Ultrafast sound production mechanism in one of the smallest vertebrates.

Motion is the basis of nearly all animal behavior. Evolution has led to some extraordinary specializations of propulsion mechanisms among invertebrates, including the mandibles of the dracula ant and the claw of the pistol shrimp. In contrast, vertebrate skeletal movement is considered to be limited by the speed of muscle, saturating around 250 Hz. Here, we describe the unique propulsion mechanism by which Danionella cerebrum, a miniature cyprinid fish of only 12 mm length, produces high amplitude sounds exceeding 140 dB (re. 1 µPa, at a distance of one body length). Using a combination of high-speed video, micro-computed tomography (micro-CT), RNA profiling, and finite difference simulations, we found that D. cerebrum employ a unique sound production mechanism that involves a drumming cartilage, a specialized rib, and a dedicated muscle adapted for low fatigue. This apparatus accelerates the drumming cartilage at over 2,000 g, shooting it at the swim bladder to generate a rapid, loud pulse. These pulses are chained together to make calls with either bilaterally alternating or unilateral muscle contractions. D. cerebrum use this remarkable mechanism for acoustic communication with conspecifics.

The larval attachment organ of the bowfin Amia ocellicauda Richardson, 1836 (Amiiformes: Amiidae) and its phylogenetic significance.

Larval attachment organs (LAOs) are unicellular or multicellular organs that enable the larvae of many actinopterygian fishes to adhere to a substrate before yolk-sac absorption and the free-swimming stage. Bowfins (Amiiformes) exhibit a sizable LAO on the snout, which was first described in the late 19th and early 20th centuries. In this study, we document the LAO of Amia ocellicauda (Richardson, 1836) using a combination of scanning electron microscopy (SEM) and light microscopy, and histochemistry. We examined material representing three stages with SEM ranging in size from 5.8 to 11.2 mm in notochord length and one stage histochemically. We compare the LAO of A. ocellicauda to that of the lepisosteid Atractosteus tropicus Gill, 1863 and show that although the LAOs of A. ocellicauda and A. tropicus are both super-organs, the two differ in the ultrastructure of the entire organ. A. ocellicauda possesses two distinct lobes, with the organs arranged on the periphery with none in the middle, whereas A. tropicus also possesses two lobes, but with the organs scattered evenly across the super-organ. The individual organs of A. ocellicauda possess adhesive cells set deep to support cells with the adhesive substance released through a pore, whereas A. tropicus possesses both support cells and adhesive cells sitting at a similar level, with the adhesive substance released directly onto the surface of the organ. We additionally provide a table summarizing vertebrate genera in which attachment organs have been documented and discuss the implications of our study for hypotheses of the homology of attachment organs in the Holostei.

Three scleral ossicles in the West African Denticle herring Denticeps clupeoides (Clupeiformes: Denticipitidae).

The eyes of teleostean fishes typically exhibit two ossifications, the anterior and posterior sclerotics, both associated with the scleral cartilage. The West African Denticle herring Denticeps clupeoides has three scleral ossifications, including the typical two associated with the scleral cartilage (anterior and posterior sclerotic) and a third ossification (Di Dario's ossicle), spatially separated from the scleral cartilage and located within the anteromedial wall of the sclera. The medial rectus muscle inserts on the medial surface of Di Dario's ossicle, suggesting that this third sclerotic may play a role in forward rotation of the eye in this surface feeding fish.

Extreme evolutionary shifts in developmental timing establish the miniature Danionella as a novel model in the neurosciences.

