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A crucial evolutionary change in vertebrate history was the Palaeozoic (Devonian 419–359 million years ago) water-to-land transition, allowed by key morphological and physiological modifications including the acquisition of lungs. Nonetheless, the origin and early evolution of vertebrate lungs remain highly controversial, particularly whether the ancestral state was paired or unpaired. Due to the rarity of fossil soft tissue preservation, lung evolution can only be traced based on the extant phylogenetic bracket. Here we investigate, for the first time, lung morphology in extensive developmental series of key living lunged osteichthyans using synchrotron x-ray microtomography and histology. Our results shed light on the primitive state of vertebrate lungs as unpaired, evolving to be truly paired in the lineage towards the tetrapods. The water-to-land transition confronted profound physiological challenges and paired lungs were decisive for increasing the surface area and the pulmonary compliance and volume, especially during the air-breathing on land. All life on Earth started out under water. However, around 400 million years ago some vertebrates, such as fish, started developing limbs and other characteristics that allowed them to explore life on land. One of the most pivotal features to evolve was the lungs, which gave vertebrates the ability to breathe above water. Most land-living vertebrates, including humans, have two lungs which sit on either side of their chest. The lungs extract oxygen from the atmosphere and transfer it to the bloodstream in exchange for carbon dioxide which then gets exhaled out in to the atmosphere. How this important organ first evolved is a hotly debated topic. This is largely because lung tissue does not preserve well in fossils, making it difficult to trace how the lungs of vertebrates changed over the course of evolution. To overcome this barrier, Cupello et al. compared the lungs of living species which are crucial to understand the early stages of the water-to-land transition . This included four species of lunged bony fish which breathe air at the water surface, and a four-legged salamander that lives on land. Cupello et al. used a range of techniques to examine how the lungs of the bony fish and salamander changed shape during development. The results suggested that the lungs of vertebrates started out as a single organ, which became truly paired later in evolution once vertebrates started developing limbs. This anatomical shift increased the surface area available for exchanging oxygen and carbon dioxide so that vertebrates could breathe more easily on land. These findings provide new insights in to how the lung evolved into the paired structure found in most vertebrates alive today. It likely that this transition allowed vertebrates to fully adapt to breathing above water, which may explain why this event only happened once over the course of evolution.

White-spotted charr ( Salvelinus leucomaenis , S . I .) is an anadromous cold water-adapted fish, distributed in the Far East. We have previously reported the complete mitochondrial DNA sequences of white-spotted chars ( S. l. imbrius and S. l. pluvius ) in Japan. In general, fish hepatocytes are useful for cellular and biochemical studies of fish. In this study, we isolated hepatocytes from the liver of white-spotted charr and used basic methods, such as enzyme digestion and low centrifugation, to analyze the molecular mechanisms involved in specific cellular responses. The isolated hepatocytes could be cultured at 5–20 °C but not 37 °C. The morphology of hepatocytes was altered in a temperature-dependent manner. The properties of hepatocyte were similar to those of living fish. Moreover, the proliferation rate and damage of isolated hepatocytes depended on the concentration of fetal bovine serum in the culture medium. Taken together, this study demonstrates that this simple method for isolation and culture of hepatocytes from white-spotted charr may be useful for other biochemical and cellular studies.

Coincident with the population increase in the 1990s, egg and larva distributional area of the Pacific stock of Japanese anchovy Engraulis japonicus expanded from the warm Kuroshio waters toward the cold Oyashio waters off northern Japan. To understand how Japanese anchovy expanded its spawning and nursery grounds, egg and larva distributions and larval growth rates were investigated in relation to temperature and flow fields in the Pacific waters off northern Japan (Pacific North, PN) in summer. Anchovy eggs occurred in the Oyashio waters (≤5 °C at a 100-m depth) warmer than or equal to 15.9 °C in sea surface temperature (SST) which is close to the known lower limit of spawning temperature. Eggs and larvae would be transported southwestward to the warmer coastal and offshore waters by the currents dominated in the study area. Temperature experienced by anchovy eggs and larvae in the northern subarctic waters was almost the same as that in the southern spawning ground in spring. Larval growth rates in the study area were comparable to the previous studies in the southern nursery areas. Thus, the subarctic PN waters would function as spawning and nursery grounds of E. japonicus in the decades of high stock abundance.

Anguilliform leptocephali of the genus Thalassenchelys Castle and Raju 1975 are remarkably large and peculiarly shaped eel larvae, whose adult form has been unknown since the discovery of the larvae in the 1950s. We found bigmouth conger Congriscus megastomus (Günther 1877 ) collected off the Pacific coasts of Japan to have mitochondrial DNA sequences (16S rDNA and COI) nearly identical to those of Thalassenchelys coheni Castle and Raju 1975 published to date and collected recently in the north Pacific. Vertebrae counts of C. megastomus were consistent with the myomere counts of T. coheni. We conclude that T. coheni, so-called larval species described by Castle and Raju ( 1975 ), is a junior synonym of C. megastomus. Therefore, the family to which the leptocephali belong must be Congridae.

