Phylogenetic relationships of Iberian cyprinids: systematic and biogeographical implications.

The phylogenetic relationships among all Iberian endemic cyprinids were inferred using the complete nucleotide sequence of the cytochrome b gene. The inferred molecular phylogeny included representatives from Central European, Asian and North African species, and is highly congruent with previous phylogenies based on osteological characters. Iberian cyprinids were grouped into only five, very speciose lineages (with the exception of the monotypic Anaecypris): Barbus, Luciobarbus, Chondrostoma, Leuciscus and Anaecypris. The existence of such a relatively small number of Iberian cyprinid lineages can be explained by the historical isolation of the Iberian Peninsula. North African and Asian barbels are the sister group of Iberian Luciobarbus, supporting a south-eastern route of colonization of the Iberian Peninsula for this subgenus. With leuciscins, Anaecypris hispanica was considered a relict species as it could not be related to any other Iberian cyprinid. The phylogenetic relationships among the main lineages of Iberian cyprinids based on cytochrome b sequence data supported the traditional division of the Cyprinidae into two subfamilies: Cyprininae and Leuciscinae.     

Evidence of a cenozoic Betic-Kabilian connection based on freshwater fish phylogeography (Luciobarbus, Cyprinidae)

The phylogenetic relationships among 26 species of the subgenus Luciobarbus were examined through comparison of the complete sequence of the mitochondrial genes ATPase 6 and 8 and cytochrome b. The monophyletic condition of the Luciobarbus subgenus was confirmed by several data treatment methods. Findings indicate a closer relationship among species inhabiting Caucasian, Greek, and North African areas than between the latter and those of the Iberian region. The isolation of these main clades is postulated to have occurred after the Messinian salinity crisis (5.5 MY ago) when the Iberian Peninsula broke away from the African continent. Based on a calibrated molecular clock proposed herein, the subsequent splitting of this subgenus corresponds to the isolation of the other three areas (Caucasian, Greek, and North African) about 4.5 MY ago, which would have interrupted any possible gene flow between lineages. However, the present data indicate that the North African Kabilies Mountain area maintained its contact with the south Iberian Peninsula for a longer period.     

Phylogeny and biogeography of the family Cyprinidae in the Middle East inferred from cytochrome b DNA- evolutionary significance of this region.

The phylogenetic relationships of cyprinid species from the Middle East and neighboring biogeographical areas were investigated using cytochrome b sequence variation in order to test hypotheses that consider the Middle Eastern area as an important interchange area or a center of speciation for the freshwater fauna. A total of 62 cyprinid species were analyzed over the complete cytochrome b fragment (1140 bp); 28 belong to the Leuciscinae subfamily and 34 to the Cyprininae. All the Leuciscinae lineage fish recorded in the Middle East are also found in Europe, which was interpreted as an important Palearctic influence in the Middle Eastern ichthyofauna consistent with the Lago Mare dispersion. However, it has also been suggested that several Danube species have their origins in the Middle East. In contrast, the Cyprininae subfamily showed three highly divergent lineages, one shared with the Euro-Mediterranean area (Barbus/Luciobarbus genus) relict of the Lago Mare dispersion, one shared with Africa (Carasobarbus/Varicorhinus subgenus), and the third shared with Asia (Garra genus). Furthermore, clades observed in the phylogenetic reconstructions are not consistent with morphometric or karyological data and disagree with previous taxonomic assumptions. Lastly, the dispersion history in the Middle East of this subfamily appears much more complicated and ancient than that of the Leuciscinae. However, taking into account Cyprininae and Leuciscinae distribution, the Middle East appears more like an important interchange area for the freshwater ichtyofauna than a center of speciation.     

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.     

Paleobiogeography of an Iberian endemic species,Luciobarbus sclateri (Günther, 1868) (Actinopterygii,Cyprinidae), inferred from mitochondrial and nuclear markers

