The impact of paleoclimatic changes on body size evolution in marine fishes.

Body size is an important species trait, correlating with life span, fecundity, and other ecological factors. Over Earth's geological history, climate shifts have occurred, potentially shaping body size evolution in many clades. General rules attempting to summarize body size evolution include Bergmann's rule, which states that species reach larger sizes in cooler environments and smaller sizes in warmer environments, and Cope's rule, which poses that lineages tend to increase in size over evolutionary time. Tetraodontiform fishes (including pufferfishes, boxfishes, and ocean sunfishes) provide an extraordinary clade to test these rules in ectotherms owing to their exemplary fossil record and the great disparity in body size observed among extant and fossil species. We examined Bergmann's and Cope's rules in this group by combining phylogenomic data (1,103 exon loci from 185 extant species) with 210 anatomical characters coded from both fossil and extant species. We aggregated data layers on paleoclimate and body size from the species examined, and inferred a set of time-calibrated phylogenies using tip-dating approaches for downstream comparative analyses of body size evolution by implementing models that incorporate paleoclimatic information. We found strong support for a temperature-driven model in which increasing body size over time is correlated with decreasing oceanic temperatures. On average, extant tetraodontiforms are two to three times larger than their fossil counterparts, which otherwise evolved during periods of warmer ocean temperatures. These results provide strong support for both Bergmann's and Cope's rules, trends that are less studied in marine fishes compared to terrestrial vertebrates and marine invertebrates.

Rapid identification of pufferfish in roast fish fillet by real-time PCR.

The purpose of this study was to develop a rapid method based on a real-time PCR assay designed to identify the presence of pufferfish in roasted fish fillet. Specific primers and probes were designed targeting Takifugu spp. and Lagocephalus spp., the most common genera in China. Specificity and sensitivity of this assay design were tested by using artificially spikes of pufferfish mixed in with other fish, such as Gadus and Thamnaconus septentrionalis,among others. Fifteen samples of retail roasted fish fillet and six samples from a 1999 poisoning event that occurred in Fujian province China were analysed for pufferfish. When the assay design was validated, no cross-reaction was observed between pufferfish and other species of fish. The limit of detection (LOD) was determined to be 0.001 ng pufferfish template, and the sensitivity of the method was 1%. Lagocephalus lunari was detected in six samples assayed from 1999 and no pufferfish was detected in the 15 retail roasted fish fillet samples tested. These results showed that the method was efficient for screening for pufferfish contamination in the roasted fish fillet and it could benefit public health protection by reducing the risk of tetrodotoxin poisoning.

Mitogenome analysis of dwarf pufferfish (Carinotetraodon travancoricus) endemic to southwest India and its implications in the phylogeny of Tetraodontidae.

The Tetraodontidae (pufferfishes), is primarily a family of marine and estuarine fishes with a limited number of freshwater species. Freshwater invasions can be observed in South America, Southeast Asia and central Africa. In the present study, we have analysed the complete mitogenome of freshwater pufferfish, Carinotetraodon travancoricus (dwarf pufferfish or Malabar pufferfish) endemic to southwest India. The genome is 16487 bp in length and consist of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region like all the other vertebrate mitogenomes. The protein-coding genes ranged from 165 bp (ATP synthase subunit 8) to 1812 bp (NADH dehydrogenase subunit 5) and comprised of 11310 bp in total, constituting 68.5% of the complete mitogenome. Some overlaps have been observed in protein-coding genes by a total of 7 bp. The AT skew (0.032166) and GC skew (-0.29746) of the mitogenome indicated that heavy strand consists equal amount of A and T, but the overall base composition was mainly C skewed. The noncoding D-loop region comprised 869 bp. The conserved motifs ATGTA and its complement TACAT associated with thermostable hairpin structure formation were identified in the control region. The phylogenetic analysis depicted a sister group relationship of C. travancoricus with euryhaline species Dichotomyctere nigroviridis and D. ocellatus with 100% bootstrap value rather than with the other freshwater members of Carinotetraodon species from Southeast Asia. The data from this study will be useful for proper identification, genetic differentiation, management and conservation of the dwarf Indian pufferfish.

