Contrasting views over a hybrid complex: Between speciation and evolutionary "dead-end"

The Squalius alburnoides complex, of hybrid origin, comprises diploid, triploid and tetraploid forms and has a widespread distribution in the Iberian waters. The southern populations of this complex, sympatric with S. pyrenaicus, show high genetic variability, diversity of forms and reproductive modes which create pathways that may allow for the establishment of a new species in the future. Here we report a contrasting view over the S. alburnoides complex: in the Mondego River basin (northern Portugal), nuclear "non-hybrid" and tetraploid forms are absent and a clearly impoverished genetic diversity is observed, contributing to a general scarcity of possibilities of generating novel genetic material. Moreover, the bisexual species involved in the maintenance of the complex in this basin (S. carolitertii) exhibits a considerably lower genetic variability, when compared with S. pyrenaicus. The observed differences suggest that, despite being originated by similar hybridization events and maintained by analogous reproductive mechanisms, different populations of the complex were exposed to distinct evolutionary constrains, which in some cases resulted in diversification and speciation while in others led to a compromising situation in terms of evolutionary potential. Additionally, and for the first time all forms were used in the calculation of genetic distances and diversity indices, widening the possibilities of analysis of the complex by allowing the inclusion of a large part of the available data, irrespective of ploidy level.     

Phylogeographical insights into the origins of the Squalius alburnoides complex via multiple hybridization events

The origin, the phylogeographical structure and divergence times of hybridrogenetic Squalius alburnoides complex were analysed based on the complete mitochondrial cytochrome b gene (1140 pb). The molecular phylogenetic analyses suggest that the S. alburnoides complex has at least five asexual lineages of independent origin. The events that produced this ancestral hybridization took place over a long period of time. There have been multiple hybridization events throughout time, beginning in the upper Pliocene and probably continuing into the present. Increased humidity caused by climate changes in the Pliocene, along with tectonic lifting and vasculation of the Iberian Peninsula, led to the formation of current river drainages which, in turn, contributed to these hybridization events. We postulate that the Northwestern (Mondego and Douro) and the Southwest (Quarteira) drainages of the Iberian Peninsula delimited the border of the maternal ancestral distribution and that vicariant events led to the disappearance of the maternal ancestor in these regions, leaving today only the hybrid species. Two hypotheses have been suggested to explain the similarities between the mtDNA diversity observed in S. alburnoides and its maternal ancestor (S. pyrenaicus). The first hypothesizes that mtDNA similarity results from the recent extinction of the paternal ancestor, while the other postulates that: 'reconstituted non hybrid males' assumed the place of the extinct bisexual paternal ancestor and produced new hybridizations with S. pyrenaicus females.     

Cytogenetic analysis of Anaecypris hispanica and its relationship with the paternal ancestor of the diploid-polyploid Squalius alburnoides complex.

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.     

Fertile triploid males: an uncommon case among hybrid vertebrates

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 genetics of maintenance of an all-male lineage in the Squalius albuvnoides complex

Squalius alburnoides is a complex of minnows common to the Iberian Peninsula, consisting of two distinct forms. The most common form is comprised of diploid and polyploid asexual hybrids heterozygous for several diagnostic allozyme loci contributed by Squalius pyrenaicus or Squalius carolitertii and a missing ancestor. The other form is diploid and homozygous for the allele contributed by the missing ancestor at these same loci. Present results from analyses of sex ratio and cytochrome b sequences are not consistent with the evolutionary distinctiveness of this non-hybrid form and suggest that it represents an all-male lineage imbedded within an almost all-female complex. This all-male lineage allowed preservation of the ancestral paternal nuclear genome after the paternal ancestor became extinct in all or most drainages, withimportant evolutionary implications.     

Reading the history of a hybrid fish complex from its molecular record

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.     

