Cichlids of the Banc d'Arguin National Park, Mauritania: insight into the diversity of the genus Coptodon.

To determine the species diversity of cichlids in the Banc d'Arguin National Park (PNBA) and their phylogenetic relationships with other species in West Africa, a morphometric and meristic and molecular phylogenetic study was conducted. Both approaches not only confirm the presence of Sarotherodon melanotheron in PNBA but also demonstrate the presence of a second species from the genus Coptodon. While morphometric characteristics match the description of the Guinean tilapia Coptodon guineensis, phylogenetic reconstructions based on three mitochondrial and one nuclear DNA fragment demonstrate that C. guineensis is paraphyletic over its range. Because different lineages of C. guineensis are allopatric, the distribution of C. guineensis should be restricted to Ghana and Côte d'Ivoire. The many other lineages of this species should be considered as C. sp. aff. guineensis.

Spatio-temporal isotopic signatures (δ13 C and δ15 N) reveal that two sympatric West African mullet species do not feed on the same basal production sources.

Potential trophic competition between two sympatric mullet species, Mugil cephalus and Mugil curema, was explored in the hypersaline estuary of the Saloum Delta (Senegal) using δ(13) C and δ(15) N composition of muscle tissues. Between species, δ(15) N compositions were similar, suggesting a similar trophic level, while the difference in δ(13) C compositions indicated that these species did not feed from exactly the same basal production sources or at least not in the same proportions. This result provides the first evidence of isotopic niche segregation between two limno-benthophageous species belonging to the geographically widespread, and often locally abundant, Mugilidae family.

Analysis of the black-chinned tilapia Sarotherodon melanotheron heudelotii reproducing under a wide range of salinities: from RNA-seq to candidate genes.

The black-chinned tilapia Sarotherodon melanotheron heudelotii is an ecologically appealing model as it shows exceptional adaptive capacities, especially with regard to salinity. In spite of this, this species is devoid of genomic resources, which impedes the understanding of such remarkable features. De novo assembly of transcript sequences produced by next-generation sequencing technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms. It also facilitates the development of quantitative real-time PCR (qPCR) assays for analysing gene expression under different environmental conditions. Nevertheless, obtaining accurate and reliable qPCR results from such data requires a number of validations prior to interpretation. The transcriptome of S. melanotheron was sequenced to discover transcripts potentially involved in the plasticity of male reproduction in response to salinity variations. A set of 54 candidate and reference genes was selected through a digital gene expression (DGE) approach, and a de novo qPCR assay using these genes was validated for further detailed expression analyses. A user-friendly web interface was created for easy handling of the sequence data. This sequence collection represents a major transcriptomic resource for S. melanotheron and will provide a useful tool for functional genomics and genetics studies.

Variation in gene expression along a salinity gradient in wild populations of the euryhaline black-chinned tilapia Sarotherodon melanotheron.

This study evaluated variation in expression of 11 genes within and among six wild populations of the black-chinned tilapia Sarotherodon melanotheron distributed along a salinity gradient from 0 to 100. Previous laboratory studies had shown that expression of these genes was sensitive to water salinity; the current study confirmed that a number of them also varied in expression in wild populations along the salinity gradient. Principal component analysis (PCA) first distinguished two, not mutually exclusive, sets of genes: trade-off genes that were highly expressed at one or other extreme of the salinity gradient and stress genes that were up-regulated at the two salinity extremes (i.e. a U-shaped expression pattern). The PCA clearly partitioned the populations into three groups based on their gene expression patterns and their position along the salinity gradient: a freshwater (GL; 0) population, four brackish and seawater (GB, HB, SM, SF; ranging from 20 to 50) populations and a hypersaline (SK, 100) population. Individual variation in gene expression was significantly greater within the populations at the extreme compared to intermediate salinities. These results reveal phenotypically plastic regulation of gene expression in S. melanotheron, and greater osmoregulatory and plasticity costs at extreme salinities, where fitness-related traits are known to be altered.

Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy.

