Phylogeography, historical demography and habitat suitability modelling of freshwater fishes inhabiting seasonally fluctuating Mediterranean river systems: a case study using the Iberian cyprinid Squalius valentinus.

The Mediterranean freshwater fish fauna has evolved under constraints imposed by the seasonal weather/hydrological patterns that define the Mediterranean climate. These conditions have influenced the genetic and demographic structure of aquatic communities since their origins in the Mid-Pliocene. Freshwater species in Mediterranean-type climates will likely constitute genetically well-differentiated populations, to varying extents depending on basin size, as a consequence of fragmentation resulting from drought/flood cycles. We developed an integrative framework to study the spatial patterns in genetic diversity, demographic trends, habitat suitability modelling and landscape genetics, to evaluate the evolutionary response of Mediterranean-type freshwater fish to seasonal fluctuations in weather. To test this evolutionary response, the model species used was Squalius valentinus, an endemic cyprinid of the Spanish Levantine area, where seasonal weather fluctuations are extreme, although our findings may be extrapolated to other Mediterranean-type species. Our results underscore the significant role of the Mediterranean climate, along with Pleistocene glaciations, in diversification of S. valentinus. We found higher nuclear diversity in larger drainage basins, but higher mitochondrial diversity correlated to habitat suitability rather than basin size. We also found strong correlation between genetic structure and climatic factors associated with Mediterranean seasonality. Demographic and migration analyses suggested population expansion during glacial periods that also contributed to the current genetic structure of S. valentinus populations. The inferred models support the significant contribution of precipitation and temperature to S. valentinus habitat suitability and allow recognizing areas of habitat stability. We highlight the importance of stable habitat conditions, fostered by typical karstic springs found on the Mediterranean littoral coasts, for the preservation of freshwater species inhabiting seasonally fluctuating river systems.

Comparative pattern of genetic structure in two Mediterranean killifishes Aphanius fasciatus and Aphanius iberus inferred from both mitochondrial and nuclear data.

In this study, genetic variation was assessed in Aphanius fasciatus and Aphanius iberus characterized by similar ecological traits but with very different distribution ranges in the Mediterranean area. Five populations of A. iberus and five of A. fasciatus were analysed using five polymorphic microsatellite loci and partial mitochondrial control region (D-loop) sequences. Congruent results were found with both nuclear and mitochondrial molecular markers. The results showed that similar levels of genetic divergence, based on mitochondrial control region sequences, are present among populations of A. iberus and among populations of A. fasciatus despite the very different geographic distance existing among the examined populations of the two species (low geographic distance in A. iberus and high in A. fasciatus). A possible explanation could be that the populations of A. iberus were isolated for a longer time than the populations of A. fasciatus supporting the hypothesis that the split in the lineage leading to A. iberus is older than the split in the lineage leading to A. fasciatus. The possibility that the wide circum-Mediterranean distribution of A. fasciatus ensures the high connectivity of its populations, preventing, in some cases, local differentiation, however, cannot be ruled out.

Comparative historical biogeography of three groups of Nearctic freshwater fishes across central Mexico.

Biogeographic patterns of the three main Nearctic groups of continental fishes inhabiting river drainages in central Mexico (livebearing goodeids, southern Mexican notropins and species of Algansea, the last two representing independent lineages of cyprinids) were obtained and compared by following two approaches: an estimate of divergence times and using a well-defined biogeographic method. Three concordant biogeographic events were identified among the three groups, showing some evidence of a partially congruent evolutionary history. The analysed groups show at least three independent colonization events into central Mexico: two western routes, followed by the Goodeinae and members of Algansea, and an early Plateau route followed by southern notropins. The most recent common ancestor (MRCA) of each of the three freshwater fish groups diversified in central Mexico in the Late Miocene. The lack of a strong congruence in their biogeographic patterns, and the differences in species richness among the three clades might be evidence for distinct patterns of diversification.

Different stocks of brook lamprey in Spain and their origin from Lampetra fluviatilis at two distinct times and places.

Using the mitochondrial non-coding region I, it was shown that the two Spanish Lampetra planeri populations (Cares-Deva and Olabidea-Ugarana) correspond to different genetic units. The Cares-Deva population is probably a recent offshoot of Lampetra fluviatilis, being the Olabidea-Ugarana population less diverse and of older origin.

