Phylogenetic relationships and intraspecific variations of loaches of the genus Lefua (Balitoridae,Cypriniformes)

Three nominal species are known in East Asian balitorid loaches of the genus Lefua, i.e. L. echigonia, L. nikkonis, and L. costata. Lefua echigonia, with large morphological variations was recently separated into two groups, L. echigonia including the holotype and L. sp., based on morphological and ecological traits. We performed protein and DNA analyses to elucidate phylogenetic relationships among loaches of the genus Lefua and to settle the taxonomic status of L. sp. We also investigated intraspecific variations in L. echigonia s. str. to shed light on the process of formation of freshwater fish fauna in Japan. Protein analyses using two-dimensional gel electrophoresis showed that genetic distances between L. sp. and L. echigonia s. str. and between L. sp. and L. nikkonis were as large as that between L. echigonia s. str. and L. nikkonis. DNA analyses of the mitochondrial D-loop region showed that L. sp. and L. echigonia s. str. were monophyletic, respectively, while neither L. nikkonis nor L. costata was monophyletic and these species formed together a clade. The results supported the specific status of L. sp. and proposed reevaluation of the taxonomic status of L. nikkonis and L. costata. DNA analyses also showed that L. sp. was more closely related to L. echigonia s. str. than to the L. nikkonis-L. costata complex, and four local populations were distinguished in L. echigonia s. str. Distribution patterns of the four local populations of L. echigonia s. str. in Japan were approximately congruent with those of the medaka, Oryzias latipes, suggesting that differentiation in the two distantly related fishes have a common historical background.     

The thoracic morphology of Nannochorista (Nannochoristidae) and its implications for the phylogeny of Mecoptera and Antliophora

External and internal thoracic structures of Nannochorista spp. are described in detail. The results are compared with conditions found in other endopterygote taxa, especially in members of Antliophora. Seventy-seven characters potentially useful for phylogenetic reconstruction are discussed, coded, presented as a data matrix and analysed cladistically. The thorax of Nannochorista shows a number of plesiomorphic characters compared with other mecopterans (except for Merope ) and members of the other antliophoran groups (e.g. presence of prospina and associated muscles). No specific affinities of thoracic features of Nannochoristidae and Diptera were found. The cladistic analysis results in strongly supported Antliophora (e.g. intraprofurcal muscle and ventral pleural arms present; bundle of M. mesonoto-pleuralis posterior originates on pleural arm). The thoracic characters do not support the monophyly of Mecoptera. This is possibly an artefact of the analysis. Several potential thoracic autapomorphies of the order are inapplicable in Boreidae, Siphonaptera and Diptera. Boreidae and Siphonaptera share a suite of characters related with flightlessness and are retrieved as sistertaxa when characters associated with wing reduction are predefined as irreversible. Merope appears exceptionally plesiomorphic in its thoracic morphology. Pistillifera (excluding Meropidae) and Panorpoidea (Panorpidae + Panorpodidae) are supported as clades. Due to the strongly modified thoracic morphology of Siphonaptera, the position of this group remains uncertain. The phylogenetic reconstruction using thoracic features alone is clearly impeded by far reaching modifications in Diptera in correlation with an advanced type of anteromotorism, and complex suites of reductional features in the secondary wingless forms.     

Genetic Differentiation among Populations of the Cave Fish Schistura Oedipus (Cypriniformes: Balitoridae)

DNA sequences were determined for portions of the mitochondrial control region and 16S rRNA genes in five populations of Schistura oedipus, a troglobitic fish from northwestern Thailand. Based on variation at seven sites out of 593 sequenced, the five populations fall into three groups, apparently isolated by altitude and distance. Nucleotide diversity within groups is on the order of 1.0×10^–3 while the extra increment of diversity among groups is on the order of 5.0×10^–3. While the groups are distinct, the level of differentiation among them is low enough to accept provisionally that the five populations represent a single species. On the basis of 16S sequence, Schistura oedipus appears to be more closely related to S. poculi than to S. reidi, a proposed sister taxon.     

