Paralog-Specific Primers for the Amplification of Nuclear Loci in Tetraploid Barbels (Barbus: Cypriniformes)

Thirty paralog-specific primers were developed, following an intron-primed exon-crossing strategy, for S7 and growth hormone genes in Barbus (subgenera Barbus and Luciobarbus). We found that paralog-specific amplification requires the use of only one paralog-specific primer, allowing their simultaneous use with universal exon-primed intron-crossing primers of broad taxonomic applicability. This hybrid annealing strategy guarantees both specificity and generality of amplification reactions and represents a step forward in the amplification of duplicated nuclear loci in polyploid organisms and members of multigene families. Assays of several representative taxa identified high levels of segregating single nucleotide polymorphisms (SNPs) and nucleotide diversity within each of these subgenera. Additionally, several insertions-deletions (indels) that are diagnostic across species are found in intronic regions. Therefore, these primers provide a reliable source of valuable nuclear SNP and indel data for population and species level studies of barbels, such as applied conservation and basic evolutionary studie

The genomic substrate for adaptive radiation in African cichlid fish

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification

Evolution: cichlid models on the runaway to speciation

Rapid speciation has fascinated biologists for a long time. A recent study shows that ecological opportunity and sex-biased color differences increase the likelihood of speciation in African cichlid fishes

Semi-permeable species boundaries in Iberian barbels (Barbus and Luciobarbus, Cyprinidae)

[Background] The evolution of species boundaries and the relative impact of selection and gene flow on genomic divergence are best studied in populations and species pairs exhibiting various levels of divergence along the speciation continuum. We studied species boundaries in Iberian barbels, Barbus and Luciobarbus, a system of populations and species spanning a wide degree of genetic relatedness, as well as geographic distribution and range overlap. We jointly analyze multiple types of molecular markers and morphological traits to gain a comprehensive perspective on the nature of species boundaries in these cyprinid fishes.[Results] Intraspecific molecular and morphological differentiation is visible among many populations. Genomes of all sympatric species studied are porous to gene flow, even if they are not sister species. Compared to their allopatric counterparts, sympatric representatives of different species share alleles and show an increase in all measures of nucleotide polymorphism (S, H d, K, π and θ). High molecular diversity is particularly striking in L. steindachneri from the Tejo and Guadiana rivers, which co-varies with other sympatric species. Interestingly, different nuclear markers introgress across species boundaries at various levels, with distinct impacts on population trees. As such, some loci exhibit limited introgression and population trees resemble the presumed species tree, while alleles at other loci introgress more freely and population trees reflect geographic affinities and interspecific gene flow. Additionally, extent of introgression decreases with increasing genetic divergence in hybridizing species pairs.[Conclusions] We show that reproductive isolation in Iberian Barbus and Luciobarbus is not complete and species boundaries are semi-permeable to (some) gene flow, as different species (including non-sister) are exchanging genes in areas of sympatry. Our results support a speciation-with-gene-flow scenario with heterogeneous barriers to gene flow across the genome, strengthening with genetic divergence. This is consistent with observations coming from other systems and supports the notion that speciation is not instantaneous but a gradual process, during which different species are still able to exchange some genes, while selection prevents gene flow at other loci. We also provide evidence for a hybrid origin of a barbel ecotype, L. steindachneri, suggesting that ecology plays a key role in species coexistence and hybridization in Iberian barbels. This ecotype with intermediate, yet variable, molecular, morphological, trophic and ecological characteristics is the local product of introgressive hybridization of L. comizo with up to three different species (with L. bocagei in the Tejo, with L. microcephalus and L. sclateri in the Guadiana). In spite of the homogenizing effects of ongoing gene flow, species can still be discriminated using a combination of morphological and molecular markers. Iberian barbels are thus an ideal system for the study of species boundaries, since they span a wide range of genetic divergences, with diverse ecologies and degrees of sympatry.This work was supported financially by Fundação para a Ciência e a Tecnologia (FCT) project grant POCTI/BSE/35121/2000 to MJA, and PhD fellowships SFRH/BD/13067/2003 from FCT (co-participated by FEDER, POCI 2010 and FSE), Proc.85547 from Fundação Calouste Gulbenkian and from the Arizona State University School of Life Sciences to HFG. This research received support from the SYNTHESYS Project ES-TAF-1605 to HFG, http://www.synthesys.info/, which is financed by European Community Research Infrastructure Action under the FP7 “Capacities” Programme, at the Museo Nacional de Ciencias Naturales (CSIC).Peer reviewe

Chondrostoma olisiponensis Gante, Santos & Alves, 2007, sp. nov.