BACKGROUND: Species of Danionella rank among the smallest of all vertebrates and their miniature size is correlated with an extreme case of progenesis, resulting in tiny, transparent sexually mature individuals. Progenesis has affected the entire skeleton of Danionella, in which 60 skeletal elements are absent, including some of the skull roofing bones. This lack of a skull roof combined with the presence of a fully formed hearing and sound producing apparatus has led to Danionella being used as an important model for neurophysiological studies. RESULTS: Using both rank based and PGi analyses we investigate sequence heterochrony in the development of the skeleton of Danionella dracula and close relatives. Extreme heterochronic shifts affect the appearance of bony elements in Danionella dracula. This includes a delay in the appearance of most chondral skull bones, and a reduction or loss of dermal bones that would otherwise form the skull roof. In contrast, formation of the anterior region of the vertebral column, including the functionally important elements of the Weberian apparatus, is greatly accelerated. CONCLUSIONS: Here we show that the anatomical conditions that favor Danionella for brain research are the result of extreme heterochronic shifts that have acted differentially across the skeleton.

The larval attachment organ of the tropical gar Atractosteus tropicus Gill, 1863 (Lepisosteiformes: Lepisosteidae).

Larval attachment organs (LAOs) are unicellular or multicellular organs that allow larvae to adhere to a substrate before yolk-sac absorption and the free-swimming stage. This study documents the LAO of tropical gar, Atractosteus tropicus, using a combination of scanning electron microscopy and light microscopy. It is shown that the LAO of A. tropicus is a super-organ surrounded by a wall and containing at its centre many smaller multicellular organ units, each comprised of attachment and support cells. Attachment cells are secretory and house large vacuoles filled with a glycoprotein. At hatching, the super-organ is well developed and occupies almost the entire anteroventral surface of the head. During subsequent development, the smaller individual units begin to regress, until at 6 days post-hatching the super-organ and its individual units are no longer visible.

Spatially restricted dental regeneration drives pufferfish beak development.

Vertebrate dentitions are extraordinarily diverse in both morphology and regenerative capacity. The teleost order Tetraodontiformes exhibits an exceptional array of novel dental morphologies, epitomized by constrained beak-like dentitions in several families, i.e., porcupinefishes, three-toothed pufferfishes, ocean sunfishes, and pufferfishes. Modification of tooth replacement within these groups leads to the progressive accumulation of tooth generations, underlying the structure of their beaks. We focus on the dentition of the pufferfish (Tetraodontidae) because of its distinct dental morphology. This complex dentition develops as a result of (i) a reduction in the number of tooth positions from seven to one per quadrant during the transition from first to second tooth generations and (ii) a dramatic shift in tooth morphogenesis following the development of the first-generation teeth, leading to the elongation of dental units along the jaw. Gene expression and 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) lineage tracing reveal a putative dental epithelial progenitor niche, suggesting a highly conserved mechanism for tooth regeneration despite the development of a unique dentition. MicroCT analysis reveals restricted labial openings in the beak, through which the dental epithelium (lamina) invades the cavity of the highly mineralized beak. Reduction in the number of replacement tooth positions coincides with the development of only four labial openings in the pufferfish beak, restricting connection of the oral epithelium to the dental cavity. Our data suggest the spatial restriction of dental regeneration, coupled with the unique extension of the replacement dental units throughout the jaw, are primary contributors to the evolution and development of this unique beak-like dentition.

Replacing the first-generation dentition in pufferfish with a unique beak.

Teleost fishes comprise approximately half of all living vertebrates. The extreme range of diversity in teleosts is remarkable, especially, extensive morphological variation in their jaws and dentition. Some of the most unusual dentitions are found among members of the highly derived teleost order Tetraodontiformes, which includes triggerfishes, boxfishes, ocean sunfishes, and pufferfishes. Adult pufferfishes (Tetraodontidae) exhibit a distinctive parrot-like beaked jaw, forming a cutting edge, unlike in any other group of teleosts. Here we show that despite novelty in the structure and development of this "beak," it is initiated by formation of separate first-generation teeth that line the embryonic pufferfish jaw, with timing of development and gene expression patterns conserved from the last common ancestor of osteichthyans. Most of these first-generation larval teeth are lost in development. Continuous tooth replacement proceeds in only four parasymphyseal teeth, as sequentially stacked, multigenerational, jaw-length dentine bands, before development of the functional beak. These data suggest that dental novelties, such as the pufferfish beak, can develop later in ontogeny through modified continuous tooth addition and replacement. We conclude that even highly derived morphological structures like the pufferfish beak form via a conserved developmental bauplan capable of modification during ontogeny by subtle respecification of the developmental module.