Anatomical characterization of the digestive system of the pufferfish (Chilomycterus spinosus spinosus)

Descriptive studies of the fish digestive system are fundamental because they provide information on the biology of the species. Thus, the objective of this study was to morphologically describe the digestive system of the pufferfish, Chilomycterus spinosus spinosus. For this, adult specimens of pufferfish (n = 10) of both sexes were used. The animals were fixed with 10% aqueous formaldehyde solution, dissected, analyzed descriptively and photographed. The results demonstrate that the pufferfish has a morphologically modified digestive system, which is adapted to the defense behavior. This species presents a pouch-shaped diverticulum, that is called abdominal pouch, which allows the expansion of the celomatic cavity and the temporary storage of food. Although it is used to store food, macroscopically the abdominal pouch does not show gastric folds. However, this absence is compensated by a small intestine containing innumerable villi.

Cucullanus marajoara n. sp. (Nematoda: Cucullanidae), a parasite of Colomesus psittacus (Osteichthyes: Tetraodontiformes) in the Marajó, Brazil / Cucullanus marajoara n. sp. (Nematoda: Cucullanidae), um parasito de Colomesus psittacus (Osteichthyes: Tetraodontiformes) no Marajó, Brasil

Abstract Cucullanus marajoara n. sp. (Cucullanidae) is reported to parasitize Colomesus psittacus (Tetraodontiformes), which is a fish species from the Marajó Archipelago, state of Pará, estuarine region of the Brazilian Amazon. The new species differs from similar species by the presence of a protruding upper lip on the cloacal opening, the distribution of the cloacal papillae: five pre-cloacal papillae pairs and 5 are ventral and located posteriorly to the pre-cloacal sucker and an unpaired papilla is located on the upper cloacal lip and five post-cloacal pairs, and a pair of lateral phasmids located between papillae pairs. Additionally, Cucullanus marajoara n. sp. is compared to other species of this genus described in Brazil, particularly Cucullanus ageneiosus and Cucullanus oswaldocruzi, which both occur in the same zoogeographic region of this study but parasitize fish of a different order (Siluriformes). Cucullanus dodsworthi and Cucullanus brevicaudatus are the only described species parasitizing fish of the order Tetradontiformes in Brazil, and the new species differs from these species by the distribution of the cloacal papillae and the host habitat. The description of Cucullanus marajoara n. sp. adds data to the biodiversity of described parasites that parasitize Tetradontiformes of the estuarine ichthyofauna in the Brazilian Amazon.

Resumo Cucullanus marajoara n. sp. (Cucullanidae) é descrita parasitando Colomesus psittacus (Tetraodontiformes) peixe do arquipélago de Marajó, Estado do Pará, região estuarina da Amazônia brasileira. A nova espécie difere de seus congêneres por apresentar lábio superior da abertura cloacal protrudente e na distribuição de papilas cloacais: 5 pares pré-cloacais, e uma papila não pareada no lábio superior da cloaca e 5 pares pós-cloacais, um par de fasmídeos laterais. Além disso, Cucullanus marajoara n. sp. é comparada com outras espécies do gênero descritas no Brasil, em especial Cucullanus ageneiosus e Cucullanus oswaldocruzi, ambos parasitos de peixes da ordem Siluriformes; porém, ocorrendo na mesma região zoogeográfica deste estudo, diferem da ordem do hospedeiro da nova espécie (Tetraodontiformes). Cucullanus dodsworthi e Cucullanus brevicaudatus são as únicas espécies descritas parasitando peixe da ordem Tetradontiformes no Brasil, e a nova espécie difere dessas na distribuição das papilas cloacais e habitat dos hospedeiros. Cucullanus marajoara n. sp. adiciona dados a biodiversidade de parasitos descritos parasitando Tetradontiformes da ictiofauna estuarina da Amazônia brasileira.

Cucullanus marajoara n. sp. (Nematoda: Cucullanidae), a parasite of Colomesus psittacus (Osteichthyes: Tetraodontiformes) in the Marajó, Brazil.