Speciation towards tetraploidization after intermediate processes of non-sexual reproduction

Polyploidy, hybridization and variation in mating systems are central issues for a deeper understanding of animal evolution. The Iberian species Squalius alburnoides represents an example combining all three phenomena. Previous studies showed that S. alburnoides populations are mainly composed of triploid and diploid hybrid forms (mainly females), and that the tetraploid forms are rare or absent. Both populations from the Douro drainage reveal a distinct scenario: tetraploid individuals represent 85.6-97.5% of the population, with no sex ratio bias observed. Based on the flow cytometry measurements of blood and spermatozoa cells, microsatellite loci and experimental crosses, we describe here, for the first time, two symmetric allotetraploid populations (CCAA) that resumed normal meiosis after undergoing intermediate processes of non-sexual reproduction to give rise to a new sexually reproducing polyploid species. Prezygotic (habitat selection and assortative mating) and postzygotic mechanisms (nonviable embryos) are responsible for the reproductive isolation from other forms of the S. alburnoides complex (e.g. CA, CAA). This example illustrates how hybrid polyploid complexes may lead to speciation.     

Hybridogenetic Reproduction and Maternal Ancestry of Polyploid Iberian Fish: The Tropidophoxinellus Alburnoides Complex

Iberian minnows collectively known as the Tropidophoxinellus alburnoides STEINDACHNER complex comprise diploid and polyploid forms with highly female biased sex ratios. Previous investigators suggested that all-female clonal reproduction and interspecific hybridization may occur in this complex. We examined nuclear (allozymes) and cytoplasmic genes (mtDNA) to assess the evolutionary origins, relationships, and reproductive modes of T. alburnoides from western Spain. The multi-locus allozyme data clearly revealed the hybrid nature of all polyploid forms of this fish and some diploid forms as well. Diagnostic markers identified fish from the genus Leuciscus as the paternal ancestor of hybrids in the Duero and Guadiana River Basins. Additionally, analysis of nuclear markers revealed that hybridogenetic reproduction occurs in the diploid and triploid hybrids. The hybrids fully express the paternal Leuciscus genome and then discard it during oogenesis. Hybridogenetic ova contain only maternal nuclear genes and mtDNA from a non-hybrid T. alburnoides ancestor. Apparently diploid and triploid hybrids of T. alburnoides persist as sperm parasites on males of a sexually reproducing Leuciscus host species.     

Evolution in action through hybridisation and polyploidy in an Iberian freshwater fish: a genetic review

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.     

The evolutionary history of the allopolyploid Squalius alburnoides (Cyprinidae) complex in the northern Iberian Peninsula

Understanding the population structure, population dynamics and processes that give rise to polyploidy and helps to maintain it is central to our knowledge of the evolution of asexual vertebrates. Previous studies revealed high genetic diversity and several reproductive pathways in the southern populations of the Squalius alburnoides hybrid complex. In contrast, lower genetic variability and the associated limited chance of introducing new genetic combinations may threaten the survival of the northern Mondego populations. We analysed the genetic diversity and structure of nine populations of S. alburnoides in the Iberian Peninsula using microsatellite loci to provide further insights on the evolutionary history of this complex. Special attention was given to the less-studied northern populations (Mondego and Douro basins). Marked population structure, a high frequency of private alleles and a high diversity of some biotypes in the Douro basin indicate that some northern populations may not be at high risk of extinction, contrary to what was expected. The genetic diversity found in the northern Douro populations contradicts the general trend of remarkable genetic impoverishment northwards that occurs in other species and regions. The results indicate the possible existence of a glacial refugium in the Rabaçal River, corroborating findings in other species of this region. Historical events seem to have affected the geographical patterns of genetic variability found among and within the northern and southern populations of this complex and contributed to different patterns of genome composition. Therefore, historical events might have a major role in the long-term persistence of some polyploid hybrid taxa.     

