, haploid eggs simultaneously, which were fertilized with S. pyrenaicus sperm. Although the large eggs were rarer (15%), they originated offspring with higher survivorship, so that tetraploids were dominant among the surviving siblings. The cross yielded apparently all female progeny. Inheritance patterns were inferred using four microsatellite markers and NORs (Nucleolus Organizer Regions) phenotypes, and suggested that haploid eggs were probably produced by an atypical hybridogenesis, in which the elimination of the unmatched genome permitted random segregation and recombination between the homospecific genomes, while the triploid eggs were clonal. The present results suggest that the occurrence of triploid unreduced eggs may be a new route for the natural tetraploidization in the complex., (Copyright 2004 Wiley-Liss, Inc.)

Two species of Odontocheila, O. confusa and O. nodicornis, from the Neotropical Region were studied regarding their karyotypes, localisation and activity of ribosomal genes and C-banding. The species, although belonging to the same genus, have quite distinct karyotypes. O. confusa has 10 pairs of autosomes and a single sex chromosome mechanism of the XY/XX type, thus a diploid value of 2n = 22 in males and females. One aneuploid male with a diploid number of 2n = 20 and one male with three B chromosomes were found in a total of eight males studied. O. nodicornis has 17 autosomal pairs and also a single chromosome system but of the X0/XX type, thus a diploid value of 2n = 35 in males and 2n = 36 in females. Fluorescence in situ hybridisation (FISH) revealed the presence of rDNA clusters in two autosomes in both species in mitotic and meiotic figures. Silver staining of male interphase nuclei confirmed the FISH results and showed that all rDNA genes were active. C-banding analysis revealed the presence of constitutive heterochromatin in the centromeres of all chromosomes in the two species plus two pairs in O. nodicornis with terminal positive C-bands. These results are discussed from the cytogenetic and evolutionary point of view.

Evidence is accumulating indicating the importance of antioxidant enzyme activity measurements in eco-toxicological studies, as they may constitute markers for exposure to a large variety of pollutants. Variation of antioxidant enzymes, superoxide dismutase (SOD) and glutathione S-transferases (GST) and the effect of heavy metals and selenium exposure on these enzymes were investigated in the livers of Iberian endemic minnows (Leuciscus alburnoides complex) captured in a copper (Cu) mining area. Higher hepatic levels of copper and selenium were always observed in fish captured at the polluted site relative to the reference area population, reflecting the environmental monitoring results. A seasonal fluctuation in zinc and selenium levels for both populations was also observed which could be related to gonad maturation. The activity of SOD did not show significant regional alterations, but a seasonal variation occurred presumably associated with the Leuciscus life cycle. The GST activity was higher in the fish population from the polluted area (except in spring) and GST variability was associated with selenium and copper levels when both regions were compared. The increased GST activity was probably a metabolic adaptation to the continuous exposure to higher levels of those elements.

Anaecypris hispanica is a cyprinid fish which is endemic to the Guadiana River basin in the Iberian Peninsula, and whose abundance and geographical range have contracted considerably during the last 20 years. We investigated mitochondrial DNA cytochrome b and control region variation among specimens representative of nine tributaries, using direct sequencing and diagnostic restriction fragment length polymorphism. The samples from the Caia, Degebe, Ardila, and Odeleite rivers exhibited haplotypes that differed by a large number of site differences, which may be indicative of population bottlenecks that have caused stochastic extinction of haplotypes. In contrast, the populations from the Xévora, Chança, Carreiras, Vascão and Foupana rivers exhibited low levels of nucleotide diversity, which together with high haplotype diversity may also be indicative of genetic bottleneck events, with subsequent population expansion. Phylogenetic analyses, a minimum spanning network, and an analysis of molecular variance revealed geographical structuring, suggesting limited or no gene flow between populations. The populations from extreme southern rivers (Foupana and Odeleite) are monophyletic entities, suggesting that they have been isolated, probably as a consequence of brackish water upstream of their confluence with the Guadiana. The results suggest that the Foupana and the Odeleite populations, and the remaining northern populations altogether should be managed as three distinct Evolutionary Significant Units (ESUs). Within the northern ESU, four Management Units (MUs) should be considered.