Chondrostoma olisiponensis sp. nov. (Figs. 3 and 4) Holotype: MB05- 2195, 1 ex., male. Rio Trancão Basin, Santo Antão do Tojal, Rio Tejo Basin, Portugal. Leg. H. F. Gante, C. D. Santos and V. Branco. 04.X. 2003. Paratypes: MB05- 2196, 2 ex., 1 male and 1 female. Rio Trancão Basin, Santo Antão do Tojal, Rio Tejo Basin, Portugal. Leg. H. F. Gante, C. D. Santos and V. Branco. 04.X. 2003. MB05- 2197, 5 ex., 3 males and 2 females. Rio Trancão Basin, Santo Antão do Tojal, Rio Tejo Basin, Portugal. Leg. H. F. Gante and C. D. Santos. 14.IV. 1999. MB05- 2198, 1 ex., female. Rio Trancão Basin, Santo Antão do Tojal, Rio Tejo Basin, Portugal. Leg. H. F. Gante, C. D. Santos and R. Matias. 26.XII. 1998. MB05- 2199, 2 ex., 1 male and 1 female. Ribeira de Almoster, Rio Maior, Rio Tejo Basin, Portugal. Leg. H. F. Gante and C. D. Santos. 05.VI. 2006. Non-type material: MB05- 1408, 1 ex., female. Vala da Azambuja, locality unknown, Rio Tejo Basin, Portugal. Leg. A. Soares. 20.I. 1979. MB05- 2026, 1 ex., male. Paúl de Magos, Salvaterra de Magos, Rio Tejo Basin, Portugal. Leg. M. J. Collares-Pereira, P. Sobral, M M. Coelho. 26.VI. 1979. MB05- 2027, 4 ex., 2 males and 2 females. Ribeira de Ulme, Monte dos Capelos (Chamusca), Rio Tejo Basin, Portugal. Leg. M. M. Coelho, C. Almaça, M. J. Collares-Pereira. 15.VI. 1980. MB05- 2031, 2 ex., 2 females. Ribeira de Ulme, Chamusca/Alpiarça, Rio Tejo Basin, Portugal. Leg. M. J. Collares-Pereira, M. M. Coelho, A. Soares. 01.IV. 1980. Diagnosis. Chondrostoma olisiponensis sp. nov. is distinguished from the other species of Chondrostoma by the following combination of characters: the new species lacks a horny blade on the lower jaw on the pronouncedly arched mouth, and lacks an intense reddish coloration at the base of the fins. Chondrostoma olisiponensis has elongated pelvic fins that reach the anus and often pass the anal-fin insertion in males. Dorsal, pelvic and anal fins usually have 8 branched rays. The new species has 36 to 43 scales in the lateral line, 7.0 to 8.5 scales above the lateral line, 13 to 16 circumpeduncular scales, denticulated (vs. smooth) grinding teeth surfaces, 6 - 5 / 5 pharyngeal teeth and 15 to 19 gill rakers. Description. LL 36 (1) 39 *(4) 40 (3) 41 (2) 43 (1); TA 7 *(2) 7.5(3) 8 (1) 8.5(5); TB 3 (2) 3.5*(4) 4 (4) 4.5(1); CP 13 *(1) 14 (4) 15 (3) 16 (3); LPT 5 *(3) 6 (8); RPT 5; DR ii(1) iii*(10), 7 (1), 8 *(10); AR ii(1) iii*(10), 7 (1) 8 (9) 9 *(1); PvR ii, 8; GR 15 (1) 16 (3) 17 (4) 18 *(2) 19 (1); PcPv 0(3) 1 (3) 2 *(5); PvA 0*(6) 1 (2) 2 (3); PvAn - 1 (1) 0(4) 1 (1) 2 (1) 3 *(4). Chondrostoma olisiponensis is a small species (largest specimen 104.4 mm in SL), with a less elongated body than many of the other species in the genus. Pre-dorsal profile is deep and convex, and becomes smoother behind the dorsal fin. Females usually have deeper bodies, while males are slender. Mouth is terminal and horseshoe-shaped, with no horny blade on the lower jaw, the upper jaw slightly overlapping the lower. Pharyngeal teeth are arranged in one row, with hooked tips. All teeth are laterally compressed, especially the mostdorsal ones and their length regularly increases from ventral to dorsal, except when there are six (in this case, the most dorsal tooth is shorter). Middle ones (and sometimes the most ventral) have projecting denticulated grinding surfaces. Dorsal ones are spoon-shaped with serrated edges (Fig. 5). The eye is located in the first half of the head and is usually longer than the snout. Dorsal fin is inserted around mid-body, always behind pelvic fin insertion. Caudal fin is moderately forked. Pectoral fins are longer than in other congeners, sometimes overlapping the pelvic fins. Pelvic fins are longer than in other congeners, reaching the anus and sometimes overlapping the anal fin. In males, pelvic fins often pass anal fin insertion while they do not pass the anus in females. Last branched ray in anal fin is often longer in males. Peritoneum is dark and gut is long and coiled. No breeding tubercles have been observed. Specific values for morphometric and meristic characters are given in Tables 1 and 2. Coloration at capture. Chondrostoma olisiponensis has a greenish coloration with yellow hues above the lateral line, and is whitish below, with metallic reflections. The transition between dorsal and ventral coloration is sometimes very marked. Other times it is less marked, with one or two rows of scales with intermediate coloration on and under the lateral line, especially beyond the dorsal-fin insertion. Rows of melanophores occur along fin rays, on the iris, and on the opercular and ventral regions. Fins usually