Incongruence between mtDNA phylogeny and morphologial and ecological characters in loaches of the genus Lefua (Balitoridae, Cypriniformes)

To elucidate the phylogenetic position of Lefua loaches from Aichi and Shizuoka Prefectures of Honshu Island, Japan, we determined their nucleotide sequences for the mitochondrial D-loop region and compared these to sequences from four other Lefua species: L. costata, L. nikkonis, L. echigonia, and L. sp. Loaches identified as L. sp. on the basis of morphology comprised a clade (the L. sp. Tokai population) that grouped together with L. echigonia; hence, the processes involved in evolution within the genus Lefua were unclear. We performed randomly amplified polymorphic DNA (RAPD) analyses to obtain genetic information on nuclear DNA. The RAPD patterns of the L. sp. Tokai population differed from those of the local L. echigonia and L. sp. populations. The L. sp. Tokai population was similar to L. echigonia with regard to mitochondrial DNA but differed from L. echigonia and L. sp. with respect to nuclear DNA; this indicated that the evolutionary background of the L. sp. Tokai population was unique. We suggest that introgression of mitochondria occurred from L. echigonia to the L. sp. Tokai population, and speculate on the process of evolution of the latter population of Lefua. As with six L. echigonia populations and two L. sp. populations, we regard the L. sp. Tokai population as an evolutionary significant unit (ESU) that qualifies for protection as an endangered loach.     

Problems of the classification and phylogeny of nemacheiline loaches of the group lacking the preethmoid I (Cypriniformes: Balitoridae: Nemacheilinae)

Phylogenetic relationships and classification of nemacheiline loaches belonging to the line with lost preethmoideum I are investigated. With consideration of the analysis of 29 apomorphous states of 23 characters, the following hypothesis of relationships within this line is suggested: Oxynoemacheilus + Indoreonectes + Dzihunia + (Seminemacheilus (Afronemacheilus + Nun)) + Iskandaria (Paracobitis + Troglocobitis) + Metaschistura + Paraschistura + (Schistura gr. B (Acanthocobitis + Nemacheilus s. str. + Schistura gr. A)))). Revision of the generic classification of loaches of this line inhabiting water bodies from the Near East and Ethiopia to middle Asia and the Indus upper reaches is made. Key to genera of this region is suggested. Four new genera are described: Afronemacheilus Golubtsov and Prok., g. n. (type species A. abyssinicus), Iskandaria Prok., g. n. (type species I. kuschakewitschi), Metaschistura Prok., g. n. (type species M. cristata), and Paraschistura Prok., g. n. (type species P. sargadensis). Independence of the genera Oxynoemacheilus and Troglocobitis is confirmed. The Oriental Schistura are subdivided into two groups (A and B). Schistura gr. A form a monophyletic line with the genera Acanthocobitis and Nemacheilus s. str. It is noted that the south-China genus Homatula cannot be related to Paracobitis or Schistura as it has no ventral axillar lobe, but it may turn out to be identical to the genus Oreias from the same region.     

Parallel evolution in eight-barbel loaches of the genus Lefua (Balitoridae, Cypriniformes) revealed by mitochondrial and nuclear DNA phylogenies

The evolutionary history of eight-barbel loaches of the genus Lefua contains important phylogenetic information that will aid in resolution of the faunal formations and evolutionary histories of Japanese and East Asian freshwater fishes. Our sequencing of the mitochondrial D-loop region in a large number of samples allowed construction of the most comprehensive phylogeny of these loaches to date; we demonstrated monophyly of five Lefua species and identified populations of Lufua. sp. and Lefua echigonia. Loaches inhabiting the Tokai region in Japan were morphologically and ecologically indistinguishable from Lefua sp. However, they were included in the L. echigonia lineage. We determined a novel phylogeny by sequencing the nuclear ribosomal S7 subunit and showed that nuclear DNA phylogeny essentially matched the mitochondrial DNA phylogeny. Loaches from the Tokai region were part of the L. echigonia lineage, indicating parallel evolution between Tokai loaches and Lefua sp. in western Japan. We presented the most robust phylogeny to date using concatenated mitochondrial and nuclear sequences. The wealth of molecular information allowed us to speculate on evolutionary processes in the genus Lefua.     