:  Quality control of imported fish products is a constant challenge for Customs officials. To help officials access the quality and integrity of imported fish products, and to enhance the effectiveness and accuracy of product screening, a method was developed to identify boiled and dried larval and juvenile sardines (Chirimen) at the genus level by direct sequencing using a partial sequence (606 bp) of mitochondrial 16S rDNA. A phylogenetic tree was constructed, based on nucleotide sequences of seven kinds of Chirimen imported from four countries with 16 standard samples belonging to the orders Clupeiformes and Salmoniformes. To identify the fish genera in imported Chirimen, bootstrap values and branch lengths of the phylogenetic tree as well as genetic distances were used. The data revealed that Chirimen imported from China and Korea contains species of the genus Engraulis. Regarding other imported Chirimen, the least that could be verified was that the sardine ingredients do not belong to regulated genera.

Anguilliform leptocephali of the genus Thalassenchelys Castle and Raju 1975 are remarkably large and peculiarly shaped eel larvae, whose adult form has been unknown since the discovery of the larvae in the 1950s. We found bigmouth conger Congriscus megastomus (Günther 1877 ) collected off the Pacific coasts of Japan to have mitochondrial DNA sequences (16S rDNA and COI) nearly identical to those of Thalassenchelys coheni Castle and Raju 1975 published to date and collected recently in the north Pacific. Vertebrae counts of C. megastomus were consistent with the myomere counts of T. coheni . We conclude that T. coheni , so-called larval species described by Castle and Raju ( 1975 ), is a junior synonym of C. megastomus . Therefore, the family to which the leptocephali belong must be Congridae.

We used mitochondrial DNA sequences to determine the phylogenetic placement of southern smelts (Retropinnidae), a group of diadromous fishes endemic to New Zealand and Australia. Our genetic data strongly support a sister group relationship between retropinnids and northern hemisphere smelts (Osmeridae), a relationship that seems consistent with the similar appearance and life history strategies of these two groups. Our analysis indicates that Retropinnidae and Osmeridae together represent the sister group to the southern hemisphere galaxiid fishes (Galaxiidae). However, this finding conflicts with several recent osteological analyses, which supported a sister relationship for Retropinnidae and Galaxiidae, giving a monophyletic southern hemisphere assemblage (Galaxioidea). We review cases of incongruence and discuss factors that might explain significant disagreement between molecular and morphological data matrices. We suggest that repeated evolutionary simplification may have undermined the accuracy of morphological hypotheses of osmeroid relationships. Although equally weighted parsimony analysis of morphological data rejects the molecular hypothesis (Osmeridae + Retropinnidae), implementation of a range of weighting schemes suggests that incongruence is nonsignificant under asymmetric character transformation models. We propose that a simple “equal transformation cost” parsimony analysis may be biologically unrealistic, especially when reductive homoplasy is widespread; as is increasingly being accepted, complex character states are more readily lost than gained. Therefore, we recommend that morphological systematists routinely implement a range of character transformation models to assess the sensitivity of their phylogenetic reconstructions. We discuss the antitropical biogeography of osmeroid fishes in the context of vicariance and transequatorial dispersal.

The authors used mitochondrial DNA sequences to determine the phylogenetic placement of southern smelts, a group of diadromous fishes endemic to New Zealand and Australia. Their genetic data strongly support a sister group relationship between retropinnids and northern hemisphere smelts.

During the past decade, a few papers were published describing techniques on making detailed larval fish illustration. However, methods described in these papers were aimed only at illustrating lateral views of larvae. Dorsal, ventral, and anterior views often reveal characters useful for identification, such as pigmentation patterns, cephalic spine formation, etc. Unfortunately, it is difficult to place and hold larval fish specimens in a position suitable for dorsal, ventral, or anterior observations. In the present paper, a larval fish clamp, a tool to retain larval fish specimens in place for such observation is described.

A crucial evolutionary change in vertebrate history was the Palaeozoic (Devonian ~400 million years ago) water-to-land transition, allowed by key morphological and physiological modifications including the acquisition of lungs. Nonetheless, the origin and early evolution of vertebrate lungs remain highly controversial, particularly whether the ancestral state was paired or unpaired. Due to the rarity of fossil soft tissue preservation, lung evolution can only be traced based on the extant phylogenetic bracket. Here we investigate, for the first time, lung morphology in extensive developmental series of key living lunged osteichthyans using synchrotron X-ray microtomography and histology. Our results shed light on the primitive state of vertebrate lungs as unpaired, evolving to be truly paired in the lineage towards the tetrapods. The water-to-land transition confronted profound physiological challenges and paired lungs were decisive for increasing the surface area and the pulmonary compliance and volume, especially during the air-breathing on land. Competing Interest Statement The authors have declared no competing interest.