darker than ventral region. As in the other species of Chondrostoma, the lateral line is pigmented but less so than in C. lusitanicum. Insertion of pectoral, pelvic and anal fins sometimes have a tint of orange. Coloration in alcohol. Dark mid-line is more intense and obvious in the caudal peduncle, and gets more diffuse below the dorsal fin and towards the anterior part of the body. Dorsal region above mid line is brownish above and amber below. Lateral-line scale pores are finely pigmented. Fins are darker than the ventral region. Remarks. Chondrostoma olisiponensis has the unusual characteristic among European cyprinids of displaying external sexual dimorphism. The function of extended paired fins in males is unknown, but we hypothesize it may play a role during courtship and spawning. Breeding tubercles were not observed in the species even though the mature specimens analyzed were collected in different seasons (winter, spring and summer). This time period should encompass the breeding season, since small (ca. 10 mm total length) specimens were observed in May 2006, for which reproduction should have occurred in the previous month. Since the presence of tubercles should be influenced by physiological conditions that may be transient, larger samples are necessary to confirm their absence. The long coiled gut and dark peritoneum suggest the species has a plant component to the diet (Moyle & Cech 2004). Chondrostoma olisiponensis has been collected along with Anguilla anguilla (L.), Carassius auratus (L.), Cyprinus carpio L., Cobitis paludica (de Buen 1930), Gambusia holbrookii Girard 1859, Gasterosteus gymnurus Cuvier 1829, Gobio lozanoi Doadrio and Madeira 2004, Liza aurata (Risso 1810), Lampetra sp., Micropterus salmoides (Lacepède 1802) and Squalius pyrenaicus (Günther 1868). Although C. lusitanicum occurs in the same sub-basins, we have not collected it together with the new species. Distribution. The species was recently collected from tributaries of the lower Rio Tejo Basin, in the vicinity of Lisbon, Portugal (Rio Trancão and Rio Maior sub-basins). Although broad sampling has been undertaken (Fig. 1), its low densities might have precluded capture in other areas where the species potentially lives. In other rivers, such as Rio Grande da Pipa and Rio de Alenquer sub-basins, collection was not attempted due to high levels of pollution. The study of museum material confirmed the species was also present in Vala da Azambuja, Paúl de Magos and Ribeira de Ulme, yet only one or a few specimens from each locality were available. The available data suggest the species is present in peripheral habitats or low-order rivers, which are more prone to human alteration. Considering its conservation status (see below) it is highly advisable that fine-scale sampling be undertaken for finer population and distribution assessment. Etymology. The specific epithet refers to the general area where the species occurs, in the proximity of Lisbon, following its archaic Latin name Olisipo. Common name. Boga-de-boca-arqueada de Lisboa. Lisbon arched-mouth nase. Conservation. Chondrostoma olisiponensis was found to be locally rare despite considerable sampling effort. Additionally, its limited and fragmented range in an area of high human impact on water resources, throughwater extraction for agriculture (especially in years of severe drought), presence of exotic species and industrial, domestic and agricultural sources of pollution and land reclamation, pose serious risks to the species’ survival. The species should be considered Critically Endangered (CR) according to IUCN criteria B 1 ab(ii,iii,iv)c(iv)+ 2 ab(ii,iii,iv)c(iv) (IUCN, 2001). Comparisons and discussion. As shown above, C. olisiponensis can be distinguished from its congeners by combinations of several characters. Specifically, it differs from species of the genei -, nasus -, polylepis -, soetta - and toxostoma -species groups by the lack of a horny blade (vs. horny blade present) on the lower jaw; it has denticulated (vs. smooth) grinding teeth surfaces, pectoral fins that may overlap with the pelvic fins (vs. non-overlapping fins), pelvic fins that may overlap with the anal fin (vs. non-overlapping fins), and external sexual dimorphism (vs. no obvious sexual dimorphism) in the form of elongated pelvic fins that reach the anus and often pass the anal fin insertion in males (vs. pelvic fins not passing the anus in females). Chondrostoma olisiponensis differs from species of the arcasii -species group by the lack of intense reddish coloration at the base of fins (especially the paired and anal fins), higher number