Evolutionary and biogeographical patterns within Iberian populations of the genus Squalius inferred from molecular data

Origins and biogeography of the chub, genus Squalius (formerly within the genus Leuciscus), in the Iberian Peninsula were inferred from comparison between patterns of geographic distribution and phylogenetic relationships among populations belonging to 14 European Squalius species. The phylogeny recovered was based on the complete sequence of the mitochondrial cytochrome b gene. Squalius species were grouped into three major clades. The basal clade included species distributed across the ancient Paratethys Sea. The second clade included species from Central and East Europe and the northern areas of the Mediterranean basin towards Minor Asia. The third clade included species from the Mediterranean Peninsulas (Iberian, Italy, and Balkans). The Iberian Squalius species do not constitutes a monophyletic group. Our data indicate that the Iberian Peninsula was colonized at least twice by two different monophyletic lineages, a meridional group and a Central Europe group. The amount of species diversity found in the Iberian Peninsula and the phylogenetic relationships among these species, together with their geographic distribution, suggest that the Central Europe lineage colonized the Iberian Peninsula at a latter time. Our data indicate that the northeastern Iberian lineage is phylogenetically close to Greek populations of Squalius cephalus, while the second lineage formed a monophyletic group including Squalius pyrenaicus, Squalius carolitertii, Squalius aradensis, and Squalius torgalensis. The speciation process that generated these species and the geographic structure of their populations, principally in S. pyrenaicus, can be attributed to paleogeographical events like the ancient endorrheism and the development of hydrographic basins.     

Variability in diet and foraging behaviour between sexes and ploidy forms of the hybridogenetic Squalius alburnoides complex (Cyprinidae) in the Guadiana River basin, Portugal

Samples of the Squalius alburnoides complex, a hybridogenetic Iberian minnow composed of diploid, triploid and, less commonly, tetraploid forms, were collected from a tributary of the Ardila River (Guadiana River basin) between February 1999 and January 2001. Seasonal variation in diet was evident, and was probably linked to prey availability. Distinct foraging behaviours between ploidy forms were found towards several prey items, suggesting that diploid adult males fed mostly near the surface, whereas diploid adult females tended to feed near the bottom and submerged vegetation. Triploid females exhibited an intermediate foraging behaviour, although there was greater affinity towards diploid male feeding behaviour. Diploid males which, in contrast to diploid and triploid females, have non‐hybrid genomes in the Guadiana drainage, exhibited a higher specialization for food. Despite considerable dietary overlap, there appeared to be spatial segregation of feeding niches between the three forms, especially during dry periods when prey availability was lower, which may be a strategy for diminishing competition for food. Therefore, considering asexual generalist and specialist hypotheses, it appears that the different ploidy levels are generalist, opportunistic feeders that partition the resources when limited.     

Evidence of extensive mitochondrial introgression with nearly complete substitution of the typical Squalius pyrenaicus‐like mtDNA of the Squalius alburnoides complex (Cyprinidae) in an independent Iberian drainage

The first occurrence of massive mitochondrial introgression of Squalius aradensis genes in Squalius alburnoides , a hybridogenetic complex that usually carries mtDNA of its maternal ancestor ( Squalius pyrenaicus ) is reported. Possible implications of such introgressions for the history of the complex are discussed.     

A polymorphic microsatellite from the Squalius alburnoides complex (Osteichthyes, Cyprinidae) cloned by serendipity can be useful in genetic analysis of polyploids

A new microsatellite locus (SAS1) for Squalius alburnoides was obtained through cloning by serendipity. The possible usefulness of this new species-specific microsatellite in genetic studies of this hybrid-species complex, was explored. The polymorphism exhibited by SAS1 microsatellite is an important addition to the set of microsatellites previously used in genetic studies in S. alburnoides complex, that mostly relied in markers described for other species. Moreover, the SAS1 microsatellite could be used to identify the parental genomes of the complex, complementing other methods recently described for the same purpose..     