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes.

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

Genetic diversity shaped by historical and recent factors in the live-bearing twoline skiffia Neotoca bilineata.

The endangered twoline skiffia Neotoca bilineata, a viviparous fish of the subfamily Goodeinae, endemic to central Mexico (inhabiting two basins, Cuitzeo and Lerma-Santiago) was evaluated using genetic and habitat information. The genetic variation of all remaining populations of the species was analysed using both mitochondrial and microsatellite markers and their habitat conditions were assessed using a water quality index (I(WQ)). An 80% local extinction was found across the distribution of N. bilineata. The species was found in three of the 16 historical localities plus one previously unreported site. Most areas inhabited by the remaining populations had I(WQ) scores unsuitable for the conservation of freshwater biodiversity. Populations showed low but significant genetic differentiation with both markers (mtDNA φ(ST) = 0.076, P < 0.001; microsatellite F(ST) = 0.314, P < 0.001). Borbollon, in the Cuitzeo Basin, showed the highest level of differentiation and was identified as a single genetic unit by Bayesian assignment methods. Rio Grande de Morelia and Salamanca populations showed the highest genetic diversity and also a high migration rate facilitated by an artificial channel that connected the two basins. Overall, high genetic diversity values were observed compared with other freshwater fishes (average N(a) = 16 alleles and loci and mean ±S.D. H(o) = 0.63 ± 0.10 and nucleotide diversity π = 0.006). This suggests that the observed genetic diversity has not diminished as rapidly as the species' habitat destruction. No evidence of correlation between habitat conditions and genetic diversity was found. The current pattern of genetic diversity may be the result of both historical factors and recent modifications of the hydrological system. The main threat to the species may be the rapid habitat deterioration and associated demographic stochasticity rather than genetic factors.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011.

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.

Exploring the effect of microsatellite size homoplasy on reconstruction of phylogenetic relationships of picote splitfin Zoogoneticus quitzeoensis.

This study explores the effects of microsatellite size homoplasies on the reconstruction of phylogenetic relationships and estimates of population parameters as the fixation index (F(ST) ) using as a case study a truncated microsatellite from the picote splitfin Zoogoneticus quitzeoensis. The results suggest that the use of imperfect microsatellites may have only a minor effect in phylogenetic and population studies.

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.

On the occurrence of Anaecypris hispanica, an extremely endangered Iberian endemism, in the Guadalquivir River basin.

The jarabugo Anaecypris hispanica, considered endemic to the Guadiana River basin, has been found in the Guadalquivir River. First genetic data showed a high degree of similarity to those of the Guadiana River populations. The genetic study recovered five different groups of haplotypes, the Guadalquivir River specimens belong to the largest and most widely extended group.

Phylogenetic analysis of peri-Mediterranean blennies of the genus Salaria: molecular insights on the colonization of freshwaters.

In this paper, the phylogenetic relationships of the marine blenny Salaria pavo and the freshwater S. fluviatilis and S. economidisi were analyzed using four molecular markers: the mitochondrial 12S rRNA, 16S rRNA, and the control region and the nuclear first intron of the S7 ribosomal protein. The monophyly of Salaria is supported, as well as that of S. pavo and that of all the freshwater members of Salaria. Thus, the present results support a single origin for all freshwater Mediterranean blenniids. Our results reject the placement of the species of Salaria in the genus Lipophrys as proposed in previous studies. Using a molecular clock calibrated with trans-Isthmian geminate blenniid species, the split between the ancestor of the freshwater lineage and the ancestor of S. pavo is tentatively placed in the Middle Miocene (well before the Messinian). The marine S. pavo displays a very low level of intraspecific sequence divergence consistent with a Pleistocene bottleneck. S. fluviatilis is a paraphyletic entity with S. economidisi nested within it. A Moroccan population of S. fluviatilis is more divergent than S. economidisi, both in nuclear and mitochondrial genes. Fish from Israel together with some Turkish samples represent the second oldest split. It is argued that these populations may represent cryptic species. Thus, further studies on the taxonomy of these freshwater blennies are urgently needed.

Body shape evolution among ploidy levels of the Squalius alburnoides hybrid complex (Teleostei, Cyprinidae).

