Patterns of nuclear and mitochondrial DNA variation in Iberian populations of <Emphasis Type="Italic">Emys orbicularis</Emphasis> (Emydidae): conservation implications

The European pond turtle (Emys orbicularis) is threatened and in decline in several regions of its natural range, due to habitat loss combined with population fragmentation. In this work, we have focused our efforts on studying the genetic diversity and structure of Iberian populations with a fine-scale sampling (254 turtles in 10 populations) and a representation from North Africa and Balearic island populations. Using both nuclear and mitochondrial markers (seven microsatellites, ∼1048 bp nDNA and ∼1500 bp mtDNA) we have carried out phylogenetic and demographic analyses. Our results show low values of genetic diversity at the mitochondrial level although our microsatellite dataset revealed relatively high levels of genetic variability with a latitudinal genetic trend decreasing from southern to northern populations. A moderate degree of genetic differentiation was estimated for Iberian populations (genetic distances, F _ST values and clusters in the Bayesian analysis). The results in this study combining mtDNA and nDNA, provide the most comprehensive population genetic data for E. orbicularis in the Iberian Peninsula. Our results suggest that Iberian populations within the Iberian–Moroccan lineage should be considered as a single subspecies with five management units, and emphasize the importance of habitat management rather than population reinforcement (i.e. captive breeding and reintroduction) in this long-lived species.     

The complete mitochondrial genome structure of snow leopard <Emphasis Type="Italic">Panthera uncia</Emphasis>

The complete mitochondrial genome (mtDNA) of snow leopard Panthera uncia was obtained by using the polymerase chain reaction (PCR) technique based on the PCR fragments of 30 primers we designed. The entire mtDNA sequence was 16 773 base pairs (bp) in length, and the base composition was: A—5,357 bp (31.9%); C—4,444 bp (26.5%); G—2,428 bp (14.5%); T—4,544 bp (27.1%). The structural characteristics [0] of the P. uncia mitochondrial genome were highly similar to these of Felis catus, Acinonyx jubatus, Neofelis nebulosa and other mammals. However, we found several distinctive features of the mitochondrial genome of Panthera unica. First, the termination codon of COIII was TAA, which differed from those of F. catus, A. jubatus and N. nebulosa. Second, tRNA^{Ser (AGY)}, which lacked the ‘‘DHU’’ arm, could not be folded into the typical cloverleaf-shaped structure. Third, in the control region, a long repetitive sequence in RS-2 (32 bp) region was found with 2 repeats while one short repetitive segment (9 bp) was found with 15 repeats in the RS-3 region. We performed phylogenetic analysis based on a 3 816 bp concatenated sequence of 12S rRNA, 16S rRNA, ND2, ND4, ND5, Cyt b and ATP8 for P. uncia and other related species, the result indicated that P. uncia and P. leo were the sister species, which was different from the previous findings.     

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris)

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) ofPanthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8–17 million years ago in the tiger and 4.6–16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular ‘fossils’ that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.     

The impact of time and field conditions on brown bear (<Emphasis Type="Italic">Ursus arctos</Emphasis>) faecal DNA amplification

To establish longevity of faecal DNA samples under varying summer field conditions, we collected 53 faeces from captive brown bears (Ursus arctos) on a restricted vegetation diet. Each faeces was divided, and one half was placed on a warm, dry field site while the other half was placed on a cool, wet field site on Moscow Mountain, Idaho, USA. Temperature, relative humidity, and dew point data were collected on each site, and faeces were sampled for DNA extraction at <1, 3, 6, 14, 30, 45, and 60 days. Faecal DNA sample viability was assessed by attempting PCR amplification of a mitochondrial DNA (mtDNA) locus (∼150 bp) and a nuclear DNA (nDNA) microsatellite locus (180–200 bp). Time in the field, temperature, and dew point impacted mtDNA and nDNA amplification success with the greatest drop in success rates occurring between 1 and 3 days. In addition, genotyping errors significantly increased over time at both field sites. Based on these results, we recommend collecting samples at frequent transect intervals and focusing sampling efforts during drier portions of the year when possible.     