Since the Cenozoic Era, the southern Iberian Peninsula has undergone a series of complex geological and climatic changes that have shaped the hydrographic configuration of the freshwater network, influencing the present‐day distribution of primary freshwater species and favoring a high level of local endemicity. The cyprinid species Luciobarbus sclateri (Günther, 1968) is an endemic species confined to the southern Iberian Peninsula and characterized by a complex evolutionary history. Previous studies linked the structure of L. sclateri populations to the effects of climate change during glaciations and were not able to explain the genetic discordance found between nuclear and mitochondrial markers. The results of this study show that the structure of L. sclateri populations is a reflection of diversification processes linked to the geological history of the region. Thus, we found three main mitochondrial phylogroups: the first one corresponding to small basins in southern Iberian Peninsula, a second one in eastern Iberian Peninsula, corresponding to Segura population, and a third one including the rest of the basins where the species is distributed. The southern group began diverging in the Pliocene as result of tectonic dynamics characterized by the emersion of the basins around the Strait of Gibraltar. The other two groups began diverging with the formation of the current Iberian hydrographic system during Pleistocene. So, the isolation of the hydrographic basins was the main factor driving intraspecific differentiation, followed by recent secondary contacts, admixture, and re‐isolation of the populations.     

Complete mitochondrial genome of the grass carp (Ctenopharyngodon idella, Teleostei): insight into its phylogenic position within Cyprinidae

Genome comparison has shed light on many fields of both basic and applied research, including the study of species phylogeny. Grass carp (Ctenopharyngodon idella) belongs to Cyprinidae, the largest freshwater fish family; but which subfamily it belongs to remains a controversial issue. In this study, the complete mitochondrial genome (mitogenome) sequence of grass carp was determined and phylogenetic analyses of all mitochondrial protein-coding genes and a nuclear gene (RAG 2) were conducted to explore the evolutionary relationship of grass carp with other cyprinid species. The mitogenome of grass carp is 16,609 bp in length. As with most other vertebrates, it contains the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one putative control region. Phylogenetic analyses using two different datasets (mitochondrial and nuclear) and three different computational algorithms (Bayesian, MP and ML) all revealed two distinct groups with high statistical support, indicating that Cyprininae and Leuciscinae are two separate, valid subfamilies. Importantly, our phylogenetic result provides strong molecular evidence in support of the placement of Ctenopharyngodon in Leuciscinae rather than in Cyprininae.     

Rapid radiation of the Mediterranean Luciobarbus species (Cyprinidae) after the Messinian salinity crisis of the Mediterranean Sea, inferred from mitochondrial phylogenetic analysis

Phylogenetic relationships of 64 freshwater Barbus s.s. species distributed in basins around the Mediterranean Sea were assessed using cytochrome b sequences. Our results are in concordance with previous morphological and genetic studies, which proposed that these species belong to two major lineages (or subgenera): Barbus and Luciobarbus . We were particularly interested in phylogenetic relationships among species of the Luciobarbus lineage that are primarily found in the southern Mediterranean region from the Iberian Peninsula to the Middle East. In the Luciobarbus lineage, species that were previously attributed to the Capoeta genus were clustered. In this study, we observed short internodes between monophyletic groups having a geographical agreement around the Mediterranean. However, groups from the opposite sides of the Mediterranean Sea (Iberian Peninsula– Capoeta , north-western Africa–Middle East) seem to be phylogenetically close. We therefore infer that rapid radiation of Luciobarbus species in the Late Miocene better fits our data rather than gradual founder events in the southern Mediterranean. We propose that the biogeographical event along an east–west route, responsible for the present distribution of Luciobarbus species, was the 'Lago Mare' phase of the Mediterranean Sea that provided a rapid dispersal route over extensive distances. This provides new insights into the speciation pattern of this group, and may be of general use in the study of freshwater species in these regions.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 207–222.     

Mitochondrial D-loop phylogeny signals two native Iberian red deer (Cervus elaphus) Lineages genetically different to Western and Eastern European red deer and infers human-mediated translocations

Native red deer (Cervus elaphus) in Western Europe might at least partially derive from refugial populations which survived in the Iberian Peninsula during the last glacial maximum, and that expanded northwards at the onset of the Holocene. However, the phylogeny and genetic structure of red deer populations in the Iberian Peninsula are still poorly known. This study was planned, in a first step, to reconstruct the phylogenetic relationship of the main red deer populations extant in Spain by the analyses of an extensive sample of mitochondrial DNA sequences. Results indicate that sequences from these populations can be assigned to one of two deeply divergent mtDNA lineages (South-Western and Central-Eastern) with molecular divergence nearby the 2 %. In each lineage were respectively found sixteen and thirteen different haplotypes. It was evidenced that they may be allopatrically distributed in Spain with 86.6 % sequences of the South-Western lineage at the South-Western side and the 65 % sequences of Central-Eastern lineage in the Central-Eastern side. These mitochondrial lineages might have originated in two distinct refugial populations during the last glacial maximum. Genetic data also reveal instances of admixture between native populations and translocated European red deer, which belong to at least three distinct subspecies. Gene introgression was mainly due to red deer from Western European populations. The genetic contribution of red deer translocated from Eastern Europe (C. e. hippelaphus) or North Africa (C. e. corsicanus, C. e. barbarus) was apparently less deep. The extant phylogenetic relationship and evidences of genetic admixture suggest that sound conservation actions for the native Iberian red deer should severely restrict the introduction of alien red deer and, when possible, avoid admixture between the South-Western and Central-Eastern mtDNA lineages.     