The family Mugilidae comprises mainly coastal marine species that are widely distributed in all tropical, subtropical and temperate seas. Mugilid species are generally considered to be ecologically important and they are a major food resource for human populations in certain parts of the world. The taxonomy and systematics of the Mugilidae are still much debated and based primarily on morphological characters. In this study, we provide the first comprehensive molecular systematic account of the Mugilidae using phylogenetic analyses of nucleotide sequence variation at three mitochondrial loci (16S rRNA, cytochrome oxidase I, and cytochrome b) for 257 individuals from 55 currently recognized species. The study covers all 20 mugilid genera currently recognized as being valid. The family comprises seven major lineages that radiated early on from the ancestor to all current forms. All genera that were represented by two species or more, except Cestraeus, turned out to be paraphyletic or polyphyletic. Thus, the present phylogenetic results generally disagree with the current taxonomy at the genus level and imply that the anatomical characters used for the systematics of the Mugilidae may be poorly informative phylogenetically. The present results should provide a sound basis for a taxonomic revision of the mugilid genera. A proportion of the species with large distribution ranges (including Moolgarda seheli, Mugil cephalus and M. curema) appear to consist of cryptic species, thus warranting further taxonomic and genetic work at the infra-generic level.

First record of Mugil capurrii (Mugilidae, Perciformes) in the Gulf of Guinea.

Leaping African mullet Mugil capurrii was caught along the Togolese coast in the Gulf of Guinea. This is the first record of this species which usually occurs from Morocco to Guinea Bissau and the southernmost point of its known distribution.

Global phylogeography of the dolphinfish (Coryphaena hippurus): the influence of large effective population size and recent dispersal on the divergence of a marine pelagic cosmopolitan species.

Pelagic fish that are distributed circumtropically are characterised by a low population structure level as a result of a high capacity for dispersion and large population sizes. Nevertheless, historical and contemporary processes, including past demographic and/or range expansions, secondary contact, dispersal, gene flow, and the achievement of large effective population sizes, may play a part in the detection of divergence signals, especially in the case of tropical pelagic species, whose distribution range depends strongly on the sea surface temperature. The connectivity and historical demography of Atlantic, Indian, Pacific and Mediterranean populations of dolphinfish (Coryphaena hippurus) was studied using partial sequences of the mitochondrial DNA NADH dehydrogenase subunit 1 (ND1). AMOVA analyses revealed significant inter-oceanic divergence with three phylogroups located in the Indo-Pacific, Eastern Atlantic, and Mediterranean Sea, the last one being the most divergent. However, it was not possible to clearly observe any genetic differentiation between the Indo-Pacific and Atlantic populations, as has been reported for most tropical pelagic species of tuna and billfishes. This supports the assumption of recent dispersal among basins facilitated by the actual continuous distribution of dolphinfish populations. Moreover, the lack of a divergence signal for populations separated by the Panamanian Isthmus reveals that genetic drift does not exert a strong influence on tropical pelagic species with large effective population sizes.

Multiplex 16S rRNA haplotype-specific PCR, a rapid and convenient method for fish species identification: an application to West African Clupeiform larvae.

A multiplex haplotype-specific polymerase chain reaction (MHS-PCR) method was developed, which identified seven Clupeiform species living in the tropical Eastern Atlantic region: Sardinella aurita, Sardinella maderensis, Ethmalosa fimbriata, Sardina pilchardus, Engraulis encrasicholus, Pellonula leonensis and Ilisha africana. 16S rRNA fragments were amplified using a species-specific set of primers, yielding species-specific size fragments, and then separated using agarose gel electrophoresis, enabling direct visual identification of targeted species. This method provides an accurate, easy and rapid tool for identifying species within large Clupeiform samples. It is suitable for investigations on early Clupeiform stages, species and identification in fishery management in the tropical Eastern Atlantic area.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 April 2010 - 31 May 2010.