of gill rakers (15 or more vs. 15 or fewer), and higher modal number of dorsal-, ventral- and anal-fin branched rays (8 vs. 7). Chondrostoma olisiponensis differs from species in the lemmingii -species group by the overall lower number of scales, lower number of gill rakers (fewer than 20 vs. more than 23), higher modal number of dorsal-fin and anal-fin branched rays (8 vs. 7), by the elongated fins, in particular the pelvic fins that reach the anus and often passanal fin insertion in males (vs. pelvic fins not passing the anus), and by the denticulated (vs. smooth) grinding teeth surfaces. The species can be readily distinguished from C. lusitanicum by most meristic characters studied (the same as for the lemmingii -species group) and almost completely by a combination of morphometric characters (Fig. 2). It differs in morphometry by having a higher body (at the head, mid-body and caudal peduncle), relatively longer head, larger eye, origins of anal and pelvic finsdis placed anteriorly, pectoral fins displaced posteriorly, and longer pelvic and pectoral fins and last anal-fin ray (Table 1). According to Mathias (1921), Collares-Pereira (1983), and Elvira (1987, 1997), all members of the genus Chondrostoma have teeth with smooth grinding surfaces. Nevertheless, Coelho (1987) observed teeth with serrated edges and replacement teeth with denticulations in members of the polylepis -species group. Additional to C. olisiponensis, we also observed several specimens of C. oligolepis (arcasii -species group) from the comparative material that showed denticulated teeth. Therefore, distribution of this character should be reevaluated in the light of the new findings – these point to a synapomorphic condition of smooth teeth in Chondrostoma and possible reversal to denticulated teeth in some species or groups. The presence of overlapping fins is a condition not seen in the genus (Mathias 1921; Elvira 1987, 1997), except for some bladeless species (Collares-Pereira 1983). In species of the lemmingii -species group, pelvic fins do not pass the anus, while they might pass the anal-fin origin in species of the arcasii -species group (Collares-Pereira 1983). This condition has been shown here to be a form of sexual dimorphism. Although species of the arcasii - and lemmingii -species groups are considered sexually non-dimorphic (Collares-Pereira 1983; Carvalho et al. 2002), Collares-Pereira (1983) referred to males of some of these species as usually having longer paired fins than do females. Therefore, it is likely that sexual dimorphism is more conspicuous in, but not restricted to C. olisiponensis. Non-overlapping fins should be considered a synapomorphy of Chondrostoma. The bladeless lower lip observed in C. olisiponensis is typical of arcasii - and lemmingii -species groups, elevated by Robalo et al. (2007) to Achondrostoma and Iberochondrostoma, respectively. Yet, the new species does not fit exclusively into any of the proposed genera using morphological characters and breaks down the combinations of traits diagnosing the newly erected genera. It shares several characters with either species groups, such as the lack of intense reddish coloration at the base of fins and presence of a 6 / 5 modal pharyngeal teeth count typical of the lemmingii -species group, and the lower numbers of scales typical of the arcasii - species group. On the other hand, C. olisiponensis shows intermediate numbers of gill rakers and different modal numbers of branched dorsal-, ventral- and anal-fin rays compared to either of these groups. Many of these characters were considered diagnostic of Achondrostoma and Iberochondrostoma (Robalo et al. 2007). We further investigated the phylogenetic relationships of C. olisiponensis using molecular markers (to be published elsewhere), which confirm its specific status while not resolving its taxonomic positioning. In fact, molecular data suggest the species has diverged during the Miocene, rejecting a possible recent hybrid origin, although molecular markers do not unequivocally place C. olisiponensis in either the arcasii - or lemmingii - species groups. Considering that the current knowledge of character distribution in the genus is fragmentary, it is advisable to maintain all of the species within Chondrostoma until further data is gathered to address the genus taxonomy.Published as part of Gante, Hugo F., Santos, Carlos D. & Alves, Maria Judite, 2007, A new species of Chondrostoma Agassiz, 1832 (Cypriniformes: Cyprinidae) with sexual dimorphism from the lower Rio Tejo Basin, Portugal, pp. 23-35 in Zootaxa 1616 on pages 29-34, DOI: 10.5281/zenodo.17909