The Iberian Peninsula: ancient history of a hot spot of mite harvestmen (Arachnida: Opiliones: Cyphophthalmi: Sironidae) diversity

The Iberian Peninsula represents a hot spot of cyphophthalmid (mite harvestman) disparity, with four of the eight genera currently recognized in the family Sironidae represented in the region – a generic diversity and morphological disparity not found in any other region of the World so far. From these, two genera (Iberosiro and Odontosiro) are monotypic, and are restricted to the western side of the peninsula. Parasiro is restricted to the north‐east region, from the Catalonian Coastal Ranges and both sides of the Eastern Pyrenees, in areas where the annual rainfall surpasses 1000 mm, and mostly restricted to areas with Paleozoic and Variscan rocks, with other species of the genus extending to Corsica, Sardinia, and the Italian Peninsula. A second species of the genus Paramiopsalis, Paramiopsalis eduardoi sp. nov. from Fragas do Eume, is described here along with a re‐diagnosis of the genus. Paramiopsalis species, together with Odontosiro, inhabit the north‐west corner of the Iberian Peninsula, an area with some of the highest recorded annual rainfall, and with Paleozoic rocks from the Iberian Massif or Variscan granitoid rocks. A phylogenetic analysis of the members of the family Sironidae using four molecular markers, despite not including all of the Iberian genera, clearly shows the non‐monophyly of the Iberian Cyphophthalmi, indicating that the Iberian Peninsula is home to multiple ancient lineages of mite harvestmen. The two Paramiopsalis species form a sister clade to the Balkan genus Cyphophthalmus, whereas Parasiro constitutes the first lineage of the sironids represented.     

Genetic and phenotypic variation among geographically isolated populations of the globally threatened Dupont's lark Chersophilus duponti

Identifying genetically and phenotypically distinct populations of threatened species is critical if we are to delineate appropriate plans for their conservation. We conducted an integrated analysis of population genetic structure, historical demographic events, current gene flow (all based on mtDNA sequences) and morphological variation of three geographically separated groups of populations of Dupont's lark Chersophilus duponti, located in the Iberian Peninsula (three populations), Morocco (two populations), and Tunisia (one population). Unusually, this lark species is the only one among the genus Chersophilus. Our results revealed the early historical divergence of an eastern Dupont's lark lineage (in Tunisia) and a western lineage (in Morocco and Spain), consistent with subspecies taxonomy and distribution. The western lineage subsequently split into two lineages, following the isolation of Iberian and African populations. Such pattern of historical differentiation caused great population genetic structure, with differences among geographic areas explaining more than 80% of total genetic variation. Mismatch distributions and coalescent estimates of divergence time showed that lineage divergence was associated with sudden population expansion events, which apparently took place during the last glaciation, when steppe habitats were widespread across the Mediterranean region. Extant populations from different geographic areas hardly shared any haplotype (only one out of 16 ND2 haplotypes was shared by Tunisian and Moroccan Dupont's larks), and consequently gene flow between geographic areas was found to be virtually absent. Apart from showing great genetic differentiation, Dupont's larks from different geographic areas were morphologically distinct, showing substantial variation in body size and feeding-related traits (length of feet and bill). We conclude that Dupont's lark populations isolated in the Iberian Peninsula, Morocco, and Tunisia are distinct evolutionary entities and should be considered as such in conservation plans. Such circumstance sets a daunting conservation challenge that exemplifies the need of incorporating knowledge of historical processes to our general understanding of the demography of threatened species.     