Cucullanus marajoara n. sp. (Cucullanidae) is reported to parasitize Colomesus psittacus (Tetraodontiformes), which is a fish species from the Marajó Archipelago, state of Pará, estuarine region of the Brazilian Amazon. The new species differs from similar species by the presence of a protruding upper lip on the cloacal opening, the distribution of the cloacal papillae: five pre-cloacal papillae pairs and 5 are ventral and located posteriorly to the pre-cloacal sucker and an unpaired papilla is located on the upper cloacal lip and five post-cloacal pairs, and a pair of lateral phasmids located between papillae pairs. Additionally, Cucullanus marajoara n. sp. is compared to other species of this genus described in Brazil, particularly Cucullanus ageneiosus and Cucullanus oswaldocruzi, which both occur in the same zoogeographic region of this study but parasitize fish of a different order (Siluriformes). Cucullanus dodsworthi and Cucullanus brevicaudatus are the only described species parasitizing fish of the order Tetradontiformes in Brazil, and the new species differs from these species by the distribution of the cloacal papillae and the host habitat. The description of Cucullanus marajoara n. sp. adds data to the biodiversity of described parasites that parasitize Tetradontiformes of the estuarine ichthyofauna in the Brazilian Amazon.

Advances in DNA Barcoding of Toxic Marine Organisms.

There are more than 200,000 marine species worldwide. These include many important economic species, such as large yellow croaker, ribbonfish, tuna, and salmon, but also many potentially toxic species, such as blue-green algae, diatoms, cnidarians, ctenophores, Nassarius spp., and pufferfish. However, some edible and toxic species may look similar, and the correct identification of marine species is thus a major issue. The failure of traditional classification methods in certain species has promoted the use of DNA barcoding, which uses short, standard DNA fragments to assist with species identification. In this review, we summarize recent advances in DNA barcoding of toxic marine species such as jellyfish and pufferfish, using genes including cytochrome oxidase I gene (COI), cytochrome b gene (cytb), 16S rDNA, internal transcribed spacer (ITS), and Ribulose-1,5-bisphosphate carboxylase oxygenase gene (rbcL). We also discuss the application of this technique for improving the identification of marine species. The use of DNA barcoding can benefit the studies of biological diversity, biogeography, food safety, and the detection of both invasive and new species. However, the technique has limitations, particularly for the analysis of complex objects and the selection of standard DNA barcodes. The development of high-throughput methods may offer solutions to some of these issues.

Neogene Proto-Caribbean porcupinefishes (Diodontidae).

Fossil Diodontidae in Tropical America consist mostly of isolated and fused beak-like jawbones, and tooth plate batteries. These durophagous fishes are powerful shell-crushing predators on shallow water invertebrate faunas from Neogene tropical carbonate bottom, rocky reefs and surrounding flats. We use an ontogenetic series of high-resolution micro CT of fossil and extant species to recognize external and internal morphologic characters of jaws and tooth plate batteries. We compare similar sizes of jaws and/or tooth-plates from both extant and extinct species. Here, we describe three new fossil species including †Chilomycterus exspectatus n. sp. and †Chilomycterus tyleri n. sp. from the late Miocene Gatun Formation in Panama, and †Diodon serratus n. sp. from the middle Miocene Socorro Formation in Venezuela. Fossil Diodontidae review included specimens from the Neogene Basins of the Proto-Caribbean (Brazil: Pirabas Formation; Colombia: Jimol Formation, Panama: Gatun and Tuira formations; Venezuela: Socorro and Cantaure formations). Diodon is present in both the Atlantic and Pacific oceans, whereas the distribution of Chilomycterus is highly asymmetrical with only one species in the Pacific. It seems that Diodon was as abundant in the Caribbean/Western Atlantic during the Miocene as it is there today. We analyze the paleogeographic distribution of the porcupinefishes group in Tropical America, after the complete exhumation of the Panamanian isthmus during the Pliocene.

Species Identification of Pufferfish Products Using RAPD Analysis.

A simple and rapid method was developed to identify the source species of pufferfish products. Randomly amplified polymorphic DNA (RAPD) analysis was applied to identify 8 species of pufferfish. Commercial kits were used for DNA extraction and amplification. Simultaneous identification was possible by polyacrylamide gel electrophoresis of PCR products. Two primers were chosen based on the result of pre-examination with 40 primers, and the PCR conditions were optimized. Characteristic RAPD patterns were obtained for each pufferfish species. The developed method was applied to identify the source species of 26 pufferfish products. The results suggest that the developed method would be useful for verification of the labeled species of pufferfish products.