Effect of crab shell size on bio-demineralization with lactic acid-producing bacterium, <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">tolerans</Emphasis> KCTC-3074

Demineralization (DM) from crab shell (CS) waste was carried out using a lactic acid-producing bacterium, Lactobacillus paracasei subsp. tolerans KCTC-3074 for 7 days at 25, 30, and 35°C. DM rates were 89∼92% and slightly affected by temperature. DM was also performed for four particle-sized shell samples (0.84∼3.35, 3.35∼10, 10∼20, and 20∼35 mm) with 10% inoculum, 5% shell, and 10% glucose at 30°C and 180 rpm for 7 days. It was found out that the shell size had a slight effect on the rate of DM. Negative relationships were found between DM and residual dry weight (r² = 0.960), and between DM and pH (r² = 0.906). Conversely, positive relationships were found between DM and medium protein (r² = 0.696), and between DM and total titratable acidity (r² = 0.630).     

Examination of the Taxonomy and Diversification of <Emphasis Type="Italic">Leontopithecus</Emphasis> using the Mitochondrial Control Region

Leontopithecus comprises 4 taxa: black lion tamarins (L. chrysopygus), golden lion tamarins (L. rosalia), black-faced lion tamarins (L. caissara), and golden-headed lion tamarins (L. chrysomelas). Endemic to the Atlantic Forest of Brazil, they are endangered (Appendix I, CITES; IUCN Critically Endangered: Leontopithecus chrysopygus, L. caissara; IUCN Endangered: L. rosalia, L. chrysomelas). The 4 taxa are differentiated morphologically and geographically and occupy different habitat types. However, it is not clear if all of them are separate species, particularly Leontopithecus caissara, or how they are related to one another evolutionarily. Therefore, we investigated lion tamarin differentiation and radiation. We sequenced the mtDNA control region and performed phylogenetic analyses, population aggregation analyses, and Mantel tests for geographic/genetic correlation. Mitochondrial genetic data suggest 3 distinct lion tamarin clades (Leontopithecus chrysomelas; L. caissara; and L. chrysopygus/L. rosalia). Phylogenetic analysis also supports: 1) the basal lion tamarin is Leontopithecus chrysomelas, and not L. chrysopygus, 2) L. caissara is not subspecific to L. chrysopygus, and 3) Quaternary forest refugia may have shaped lion tamarin diversification via a pattern that does not follow the theory of metachromism. Even though mitochondrial genetic analyses do not unequivocally support the 4 lion tamarins as separate species, one should consider the 4 lion tamarins, with equal conservation priority based on the combination of morphological, genetic, and habitat differentiation. Each of them is an extremely valuable flagship species that focuses attention on the diminishing, highly endemic Atlantic Forest of Brazil.     

Evidence of olive ridley mitochondrial genome introgression into loggerhead turtle rookeries of Sergipe, Brazil

The coastline of Sergipe state hosts the main Brazilian nesting sites of Lepidochelys olivacea (Eschscholtz, 1829). The second most abundant species of turtles in Sergipe is Caretta caretta (Linnaeus, 1758). Both sea turtle species, respectively known as olive ridley and loggerhead, are currently listed as endangered by the International Union for the Conservation of Nature and Natural Resources. The genetic diversity of the Sergipe loggerhead population (N = 51) was assayed by analyzing 627 bp from the control region of mitochondrial DNA in nesting females. Three haplotypes were identified: CC-A4, CC-A24 and CC × LO. The last one was recorded for specimens considered hybrids because they represent L. olivacea’s mtDNA, but had the external morphology of C. caretta or of a mixture of both species. Based on the two types of hybrids, it was hypothesized that at least two hybridization events had occurred: a more ancient hybridization event, accompanied by introgression (F2 or later backcrosses), and a recent one (F1), both of which involving the same L. olivacea haplotype. The incidence of L. olivacea mitochondrial genome introgression into the C. caretta rookeries was only observed in Sergipe, which could be related to the large numbers of L. olivacea in this region and an overlap of reproduction periods and distribution areas of both species. This may also be associated to global warming since it might alter the sex ratio of sea turtles, thus facilitating interspecific mating. Awareness of gene flow between these species will significantly influence the development and implementation of adequate management strategies.     