Hybridization, ploidy level and genomic constitution may be important to respond to different environments, by producing different phenotypes and thus reducing competitive interaction. Through geometric morphometrics, we examined variation in body size and shape among biotypes of the Squalius alburnoides hybrid complex and their sperm donor (Squalius carolitertii). Results showed that S. carolitertii is significantly larger in size than the biotypes of the complex. No significant relationship was observed between ploidy and body size among S. alburnoides biotypes. Significant variation in body shape was found between S. carolitertii and S. alburnoides, and between tetraploids and the other biotypes. These differences in biotypes may reduce resource competition, highlighting the potential importance of resource availability favouring one biotype over another. In S. alburnoides, the adaptation to different trophic niches through modification of trophic morphology, body shapes, and feeding behaviour, may result from an increase in ploidy and genomic constitution. This adaptation may account also for the formation and maintenance of this nonsexual complex.

Insights on speciation patterns in the genus Iberochondrostoma (Cyprinidae): evidence from mitochondrial and nuclear data.

In this paper, the patterns of cladogenesis in the cyprinid fish genus Iberochondrostoma were analysed using a mitochondrial (cytochrome b) and a nuclear (beta-actin) gene fragment. The two genes yielded discordant results. While the cytochrome b gene yielded a fully dichotomous tree, where all species of the genus are monophyletic, the much slower beta-actin gene yielded star-like relationships. However, when information from both genes was considered together, the data suggested the persistence of a very large central unit from which at least two peripheral clades arose at different times. This pattern which is akin to peripatric speciation was shown to be compatible with the paleogeographical information available. It is suggested that combining the techniques of phylogeny and phylogeography and the use of multiple markers varying in their rate of evolution may enrich our understanding of speciation and evolution of clades beyond species level.

Evolutionary history of the synbranchid eels (Teleostei: Synbranchidae) in Central America and the Caribbean islands inferred from their molecular phylogeny.

Swamp eels of the genera Synbranchus and Ophisternon are secondary freshwater fishes whose biogeography provides evidence of their long residence in Mesoamerica, while their impoverished species-level taxonomy might suggest a more recent diversification or a conservative morphology. We have inferred the phylogenetic relationships of Synbranchus marmoratus and Ophisternon aenigmaticum from 45 drainages throughout South, Central America, and Cuba based on mitochondrial genes (cytochrome b and ATPase 8/6). Phylogeographic analysis supported the monophyly of Mesoamerican O. aenigmaticum although our results suggest that S. marmoratus is not a monophyletic group. We found a evolutionary differentiated Synbranchus mtDNA lineage inhabiting Las Perlas islands (Pacific Panama) that appeared to be taxonomically distinct and separated for a long period of time from the main Synbranchus clade. Major synbranchid clades were also corroborated with the nuclear RAG-1 gene (1171-bp). Application of two fish-based mtDNA clocks (1.05-1.3% pairwise divergence/million year (Ma)), is in accordance with the Gondwanian origin suggested for the Synbranchidae. The mtDNA lineages exhibited a remarkable geographic structure in Central America suggesting that vicariance has most likely promoted the Synbranchus and Ophisternon mtDNA diversification. Although our data indicate the importance of the Pacific area in Synbranchus differentiation, the mtDNA divergence between South and Central American Synbranchus is too small to support Cretaceous colonization via the proto-Antillean bridge suggested by Rosen [Syst. Zool. 24 (1976) 431]. Instead, our phylogeographic results suggest that Ophisternon and Synbranchus mtDNA clades most likely colonized Central America during the Miocene (12.7-23Ma) prior the final closure of the Isthmus of Panama (3.3Ma).

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.

Pleistocene effects on the European freshwater fish fauna: double origin of the cobitid genus Sabanejewia in the Danube basin (Osteichthyes: Cobitidae).