First confirmed record of Mola sp. A in the western Mediterranean Sea: morphological, molecular and parasitological findings.

Recent molecular and morphological studies suggest the existence of at least three species of Mola (Mola spp. A, B and C). Currently, only Mola mola and Mola ramsayi are formally accepted and species A, B or C have not been assigned to these thus far. In this study, a large ocean sunfish in the western Mediterranean Sea was analysed molecularly and morphologically, identified as Mola sp. A and a detailed account of the specimen's parasite load is reported.

Phylogenetic relationships and the evolution of BMP4 in triggerfishes and filefishes (Balistoidea).

The triggerfishes (family Balistidae) and filefishes (family Monacanthidae) comprise a charismatic superfamily (Balistoidea) within the diverse order Tetraodontiformes. This group of largely marine fishes occupies an impressive ecological range across the world's oceans, and is well known for its locomotor and feeding diversity, unusual body shapes, small genome size, and ecological and economic importance. In order to investigate the evolutionary history of these important fish families, we used multiple phylogenetic methods to analyze molecular data from 86 species spanning the extant biodiversity of Balistidae and Monacanthidae. In addition to three gene regions that have been used extensively in phylogenetic analyses, we include sequence data for two mitochondrial regions, two nuclear markers, and the growth factor gene bmp4, which is involved with cranial development. Phylogenetic analyses strongly support the monophyly of the superfamily Balistoidea, the sister-family relationship of Balistidae and Monacanthidae, as well as three triggerfish and four filefish clades that are well resolved. A new classification for the Balistidae is proposed based on phylogenetic groups. Bayesian topology, as well as the timing of major cladogenesis events, is largely congruent with previous hypotheses of balistid phylogeny. However, we present a novel topology for major clades in the filefish family that illustrate the genera Aluterus and Stephanolepis are more closely related than previously posited. Molecular rates suggest a Miocene and Oligocene origin for the families Balistidae and Monacanthidae, respectively, and significant divergence of species in both families within the past 5 million years. A second key finding of this study is that, relative to the other protein-coding gene regions in our DNA supermatrix, bmp4 shows a rapid accumulation of both synonymous and non-synonymous substitutions, especially within the family Monacanthidae. Overall substitution patterns in bmp4 support the hypothesis of stabilizing selection during the evolutionary history of regulatory genes, with a small number of isolated examples of accelerated non-synonymous changes detected in our phylogeny.

Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry.

A method based on liquid chromatography-electrospray mass spectrometric analysis of the enzymatically digested amplicons derived from the mitochondrial 16S rRNA gene was established for the discrimination of toxic pufferfish. A MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q Exactive mass spectrometer (Thermo Fisher) were employed for separation and detection, respectively. Monoisotopic masses of the oligonucleotides were calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm on average) and precision (relative standard deviation, 0.49 ppm on average) were achieved, and a mass accuracy of <2.8 ppm was maintained for at least 180 h without additional calibration. The present method was applied to 29 pufferfish samples, and results were consistent with Sanger sequencing.

Tetrodotoxin detection and species identification of pufferfish in retail roasted fish fillet by DNA barcoding in China.

This study identifies the pufferfish species and detects tetrodotoxin (TTX) in roasted fish fillet samples collected in Beijing, Qingdao and Xiamen, China. The cytochrome c oxidase I (COI) gene was used as the target gene for identification of the pufferfish species in the samples. Enzyme-linked immunosorbent assay (ELISA) screened the TTX levels in samples that had been detected as containing pufferfish by DNA barcode. A total of 125 samples were identified by DNA barcodes; 32 (26%) samples contained pufferfish composition and, among them, 26 (81%) were the highly toxic species Lagocephalus lunaris. All 32 samples containing the pufferfish composition were positive for TTX with levels ranging from 100 to 63,800 ng g(-1). Most of the 32 samples contained the highly toxic L. lunaris. Based on the results, we suggest that the monitoring of roasted fish fillet should be strengthened and the processing procedures should be standardised to minimise TTX poisoning caused by pufferfish.

Characterization and evolutionary analysis of tributyltin-binding protein and pufferfish saxitoxin and tetrodotoxin-binding protein genes in toxic and nontoxic pufferfishes.

