Phylogenetic relationships within the South American fish family Anostomidae (Teleostei,Ostariophysi, Characiformes)

Analysis of a morphological dataset containing 152 parsimony‐informative characters yielded the first phylogenetic reconstruction spanning the South American characiform family Anostomidae. The reconstruction included 46 ingroup species representing all anostomid genera and subgenera. Outgroup comparisons included members of the sister group to the Anostomidae (the Chilodontidae) as well as members of the families Curimatidae, Characidae, Citharinidae, Distichodontidae, Hemiodontidae, Parodontidae and Prochilodontidae. The results supported a clade containing Anostomus, Gnathodolus, Pseudanos, Sartor and Synaptolaemus (the subfamily Anostominae sensu Winterbottom) albeit with a somewhat different set of relationships among the species within these genera. Anostomus as previously recognized was found to be paraphyletic and is split herein into two monophyletic components, a restricted Anostomus and the new genus Petulanos gen. nov. , described herein. Laemolyta appeared as sister to the clade containing Anostomus, Gnathodolus, Petulanos, Pseudanos, Sartor and Synaptolaemus. Rhytiodus and Schizodon together formed a well‐supported clade that was, in turn, sister to the clade containing Anostomus, Gnathodolus, Laemolyta, Petulanos, Pseudanos, Sartor and Synaptolaemus. Anostomoides was sister to the clade formed by these nine genera. Leporinus as currently defined was not found to be monophyletic, although certain clades within that genus were supported, including the species with subterminal mouths in the former subgenus Hypomasticus which we recognize herein as a genus. Abramites nested in Leporinus, and Leporellus was found to be the most basal anostomid genus. The presence of cis‐ and trans‐Andean species in Abramites, Leporellus, Leporinus and Schizodon, all relatively basal genera, suggests that much of the diversification of anostomid species pre‐dates the uplift of the Andean Cordilleras circa 11.8 million years ago. Several important morphological shifts in anostomid evolution are illustrated and discussed, including instances of convergence and reversal. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154 , 70–210.     

Testing monophyly of the freshwater fish Leporinus (Characiformes,Anostomidae) through molecular analysis

Monophyly of the genus Leporinus (Characiformes: Anostomidae) was tested by sequencing and analysing a total of 4732 bp, including two mitochondrial [cytochrome oxidase subunit 1 (CO1) and cytochrome b (Cytb)] and three nuclear [myosin heavy chain 6 cardiac muscle alpha (Myh6), recombination activating gene 1 (RAG1) and recombination activating gene 2 (RAG2)] loci for 22 species of Leporinus, or c. 25% of all described species in the genus. Phylogenetic tree analyses (maximum parsimony, maximum likelihood and Bayesian species tree) indicate Leporinus to be paraphyletic, with monophyly being rejected by both Kishino–Hasegawa and Shimodaira–Hasegawa tests. The sequenced species of Leporinus are distributed across five clades that are interleaved among other anostomid genera. Several taxonomic changes are suggested as being necessary to restore monophyly for the group. The clade containing the type species, Leporinus fasciatus, should be considered Leporinus sensu stricto and at least three new genera should be described for other species currently considered part of Leporinus.     

Heterochromatin characterization of four fish species of the family Loricariidae (Siluriformes)

The karyotypic structures and the composition and distribution of the heterochromatin in the karyotypes of four catfish species belonging to four Loricariidae subfamilies were analysed, namely: Neoplecostomus microps (Neoplecostominae) with 2n=54 chromosomes, Harttia loricariformis (Loricariinae) with 2n=56 chromosomes, Hypostomus affinis (Hypostominae) with 2n=66 chromosomes and Upsilodus sp. (Upsilodinae) with 2n=96 chromosomes. The amount and composition of heterochromatin was quite unequal among the studied species, being copious and mainly GC-rich in Upsilodus sp. and scarce and balanced in H. loricariformis. All of the H. affinis heterochromatin is GC-rich and related with nucleolar organizing regions. N. microps show low quantity of interstitial and GC-rich heterochromatin, one of them being related with NORs. Trends in the macrokaryotypic diversification as well as in the distribution pattern of the heterochromatin are discussed.     