The comparative osteology and phylogenetic relationships of African and South American lungfishes (Sarcopterygii: Dipnoi)

Lepidosirenidae is a clade of freshwater lungfishes that include the extant South American Lepidosiren paradoxa Fitzinger, 1837 and African species of the genus Protopterus. These genera have been geographically separated since the break‐up of Gondwana in the Early Cretaceous, but they display similar biology and morphology. Species were distinguished by a combination of features such as head‐to‐body ratios, the number of pairs of ribs, and the presence of external gills, but no discrete skeletal characters were identified, and no comparative studies including all extant species have been published. I used computed tomography (CT), X‐ray photography, and specimens from museum collections to describe the skeletal morphology of all species of lepidosirenid in a comparative context. I digitally disarticulated the bones in each specimen to compile a comparative atlas of the cranial and pectoral elements of all extant lungfishes, which has the potential to increase the correct identifications of specimens in museum collections. The morphology of the frontoparietal, parasphenoid, supraorbital, and suboperculum differ between species. I used those characters, along with molecular sequence data from the ribosomal RNA gene 16S, to run combined morphological and molecular phylogenetic analyses. Lepidosirenidae is monophyletic in all analyses, but the interrelationships of the species of Protopterus vary with the different sources of character data. © 2015 The Linnean Society of London     

犁头鳅属鱼类物种地理分化过程

犁头鳅属为中国的特有属,包括犁头鳅和长鳍犁头鳅两个物种,犁头鳅广泛分布于长江中上游,而长鳍犁头鳅则局限分布于闽江水系。本研究对采自长江中上游的犁头鳅20个个体和闽江水系的长鳍犁头鳅8个个体的线粒体细胞色素b（cyt b）基因序列进行分析,以期对犁头鳅属鱼类的物种地理分化过程进行研究。结果显示,犁头鳅不同单倍型之间的遗传变异水平为0.1%—1.8％,平均为0.7%;长鳍犁头鳅各单倍型之间的遗传变异水平为0.6%—1.3%,平均为0.7%;犁头鳅和长鳍犁头鳅单倍型之间的遗传差异也较小,仅为0.2%—1.8%,平均为0.9%。采用邻接(NJ)法和贝叶斯(BI)法构建的分子系统发育树一致显示,该研究中的犁头鳅属鱼类构成一个单系;所有长鳍犁头鳅样本构成一个单系,位于系统发育树的顶部位置;犁头鳅的样本不构成单系,而是形成并系。从形态上看,犁头鳅和长鳍犁头鳅均为有效种。由此推测,在第四纪冰期,长江中下游的犁头鳅沿东海大陆架向南扩散到东南沿海水系,长鳍犁头鳅可能是犁头鳅的一个种群扩散到闽江水系后,由于适应新的环境条件而分化成的一个新物种,剩下的长江流域的犁头鳅种群则构成一个并系类群。本文对于类似的地理物种形成方式也进行了讨论。     

Mitochondrial capture and incomplete lineage sorting in the diversification of balitorine loaches (Cypriniformes,Balitoridae) revealed by mitochondrial and nuclear genes

