M. M. Coelho

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.

Benthic macroinvertebrate communities of intermittent streams in the middle reaches of the Guadiana Basin (Portugal).

The Guadiana River has an irregular hydrological regime, with severe droughts and floods, but little is known about how aquatic fauna respond to these natural events. Macroinvertebrate data and environmental information were collected at seven sites from three tributaries in the middle reaches of the Guadiana River, approximately every 3 months from April 1995 to April 1997. Despite considerable annual variation in discharge (related to duration of flood and drought periods), the number of macroinvertebrates found was consistently high. Diptera represented the major proportion of the benthic fauna (73.2%) followed by Ephemeroptera (10.3%), Coleoptera (4.1%) and Trichoptera (3.1%). Canonical correspondence analysis (CCA) was used to evaluate the relationships between taxa density and habitat variables. Generally, Plecoptera and Ephemeroptera were found in the upstream sampling sites. Wider and deeper sites were associated with the presence of Diptera and were least diverse. High values for both the Shannon-Wiener diversity index and the average score per taxon were usually found at upstream sites where Ephemeroptera and Plecoptera predominated. The data suggest that macroinvertebrates have a great capacity to recover rapidly from severe drought periods, both in terms of taxonomic diversity and number of individuals.

Microsatellite analysis of genetic population structure of the endangered cyprinid Anaecypris hispanica in Portugal: implications for conservation

Abstract The endangered fish species Anaecypris hispanica is restricted to eight disjunct populations in the Portuguese Guadiana drainage. The genetic structure of these populations was studied in order to determine levels of genetic variation within and among populations and suggest implications for conservation of the species. Based on five microsatellite loci, the null hypothesis of population homogeneity was tested. Tests for genetic differentiation revealed highly significant differences for pairwise comparisons between all populations, and substantial overall population subdivision ( F ST =0.112). All sampled populations contained unique alleles. Our findings indicate marked genetic structuring and emphasise limited dispersal ability. The high levels of genetic diversity detected within and among A. hispanica populations suggest, however, that the observed fragmentation and reduction in population size of some populations during the last two decades, has impacted little on levels of genetic variability. Data imply that most A. hispanica populations should be managed as distinct units and that each has a high conservation value containing unique genetic variation. It is argued that geographic patterns of genetic structuring indicate the existence of eight management units.

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 Φ‐/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.

Dosage Compensation by Gene-Copy Silencing in a Triploid Hybrid Fish

In mammals, the increase in gene dosage, in the form of polyploidy or involving chromosomal fragments, has deleterious effects [1]. Regulation of appropriate gene product amounts has to be warranted by complex dosage-compensation mechanisms. Lower vertebrates, on the other hand, cope very well with ploidy increase [2-4], implying either effective compensation or a lack of necessity for such mechanisms. Unfortunately, nothing is known about the genetic and molecular mechanisms underlying this phenomenon. For an experimental approach, we have studied gene expression in the allotriploid form of Squalius alburnoides. In these organisms, different genomes are joined through hybridization; thus, sequence differences can be used to follow expression of different alleles [5, 6]. We found that a compensation mechanism exists, reducing transcript levels to the diploid state. Our data suggest a silencing of one of the three alleles. Unexpectedly, it is not a whole haplome that is inactivated. The allelic expression patterns differ between genes and between different tissues for one and the same gene. Our data provide the first evidence of a regulation mechanism involving gene-copy silencing in a triploid vertebrate.

Introgressive hybridization between two Iberian endemic cyprinid fish: a comparison between two independent hybrid zones

Pseudochondrostoma duriense and Achondrostoma oligolepis are two Iberian endemic cyprinid fish species that occur in sympatry over most of their distribution range and that are suspected to hybridize in nature. Here, we employed a combination of mitochondrial and microsatellite markers to explore the extent of introgressive hybridization between these fishes. Two natural hybrid zones were identified in different river basins. Introgression was bi‐directional and both hybrid zones consisted mostly of parental genotypes/phenotypes (i.e. bimodal hybrid zones). Yet, they appeared to differ in the extent and direction of introgression, which supports the view that they constitute independent outcomes of different hybridization processes probably influenced by environmental features. Several discordances were found between mtDNA and microsatellite results, suggesting that this hybridization process has complex consequences and illustrating the importance of using independent markers to define accurately the hybrid status of individuals in the presence of high levels of backcrossing.

Mitochondrial DNA variation in the highly endangered cyprinid fish Anaecypris hispanica: importance for conservation

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.

River basin-related genetic structuring in an endangered fish species, Chondrostoma lusitanicum, based on mtDNA sequencing and RFLP analysis.

Chondrostoma lusitanicum is a Portuguese endemic cyprinid with a restricted distribution and reduced numbers in some basins, justifying its status as a threatened species. We examined genetic population structure using samples from throughout its geographical range in Portugal, using sequencing of b cytochrome and restriction fragment length polymorphism analysis of the NADH subunits 5 and 6. There was reduced within-population genetic variability but considerable among-population differentiation, particularly marked between both the Mira and Arade basins in the extreme south and other populations. These results confirm phylogeographic relationships suggested by previous fragmentary allozyme studies for C. lusitanicum, and are in accordance with allozyme and mitochondrial DNA on phylogeography of coexisting cyprinid species of the genus Leuciscus. The levels of genetic divergence revealed by sequence and RFLP data showed strongly concordant patterns: geographical genetic structuring, with the definition of three distinct groups, was observed. The high values of nucleotide divergence and pairwise sequence divergence of the Mira and Arade groups, when compared with all other samples, support a distinct taxonomic status probably at the species level. Results are also discussed in relation to conservation of this highly fragmented species, in terms of Evolutionary Significant Units and Management Units.

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.

MATERNAL ANCESTRY OF THE RUTILUS ALBURNOIDES COMPLEX (TELEOSTEI, CYPRINIDAE) AS DETERMINED BY ANALYSIS OF CYTOCHROME B SEQUENCES

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.