Biogeographical hypotheses of European freshwater fishes were inferred using phylogeographic analysis of the complete cytochrome b and ATP synthase 8 and 6 mitochondrial genes (1982bp). To test the relative importance of drainage origin versus Pleistocene glaciations in the origin of primary freshwater fishes in Europe, we reconstructed the phylogenetic relationships of the genus Sabanejewia which is distributed in European waters. The phylogenetic relationships recovered for the genus Sabanejewia (n=75) provide support for the monophyly of six main evolutionary mtDNA lineages: Sabanejewia larvata, Sabanejewia romanica, Sabanejewia aurata/Sabanejewia caucasica, Sabanejewia kubanica, Sabanejewia baltica, and the Danubian-Balkanian complex. The Caucasian-Caspian mtDNA lineages, S. kubanica, S. aurata/S. caucasica, and the Northern European S. baltica represents the sister group of the Danubian-Balkanian complex mtDNAclade, supporting a Caucasian-Northern European origin of most of mtDNA lineages of the Central European freshwater fish fauna. The mtDNA divergence observed between the Danubian Sabanejewia species is too dissimilar to support their contemporary origin. Rather, the mtDNA data suggest that the Danubian Sabanejewia lineages most likely have a double origin, indicating that the European Sabanejewia lineages have experienced different historical processes for the following reasons. First, the origin of the S. larvata and S. romanica mtDNA clades predates the origin of the Danubian-Balkanian complex, and our results showed that the completion of the Alps and the origin of the Danube drainage seem to have promoted the speciation of the earliest Sabanejewia clades in the Miocene. Second, small genetic distances and the geographical pattern found within the Danubian-Balkanian complex clade indicate that the lineages included in this clade spread recently across the Danube and Greek river drainages. The inclusion of the S. balcanica species within all mtDNA lineages suggests that cyclical cold periods during the Pleistocene glaciations have favoured its rapid expansion and genetic homogenisation across Central European and Greek waters.

Haplotype diversity and phylogenetic relationships among the Iberian barbels (Barbus, Cyprinidae) reveal two evolutionary lineages.

The phylogenetic relationships and haplotype diversity of all Iberian barbels were examined by analyzing the complete mitochondrial cytochrome b gene sequence (1141 bp) of 72 specimens from 59 Iberian localities. Phylogenetic findings demonstrated a clear distinction between two mitochondrial lineages and confirmed the existence of two previously considered subgenera: Barbus and Luciobarbus: The first subgenus, Barbus, is represented on the Iberian Peninsula by Barbus haasi and Barbus meridionalis. The second subgenus, Luciobarbus, includes the remaining endemic Iberian species: Barbus comizo, Barbus bocagei, Barbus microcephalus, Barbus sclateri, Barbus guiraonis, and Barbus graellsii. Mean haplotype divergence between these subgenera was 10.40%, providing evidence of a clear subdivision within the Iberian barbels. Our results conflict with those reported in a recent study, based on 307 cytochrome b base pairs, that failed to identify any division within the genus Barbus in the Iberian Peninsula. The inclusion of nine further species belonging to this genus (used as outgroups) allowed us to establish a closer relationship of the Iberian species of the subgenus Barbus with other European taxa than with the Iberian Luciobarbus, which was found to cluster with North African, Caucasian, and Greek species. At the population level, no biogeographic structure was shown by specimens of each species (only 5.98% of the variation was attributable to differences among populations of each species). Given the discrete amount of divergence found among the Luciobarbus species, the formation of current hydrographic basins during the Plio-Pleistocene seems to have played a major role in their isolation and evolution.

Nuclear and mitochondrial data reveal high genetic divergence among Atlantic and Mediterranean populations of the Iberian killifish Aphanius iberus (Teleostei: Cyprinodontidae).

The molecular divergence and phylogenetic relationships of the Iberian populations of Aphanius iberus were established using allozymes and the complete cytochrome b gene sequence. Congruent results were found with both nuclear and mitochondrial molecular markers. The Mediterranean and Atlantic populations are clearly differentiated into two independent lineages. Their high molecular divergence suggests an early isolation, and the absence of gene flow among the populations indicates their independent evolution. The nuclear and mitochondrial data reveal monophyletic clustering of the two geographical lineages, but provide weak support for the population relationships. However, the mitochondrial results differentiated the Villena population as a distinct mitochondrial unit within the Mediterranean group. Geographically broad studies across the distribution range of A. iberus have helped to elucidate the patterns of diversification of this species. The genetic divergence found between the Atlantic and the Mediterranean populations is of the same order as those found among recognized species of cyprinodontids. The identification of two discrete evolutionary lineages has important implications for the conservation of this species, since its recovery requires the recognition and preservation of natural diversity. The Mediterranean and Atlantic lineages should be managed separately to avoid loss of their genetic identity, and the genetic uniqueness of the populations should be preserved by using wild stocks as the source of genetic diversity in captive breeding programmes.