Phylogeography of the cyprinid Squalius aradensis and implications for conservation of the endemic freshwater fauna of southern Portugal

The Iberian cyprinid fauna, characterized by the presence of numerous endemic species, has suffered from significant habitat degradation. The critically endangered Squalius aradensis is restricted to small drainages of southern Portugal, habitats that typically exhibit a characteristic Mediterranean-type heterogeneous hydrological system throughout the year, including alternation of flooding events during winter and complete drought in large river sections during summer. To assess the effect of historical and recent processes on genetic diversity in S. aradensis we examined within- and among-population variability in cytochrome b and six polymorphic microsatellite loci. Estimates of genetic diversity in time and space through the combined use of traditional Phi-/F-statistics, phylogenetic trees, ordination methods and nested clade analysis indicated significant and congruent structuring among populations. Data suggest that the Arade drainage represent the evolutionary centre of the species, with subsequent allopatric fragmentation across drainages. Factors other than isolation by distance strongly affected the within-drainage genetic differentiation observed in these Mediterranean-type drainages, including recent population expansion from a bottleneck event and restricted gene flow imposed by a long-term barrier (brackish water area). Significant correlation was found between S. aradensis allelic diversity and upstream drainage area. The relevance of findings for conservation issues is discussed in relation to local intermittent hydrological conditions, the highly restricted distribution and the critically endangered status of the species.     

Simultaneous production of triploid and haploid eggs by triploid Squalius alburnoides (Teleostei: Cyprinidae)

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.     

Gene expression regulation and lineage evolution: the North and South tale of the hybrid polyploid Squalius alburnoides complex

The evolution of hybrid polyploid vertebrates, their viability and their perpetuation over evolutionary time have always been questions of great interest. However, little is known about the impact of hybridization and polyploidization on the regulatory networks that guarantee the appropriate quantitative and qualitative gene expression programme. The Squalius alburnoides complex of hybrid fish is an attractive system to address these questions, as it includes a wide variety of diploid and polyploid forms, and intricate systems of genetic exchange. Through the study of genome-specific allele expression of seven housekeeping and tissue-specific genes, we found that a gene copy silencing mechanism of dosage compensation exists throughout the distribution range of the complex. Here we show that the allele-specific patterns of silencing vary within the complex, according to the geographical origin and the type of genome involved in the hybridization process. In southern populations, triploids of S. alburnoides show an overall tendency for silencing the allele from the minority genome, while northern population polyploids exhibit preferential biallelic gene expression patterns, irrespective of genomic composition. The present findings further suggest that gene copy silencing and variable expression of specific allele combinations may be important processes in vertebrate polyploid evolution.     

Reassessment of the generic position of the Iberian cyprinid alburnoides complex: its return to the genus Leuciscus

The generic position of the Iberian unisexual fish complex alburnoides is reassessed based on genetic evidence of a hybrid ancestry and of a monophyletic relationship with endemic Leuciscus taxa. There is a continuous movement of genes between the different forms of the complex and the main mode of reproduction is by meiotic hybridogenesis. Thus its return to the original generic position Leuciscus is recommended. © 1999 The Fisheries Society of the British Isles     

Polymorphism of major ribosomal gene chromosomal sites (NOR-phenotypes) in the hybridogenetic fish <Emphasis Type="Italic">Squalius alburnoides</Emphasis> complex (Cyprinidae) assessed through crossing experiments

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. alburnoidescomplex 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. alburnoidescomplex 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.alburnoidescomplex 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.     

Diploids v . triploids of Rutilus alburnoides: spatial segregation and morphological differences

Rutilus alburnoides complex is a common and widely distributed Iberian cyprinid, whose natural populations include mainly diploid and triploid forms. The Guadiana populations of R. alburnoides were studied to determine whether habitat segregation and morphological differences exist between these forms. The ploidy level of each specimen was determined by measuring erythrocyte DNA content using flow cytometry. Evidence of spatial segregation between diploid males and the two female forms was found. Diploid males were best represented in the River Degebe, which was shallow, with higher temperatures (especially during the spring and summer), and silt and sandy substrate. Diploid females were found in deeper water, on steeper gradients and coarse substrata, while triploid females preferred higher current velocity and a high proportion of instream cover, especially during the spring. The ecological differences may reduce competitive interactions, and should promote a stable coexistence of the different forms. Morphological distinction between fish of different ploidy levels was not established, but differences were found between the males and females. Discriminant analysis allowed, with a 10% error, the separation of both sexes through six morphological characteristics that could be recorded in the field.