Different on the inside: extreme swimbladder sexual dimorphism in the South Asian torrent minnows.

The swimbladder plays an important role in buoyancy regulation but is typically reduced or even absent in benthic freshwater fishes that inhabit fast flowing water. Here, we document, for the first time, a remarkable example of swimbladder sexual dimorphism in the highly rheophilic South Asian torrent minnows (Psilorhynchus). The male swimbladder is not only much larger than that of the female (up to five times the diameter and up to 98 times the volume in some cases), but is also structurally more complex, with multiple internal septa dividing it into smaller chambers. Males also exhibit a strange organ of unknown function or homology in association with the swimbladder that is absent in females. Extreme sexual dimorphism of non-gonadal internal organs is rare among vertebrates and the swimbladder sexual dimorphisms that we describe for Psilorhynchus are unique among fishes.

Channa andrao, a new species of dwarf snakehead from West Bengal, India (Teleostei: Channidae).

Channa andrao, new species, from Lefraguri swamp, West Bengal, India, differs from all its congeners except, C. asiatica, C. bleheri and C. burmanica and the recently described C. hoaluensis and C. ninhbinhensis by the absence of pelvic fins. It can be distinguished from all other pelvic fin-less species of snakeheads by its colour pattern, and differs further in its number of vertebrae, dorsal- and anal-fin rays, and lateral-line scales from individual snakehead species in this complex. Channa andrao raises the number of snakehead species endemic to the Eastern Himalaya biodiversity hotspot to ten, representing almost one third of the known species in the genus.

Dario kajal, a new species of badid fish from Meghalaya, India (Teleostei: Badidae).

Dario kajal, new species, is described from Seinphoh stream in the Jaintia Hills of Meghalaya, India. It can be distinguished from all other congeners by the presence of a postorbital stripe that continues behind the eye in line with the preorbital stripe and by the presence in males of a series of double bars restricted to the upper half of the body. The discovery of D. kajal in the Meghna River drainage raises the number of Dario species to five and raises interesting questions about the biogeography of the genus.

Dario tigris and Dario melanogrammus, two new species of miniature chameleon fishes from northern Myanmar (Teleostei: Badidae).

Dario tigris, new species, is described from mountain streams south of Mogaung, in the Ayeyarwaddy River basin, Myanmar. It differs from congeneric species by its unique colour pattern, which consists of a series of eight straight vertical bars, the first two of which in males are ash-grey and the subsequent six are orange-red in life, combined with a series of small black spots arranged in three to four rows dorsolaterally on the head and nape. It differs further from its close relative Dario hysginon, with which it may occur syntopically, by fewer dorsal-fin spines (modally 14 vs modally 15), and fewer vertebrae (modally 12+12=24 vs modally 12+13= 25). Dario melanogrammus, new species, is another barred Dario from the Chindwin River basin. It differs from all other species of the genus by its zigzagging dark vertical bars; amongst Myanmar species, it can be further distinguished from D. hysginon and D. tigris by more vertebrae (modally 26 vs 25 in D. hysginon, and 24 in D. tigris).

Evolutionary divergence of locomotion in two related vertebrate species.

Locomotion exists in diverse forms in nature; however, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. Here, we investigate this question by comparing divergent swimming pattern in larval Danionella cerebrum (DC) and zebrafish (ZF). We show that DC displays long continuous swimming events when compared with the short burst-and-glide swimming in ZF. We reveal that mesencephalic locomotion maintenance neurons in the midbrain are sufficient to cause this increased swimming. Moreover, we propose that the availability of dissolved oxygen and timing of swim bladder inflation drive the observed differences in the swim pattern. Our findings uncover the neural substrate underlying the evolutionary divergence of locomotion and its adaptation to their environmental constraints.