Tang, Q.‐Y., Liu, S.‐Q., Yu, D., Liu, H.‐Z. & Danley, P.D. (2012) Mitochondrial capture and incomplete lineage sorting in the diversification of balitorine loaches (Cypriniformes, Balitoridae) revealed by mitochondrial and nuclear genes. —Zoologica Scripta, 41, 233–247. Understanding the diversification of species is a central goal of evolutionary biological studies. One powerful tool to investigate the speciation process is molecular systematics. Here, we use molecular methods to investigate the evolution of balitorine loaches belonging to two genera, Lepturichthys and Jinshaia. Both genera contain only two species (Lepturichthys fimbriata, Lepturichthys dolichopterus and Jinshaia sinensis and Jinshaia abbreviata), all of which are endemic to China. These species share many morphological and ecological characters and exhibit overlapping distributions in the Upper Yangtze River. In this study, we used two mitochondrial genes (Cytb and COI) and one nuclear gene (RAG1) to investigate the phylogenetic relationships within and between these two genera. Phylogenetic analyses and network construction based on mitochondrial and nuclear genes consistently supported the monophyly of Jinshaia. In contrast, the mitochondrial and nuclear genes yielded conflicting results in Lepturichthys. The phylogenetic analyses of mitochondrial sequences identify two distinct Lepturichthys lineages, Lepturichthys A and Lepturichthys B. Lepturichthys A includes most of L. fimbriata individuals from the Upper Yangtze River and is the sister group to all Jinshaia species. Lepturichthys B consists of the remaining L. fimbriata individuals from the Upper and Middle Yangtze River, and all L. dolichopterus individuals from the Minjiang River in Southeastern China. However, the analysis of the nuclear sequence indicates that the genus Lepturichthys is monophyletic and is only distantly related to Jinshaia. This incongruence suggests that introgressive hybridization might have occurred between L. fimbriata (Lepturichthys A) and Jinshaia species. As a result of this hybridization event, L. fimbriata captured the mitochondrial genome of the sympatric Jinshaia species. This capture event appears to have occurred at least 1.74 million years ago. Additionally, L. fimbriata appears to be paraphyletic; the nuclear data indicated that L. dolichopterus forms a monophyletic clade nested within L. fimbriata. Because L. dolichopterus and L. fimbriata are allopatric and hybridization may not be possible, we suggest that the observed paraphyly of L. fimbriata is a product of incomplete lineage sorting. In addition, the reciprocal monophyly of J. sinensis and J. abbreviata could not be resolved. This may be the result of interspecific hybridization as these species occur sympatrically. However, incomplete lineage sorting may have caused the observed topology of the Jinshaia species. The data presented here illustrate the complex evolutionary history of the balitorine loach species: intergeneric hybridization and interspecific hybridization have likely occurred in this lineage. In addition, possible incomplete lineage sorting may further obscure the evolutionary history of this group. The complex relationships of the balitorine loaches provide a rich evolutionary system to study the creation of sympatric and sister species.     

Using the whole body as a sucker: Combining respiration and feeding with an attached lifestyle in hill stream loaches (Balitoridae,Cypriniformes)

Small fishes living in fast‐flowing rivers face a harsh environment as they can easily be swept away by the rapid currents. To survive such circumstances, teleosts evolved a wide variety of attachment mechanisms, based on friction, negative pressure or both. Balitorinae (Balitoridae, Cypriniformes) are exceptional in using their whole body as an adhesive apparatus. We investigated the morphological adaptations of Balitorinae by studying the osteology and myology of four species (Beaufortia leveretti, Sewellia lineolata, Pseudogastromyzon myersi, and Gastromyzon punctulatus) using clearing and staining, serial cross‐sections and CT‐scanning. A kinematic analysis was performed to study the respiration and feeding mechanisms and to identify key structures in these mechanisms. Our research showed that the whole body of Balitorinae acts as a suction disc, with friction‐enhancing structures (unculi) on the thickened anterior rays of the paired fins. The abruptly rising head profile, supported by the extremely enlarged lacrimal bone and the flat ventral body surface facilitate effective substrate attachment. During attachment, the pelvic girdle is pulled anterodorsally, suggesting the formation of a negative pressure underneath the body. Detachment by water inflow underneath the body is prevented by three mechanisms. 1) Barbels control the water inflow by detachment and reattachment to the substrate. 2) Most water present underneath the body is removed during inspiration. 3) Excess water is regularly removed by movements of the posterior pectoral fin rays. The balitorine body is thus modified as such that it allows effective attachment, while not impairing respiration. Comparison with other teleosts living in similar environments shows that most species use more locally concentrated modifications of the paired fins and/or the mouth for attachment. The high diversity in teleostean adhesive apparatuses and associated myological modifications suggest a substantial functional convergent evolution, without necessarily highly convergent anatomical adaptations. J. Morphol. 275:1066–1079, 2014. © 2014 Wiley Periodicals, Inc.     

