Anabel Perdices

Evolutionary history of the genus Rhamdia (Teleostei: Pimelodidae) in Central America.

We constructed phylogenetic hypotheses for Mesoamerican Rhamdia, the only genus of primary freshwater fish represented by sympatric species across Central America. Phylogenetic relationships were inferred from analysis of 1990 base pairs (bp) of mitochondrial DNA (mtDNA), represented by the complete nucleotide sequences of the cytochrome b (cyt b) and the ATP synthase 8 and 6 (ATPase 8/6) genes. We sequenced 120 individuals from 53 drainages to provide a comprehensive geographic picture of Central American Rhamdia systematics and phylogeography. Phylogeographic analysis distinguished multiple Rhamdia mtDNA lineages, and the geographic congruence across evolutionarily independent Rhamdia clades indicated that vicariance has played a strong role in the Mesoamerican diversification of this genus. Phylogenetic analyses of species-level relationships provide strong support for the monophyly of a trans-Andean clade of three evolutionarily equivalent Rhamdia taxa: R. guatemalensis, R. laticauda, and R. cinerascens. Application of fish-based mitochondrial DNA clocks ticking at 1.3-1.5% sequence divergence per million years (Ma), suggests that the split between cis- and trans-Andean Rhamdia extends back about 8 Ma, and the three distinct trans-Andean Rhamdia clades split about 6 Ma ago. Thus the mtDNA divergence observed between cis- and trans-Andean Rhamdia species is too low to support an ancient colonization of Central America in the Late Cretaceous or Paleocene as had been hypothesized in one colonization model for Mesoamerican fishes. Rather the mtDNA data indicate that Rhamdia most likely colonized Central America in the late Miocene or Pliocene, promoting a strong role for the Isthmus of Panamá in the Mesoamerican expansion of this genus. Basal polytomies suggest that both the R. laticauda and R. guatemalensis clades spread rapidly across the Central American landscape, but differences in the average mtDNA genetic distances among clades comprising the two species, indicate that the R. laticauda spread and diversified across Mesoamerica about 1 million years before R. guatemalensis.

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.

Allozymic variation of the endangered killifishAphanius iberus and its application to conservation

SynopsisGenetic differentiation and patterns of variability in the endangered Iberian endemic,Aphanius iberus, were analyzed by allozyme electrophoresis as a valuable database for conservation purposes. Genetic variability values expressed as heterozygosity (H = 0.015–0.097) were close to the values found in other members of Cyprinodontidae (H = 0.012–0.123). Polymorphism values (P = 0.125–0.542) were higher than reported in the literature (P = 0.036–0.150) for Cyprinodontidae. Significant correlation existed between salinity values and genetic variability expressed as heterozygosity (r = − 0.76, p < 0.01) and polymorphism (r = − 0.60, p = 0.04). Low genetic variability values (H = 0.024–0.055, P = 0.125–0.292) were exhibited by populations which inhabit salty lagoons. The highest values were found in populations occurring in marshes and irrigation channels (H = 0.051–0.097, P = 0.250–0.542). Associations among genetic, geographic and ecological parameters were tested using a Mantel test indicated that most of the genetic distances were explained by geographic distances but not by ecological factors, suggesting that isolation by distance could be the main factor explaining the differentiation between sites. According to the genetic distances obtained, two mainA. iberus groups were discernible: the Atlantic and Mediterranean. Genetic distances between both groups (DRogers = 0.179–0.261) were higher than values between recognized species of other cyprinodontids (DRogers = 0.11–0.27). On the basis of genetic distances we have dated the fragmentation of both populations to the Upper Miocene-Pliocene when most of the Mediterranean sea dried up. Subsequently, gene flow between Mediterranean and Atlantic populations was interrupted. The results of our genetic analyses suggested the existence of five operational conservation units (OCUs) forA. iberus. These units are defined as a continuous area limited by geographical boundaries, and inhabited by one or more populations sharing the same genetic pattern.

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.

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.