Phylogeny and biogeography of highly diverged freshwater fish species (Leuciscinae,Cyprinidae, Teleostei) inferred from mitochondrial genome analysis

The distribution of freshwater taxa is a good biogeographic model to study pattern and process of vicariance and dispersal. The subfamily Leuciscinae (Cyprinidae, Teleostei) consists of many species distributed widely in Eurasia and North America. Leuciscinae have been divided into two phyletic groups, leuciscin and phoxinin. The phylogenetic relationships between major clades within the subfamily are poorly understood, largely because of the overwhelming diversity of the group. The origin of the Far Eastern phoxinin is an interesting question regarding the evolutionary history of Leuciscinae. Here we present phylogenetic analysis of 31 species of Leuciscinae and outgroups based on complete mitochondrial genome sequences to clarify the phylogenetic relationships and to infer the evolutionary history of the subfamily.     

Molecular evidence for the monophyly of East Asian groups of Cyprinidae (Teleostei: Cypriniformes) derived from the nuclear recombination activating gene 2 sequences

The family Cyprinidae is one of the largest families of fishes in the world and a well-known component of the East Asian freshwater fish fauna. However, the phylogenetic relationships among cyprinids are still poorly understood despite much effort paid on the cyprinid molecular phylogenetics. Original nucleotide sequence data of the nuclear recombination activating gene 2 were collected from 109 cyprinid species and four non-cyprinid cypriniform outgroup taxa and used to infer the cyprinid phylogenetic relationships and to estimate node divergence times. Phylogenetic reconstructions using maximum parsimony, maximum likelihood, and Bayesian analysis retrieved the same clades, only branching order within these clades varied slightly between trees. Although the morphological diversity is remarkable, the endemic cyprinid taxa in East Asia emerged as a monophyletic clade referred to as Xenocypridini. The monophyly for the subfamilies including Cyprininae and Leuciscinae, as well as the tribes including Labeonini, Gobionini, Acheilognathini, and Leuciscini, was also well resolved with high nodal support. Analysis of the RAG2 gene supported the following cyprinid molecular phylogeny: the Danioninae is the most basal subfamily within the family Cyprinidae and the Cyprininae is the sister group of the Leuciscinae. The divergence times were estimated for the nodes corresponding to the principal clades within the Cyprinidae. The family Cyprinidae appears to have originated in the mid-Eocene in Asia, with the cladogenic event of the key basal group Danioninae occurring in the early Oligocene (about 31-30 MYA), and the origins of the two subfamilies, Cyprininae and Leuciscinae, occurring in the mid-Oligocene (around 26 MYA).     

Partial combination applied to phylogeny of European cyprinids using the mitochondrial control region

Previous molecular phylogenies of European cyprinids led to some solid facts and some uncertainties. This study is based on a stretch of more than 1 kb in the mitochondrial control region newly sequenced for 35 European cyprinids and on previous cytochrome b and 16S rDNA data. The trees based on the control region are more accurate and robust than those obtained from the two other genes. Character incongruence among the three genes was tested using the incongruence length difference (ILD) test. Iterative removals of individual sequences followed by new ILD tests identified two sequences responsible for statistically significant incongruence. A partial combination was conducted, that is, a combination of the three data sets, removing the two sequences previously identified. The phylogenetic analysis of this partial combination gives a more robust and resolved picture of subfamilial interrelationships. The Rasborinae are the sister group of all other cyprinids. The monophyletic Cyprininae emerges next. Tinca tinca first and then Rhodeus are the sister groups of all the remaining nonrasborine and noncyprinine species. Gobio is the sister group of the Leuciscinae, in which the Phoxinini are the sister group of the Leuciscini. Within the Leuciscini, the genus Leuciscus and the subfamily Alburninae are both paraphyletic. The Rasborinae are the most basal cyprinid subfamily and the Tincinae are not the sister group of the Cyprininae. These two results challenge only two anatomical characters, which need to be reinterpreted or considered as homoplastic in cyprinid evolution: the modification of the first pleural rib and its parapophysis and the bony composition of the interorbital septum.     

Isolation and characterization of major histocompatibility class IIβ genes in an endangered North American cyprinid fish, the Rio Grande silvery minnow (Hybognathus amarus)