Balitora eddsi,a new species of hillstream loach (Ostariophysi: Balitoridae) from Nepal

A new species of hillstream loach Balitora eddsi is described from the Karnali River drainage in south‐western Nepal. The new species is distinguished from all its congeners by possessing the following combination of characters: six to seven unbranched pectoral‐fin rays, pelvic‐fin length 12–14% standard length (L_S), dorsal surface without circular or irregular shaped dark blotches, snout pointed, median lobe between anterior rostral barbels pointed posteriorly, dorsal‐fin origin posterior to pelvic‐fin origin, lateral line scales 66–67, caudal peduncle length 22–23·2% L_S, caudal peduncle depth 4·1–4·2 times its length.     

Four fish species new to the Omo-Turkana basin,with comments on the distribution ofNemacheilus abyssinicus (Cypriniformes: Balitoridae) in Ethiopia

Four fish species,Pollimyrus isidori (Mormyridae),Barbus paludinosus, Labeo forskalii (Cyprinidae), andNemacheilus abyssinicus (Balitoridae), new to the Omo-Turkana basin, were recorded from the Gojeb River, a tributary of the Omo River (south-western Ethiopia). Occurrence of the latter species in the upper reaches of the Blue Nile and of the Omo drainage substantiates the belonging of the upper parts of these water systems to the Abyssinian highlands ichthyofaunal province.     

Musculoskeletal morphology of the pelvis and pelvic fins in the lungfish Protopterus annectens

The West African lungfish (Protopterus annectens) performs benthic, pelvic fin‐driven locomotion with gaits common to tetrapods, the sister group of the lungfishes. Features of P. annectens movement are similar to those of modern tetrapods and include use of the distal region of the pelvic fin as a “foot,” use of the fin to lift the body above the substrate and rotation of the fin around the joint with the pelvis. In contrast to these similarities in movement, the pelvic fins of P. annectens are long, slender structures that are superficially very different from tetrapod limbs. Here, we describe the musculoskeletal anatomy of the pelvis and pelvic fins of P. annectens with dissection, magnetic resonance imaging, histology and 3D‐reconstruction methods. We found that the pelvis is embedded in the hypaxial muscle by a median rostral and two dorsolateral skeletal projections. The protractor and retractor muscles at the base of the pelvic fin are fan‐shaped muscles that cup the femur. The skeletal elements of the fin are serially repeating cartilage cylinders. Along the length of the fin, repeating truncated cones of muscles, the musculus circumradialis pelvici, are separated by connective tissue sheets that connect the skeletal elements to the skin. The simplicity of the protractor and retractor muscles at the base of the fin is surprising, given the complex rotational movement those muscles generate. In contrast, the series of many repeating segmental muscles along the length of the fin is consistent with the dexterity of bending of the distal limb. P. annectens can provide a window into soft‐tissue anatomy and sarcopterygian fish fin function that complements the fossil data from related taxa. This work, combined with previous behavioral examination of P. annectens, illustrates that fin morphologies that do not appear to be capable of walking can accomplish that function, and may inform the interpretation of fossil anatomical evidence. J. Morphol. 275:431–441, 2014. © 2013 Wiley Periodicals, Inc.     