This article documents the addition of 396 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anthocidaris crassispina, Aphis glycines, Argyrosomus regius, Astrocaryum sciophilum, Dasypus novemcinctus, Delomys sublineatus, Dermatemys mawii, Fundulus heteroclitus, Homalaspis plana, Jumellea rossii, Khaya senegalensis, Mugil cephalus, Neoceratitis cyanescens, Phalacrocorax aristotelis, Phytophthora infestans, Piper cordulatum, Pterocarpus indicus, Rana dalmatina, Rosa pulverulenta, Saxifraga oppositifolia, Scomber colias, Semecarpus kathalekanensis, Stichopus monotuberculatus, Striga hermonthica, Tarentola boettgeri and Thermophis baileyi. These loci were cross-tested on the following species: Aphis gossypii, Sooretamys angouya, Euryoryzomys russatus, Fundulus notatus, Fundulus olivaceus, Fundulus catenatus, Fundulus majalis, Jumellea fragrans, Jumellea triquetra Jumellea recta, Jumellea stenophylla, Liza richardsonii, Piper marginatum, Piper aequale, Piper darienensis, Piper dilatatum, Rana temporaria, Rana iberica, Rana pyrenaica, Semecarpus anacardium, Semecarpus auriculata, Semecarpus travancorica, Spondias acuminata, Holigarna grahamii, Holigarna beddomii, Mangifera indica, Anacardium occidentale, Tarentola delalandii, Tarentola caboverdianus and Thermophis zhaoermii.

Phylogeography of the flathead mullet Mugil cephalus in the north-west Pacific as inferred from the mtDNA control region.

The population genetic structure and historical demography of the flathead mullet Mugil cephalus were investigated using the mtDNA control region (CR) sequences (909-1015 bp) of 126 individuals collected from seven locations in the north-west Pacific between 2005 and 2007. Haplotype diversity (h = 0.9333-1.000) and nucleotide diversity (pi = 0.0046-0.1467) varied greatly among the sampling locations. Phylogenetic analysis of the CR sequences indicated that M. cephalus in the north-west Pacific belongs to two highly divergent lineages (lineages 1 and 2), with the inferred population structure being closely associated with the distribution of both lineages. Two populations were identified, one from the East China Sea and the other from the South China Sea. The former samples were obtained from Taiwan and Qingdao of north China and associated with lineage 1 haplotypes. The latter samples were collected from the Philippines, Pearl River of South China and two samples from Japan, all of which were associated with lineage 2. Japanese samples from Okinawa and Yokosuka had different degrees of mixing between lineages 1 and 2. Historical demographic variables in both populations indicated that Pleistocene glaciations had a strong impact on M. cephalus in the north-west Pacific, resulting in a recent demographic decline of the East China Sea population but in demographic equilibrium for the South China Sea population. Japan appears to be a contact zone between lineages 1 and 2, but it may also be indicative of coexistence between resident and migratory populations. Further global studies are required to clarify the taxonomic status of this cosmopolitan species.

Molecular systematics, phylogeny and biogeography of roaches (Rutilus, Teleostei, Cyprinidae).

In this study we used sequence data from the entire mtDNA cytochrome b gene to reconstruct patterns and times of diversification in the roach genus Rutilus. The genus is present with numerous endemic species in the Eastern peri-Mediterranean area and with a few widespread species in Central Europe. Our phylogenetic results do not support the subdivision into two subgenera proposed on morphological grounds. Within R. pigus and R. rutilus we identify highly divergent and allopatric mitochondrial lineages. The deeper splits in the genus phylogeny date back to the middle Miocene; the main diversification took place at the Miocene-Pliocene boundary.

Growth hormone and Prolactin-1 gene transcription in natural populations of the black-chinned tilapia Sarotherodon melanotheron acclimatised to different salinities.

The effects of salinity on the expression of genes coding for growth hormone (GH) and prolactin-1 (PRL1) were studied in various natural populations of the black-chinned tilapia Sarotherodon melanotheron from West Africa. Individuals were sampled in June 2005 in six locations in Senegal and the Gambia, at various salinities between 0 and 101. The poorest condition factors were recorded in the most saline sampling site and the best growth in the fish from a marine environment. The pituitary GH mRNA levels were significantly higher in fish adapted to seawater, whereas the PRL1 mRNA levels were highest in fish adapted to fresh- and brackish water. These results show that the PRL1 mRNA levels seem to reflect relatively well the differences in environmental salinity, in contrast to those of GH, which would tend instead to reflect the individual growth in each environment. However, no relation could be found between growth in the hypersaline areas and the expression profile of GH. Although the fish analysed were morphologically identical, the expression of genes coding for GH and PRL1 showed large differences between individuals. This inter-individual variation in gene expression remains poorly understood.

Discrepancies in phylogeographical patterns of two European anglerfishes (Lophius budegassa and Lophius piscatorius).