Potential impacts of gravel extraction on Spanish populations of river blennies Salaria fluviatilis (Pisces, Blenniidae)

River blennies Salaria fluviatilis have a wide circum-Mediterranean distribution, but they are mostly confined to small, very localised populations. In the Iberian Peninsula, they are endangered due to a variety of causes, including gravel extraction. This study identified the breeding requirements of river blennies at a site where gravel extraction takes place and at three other sites in different drainage basins in Spain. Breeding males chose nest stones that were significantly larger than other stones available in the immediate vicinity. Although clutch area was significantly related to stone size in two of three populations, male size was not. Stone size appeared to be the main correlate of clutch size, and stone sizes were significantly smaller at sites where gravel had been extracted. The potential effects of stone and gravel removal on nesting density and egg productivity were simulated, and it was found that a 75% reduction in stone size, as observed in this study, could result in a 47% decrease in nesting density. Because of the relationship between clutch size and nest stone size, egg production would be reduced even further, to 25% of its initial level. Removal of stones and gravel from the river bed also causes structural alterations which may render the habitat unsuitable for breeding blennies despite the presence of apparently suitable nest stones. Our results may be applicable to the conservation of other substrate-spawning fish.

Molecular phylogeny and intraspecific structure of loaches (genera Cobitis and Misgurnus) from the Far East region of Russia and some conclusions on their systematics

Russian Far East loaches of the genera Cobitis and Misgurnus are among members of the family Cobitidae with poorly understood systematics. In this study we present phylogenetic hypotheses based on mitochondrial (cytochrome b) and nuclear (RAG-1) sequences. All analyses recovered comparable topological phylogenies, and all data sets supported the non-monophyly of the genera Cobitis and Misgurnus. Both genera are represented by multiple lineages that in some cases do not correspond to the species described. We found some phylogenetic incongruities for the genus Misgurnus (M. mohoity and M. anguillicaudatus) that are explained by ancient hybridization, as was suggested previously for M. anguillicaudatus. The revealed phylogenetic relationships suggest that Paramisgurnus should be treated as a synonym of Misgurnus and M. bipartitus as a synonym of M. mohoity. All analyses recovered C. choii as a member of the genus Cobitis, confirming previous taxonomic conclusions. Most of the molecular lineages found follow currently recognized taxa with some exceptions, such as M. anguillicaudatus and C. lutheri. Phylogenetic relationships recover several unrelated lineages of M. anguillicaudatus and suggest additional studies to solve current taxonomic uncertainty. We found that C. lutheri is a non-natural group that contains two unrelated lineages: specimens of C. lutheri from the Far East of Russia collected close to the type locality and a second lineage with specimens of C. lutheri from Korea, the identification of which must be revised. The study provides evidence of the presence of the Far East species M. nikolskyi in Sakhalin Island, but simultaneously shows conspicuous genetic distinctiveness between the island and the mainland populations.

Genetic Divergence and Origin of Mediterranean Populations of the River Blenny Salaria fluviatilis (Teleostei: Blenniidae)