Isolation and characterization of 20 polymorphic microsatellite loci in the migratory freshwater fish Leporinus obtusidens (Characiformes: Anostomidae) using 454 shotgun pyrosequencing

Twenty polymorphic microsatellite markers were developed for the Neotropical fish Leporinus obtusidens using a next generation sequencing approach and tested in two other characifomes species, Schizodon platae and Prochilodus lineatus. Microsatellite loci alleles in L. obtusidens ranged between 2 and 20 alleles per locus (mean = 5·7), with expected heterozygosity values ranging from 0·097 to 0·956 (mean = 0·578) and observed heterozygosity values ranging from 0·000 to 0·800 (mean = 0·400) in a sample of 20 specimens from the lower Paraná River (Argentina). Most of these markers will be a valuable tool for captive breeding and stocking programmes, as well as for analyses of population connectivity and genetic structure in this broadly distributed Neotropical migratory fish.     

Recent chromosome diversification in the evolutionary radiation of the freshwater fish family Curimatidae (Characiformes)

Neotropical Curimatidae fishes include 97 species in eight genera. Basic cytogenetic studies show a karyotype of 2 n = 54 chromosomes in most species. Karyotype divergence of the nucleolus organizing regions between species has been reported, and these regions appear to be good cytotaxonomic markers. In the present work, karyotype, heterochromatin and Ag-NOR variability in 13 species were investigated to analyse the chromosome diversification in view of the biogeographic history of this group. Only Cyphocharax platanus showed a karyotype with 2 n = 58 chromosomes. Ag-NOR and C-banding patterns were quite divergent among the species studied. All species whose C-bands were analysed had heterochromatic blocks associated with the nucleolus organizing regions. Species with multiple Ag-NORs also showed an increase in NOR-associated heterochromatic blocks. C-banding showed considerable differentiation among species, revealing a pronounced chromosome diversification in this group. Karyotypic variability corroborates the hypothesis that these fishes in Amazon region show various discrete patterns of species endemism. Chromosome diversification in curimatids has a recent origin and appears to be accompanying the post-Andean speciation responsible for the diversity of species in the family.     

Ontogenesis and embryogenesis of Megaleporinus elongatus larvae (Characiformes: Anostomidae) from the Jequitinhonha River Basin

Five females and five males of Megaleporinus elongatus captured in the Jequitinhonha River were transferred to hypophysation tanks with a constant water flow at 26°C at the Machado Mineiro Fish Farm. They were submitted to induced reproduction by the hypophysation method. Crude carp pituitary extract (Cyprinus carpio) was injected into the coelomic cavity in two doses (2.5 and 5.0 mg/kg) for males and 3 doses (2.5, 5.0, and 2.5 mg/kg) for females. The doses were applied with a 24‐hr interval between the 1st and 2nd doses and a 12‐hr interval between the 2nd and 3rd ones. Oocytes and semen were obtained by manual extrusion, and the dry method was used for fertilisation. The eggs were kept in funnel type 20‐litre incubators with constant water flow at 23°C. Egg samples were collected every 10 min and photographed under a stereomicroscope for analysis of embryonic development until hatching. The main events of embryogenesis and their respective duration times were: cleavage (45 min), blastula (3 hr 14 min), gastrula (5 hr 25 min), closure of blastopore (6 hr 34 min), differentiation of somites (13 hr 20 min), tail release (17 hr 50 min), and hatching (24 hr 50 min). During the larval ontogenesis, the yolk sac gradually decreased until its complete reabsorption on the 7th day after hatching. The opening of the mouth and digestive tract occurred from the 5th to the 7th day, indicating that larvae could feed exogenously. These results provide information to improve the techniques of induced spawning and cultivation of M. elongatus.     

Henneguya schizodon n. sp. (Myxozoa, Myxobolidae), a parasite of the Amazonian teleost fish Schizodon fasciatus (Characiformes, Anostomidae)

A new histozoic species of myxosporean (Henneguya schizodon n. sp.) is described from the Amazon River teleost fish Schizodon fasciatus Spix & Agassiz, 1892 (Characiformes, Anostomidae). The plasmodia, which showed asynchronous development, were located in the kidney of the host. The spore body was ellipsoidal and was 13.1 (12-14) micron long by 3.3 (3-4) micron wide. The total length of the spore was 28.9 (27-30) micron, and each value had a caudal process measuring 16.3 (15-17) micron. The polar capsules were 5.4 (5-6) micron long by 1.3 (1-1.5) micron wide, and each had a polar filament with 8-10 coils. The characteristics of the species were compared with nearly all the species described so far, including all the species reported from South American fishes. This comparison allows to consider the materials as a new species, and the name Henneguya schizodon n. sp. is proposed.     