Data from: Semi-permeable species boundaries in Iberian barbels (Barbus and Luciobarbus, Cyprinidae)

Background: The evolution of species boundaries and the relative impact of selection and gene flow on genomic divergence are best studied in populations and species pairs exhibiting various levels of divergence along the speciation continuum. We studied species boundaries in Iberian barbels, Barbus and Luciobarbus, a system of populations and species spanning a wide degree of genetic relatedness, as well as geographic distribution and range overlap. We jointly analyze multiple types of molecular markers and morphological traits to gain a comprehensive perspective on the nature of species boundaries in these cyprinid fishes. Results: Intraspecific molecular and morphological differentiation is visible among many populations. Genomes of all sympatric species studied are porous to gene flow, even if they are not sister species. Compared to their allopatric counterparts, sympatric representatives of different species share alleles and show an increase in all measures of nucleotide polymorphism (S, H d, K, π and θ). High molecular diversity is particularly striking in L. steindachneri from the Tejo and Guadiana rivers, which co-varies with other sympatric species. Interestingly, different nuclear markers introgress across species boundaries at various levels, with distinct impacts on population trees. As such, some loci exhibit limited introgression and population trees resemble the presumed species tree, while alleles at other loci introgress more freely and population trees reflect geographic affinities and interspecific gene flow. Additionally, extent of introgression decreases with increasing genetic divergence in hybridizing species pairs. Conclusions: We show that reproductive isolation in Iberian Barbus and Luciobarbus is not complete and species boundaries are semi-permeable to (some) gene flow, as different species (including non-sister) are exchanging genes in areas of sympatry. Our results support a speciation-with-gene-flow scenario with heterogeneous barriers to gene flow across the genome, strengthening with genetic divergence. This is consistent with observations coming from other systems and supports the notion that speciation is not instantaneous but a gradual process, during which different species are still able to exchange some genes, while selection prevents gene flow at other loci. We also provide evidence for a hybrid origin of a barbel ecotype, L. steindachneri, suggesting that ecology plays a key role in species coexistence and hybridization in Iberian barbels. This ecotype with intermediate, yet variable, molecular, morphological, trophic and ecological characteristics is the local product of introgressive hybridization of L. comizo with up to three different species (with L. bocagei in the Tejo, with L. microcephalus and L. sclateri in the Guadiana). In spite of the homogenizing effects of ongoing gene flow, species can still be discriminated using a combination of morphological and molecular markers. Iberian barbels are thus an ideal system for the study of species boundaries, since they span a wide range of genetic divergences, with diverse ecologies and degrees of sympatry

Woundsnow

Reflectance data of wound snow and wound spray (KOI MED®) used to manipulate the horizontal facial stripe of Neolamprologus brichardi