Evolutionary patterns in the antR-Cor gene in the dwarf dogwood complex (Cornus, Cornaceae)

The evolutionary pattern of the myc-like anthocyanin regulatory gene antR-Cor was examined in the dwarf dogwood species complex (Cornus Subgenus Arctocrania) that contains two diploid species (C. canadensis and C. suecica), their putative hybrids with intermediate phenotypes, and a tetraploid derivative (C. unalaschkensis). Full-length sequences of this gene (∼4 kb) were sequenced and characterized for 47 dwarf dogwood samples representing all taxa categories from 43 sites in the Pacific Northwest. Analysis of nucleotide diversity indicated departures from neutral evolution, due most likely to local population structure. Neighbor-joining and haplotype network analyses show that sequences from the tetraploid and diploid intermediates are much more strongly diverged from C. suecica than from C. canadensis, and that the intermediate phenotypes may represent an ancestral group to C. canadensis rather than interspecific hybrids. Seven amino acid mutations that are potentially linked to myc-like anthocyanin regulatory gene function correlate with petal colors differences that characterize the divergence between two diploid species and the tetraploid species in this complex. The evidence provides a working hypothesis for testing the role of the gene in speciation and its link to the petal coloration. Sequencing and analysis of additional nuclear genes will be necessary to resolve questions about the evolution of the dwarf dogwood complex.     

Genetic identification of units for conservation in tomato frogs, genus Dyscophus

Dyscophus antongilii and D. guineti are two morphologically very similar microhylid frogs from Madagascar of uncertain taxonomy. D. antongilii is currently included in Appendix I of the Convention on the International Trade in Endangered Species (CITES) and its exportation is banned completely. In contrast, D. guineti does not receive any legal protection and it is regularly exported. Field data on ecology and behaviour are to a large extent lacking. Here we report on a genetic survey of D. antongilii and D. guineti using nuclear and mitochondrial DNA markers. Sequences of a fragment of 501 bp of the mitochondrial cytochrome b gene from one population of D. antongilii and two populations of D. guineti resulted in a single haplotype network, without haplotype sharing among the populations. However, haplotypes of D.␣guineti were only 1–4 mutational steps from those of D. antongilii, and did not form a clade. The analysis of eight microsatellites newly developed and standardized for D. antongilii revealed an excess of homozygotes and the absence of Hardy–Weinberg equilibrium. The microsatellite data clearly distinguished between D. antongilii and D. guineti, and fixed differences were observed at one locus. Although confirmation of the status of Dyscophus antongilii and D. guineti as separate species requires further data, our study supports the definition of these two taxa as different evolutionary significant units under the adaptive evolutionary conservation concept.     

Phylogeny and phylogeography of the Mediterranean species of the parasitic ant genus <Emphasis Type="Italic">Chalepoxenus</Emphasis> and its <Emphasis Type="Italic">Temnothorax</Emphasis> hosts

We analysed the phylogenetic and phylogeographic relationships of four Mediterranean species of the rare slave-making ant genus Chalepoxenus and eleven of its about 20 Temnothorax host species by sequencing the mitochondrial Cytochrome Oxidase I and II genes. Neighbour-Joining, Maximum Parsimony and Bayesian analyses based on 1320 bp indicate that the genus Chalepoxenus constitutes a monophylum. In all three analyses, C. kutteri from Southwest Europe and the workerless, “degenerate slavemaker” C. brunneus from North Africa form a monophyletic group. C. muellerianus and C. tauricus, distributed in Southern Europe and Ukraine, respectively, form a monophylum in the Neighbour-Joining and the Maximum Parsimony analysis. In our limited set of only 11 of several hundred Temnothorax species, T. flavicornis forms the sister group of Chalepoxenus. Our study further indicates paraphyly of the genus Temnothorax with respect to Chalepoxenus. Moreover, the results suggest that speciation in this slave-making genus is possibly caused by the formation of host races as different Chalepoxenus species use different hosts, and some samples seem to cluster by host species rather than by geographical distance. Received 5 September 2006; revised 17 March 2007; accepted 23 March 2007.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