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.

An evaluation of fossil tip-dating versus node-age calibrations in tetraodontiform fishes (Teleostei: Percomorphaceae).

Time-calibrated phylogenies based on molecular data provide a framework for comparative studies. Calibration methods to combine fossil information with molecular phylogenies are, however, under active development, often generating disagreement about the best way to incorporate paleontological data into these analyses. This study provides an empirical comparison of the most widely used approach based on node-dating priors for relaxed clocks implemented in the programs BEAST and MrBayes, with two recently proposed improvements: one using a new fossilized birth-death process model for node dating (implemented in the program DPPDiv), and the other using a total-evidence or tip-dating method (implemented in MrBayes and BEAST). These methods are applied herein to tetraodontiform fishes, a diverse group of living and extinct taxa that features one of the most extensive fossil records among teleosts. Previous estimates of time-calibrated phylogenies of tetraodontiforms using node-dating methods reported disparate estimates for their age of origin, ranging from the late Jurassic to the early Paleocene (ca. 150-59Ma). We analyzed a comprehensive dataset with 16 loci and 210 morphological characters, including 131 taxa (95 extant and 36 fossil species) representing all families of fossil and extant tetraodontiforms, under different molecular clock calibration approaches. Results from node-dating methods produced consistently younger ages than the tip-dating approaches. The older ages inferred by tip dating imply an unlikely early-late Jurassic (ca. 185-119Ma) origin for this order and the existence of extended ghost lineages in their fossil record. Node-based methods, by contrast, produce time estimates that are more consistent with the stratigraphic record, suggesting a late Cretaceous (ca. 86-96Ma) origin. We show that the precision of clade age estimates using tip dating increases with the number of fossils analyzed and with the proximity of fossil taxa to the node under assessment. This study suggests that current implementations of tip dating may overestimate ages of divergence in calibrated phylogenies. It also provides a comprehensive phylogenetic framework for tetraodontiform systematics and future comparative studies.

[Study on tetrodotoxin detection and toxic puffer fish identification of roasted fish fillet at the retail in Beijing and Qingdao].

OBJECTIVE: The roasted fish fillet sample at the retail collected in Beijing and Qingdao were detected for TTX, and the TTX positive samples was analyzed for fish species identification. METHODS: TTX was tested by EUSA method and the cytochrome c oxidase I (COI) genome of TTX-positive samples was extracted and identified by DNA barcode. RESULTS: Totally, 90 samples were tested by EUSA and 58 (64.4%) samples were positive for TTX with the levels ranging from 0.10 mg/kg to 63.81 mg/kg. Among the TTX positive samples, 24 (41.3%) were identified containing toxic puffer fish and 21 (87.5%) were Lagocephalus lunaris, the highly toxic puffer fish. CONCLUSION: Some roasted fish fillet samples obtained from the retail in two cities were positive for TTX and contained toxic puffer fish. Based on these results, we suggest that roasted fish fillet producers should prevent toxic puffer fish from mixing in the raw material and the I regulators should strengthen the TTX surveillance and product labeling supervision of roasted fish fillet.

A new cryptic species of South American freshwater pufferfish of the genus Colomesus (Tetraodontidae), based on both morphology and DNA data.

The Tetraodontidae are an Acantomorpha fish family with circumglobal distribution composed of 189 species grouped in 19 genera, occurring in seas, estuaries, and rivers between the tropical and temperate regions. Of these, the genus Colomesus is confined to South America, with what have been up to now considered only two species. C. asellus is spread over the entire Amazon, Tocantins-Araguaia drainages, and coastal environments from the Amazon mouth to Venezuela, and is the only freshwater puffers on that continent. C. psittacus is found in coastal marine and brackish water environments from Cuba to the northern coast of South America as far south as to Sergipe in Brazil. In the present contribution we used morphological data along with molecular systematics techniques to investigate the phylogeny and phylogeography of the freshwater pufferfishes of the genus Colomesus. The molecular part is based on a cytochrome C oxidase subunit I dataset constructed from both previously published and newly determined sequences, obtained from specimens collected from three distinct localities in South America. Our results from both molecular and morphological approaches enable us to identify and describe a new Colomesus species from the Tocantins River. We also discuss aspects of the historical biogeography and phylogeography of the South American freshwater pufferfishes, suggesting that it could be more recent than previously expected.