Phylogeny of the Southeast Asian freshwater fish genus Pangio (Cypriniformes; Cobitidae).

The genus Pangio is one of the most species-rich of the loach family Cobitidae and widespread across South and Southeast Asia. Its species diversity has never been studied under a clear phylogenetic approach, but four 'species-groups' were proposed according to the most obvious morphological characters. We present here phylogenetic analyses of the genus Pangio based on sequence data of the mitochondrial cytochrome b gene, the nuclear recombination-activating gene 1 (RAG 1) and a combined dataset of 109 specimens from 18 morphologically identified species across the whole distribution area of the genus. Our data reveal the existence of three major lineages within Pangio. Two of our major lineages were congruent with formerly proposed species-groups, the remaining two species-groups together formed the third major lineage; herein we refer to the lineages as to anguillaris-group, kuhlii-oblonga group and shelfordii-group. The application of a molecular clock dated the age of the three lineages to 33-29 million years. At the species level, our data suggest about 30 distinct lineages, indicating that there is a high number of undescribed species within Pangio. The use of Pangio to address biogeographic questions is demonstrated with the example of the shelfordii-group, which is distributed across Sundaland.

Poor groundwater governance threatens ancient subterranean fishes.

Groundwater depletion is a significant global issue, but its impact on the often-enigmatic subterranean biodiversity and its conservation remains poorly understood. In the Western Ghats biodiversity hotspot of India, poor governance of groundwater resources is threatening its evolutionarily distinct subterranean freshwater fauna, some taxa of which represent Gondwanan relics.

The emerging vertebrate model species for neurophysiological studies is Danionella cerebrum, new species (Teleostei: Cyprinidae).

The four described species of Danionella are tiny, transparent fishes that mature at sizes between 10-15 mm, and represent some of the most extreme cases of vertebrate progenesis known to date. The miniature adult size and larval appearance of Danionella, combined with a diverse behavioral repertoire linked to sound production by males, have established Danionella as an important model for neurophysiological studies. The external similarity between the different species of Danionella has offered an important challenge to taxonomic identification using traditional external characters, leading to confusion over the identity of the model species. Using combined morphological and molecular taxonomic approaches, we show here that the most extensively studied species of Danionella is not D. translucida, but represents an undescribed species, D. cerebrum n. sp. that is externally almost identical to D. translucida, but differs trenchantly in several internal characters. Molecular analyses confirm the distinctiveness of D. cerebrum and D. translucida and suggest that the two species are not even sister taxa. Analysis of the evolution of sexual dimorphisms associated with the Weberian apparatus reveals significant increases in complexity from the simpler condition found in D. dracula, to most complex conditions in D. cerebrum, D. mirifica and D. translucida.

A note on emMuraena/em emalba/em Zuiew, 1793 (Teleostei: Synbranchidae).

Originally described as Muraena alba by the Russian ichthyologist Basilius Zuiew (1793) [Vasilij Fyodorovich Zuev'], the name Monopterus albus has long been used for a species of swamp eel (Synbranchidae) with a reportedly widespread occurrence in Asia (Rosen Greenwood 1976, Kottelat 2013). In recent years molecular studies have shown that Monopterus albus of authors is a species complex and several authors have recommended that up to three (Collins et al. 2002, Matsumoto et al. 2010, Kottelat 2013, Nico et al. 2019) or even five (Arisuryanti 2016) different species can be recognized. Kottelat (2013) referred to the eastern Asian clade of Matsumoto et al. (2010) as Monopterus albus and the Southeast Asian clade as Monopterus javanensis La Cepède, 1800, noting that no name is available for the clade on the Ryukyu Islands.

Aenigmachannidae, a new family of snakehead fishes (Teleostei: Channoidei) from subterranean waters of South India.