The world’s economically most important chelonians represent a diverse species complex (Testudines: Trionychidae: <Emphasis Type="Italic">Pelodiscus</Emphasis>)

Pelodiscus is one of the most widely distributed genera of softshell turtles, ranging from south-eastern Siberia and Korea over central and southern China to Vietnam. Economically, Pelodiscus are the most important chelonians of the world and have been bred and traded in high numbers for centuries, resulting in many populations established outside their native range. Currently, more than 300 million turtles per year are sold in China alone, and the bulk of this figure comprises farmed Pelodiscus. Due to easy availability, Pelodiscus also constitutes a model organism for physiological and embryological investigations. Yet, diversity and taxonomy of Pelodiscus are poorly understood and a comprehensive investigation using molecular tools has never been published. Traditionally, all populations were assigned to the species P. sinensis (Wiegmann, 1834); in recent years up to three additional species have been recognized by a few authors, while others have continued to accept only P. sinensis. In the present study, we use trade specimens and known-locality samples from Siberia, China, and Vietnam, analyze 2,419 bp of mtDNA and a 565-bp-long fragment of the nuclear C-mos gene to elucidate genetic diversity, and compare our data with sequences available from GenBank. Our findings provide evidence for the existence of at least seven distinct genetic lineages and suggest interbreeding in commercial turtle farms. GenBank sequences assigned to P. axenaria (Zhou, Zhang & Fang, 1991) are highly distinct. The validity of P. maackii (Brandt, 1857) from the northernmost part of the genus’ range is confirmed, whereas it is unclear which names should be applied to several taxa occurring in the central and southern parts of the range. The diversity of Pelodiscus calls for caution when such turtles are used as model organisms, because the respective involvement of more than a single taxon could lead to irreproducible and contradictory results. Moreover, our findings reveal the need for a new assessment of the conservation status of Pelodiscus. While currently all taxa are subsumed under ‘P. sinensis’ and listed as ‘vulnerable’ by the IUCN Red List of Threatened Species, some could actually be endangered or even critically endangered.     

Multi-locus phylogeny of the family Acrocephalidae (Aves: Passeriformes) – The traditional taxonomy overthrown

We present the first study of the warbler family Acrocephalidae based on one mitochondrial and three nuclear DNA loci, in total 2900 bp, including most or all of the species in three (Acrocephalus, Hippolais and Chloropeta) of the four genera and one species in the fourth genus (Nesillas) in this family. All three genera were suggested to be non-monophyletic, although the non-monophyly of Acrocephalus is not fully convincingly demonstrated. Six major clades were found, which agreed largely with the results from two earlier mitochondrial studies, and for which the names Hippolais, Iduna, Acrocephalus, Calamocichla, Notiocichla and Calamodus have been used. However, the results also revealed some new constellations, due to better resolution of deeper nodes and the inclusion of more taxa. The taxonomic implications are discussed.     