In order to investigate the relative importance of historical processes and life-history traits in shaping the present-day genetic structure of European anglerfishes, 382 Lophius piscatorius and 134 Lophius budegassa were sequenced on the 5' end of the mitochondrial control region. Both species showed a limited genetic structure and some evidence of a demographic expansion that probably occurred not at the end of the Last Glacial Maximum, but earlier in the Pleistocene. The main discrepancy between the two anglerfishes concerned the genetic structure between Atlantic and Mediterranean populations, with weak but significant differentiation observed only in L. budegassa. This genetic structure was congruent with the existence of a phylogeographical break previously reported in several marine species across the Almeria-Oran front. The contrast observed between both anglerfishes was supposed to be induced by a possible more ancient (re)colonisation of the Mediterranean Sea by L. budegassa. Finally, the limited genetic structure and lack of isolation by distance observed in both species suggested high larval dispersal capacities that probably overwhelm the influence of the bathymetric distribution range on migrations of adults and juveniles.

Impact of glaciations and geographic distance on the genetic structure of a tropical estuarine fish, Ethmalosa fimbriata (Clupeidae, S. Bowdich, 1825).

We assayed the mtDNA phylogeography [196 base pairs (bp) of the cytochrome b] and population structure (n = 680) in the estuarine fish, Ethmalosa fimbriata, from its whole distribution range: 14 locations along the West African coasts were sampled. Specifically, we considered Pleistocene glaciations as well as the hydrodynamics and climatic conditions of the estuarine environments in order to identify the main evolutionary forces that have shaped the genetic variation in mtDNA, i.e., the contemporary or the historical gene flow. There was an overall significant population differentiation among estuaries (Fst = 0.10). Although E. fimbriata showed a significant pattern of isolation by distance over the entire sampled range, this genetic structure did not mirror contemporary gene flow but the colonization sequence of the present distribution range. Finally, the mtDNA genetic structure traced the past historic dispersion that occurred at the end of the Pleistocene glaciations. The central part of the present distribution area was probably the species origin and due to difference in the historic migration rate northward and southward, isolation of a South group occurred first, 110,000-190,000 years ago, before the divergence of the North group 47,000-82,000 years ago.

Genetic structure of fragmented populations of a threatened endemic percid of the Rhône river: Zingel asper.

Zingel asper is an endemic percid of the Rhône basin considered to be critically endangered. This species was continuously distributed throughout the Rhône in 1900, but today only occupies 17% of its initial area. In the present study, five microsatellite loci were used to assess the level of genetic variability within and among populations localized in different sub-basins. Contrasting results were obtained for the three main populations from the Rhône. A reduced allelic diversity was observed for the two populations displaying the lowest patch sizes (length of the river system occupied); of these, a recent genetic bottleneck was detected for the population showing a particularly low density. However, the third population was characterized by a relatively large spatial extent, high local fish concentrations and an allelic diversity that was twice as high and associated with an equilibrium between mutation and drift. Thus, this population shows an apparently better evolutionary potential for long-term survival. Since 1930, a marked fragmentation of the whole Rhône system has appeared, related to the development of dams, and we assume that the significant genetic differentiation detected between the populations could mainly reflect the impact of this fragmentation. The high turnover of the Z. asper populations, and the major role of dispersal in population persistence (highlighted in a recent population dynamics study), indeed suggest that the differentiation observed could mainly have arisen from habitat fragmentation in recent history.

Insight into the origin of endemic Mediterranean ichthyofauna: phylogeography of Chondrostoma genus (Teleostei, Cyprinidae).

The Chondrostoma genus is widespread in Europe, with numerous endemic species in northern Mediterranean rivers. We reconstructed the phylogenetic relationships of this genus, using the whole cytochrome b sequence and compared the two freshwater fish dispersion hypotheses: (1) dispersion around the Mediterranean Sea during the Lago Mare phase of the Messinian salinity crisis (Bianco's hypothesis) and (2) an older and more gradual colonization of the Mediterranean rivers (Banarescu's hypothesis). All phylogenetic analyses identified two levels of divergences, implying two radiation events in the Chondrostoma genus. The first radiation mainly concerned Mediterranean species, whereas the second one includes Danubian and Mesopotamian species. This phylogeographic pattern was already observed for the genus Squalius, which exhibits a similar geographic range distribution in Europe and probably is shared with several other Mediterranean genera, such as Scardinius, Rutilus, and TELESTES: Furthermore, assuming a molecular clock of 1% per million years, the first radiation appears consistent with a Messinian dispersion during the Lago Mare, 5.3 million years ago, whereas the second one may correspond to a Mesopotamian dispersion through the Black Sea to the Danube system. According to our results, the Lago Mare theory is strengthened, and a more recent and pre-Pleistocene colonization of the Danube from Mesopotamian freshwater fishes is suggested.