Pronounced organism-wide morphological stasis in evolution has resulted in taxa with unusually high numbers of primitive characters. These 'living fossils' hold a prominent role for our understanding of the diversification of the group in question. Here we provide the first detailed osteological analysis of Aenigmachanna gollum based on high-resolution nano-CT scans and one cleared and stained specimen of this recently described snakehead fish from subterranean waters of Kerala in South India. In addition to a number of derived and unique features, Aenigmachanna has several characters that exhibit putatively primitive conditions not encountered in the family Channidae. Our morphological analysis provides evidence for the phylogenetic position of Aenigmachanna as the sister group to Channidae. Molecular analyses further emphasize the uniqueness of Aenigmachanna and indicate that it is a separate lineage of snakeheads, estimated to have split from its sister group at least 34 or 109 million years ago depending on the fossil calibration employed. This may indicate that Aenigmachanna is a Gondwanan lineage, which has survived break-up of the supercontinent, with India separating from Africa at around 120 mya. The surprising morphological disparity of Aenigmachanna from members of the Channidae lead us to erect a new family of snakehead fishes, Aenigmachannidae, sister group to Channidae, to accommodate these unique snakehead fishes.

Unexpected species diversity within Sri Lanka's snakehead fishes of the Channa marulius group (Teleostei: Channidae).

The taxonomic status of the large snakeheads of the Channa marulius group that occur in Sri Lanka is reviewed and clarified. Two species are recognized from the island, based on both morphological and molecular (cytochrome c oxidase subunit 1: cox1) differentiation: C. marulius Hamilton from the northern dry zone and C. ara Deraniyagala from the middle and lower regions of the Mahaweli basin. Channa ara is endemic to Sri Lanka and can be distinguished from its Marulius group congeners, C. marulius, C. aurolineata and C. auroflammea, by having fewer dorsal- and anal-fin rays, fewer lateral-line scales and fewer vertebrae; from C. marulioides by a different adult colour pattern; and from C. pseudomarulius by having more vertebrae. At the cox1 barcoding locus, Channa ara is at least 3.6% genetically different from C. marulius, and at least 8% different from the other described species in the group. Specimens collected from the southwestern wet zone in Sri Lanka are a puzzling third component of the Marulius group's diversity, uncovered in this study, and identified here as C. cf. ara. Whilst genetically more similar to C. marulius, C. cf. ara possesses fewer dorsal- and anal-fin rays, fewer lateral-line scales and fewer vertebrae and is therefore morphologically more similar to C. ara. Channa ara can be distinguished from C. cf. ara, however, by differences in circumpeduncular scale count, adult colour pattern, and by an uncorrected pairwise genetic distance of 3.7% in cox1 sequences. A neotype is designated for Ophicephalus marulius ara Deraniyagala.

Spectacular morphological novelty in a miniature cyprinid fish, Danionella dracula n. sp.

Danionella dracula is a new species of sexually dimorphic, miniature and highly developmentally truncated cyprinid fish. Compared with its close relative, the zebrafish Danio rerio, it lacks 44 bones or parts thereof and represents one of the most developmentally truncated vertebrates. Absence of the majority of bones appears to be due to developmental truncation via terminal deletion. In contrast to these larval-like features, D. dracula also shows several hyperossifications. Uniquely, among carp-like fishes, male D. dracula have a series of long, pointed odontoid processes on the jaws greatly resembling the jaw dentition of teleosts with true teeth. The anterior-most process in each jaw is extended as a canine-like fang projecting through the epithelium. True jaw teeth are absent from all 3700 species of cypriniforms and were lost at least in the Upper Eocene. It remains to be investigated, however, whether the conserved pathways to regulate tooth development in cypriniforms have been used in D. dracula to form and pattern the odontoid processes. This new species represents a remarkable example linking progenetic paedomorphosis via heterochronic change in developmental timing to the evolution of morphological novelties.