Anatomical features and histology of the digestive tract of two related neotropical omnivorous fishes (Characiformes; Anostomidae)

The estimated mean intestinal coefficients of Leporinus friderici Bloch, 1794 and L. taeniofasciatus Britski, 1997, from the Upper Tocantins River, central-western Brazil, were 1·25±0·15 and 1·14±0·07, respectively. The external surfaces of the lips have taste buds. The histological structure is composed of four layers: mucosa, submucosa, muscular, and serosa. No muscularis mucosa is detected. The oesophageal epithelium is stratified in L. friderici and pseudostratified in L. taeniofasciatus , the latter with PAS+ basal cells and eosinophilic apical cells. A thick layer of adipose tissue was found within the submucosa. Leporinus taeniofasciatus has a sphincter separating the cardiac and fundic portions of the stomach. In both species, gastric glands are present in the stomach, except in its pyloric portion. The pyloric caeca possess a simple columnar epithelium with brush border and three types of goblet cells. Rodlet cells were found in L. friderici . The three portions of the intestine showed no histological differences. Anatomical and histological features of the digestive tract are consistent with the omnivorous habit of both these species.     

Spermiogenesis and fine structure of the spermatozoon in a headstander, Chilodus punctatus (Teleostei, Characiformes, Anostomidae)

The main characteristic features of spermiogenesis in Chilodus punctatus (Characiformes) are rotation of the nucleus, development of a nuclear fossa, which extends as a narrow invagination deep into the nucleus and the way in which flagellum is formed. The chromatin condensation proceeds during the spermiogenesis from heterogeneous through homogenous and granular to a highly compact one present in the mature spermatozoon. Mature Ch. punctatus spermatozoon shows a spherical nucleus, short midpiece and flagellum with lateral fins. The centrioles are in perpendicular arrangement and are located in the deep nuclear fossa, which extends towards the anterior pole of the nucleus. The midpiece contains a few mitochondria, which are separated from the anterior fragment of flagellum by the cytoplasmic channel. Spermiogenesis and spermatozoon ultrastructure conform to the pattern observed in other ostariophysans, but for the first time the presence of lateral fins along flagellum has been documented in a representative of Characiformes.     

Heterochromatin,colchicine, and karyotype

Summary Species of Chilocorus differ in chromosome number owing to centric fusion of metacentric chromosomes. The concomitant loss of arms is tolerated because in all unfused chromosomes one arm is completely heterochromatic, the other euchromatic. Under the influence of colchicine, the arms of unfused and fused chromosomes contract differentially. Unless the distribution of heterochromatin is known, karyotype analysis is destined to failure.Contribution No. 1138, Forest Entomology and Pathology Branch, Department of Forestry, Ottawa, Canada.Dedicated to Professor H. Bauer on the occasion of his sixtieth birthday.     

Metazoan parasite communities of wild Leporinus friderici (Characiformes: Anostomidae) from Amazon River system in Brazil

This study compared the structure of metazoan parasite communities in two populations of Leporinus friderici from two rivers of the Amazon River system in Brazil. Jainus leporini, Urocleidoides paradoxus, Urocleidoides sp., Tereancistrum parvus, Tereancistrum sp., Clinostomum marginatum, Procamallanus (Spirocamallanus) inopinatus, Contracaecum sp., Octospiniferoides incognita and Ergasilus sp. were found in both hosts. There were differences in species richness of parasites, Shannon index and the evenness between both host populations, because the parasite community showed a similarity of only 33%. Parasites of both host populations had an aggregated dispersion. Host size also contributes to the differences between the populations of hosts investigated, and the feeding habit contributes to the occurrence of endoparasites in L. friderici, an intermediate host for these parasites. The existence of variation in infracommunity and community of parasites for the same host species from different localities indicates the presence of an uneven distribution in terms of species and density of parasites, which are intermediate hosts, and some of them constitute food items for L. friderici in the localities surveyed.     

A Novel ZZ/ZW Sex Chromosome System for the Genus Leporinus (Pisces, Anostomidae, Characiformes)

A wide range of sex chromosome mechanisms, including simple and multiple chromosome systems is characteristic of fishes. The Leporinus genus represent a good model to study sex chromosome mechanisms, because an unambiguous ZZ/ZW sex chromosome system was previously described for seven species, while the remaining studied species of the genus do not show differentiated sex chromosomes. The occurrence of sex chromosomes in Leporinus trifasciatus and Leporinus sp2 from the Araguaia river, Amazon basin, Brazil, was here investigated. ZZ/ZW sex chromosomes were detected for both species. The Z and W chromosome morphology of L. trifasciatus is the same as described for other species of the genus Leporinus. However, the Z and W chromosomes of L. sp2 were quite different in their morphology and banding pattern suggesting that the ZW system of this species have originated independently from the ZW system previously described for other Leporinus.     