Analysis of mitochondrial DNA: Taxonomic and phylogenetic relationships in two fish taxa (Pisces: Mugilidae and Cyprinidae)

To solve some systematic questions as well as to study genetic variability and evolutionary relationships in two groups of fish belonging to the Mugilid (Mugilidae) and Cyprinid (Cyprinidae) families, we have used restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction. The analysis of three mtDNA fragments of 7220 bp total length of six Mugilid species has shown that Mediterranean Liza aurata, L. ramada, L. saliens, and Chelon labrosus form a common cluster, L. aurata and C. labrosus being the closest relatives, whereas L. haematocheilus (syn. C. haematocheilus) of the Sea of Japan forms a sister group to the Mediterranean cluster. It was found that Chelon and Liza genera are paraphyletic, and therefore their division into two genera is unnatural and they should be synonymized. According to priority, Liza species should be ascribed to Chelon genus. Mugil cephalus is the most distant compared to the rest of the species studied. The level of genetic divergence between allopatric samples of M. cephalus from the Sea of Japan and the Mediterranean Sea has proved to be very high—4.5% of nucleotide substitutions. The analysis of four mtDNA fragments of 9340 bp total length of six Cyprinid species has shown that L. waleckii is the most genetically distant. Pseudaspius leptocephalus is a sister group to Tribolodon species. All Tribolodon species form a common cluster with T. sachalinensis as a root. The remaining species form two branches, one of which includes T. nakamurai and T. brandtii, another one combines T. hakonensis and a new form of Tribolodon revealed that is close to T. hakonensis by its mtDNA (2.4% of nucleotide substitutions). This new form might be an independent species. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 12, pp. 1651–1658.     

Genetic differentiation between populations of swimming crab <Emphasis Type="Italic">Portunus trituberculatus</Emphasis> along the coastal waters of the East China Sea

Portunus trituberculatus is a commercially important species widely spread in the East China Sea. Intraspecific variation of the mitochondrial DNA cytochrome oxidase subunit I (mtDNA COI) gene was investigated in 213 individuals from six localities (Changjiang Estuary, Shengsi Islands, Zhoushan Islands, Dongtou Islands, Dinghai Bay, and Quanzhou Bay) ranging from north (31°21′N) to south (24°55′N) coastal waters of the East China Sea. Overall, a total of 27 mtDNA haplotypes and 21 variable sites were detected in the 787 bp segment of COI gene. Analysis of mtDNA COI sequence data revealed that crabs from the six localities were characterized by moderately high haplotypic diversity (h = 0.787 ± 0.026), while sequence divergence values between haplotypes were relatively low (π = 0.00241 ± 0.00098). Each population was characterized by a single most frequent haplotype, shared among all six localities, and a small number of rare ones, typically present in only one or two individuals and representative of a specific population. However, neither the neighbor-joining tree nor the minimum spanning network (MSN) based on the haplotype data exhibited geographical patterns of the six populations. Mismatch distribution analysis of P. trituberculatus individuals sampled from the six localities suggested that sudden population expansion might have occurred in CJ and SS population that might be consistent with over-exploitation of the swimming crab. Analysis of molecular variance (AMOVA) and F _ST statistics showed that significant genetic differentiation existed among the SS, ZS, DT, DH, and QZ populations, suggesting that gene flow might be reduced, even between the geographically close sites, despite the high potential of dispersal. The possible causes of the observed genetic heterogeneity among the P. trituberculatus populations and the potential applications of the mtDNA COI marker in the artificial breeding and fisheries management are discussed. Handling editor: C. Sturmbauer     

Phylogeny and phylogeography of critically endangered Gyps species based on nuclear and mitochondrial markers

Populations of Oriental White-backed Vulture (Gyps bengalensis) and Long-billed Vulture (Gyps indicus) dramatically declined by 95–100% on the Indian subcontinent in mid-1990s. The present study was conducted to discover the phylogeny and phylogeography of Gyps species based on nuclear (recombination activating gene, RAG-1) and mitochondrial (cytochrome b, cytb) markers. Gyps species showed monophyly and no geographic partition was observed within the three groups of Gyps species (G. bengalensis, G. indicus and G. fulvus) despite the large sample size available (n = 149). Our study supports the treatment of G. indicus and G. tenuirostris as separate species. In all analyses, the earliest divergence separated G. bengalensis from all other Gyps taxa while a sister relationship was supported between G. fulvus and G. rueppellii, and these two taxa together were sister group to a clade consisting of G. indicus, G. tenuirostris and G. coprotheres. Molecular clock estimates of both nuclear and mitochondrial (RAG-1, cytb) genes indicated a rapid and recent diversification within the Gyps species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mitochondrial diversity of European pond turtles (Emys orbicularis) in Anatolia and the Ponto-Caspian Region: Multiple old refuges, hotspot of extant diversification and critically endangered endemics