The Iberian minnow Leuciscus alburnoides represents a complex of diploid and polyploid forms with altered modes of reproduction. In the present paper, we review the recent data on the origin, reproductive modes, and inter-relationships of the various forms of the complex, in order to predict its evolutionary potential. The complex follows the hybrid-origin model suggested for most other asexual vertebrates. Diploid and triploid females from the southern river basins exhibit reproductive modes that cannot be conveniently placed into the categories generally recognised for these vertebrate complexes, which imply continuous shifting between forms, where genomes derived from both parental ancestors are cyclically lost, gained or replaced. Replacement of nuclear genomes allow the introduction of novel genetic material, that may compensate for the disadvantages of asexual reproduction. Contrasting with most other vertebrate complexes, L. alburnoides males are fertile and play an important role in the dynamics of the complex. Moreover, diploid hybrid males may have initiated a tetraploidization process, when a diploid clonal sperm fertilised a diploid egg. This direct route to tetraploidy by originating fish with the right constitution for normal meiosis (symmetric), may eventually lead to a new sexually reproducing polyploid species. This case-study reinforces the significance of hybridisation and polyploidy in evolution and diversification of vertebrates.

Chromosomal polymorphism regarding the number of chromosomal NOR sites in the cyprinid fish Chondrostoma lusitanicum reported previously (Rodrigues & Collares-Pereira, 1996) was re-examined using fluorescence in situ hybridization (FISH) with a ribosomal DNA (rDNA) probe. All positive CMA3-bands contained ribosomal DNA documented by either two or four FISH positive signals in the respective karyotypes. This polymorphism suggests the occurrence of structural rearrangements of translocation type in rDNA region from one ancestral NOR-bearing chromosome pair ubiquitous among leuciscine cyprinid fishes to another pair. The absence of individuals heterozygous for this polymorphism is discussed.

The hybrid minnow Rutilus alburnoides comprises diploid and polyploid females and males. Previous studies revealed that diploid and triploid females exhibit altered oogenesis that does not involve random segregation and recombination of the genomes of the two ancestors, constituting unisexual lineages. In the present study, we investigated the reproductive mode of hybrid males from the Tejo basin, using experimental crosses and flow cytometric analysis of blood and sperm. The results suggest that diploid hybrids produced fertile unreduced sperm, transmitting their hybrid genome intact to offspring. Triploid hybrids also produced unreduced sperm, but it was not possible to obtain data concerning their fertility. Finally, tetraploid hybrids produced fertile diploid sperm, which exhibited Mendelian segregation. Tetraploid R. alburnoides may reestablish biparental reproduction, as individuals of both sexes with the appropriate constitution for normal meiosis (two haploid genomes from each parental species) are likely to occur in natural populations. Tetraploids probably have arisen from syngamy of diploid eggs and diploid sperm produced by diploid hybrid males. Diploid hybrid males may therefore play a significant role in the dynamics of the complex, starting the evolutionary process that may ultimately lead to a new sexually reproducing species.

Cytochrome b (cyt b) sequences from specimens of the Rutilus alburnoides unisexual complex and five bisexual species were compared to examine hypotheses regarding the origin and maternal ancestry of this complex. Phylogenetic analysis revealed a monophyletic relationship among unisexuals and Leuciscus pyrenaicus, clearly identifying this species as the maternal ancestor. Considerable mtDNA diversity exists among R. alburnoides populations, with many localities exhibiting unique haplotypes. The topology recovered from analysis of cyt b variation among populations suggested that R. alburnoides is polyphyletically derived from their sympatric L. pyrenaicus populations, indicating that unisexual lineages have been generated through multiple hybridization events. Although much less abundant, R. alburnoides is present outside the range of L. pyrenaicus, suggesting that it may have dispersed from the Tejo drainage into the northern basins. In this region, Leuciscus carolitertii is most likely the sexual host for the unisexual complex., (© 1997 The Society for the Study of Evolution.)

Cellular suspensions of germinal tissues of Cylindera trisignata provided the definition of its karyotype: 2n = 23 and 2n = 26 for the males and females respectively. This Palearctic species has a sex chromosome system of the X1X2X3X4Y/X1X1X2X2X3X3X4X4 type, only found until now in Cicindela maroccana pseudomaroccana. The heterosomes are not well-differentiated from the general morphological standpoint. To explain the origin of the 4X condition, a mechanism of dissociation of the X chromosomes rather than an incorporation of autosomal segments is proposed. However, based on the occurrence of distinct ploidy levels, both in male and female cells, with a relatively high incidence for the tetraploid condition with regular autosomal bivalents, the hypothesis of a possible evolutionary role of polyploidy is suggested.