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.

Identifying main evolutionary mechanisms shaping genetic variation of Leuciscus cephalus L. 1758 (Cyprinidae) in western Greece: discordance between methods.

Numerous methods can be used in intraspecific phylogeographic studies to infer the evolutionary mechanisms that shaped observed genetic variation in populations. However, these methods are scarcely used jointly, and the evolutionary outcomes they could propose are not fully compared. In this study, using a chub (Leuciscus cephalus; Cyprinidae) mitochondrial DNA data set (13 populations in Western Greece, 14 haplotypes), we compare three distinct 'historical' methods that could possibly infer relative importance of basic evolutionary mechanisms (isolation vs migration) shaping genetic variation: the nested clade analysis, the psi-test and the 'mismatch distributions'. Taking together, interpretations of these analyses allow to draw a picture of the evolutionary history of chub in Western Greece based on isolation and genetic drift for higher clades. However, results issued each method can differ for low differentiated clades. We discuss such differences and suggest that methods should be used jointly in phylogeographic studies for a better evaluation of the evolutionary mechanisms that shaped genetic variation.

Origin, radiation, dispersion and allopatric hybridization in the chub Leuciscus cephalus.

The phylogenetic relationships of 492 chub (Leuciscus cephalus) belonging to 89 populations across the species' range were assessed using 600 base pairs of cytochrome b. Furthermore, nine species belonging to the L. cephalus complex were also analysed (over the whole cytochrome b) in order to test potential allopatric hybridization with L. cephalus sensu stricto (i.e. the chub). Our results show that the chub includes four highly divergent lineages descending from a quick radiation that took place three million years ago. The geographical distribution of these lineages and results of the nested clade analysis indicated that the chub may have originated from Mesopotamia. Chub radiation probably occurred during an important vicariant event such as the isolation of numerous Turkish river systems, a consequence of the uplift of the Anatolian Plateau (formerly covered by a broad inland lake). Dispersion of these lineages arose from the changes in the European hydrographic network and, thus, the chub and endemic species of the L. cephalus complex met by secondary contacts. Our results show several patterns of introgression, from Leuciscus lepidus fully introgressed by chub mitochondrial DNA to Leuciscus borysthenicus where no introgression at all was detected. We assume that these hybridization events might constitute an important evolutionary process for the settlement of the chub in new environments in the Mediterranean area.

Phylogeography and postglacial dispersion of the chub (Leuciscus cephalus) in Europe.

A phylogeographic analysis of mitochondrial DNA variation was performed in order to test the hypothesis of a postglacial recolonization of mid- and north-European rivers from a Danubian refuge. Over 345 chub specimens from European rivers covering most of the species' native range were investigated using 600 bp of the cytochrome b gene. Chub in European rivers belong to four highly divergent mitochondrial groups (lineages) differing by mean divergence estimates from 5.2% to 7.89%. These four lineages have a largely allopatric distribution, implying four geographical sets: two Mediterranean, and two north-European sets. This pattern provided strong evidence for: (i) the eradication of this species from most of Europe during maximum ice extent; (ii) its survival in four refugia (Adriatic side of the Balkans, eastern Greece (Aegean Rivers), southern tributaries of the Danube, and periphery of Black and Caspian Seas); (iii) a differential postglacial recolonization of mid- and northern Europe from the last two refugia only; (iv) the occurrence of this recolonization in two steps for the Danubian (western) lineage that entered western Europe (Rhine-Rhone-Loire drainages) during the Riss-Würm interglacial period and survived the last glaciation there before colonizing Garonne, UK and German drainages up to the Elbe during the Holocene; and (v) the occurrence of this recolonization in a single step for the Ponto-Caspian (eastern) lineage that entered the Baltic area as far as the Oder in the Holocene. Both lineages came into contact in the River Elbe without evident mixing.