The European pond turtle, Emys orbicularis (Linnaeus, 1758), is one of the world's most widely distributed chelonian species. We investigated its mitochondrial phylogeography and demography using ∼1300 cyt b sequences from the entire range, with a focus on the eastern part, in particular on Turkey, where the species currently suffers massive losses. Coloration data from >1450 turtles were compared with mtDNA differentiation to assess the validity of the currently accepted subspecies from Turkey, the Black Sea Region, the Transcaucasus, and Iran. Our study region harbors considerable part of the mtDNA diversity of Emys, including a newly discovered lineage and 16 new haplotypes. In this area corresponding to approximately one-third of the entire distribution range, six out of the ten mitochondrial lineages and about half of all 72 haplotypes occur. Two mitochondrial lineages (VIII, X) are confined to small ranges along the southern coast of Turkey, another lineage (I) occupies the remainder of Turkey, the entire Black Sea Region, and the north-eastern part of the species’ range. In the south-western corner of the Black Sea and in the Aegean Region, two lineages (II, IV) occur that have their main distribution areas farther west. In the Transcaucasus and northern Iran, another endemic lineage (VII) is found. Lineage I is the largest and most diverse of all lineages and has its greatest diversity in Anatolia. Phylogeographic and demographic data suggest Anatolia as an ancient glacial refuge for turtles harboring mitochondrial lineages I, VIII and X, and that Anatolia and the Black Sea coasts constitute a hotspot for a younger burst of diversification within lineage I. These two regions correspond to the glacial refuge from which lineage I turtles recolonized more northerly parts of the range in the Holocene; lineage II represents an off-shoot of lineage I that became isolated in a westward-located refuge in the south-eastern Balkans during a previous Pleistocene glacial. Our data on coloration indicate that such characters have only limited value for delineating evolutionarily significant units. We propose to reduce the number of subspecies using mtDNA lineages as arbiter, and to recognize three subspecies as valid in Turkey, the Black Sea Region, the Transcaucasus and Iran: Emys orbicularis orbicularis (mtDNA lineage I); E. o. eiselti Fritz et al., 1998 (X); and E. o. persica Eichwald, 1831 (VII). However, the southern Turkish lineage VIII most probably represents an additional undescribed subspecies. Both southern Turkish endemics are critically endangered, with only three surviving populations of fewer than 30 adults each. We recommend establishing sanctuaries for them, and including them in the IUCN Red List.     

Molecular cloning, expression and characterization of the zebrafish bram1 gene, a BMP receptor-associated molecule

Summary We have identified a cDNA clone encoding BMP receptor-associated molecule 1 (BRAM1) from the zebrafish expressed sequence tag (EST) database. The 2606 bp full-length bram1 cDNA was cloned, and further confirmed by nucleotide sequencing. The zebrafish sequence encodes a protein of 195 amino acids with an evolutionarily conserved MYND domain, which displays ∼ ∼98% homology with human and mouse BRAM1, and ∼ ∼64% homology with C. elegans BRA-1 and BRA-2. The bram1 gene, composed of five exons and four introns, spans ∼ ∼14 kb on linkage group 14 of the zebrafish genome. RT-PCR and whole mount in situ hybridization analyses disclosed that zebrafish BRAM1 is a maternal factor. The protein interacts directly with zebrafish BMP Receptor type IA, as observed from GST-pull down and co-immunoprecipitation assays. Furthermore, cotransfection of zebrafish BRAM1 with the corresponding BMP receptor resulted in down-regulation of BMP-mediated signaling. Our results collectively indicate that BRAM1 plays a biological role during zebrafish development.