The major histocompatibility complex (MHC) is a critical component of the adaptive immune response in vertebrates. Due to the role that MHC plays in immunity, absence of variation within these genes may cause species to be vulnerable to emerging diseases. The freshwater fish family Cyprinidae comprises the most diverse and species-rich group of freshwater fish in the world, but some are imperiled. Despite considerable species richness and the long evolutionary history of the family, there are very few reports of MHC sequences (apart from a few model species), and no sequences are reported from endemic North American cyprinids (subfamily Leuciscinae). Here we isolate and characterize the MH Class II beta genes from complementary DNA and genomic DNA of the non-model, endangered Rio Grande silvery minnow (Hybognathus amarus), a North American cyprinid. Phylogenetic reconstruction revealed two groups of divergent MH alleles that are paralogous to previously described loci found in deeply divergent cyprinid taxa including common carp, zebrafish, African large barb and bream. Both groups of alleles were under the influence of diversifying selection yet not all individuals had alleles belonging to both allelic groups. We concluded that the general organization and pattern of variation of MH class II genes in Rio Grande silvery minnow is similar to that identified in other cyprinid fishes studied to date, despite distant evolutionary relationships and evidence of a severe genetic bottleneck.     

Molecular phylogeny of two lineages of Leuciscinae cyprinids (Telestes and Scardinius) from the peri-Mediterranean area based on cytochrome b data

We examined phylogenetic relationships in two lineages of Leuciscinae cyprinid fishes based on the sequence data of the complete mitochondrial DNA region coding for the cytochrome b gene (1140 bp). Telestes includes obligate riverine, moderately cold water-adapted species whereas Scardinius comprises warm-adapted species living in lowland lakes and still waters of rivers and streams. We also analysed selected representatives of Leuciscus and Phoxinellus because the taxonomic status of some species belonging to these genera is dubious and they could be placed in the genus Telestes. The study includes 18 species, 43 populations, and 111 individuals from 9 of the 14 peri-Mediterranean ichthyogeographic districts. Clades recovered from the phylogenetic analyses do not support previous taxonomic assumptions based on morphology. Telestes, Leuciscus, and Phoxinellus do not form monophyletic assemblages; phylogenetic analyses suggest that L. polylepis, L. turskyi, P. croaticus, and P. metohiensis should be included in Telestes. Similarly, populations of Scardinius erythrophthalmus do not cluster together and the endangered S. scardafa, endemic to central Italy and surviving in a single locality, is nested within them. The radiations of Telestes and Scardinius occurred in different time periods. A major diversification of Telestes is consistent with a sea dispersal during the freshwater Messinian "Lago Mare" phase of the Mediterranean Sea. Cladogenetic events within Scardinius are likely related to the extension and confluence of river drainages in lowlands following multiple lowering of the sea level during the Quaternary glaciations.     

Weight–length relationships of cyprinid fishes of the Iberian Peninsula

Weight–length (W–L) relationships for 28 cyprinid species collected on the Iberian Peninsula are presented. Values of the exponent b in the W–L relationships ranged from 2.78 to 3.47. These are the first W–L parameters reported for 11 of the species, all of them endemic to the Iberian Peninsula.     

Contributions to the karyological study of the genus Ranunculus L. subgenus Batrachium (DC.) A Gray from the Iberian Peninsula

DIOSDADO. J. C, PASTOR, J. E. & VALDÉS, B. 1993. Contributions to the karyological study of the genus Ranunculus L. subgenus Batrachium (DC.) A. Gray from the Iberian Peninsula. The somatic and gametic chromosome numbers and detailed chromosome morphology are presented for eight taxa of aquatic ranunculi from the Iberian Peninsula. Within the subgenus Batrachium diploid and tetraploid levels have been observed (2 n = 16, 32) the commoner of which is the diploid. From the karyological data relationships have been established between the subgenus Batrachium and subgenus Ranunculus.     

The European Far West: Miocene mammal isolation,diversity and turnover in the Iberian Peninsula

Aim To analyse the diversity dynamics of Miocene mammalian faunas in the Iberian Peninsula in order to determine whether the patterns are related to the dispersal of taxa from other areas into this region. Location Mainly the Iberian Peninsula, but two close geographical areas (Central Europe and the Eastern Mediterranean) are also considered in some of our calculations. Methods Genus‐level faunal lists for a total of 299 localities from the Iberian Peninsula, covering 10 successive biochronological units [Mammal Neogene (MN) zones] that span from the latest Early Miocene to the early Pliocene (about 17–4 Ma), were compiled. The dataset was expanded with a further 331 localities in Central Europe and the Eastern Mediterranean for the same time span. Next, a taxonomically standardized database was used to create composite faunal lists of micro‐ and macromammalian genera present during each MN zone. Separate genera‐by‐MN‐zone matrices for both micro‐ and macromammals were built for each region. Mean standing diversity as well as origination and extinction rates were calculated for the Iberian Peninsula, and their correlation with preservation rates is discussed. Simpson’s coefficient of faunal similarity with Central Europe and the Eastern Mediterranean was calculated in order to evaluate whether diversity patterns were related to changes in the affinity of the Iberian mammalian faunas with those of other regions. Results Diversity changes in the Iberian macromammalian faunas coincide with periods of increased faunal similarity with other regions, suggesting a relationship to the expansions and contractions of the geographical ranges of the constituent taxa. This pattern is not recognized for micromammals; that is, their diversity trends are not related to changes in geographical ranges. Main conclusions Climatic shifts result in expansions or contractions in the geographical ranges of macromammals, owing to changes in the distribution of their preferred habitats. The lower dispersal ability of micromammals results in a higher extinction risk when habitat fragmentation confines their populations to relatively small environmental patches. Hence, they are more severely affected by climatic changes. Our results thus emphasize the role of climatic forcing in mammalian biogeography and diversity.     