A new fish species Hemibrycon (Characiformes: Characidae)

Hemibrycon pautensis (Characiformes, Characidae), a new fish species from Paute River, eastern Ecuador is described. Diagnostic characteristics: eight to nine branched rays in the dorsal fin (vs. six to seven), and 27 - 28 in the anal fin (vs. 16 - 26, except in H. dariensis which presents 22 - 27, in H. metae 26 - 31 and H. jabonero 23 - 28); a no occurrence of dorsal pharyngeal plate (vs. occurrence); a cartilaginous and divided-in-two basihial (vs. an osseous base and a cartilaginous upper part). Hemibrycon pautensis resembles H. metae by its oblique external edge of the pelvic fins. They can be distinguished by the position of the pectoral fins in relation to the snout (38.24-41.6% in H. pautensis vs. 21.21-25.87) and by the position of the pectoral fins in relation to the origin of the dorsal fin (20.95-24.30 in Hemibrycon pautensis vs. 35.89-42.63), and by the number of proximate radials in the pectoral girdle (five in Hemibrycon pautensis vs. three to four). In addition, the geographic distribution of H. metae is restricted to the upper part of the Meta River in Colombia and can be distinguished of H. boquiae by: the number of scales between the lateral-line and the origin of the dorsal fin (eight in H. pautensis vs. 5-7); the distance between the snout and the pelvic fins (38.00-42.90 % in H. pautensis vs. 42.9-46.19%); the pelvic fins length (13.77-17.96% in H. pautensis vs. 10.72-13.21%); and the snout length (21.34-27.88 in H. pautensis vs. 26.92-33.66%).     

A new endangered species of Megaleporinus (Characiformes: Anostomidae) from the Rio de Contas basin,eastern Brazil

A new species of Megaleporinus is described from the Rio de Contas, a coastal drainage of eastern Brazil, and its phylogenetic relationships are studied using molecular data. The new species is unique among Anostomidae by possessing two exclusive features: an irregular dark longitudinal stripe from supracleithrum to second midlateral blotch and anterior cranial fontanel partially closed. In addition, the new species is diagnosed by having three premaxillary teeth, three dentary teeth, 37 or 38 scales in lateral line, 16 scale rows around caudal peduncle, three dark midlateral blotches on body, and red fins in life. The new species is closely related to M. obtusidens from the São Francisco basin, corroborating previous studies that indicated that the latter represents a species complex as currently defined. The new species exhibits the first rib enlarged in mature males, a feature described for some congeners. The new species is herein considered to be Endangered under the IUCN criteria.     

Isolation and characterization of eight microsatellite loci in Leporinus macrocephalus (Characiformes: Anostomidae) and cross‐species amplification

Leporinus macrocephalus is a species endemic to the Paraguay River basin and an important fishery resource, as well as a valuable species in aquaculture programs. A total of eight polymorphic microsatellite loci were isolated and characterized. A population survey was conducted involving 45 specimens whereby a large number of alleles (range 5–17 among loci), a highly observed (0.1667–0.6129) and an expected (0.6967–0.9448) heterozygosity was detected, indicating its usefulness in population genetics studies. Cross‐species amplification was successful in eight Characiformes species.     

Cryptic species in the Neotropical fish genus Curimatopsis (Teleostei,Characiformes)

Detritivores of the fish family Curimatidae are assigned to eight genera, one of which, the Curimatopsis, with only five species, is the least speciose genus and sister to other seven genera in the family. Ongoing morphological investigations reveal, however, the likely existence of additional species. In this study, fifty‐one specimens of Curimatopsis from multiple rivers of the Amazon, Paraguay and Suriname drainages were identified morphologically according to the present species concepts and then barcoded using the universal cytochrome c oxidase subunit I (COI) mitochondrial marker. Species delimitation analyses were conducted using Bayesian methods through the general mixed Yule‐coalescent analysis combined with conventional likelihood, genetic distance and haplotypic diversity approaches. We found eleven well‐supported clusters that represent four of the named species and seven cryptic, undescribed species of Curimatopsis. Our results show a clear delimitation of species boundaries constrained by distinct Amazonian river ecotones that may have promoted intrageneric lineage diversification. This is the first of a series of genetic studies applicable to future taxonomic, phylogenetic and evolutionary studies across the Curimatidae.     