The cranial morphology of Greererpeton burkemorani Romer (Amphibia: Temnospondyli)

The skull of Greererpeton burkemorani Romer, a temnospondyl amphibian from the Upper Mississippian at Greer, West Virginia is described. A detailed account of the stapes of a Mississippian amphibian is given for the first time and its function is discussed. It is suggested that the stapes formed the principal element of support for the back of the braincase and resisted potential dislocation of the otico-occipital region from the skull roof during contraction of the hypaxial musculature.
Greererpeton is included in the Colosteidae and an amended diagnosis of the family is given. Erpetosaurus differs from Colosteus, Greererpeton and Pholidogaster in the pattern of bones in the skull roof and palate, the dentition and the otic region and, consequently, it is removed from the Colosteidae. The Temnospondyli are considered to be a monophyletic group characterized by the development of a connection between the dorsal portion of the occipital arch, the exoccipital bones, and the skull roof. The loxommatids are removed from the Temnospondyli as they retain the plesiomorphic condition of braincase attachment which relies exclusively on derivatives of the auditory capsules.
On the basis of similarities in the structure of the braincase, palate and manus it is suggested that microsaurs are the collateral descendants (sister group) of temnospondyls. This relationship may account for the large number of similarities in the three living groups of Amphibia: Anura are generally believed to have descended from temnospondyls, while the Urodela and Apoda are often considered to have descended from microsaurs. These systematic conclusions endorse the recent suggestions that neither the Lepospondyli nor the Labyrinthodontia are natural groups, and both terms should be abandoned.     

Functional morphology of the anchorage system and food scrapers of a hillstream fish, Garra lamta (Ham.) (Cyprinidae, Cypriniformes)

The functional morphology of the anchorage system and food scrapers of a hillstream fish, Garra lamta (Ham.) is described in relation to their role in the swift water current of the hillstreams (Ranchi, Bihar, India).     

Sperm morphology of salamandrids (Amphibia, Urodela): implications for phylogeny and fertilization biology

Mature spermatozoa belonging to four salamander species, Salamandrina terdigitata, Triturus alpestris, Triturus carnifex and Triturus vulgaris, have been investigated by electron microscopy. The sperm ultrastructure of these species was compared with that of previously examined urodeles (36 species and 20 genera) and with that of anurans and caecilians. Many phylogenetic considerations may be inferred as a consequence of comparative spermatology. Urodela appears to be a monophyletic order characterized by three sperm synapomorphies: the acrosomal barb, nuclear ridge and marginal filament. Cryptobranchoidea are confirmed to form a monophyletic suborder having two synapomorphic characters: absence of mitochondria in the tail, and cylindrical shape of the tail axial rod. Within the family Salamandridae, sperm morphology confirms the phylogenetic distance between Salamandrina and Triturus, as already pointed out on the basis of molecular and morphological characters. The very complex ultrastructure of spermatozoa confirms a previous opinion that internal fertilization is the ancestral condition of the Amphibia.     

Automated pelvic anatomical coordinate system is reproducible for determination of anterior pelvic plane

Most of computer-assisted planning systems need to determine the anatomical axis based on the anterior pelvic plane (APP). We analysed that our new system is more reproducible for determination of APP than previous methods. A pelvic model bone and two subjects suffering from hip osteoarthritis were evaluated. Multidetector-row computed tomography (MDCT) images were scanned with various rotations by MDCT scanner. The pelvic rotation was calibrated using silhouette images. APP was determined by an optimisation technique. The values of variation of APP caused by pelvic rotation were analysed with statistical analysis. APP determination with calibration and optimisation was most reproducible.The values of variance of APP were within 0.05° in model bone and 0.2° even in patient pelvis. Furthermore, the values of variance of APP with calibration/optimisation were significantly lower in comparison without calibration/optimisation. Both calibration and optimisation are actually required for determination of APP. This system could contribute to the evaluation of hip joint kinematics and computer-assisted surgery.