Effects of habitat transition on the evolutionary patterns of the microgastropod genus Pseudamnicola (Mollusca,Hydrobiidae)

Molecular phylogenies of extant species are considered effective tools to infer mechanisms of speciation. Here, we benefit from this utility to investigate the evolutionary history of an organismal group linked to different aquatic ecosystems, the microgastropod genus Pseudamnicola (family Hydrobiidae). Previous studies have found around 45 species of the nominal subgenus P. (Pseudamnicola), most of them in coastal stream localities of several Mediterranean islands and mainland territories, whereas only 12 species of the other subgenus, P. (Corrosella), have been collected from springs and headwaters of mountainous regions of the Iberian Peninsula and south of France. As springs often act as isolated habitats affecting dispersion and constraining gene flow, we supposed that the temporal history and mode of diversification of species from both subgenera should differ and therefore be reflected in their phylogenetic patterns. To assess this hypothesis, we performed a molecular phylogeny based on mitochondrial and nuclear DNA sequences and later conducted an independent analysis to examine the potential effect of certain geographic and ecological variables in the genetic divergences of the subgenera. Additionally, we estimated the ancestral area of diversification of both groups. Published anatomical revisions and our molecular analyses suggest that the genus Pseudamnicola should be divided into three genera: the two previous subgenera plus a new one described here. As postulated, the evolution of the spring organisms was strongly related to habitat fragmentation and isolation, whereas dispersal followed by divergence seem to have been the most common speciation processes for euryhaline species inhabiting coastal streams and low river stages in which waters remain connected. On the contrary, rather than habitat fragmentation or dispersion, environmental conditions have played a larger role during the deep divergent split leading to the three genera.     

Genetic divergence and phylogenetic independence of Far Eastern species in subfamily Leuciscinae (Pisces: Cyprinidae) inferred from mitochondrial DNA analyses

Classification of freshwater fish in the subfamily Leuciscinae, Cyprinidae is hampered by complexity or lack of morphological diversity. In this study, analyses based on mtDNA sequences were undertaken to clarify phylogenetic relationships among Far Eastern, North American and European species in the Leuciscinae. Evolutionary rate in cytochrome b gene (Cyt-b) and D-loop sequences appear to be almost constant in Leuciscinae. The topology of phylogenetic trees generated by neighbor-joining (NJ) and maximum likelihood (ML) methods based on Cyt-b gene and D-loop sequences was similar. Five major clades, designated clades 1-5, and a minor clade were discriminated. Most of the Far Eastern, North American and European species were included in the major clades. Clade 1, comprised almost entirely of Far Eastern phoxinins, is monophyletic and greatly diverged from the other species of Leuciscinae. From the present phylogenetic relationships and the previous studies, we present the following hypotheses with respect to the evolutionary history of the Far Eastern phoxinins. The Far Eastern species should be classified into Far Eastern-specific genera, although ichthyologists have still insisted that the species should be included in the European genera. The Far Eastern clade 1 consists of two subclades, including genera Pseudaspius-Tribolodon and Far Eastern Phoxinus species. According to our phylogenetic analyses, Pseudaspius leptocephalus and Tribolodon species should be reclassified into the same genus. On the basis of evolutionary rate in Cyt-b gene in Cyprinidae, it is estimated that the Far Eastern lineage diverged approximately 10-14 million years ago (mya) from the common ancestor of Leuciscinae. It is deduced that speciation of the Far Eastern species occurred until approximately 4 mya, in relation to the formation of the Sea of Japan and the Japanese Islands.