A new phylogeny of tetraodontiform fishes (Tetraodontiformes, Acanthomorpha) based on 22 loci.

Tetraodontiform fishes represent one of the most peculiar radiations of teleost fishes. In spite of this, we do not currently have a consensus on the phylogenetic relationships among the major tetraodontiform lineages, with different morphological and molecular datasets all supporting contrasting relationships. In this paper we present the results of the analysis of tetraodontiform interrelationships based on two mitochondrial and 20 nuclear loci for 40 species of tetraodontiforms (representing all of the 10 currently recognized families), as well as three outgroups. Bayesian and maximum likelihood analyses of the concatenated dataset (18,682 nucleotides) strongly support novel relationships among the major tetraodontiform lineages. Our results recover two large clades already found in mitogenomic analyses (although the position of triacanthids differ), while they strongly conflict with hypotheses of tetraodontiform relationships inferred by previous studies based on morphology, as well as studies of higher-level teleost relationships based on nuclear loci, which included multiple tetraodontiform lineages. A parsimony gene-tree, species-tree analysis recovers relationships that are mostly congruent with the analyses of the concatenated dataset, with the significant exception of the position of the pufferfishes+porcupine fishes clade. Our findings suggest that while the phylogenetic placement of some tetraodontiform lineages (triacanthids, molids) remains problematic even after sequencing 22 loci, an overall molecular consensus is beginning to emerge regarding the existence of several major clades. This new hypothesis will require a re-evaluation of the phylogenetic usefulness of several morphological features, such as the fusion of several jaw bones into a parrot-like beak, or the reduction and loss of some of the fins, which may have occurred independently more times than previously thought.

A new multi-locus timescale reveals the evolutionary basis of diversity patterns in triggerfishes and filefishes (Balistidae, Monacanthidae; Tetraodontiformes).

Balistoid fishes (triggerfishes, filefishes, leatherjackets) represent one of the most successful radiations of tetraodontiform fishes across the world's oceans. Balistids (triggerfishes) are largely circumtropical in coral reef environments while most monacanthids (filefishes, leatherjackets) are distributed across reef and non-reef habitats in the Indo-western Pacific. Although members of these clades share a distinctive mode of locomotion that relies upon coordinated oscillation or undulation of enlarged dorsal and anal fins, species richness as well as morphologial and ecological diversity are generally considered to be higher in monacanthids than in triggerfishes. Explicit evolutionary comparisons of diversity patterns between these sister clades have been hampered by the paucity of systematic studies of filefishes relative to triggerfishes. Furthermore, a well-sampled molecular timescale for balistoids is lacking, hindering our understanding of the evolutionary history of these fishes. Here, we produce the largest balistoid molecular dataset to date, based on two mitochondrial and three nuclear loci, for a total of 86 species, and we time-calibrate it using three tetraodontiform fossils. We show that several of the traditional monacanthid genera are not monophyletic and that the balistid Xenobalistes tumidipectoris is nested within the genus Xanthichthys, and suggest that the generic name Xenobalistes be dissolved. Our timetree reveals a Late Miocene origin of balistids, in accordance with previous studies, but a Late Eocene age for the crown monacanthids, which experienced significant diversification during the Late Oligocene and Early Miocene. Comparative analyses reveal no significant family-level differences in rates of speciation or body size evolution, suggesting that the greater diversity of filefishes can be attributed to their more ancient crown age compared to triggerfishes.

Genetic relatedness and phylogenetics of five Indian pufferfishes.

The taxonomy and phylogeny of the pufferfishes belonging to the family Tetraodontidae found in India are poorly understood. We investigated five species of freshwater and marine pufferfishes using partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) of mitochondrial genes. The sequence alignment of 16S rRNA yielded 573 bp, whereas COI gene sequence alignment yielded 614 bp. The sequence analysis of the genes revealed two distinct groups of freshwater and marine origin, which are genetically distinct from each other and exhibit identical phylogenetic resolution. The partial sequences of both the genes provided sufficient phylogenetic resolution to distinguish all the five species of pufferfishes. The COI sequences could be used as DNA barcodes for identification of the pufferfishes.