Abstract The current distribution of the River Blenny Salaria (= Blennius) fluviatilis, one of the two freshwater representatives of a large, cosmopolitan marine fish family, poses an interesting biogeographical problem because this species inhabits widely separate circum-Mediterranean watersheds. Potential scenarios of its dispersal were examined using allozyme analysis of several populations from the Iberian and Greek peninsulas. Based on Nei genetic distances, the most divergent populations were the populations inhabiting lakes, Lake Trichonis in Greece, and Ruidera Lakes in Spain. Their high divergence suggests their early isolation from the main ingroup populations. In contrast, low genetic distances were found among river populations regardless of geographic location. There was a correlation between genetic distance and geographic distance among Iberian river populations, suggesting that dispersal following the colonization of fresh water occurred via the sea to nearby, unconnected river basins. The ancestor of S. fluviatilis may have been a euryhaline species, allowing incursions into fresh water and subsequent dispersal via the sea. This dispersal scenario could theoretically be combined with multiple colonization episodes. The two old lake populations shared a unique allele at the Pgdh-A locus in high frequency with its closest relative S. pavo, which was absent from other populations. This may indicate two initial incursions into fresh water by a wide-ranging marine ancestor that possessed this allele. Differential selection on this allele in lake habitats or convergence are less likely possibilities. Thus, the present distribution of S. fluviatilis appears to stem from a combination of “raceme” origins (i.e., more than one colonization episode) and subsequent dispersal and divergence in new watersheds. La distribución actual del blenio de río, Salaria (= Blennius) fluviatilis, uno de los dos representantes de agua dulce de una gran familia cosmopolita de peces marinos, plantea un interesante problema biogeográfico ya que esta especie vive en aguas circummediterráneas ampliamente separadas. Mediante el estudio de aloenzimas de varias poblaciones de la península Ibérica y Grecia se han analizado los escenarios potenciales para su dispersión. Las poblaciones más divergentes, basándonos en las distancias genéticas de Nei, fueron las pertenecientes a lagos, Lago Trichonis en Grecia y las Lagunas de Ruidera en España. Su gran divergencia sugiere su aislamiento temprano del resto de las poblaciones. Contrariamente, las poblaciones de los ríos mostraron distancias genéticas bajas independientemente de su localización geográfica. En las poblaciones ibéricas se encontró correlación entre las distancias genéticas y geográficas sugiriendo que la dispersión posterior a la colonización del agua dulce entre cuencas independientes se produjo a través del mar. El ancestro de S. fluviatilis podría haber sido una especie eurihalina, lo que le permitiría incursiones en el agua dulce y una posterior dispersión a través del mar. Este escenario podría teóricamente combinarse con múltiples episodios de colonización. Las dos poblaciones antiguas de los lagos comparten con su especie más cercana, Salaria pavo, un alelo único en el locus Pgdh-A en alta frecuencia, no presente en el resto de las poblaciones. Esto podría indicar dos incursiones iniciales en el agua dulce de un ancestro marino ampliamente distribuido que poseyera este alelo. La selección diferencial en hábitats lacustres o la convergencia de este alelo son hipótesis menos probables. Así pues, la distribución actual de Salaria fluviatilis parece ser una combinación de un origen en “racimo” (i.e., más de un episodio de colonización), y una posterior dispersión y divergencia en nuevas masas de agua.

Phylogeny, diversity, and species delimitation of the North American Round-Nosed Minnows (Teleostei: Dionda), as inferred from mitochondrial and nuclear DNA sequences

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts.

Phylogenetic relationships of North American western chubs of the genus Gila (Cyprinidae, Teleostei), with emphasis on southern species

Species of Gila comprise a heterogeneous and widespread group of freshwater fishes inhabiting drainage systems of western North America. The classification of species of Gila and relatives has been complicated and sometimes compromised by differences in body shapes, sizes, habitats, variable taxonomic placement by early taxonomists, and instances of hypothesized hybridization. While most attention on Gila has focused on hybridization in USA, little is actually know about their intra and intergeneric relationships. We present a molecular phylogeny using 173 specimens for all 19 recognized species of Gila, covering their entire distributions in 31 major drainages. Using one mitochondrial and three nuclear genes, specimens of Gila were analyzed with 10 other North American genera that comprise the Revised Western Clade. All analyses identified most species of Gila in a lineage that always included the monotypic genera Moapa and Acrocheilus, and we recommend the synonymy of both genera with Gila. The composition of this Gila lineage varied depending on the genes analyzed. Within the Gila lineage, similar morphotypes (forms adapted to fast currents vs. general forms) were not resolved as closest relatives. Analyses of mitochondrial DNA resolved all species of Gila from Mexico in reciprocally monophyletic clades except G. modesta. Most species of Gila in the USA were nested in 3 major clades, potentially indicating some level of historic or contemporary interspecific hybridization. Herein, we redefine the ranges for all species of Gila in Mexico. Relevant taxonomic and conservation implications stemming from the results are discussed.