Trends in the karyotype evolution of Loricariidae fish (Siluriformes)

Six species of Loricariidae belonging to the subfamilies Hypostominae (Hypostomus emarginatus, Rhinelepsis aspera, Pogonopoma wertheimeri), Ancistrinae (Panaque cf. nigrolineatus, Hemiancistrus sp.) and Loricariinae (Sturisoma cf. nigrirostrum) were studied cytogenetically. The results show that 2n = 54 represents the basal diploid number for this fish family. Different trends in the karyotypic evolution can be seen among the subfamilies: Hypostominae and Loricariinae species present diversified karyotypic macrostructures, while the Ancistrinae appear to show more conserved karyotypes. Among the Hypostominae, the genus Hypostomus had a wide karyotypic variation (2n = 52 to 80), where centric fissions seem to play an important role in this chromosomal divergence. The nucleolar organizing regions were diversified, and occurrence of multiple NORs was frequent. Heteromorphic chromosomes belonging to distinct sex chromosome systems can also occur infrequently among the Loricariidae.     

Two new species of Leporinus (Characiformes: Anostomidae) from the Brazilian Amazon,and redescription of Leporinus striatus Kner 1858

Two new species of Leporinus are described from tributaries of the Rio Amazonas in Brazil. One species is known from the Jari and Tapajós River basins, and is identified on the basis of a gas bladder reduced in size, a dark midlateral stripe on the body, dark transverse bars on the dorsum, a subinferior mouth, three teeth on the premaxilla, four teeth on the dentary and 16 scale rows around the caudal peduncle. The second new species is known from the Tocantins, Xingu and Tapajós River basins, and is identified on the basis of three dark longitudinal stripes on the body, a subinferior mouth, three teeth on the premaxilla, four teeth on the dentary and 12 scale rows around the caudal peduncle. In addition, Leporinus striatus is redescribed based on type and additional specimens from the Río de La Plata, Amazonas, Orinoco, Atrato, Magdalena and Sinu River basins. Leporinus striatus is identified on the basis of four dark longitudinal stripes on the body, a subterminal mouth, three teeth on the premaxilla, four teeth on the dentary and 16 scale rows around the caudal peduncle.     

Isolation and characterization of microsatellite DNA in the piracema fish Prochilodus lineatus (Characiformes)

We described five novel microsatellite loci for the piracema fish species Prochilodus lineatus (Characiformes), endemic to South America and of extreme importance to both commercial and artisanal fisheries across its occurrence area. A primary, unenriched genomic library was constructed and radioactively screened for repetitive motifs. Positive clones were automatically sequenced and based on the design of new primers, polymerase chain reaction assays were carried out to determine optimum reaction and electrophoretic conditions for each characterized locus. We evaluated its usefulness in population genetic studies by determining Hardy-Weinberg equilibrium, FIS and a jackknife estimate of the number of alleles for a sample of fish caught below the Funil Hydroelectric Power Plant dam (N = 95), Grande River, Brazil. The number of alleles varied from 3 to 21 and expected heterozygosities ranged from 0.58 to 0.91. Two of five loci were in Hardy-Weinberg equilibrium. Jackknife estimates of the number of alleles were higher than the observed number of alleles for three loci and could provide a measure of sampling bias. These markers should provide important tools for the determination of genetic structure, stock delimitation and reservoir fish management in the Grande River as well as to improve hatchery practices for environmental mitigation measures and to help sustain fisheries in the river.     

Weighted parsimony phylogeny of the family Characidae (Teleostei: Characiformes)

The family Characidae, including more than 1000 species, lacks a phylogenetic diagnosis, with many of its genera currently considered as incertae sedis . The aims of the present study are to propose a phylogenetic diagnosis and to assess higher-level relationships of and within Characidae. In this regard, 360 morphological characters are studied for 160 species of Characidae and related families. Phylogenetic analyses under implied weighting and self-weighted optimization are presented, exploring a broad range of parameters. The analysis under self-weighted optimization is innovative for this size of matrices. Familial status of Serrasalmidae is supported, and Acestrorhynchidae and Cynodontidae are included in a monophyletic Characidae. Engraulisoma taeniatum is transferred from Characidae to Gasteropelecidae. Thus constituted, the monophyly of Characidae is supported by seven synapomorphies. A new subfamily, Heterocharacinae, is proposed, and the subfamilies Aphyocharacinae, Aphyoditeinae, Characinae, Gymnocharacinae, and Stevardiinae are redefined. The Glandulocaudinae are included in Stevardiinae together with remaining members of "clade A" ( sensu Malabarba and Weitzman, 2003 . Comun. Mus. Ciênc. Tecnol. PUCRS, Sér. Zool. 16, 67–151.) and the genera Aulixidens and Nantis . Most incertae sedis genera are assigned, at least tentatively, to a phylogenetically diagnosed clade.