Phylogeny of the Acipenseriformes: cytogenetic and molecular approaches

The review of the data on karyology and DNA content in Acipenseriformes shows that both extant families, the Polyodontidae and Acipenseridae, originated from a tetraploid ancestor which probably had a karyotype consisting of 120 macro- and microchromosomes and DNA content of about 3.2–3.8 pg per nucleus. The tetraploidization of the presumed 60-chromosome ancestor seems to have occurred at an early time of evolution of the group. The divergence of the Acipenseridae into Scaphirhyninae and Acipenserinae occurred without polyploidization. Within the genus Acipenser, polyploidization was one of the main genetic mechanisms of speciation by which 8n and 16n-ploid species were formed. Individual gene trees constructed for sequenced partial fragments of the 18S rRNA (230 base pairs, bp), 12S rRNA (185 bp), 16S rRNA (316 bp), and cytochrome b (270 bp) genes of two Eurasian (A. baerii and A. ruthenus) and two American (A. transmontanus and A. medirostris) species of Acipenser, Huso dauricus, Pseudoscaphirhynchus kaufmanni, Scaphirhynchus albus, and Polyodon spathula showed a low level of resolution; the analysis of a combined set of data for the four genes, however, gave better resolution. Our phylogeny based on molecular analysis had two major departures from existing morphological hypotheses: Huso dauricus is a sister-species to Acipenser instead of being basal to all acipenseriforms, and Scaphirhynchus and Pseudoscaphirhynchus do not form a monophyletic group. The phylogenetic tree constructed for the cytochrome b gene fragments (with inclusion of 7 additional species of Acipenser) supported the conclusion that octoploid species appeared at least three times within Acipenser.     

An overview of Acipenseriformes

Acipenseriformes occupy a special place in the history of ideas concerning fish evolution, but in many respects, phylogenetic studies of the group remain in their infancy. Even such basic questions as the monophyly of Acipenser (the largest genus) are unanswered. We define relationships based on comparative osteology, which allows us to incorporate well-preserved fossils into analyses. Acipenseriformes has existed at least since the Lower Jurassic (approximately 200 MYBP), and all fossil and recent taxa are from the Holarctic. Phylogenetic relationships among Paleozoic and Early Mesozoic actinopterygians are problematic, but most workers agree that Acipenseriformes is monophyletic and derived from some component of paleonisciform fishes. (Paleonisciformes is a grade of primitive non-neopterygian actinopterygians, sensu Gardiner 1993.) Taxa discussed in comparison here are: Cheirolepis, Polypterus, Mimia, Moythomasia, Birgeria, Saurichthys, Lepisosteus and Amia. We review generic diversity within the four nominal families of fossil and recent Acipenseriformes (Chondrosteidae, Peipiaosteidae, Polyodontidae, and Acipenseridae), and provide a cladogram summarizing osteological characters for those four groups. Monophyly of the two extant families is well-supported, but there are no comprehensive studies of all of the known species and specimens of Chondrosteidae and Peipiaosteidae. As a result, sister-group relationships among Chondrosteidae, Peipiaosteidae, and Acipenseroidei (= Polyodontidae + Acipenseridae) are unresolved. We discuss five features fundamental to the biology of acipenseriforms that benefit from the availability of our new phylogenetic hypothesis: (1) specializations of jaws and operculum relevant to jaw protrusion, feeding, and ram ventilation; (2) anadromy or potamodromy and demersal spawning; (3) paedomorphosis and evolution of the group; (4) the bioégeography of Asian and North American polyodontids and scaphirhynchines; and (5) the great abundance of electroreceptive organs in the rostral and opercular regions. Finally, we summarize our nomenclatural recommendations.     

Complete mitochondrial genome sequence and phylogenetic position of the Amu Darya sturgeon,Pseudoscaphirhynchus kaufmanni (Acipenseriformes: Acipenseridae)

In this study, we successfully assembled the complete mitochondrial genome of the Amu Darya sturgeon Pseudoscaphirhynchus kaufmanni. Based on this mitochondrial genome and previously published mitochondrial genomes of members of the Acipenseridae family, we assessed the phylogenetic position of P. kaufmanni using maximum likelihood and Bayesian inference for phylogeny reconstruction. The resultant phylogenetic trees were well-resolved, with congruence between different phylogenetic methods. This robust phylogenetic analysis elucidated the relationship among the four acipenserid genera and strongly supported the division of the family into three main clades. Evaluation of molecular phylogeny using maximum likelihood and Bayesian analysis led to the following conclusions: (a) the most basal position within the Acipenseridae remains in the clade containing Acipenser oxyrinchus and Acipenser sturio; (b) the genus Scaphirhynchus belongs to the Atlantic clade and is a sister group of the remaining species of the clade; and (c) the close relationship between P. kaufmanni and Acipenser stellatus is well supported.     

基于线粒体基因组全序列的鲟形目鱼类(Pisces: Acipenseriformes)的分子系统发育重建

为厘清鲟形目鱼类的系统发育, 研究新测定了中华鲟(Acipenser sinensis)、长江鲟(A. dabryanus)、短吻鲟(A. brevirostrum)、纳氏鲟(A. naccarii)、鳇(Huso dauricus)和匙吻鲟(Polyodon spathula)共6种鲟类的线粒体全基因组序列。联合已测的17种鲟类的线粒体基因组数据, 利用最大似然法和贝叶斯法重建了鲟形目鱼类的分子系统发育关系, 并采用似然值检验对不同的树拓扑结构进行了评价。结果表明, 6种新测鲟类的线粒体基因组大小为16521—16766 bp, 编码13个蛋白质编码基因、22个转运RNA基因和2个核糖体基因, 与大多数已测的鲟类的线粒体基因组结构高度相似。基于23种鲟形目鱼类线粒体基因组数据, 系统发育分析的结果表明: (1)鲟形目的两个科, 匙吻鲟科(Polyodontidae)和鲟科(Acipenseridae)均为单系; (2)鲟科的内部亲缘关系复杂, 鲟属和鳇属的物种均不构成单系群。鲟科鱼类按分子系统发育重建结果可以分为3个类群: 尖吻鲟类(A. sturio - A. oxyrinchus clade)、大西洋鲟类(Atlantic clade)和太平洋鲟类(Pacific clade)。树拓扑结构的检验结果表明, 鲟科的系统发育关系为(尖吻鲟类(太平洋鲟类, 大西洋鲟类))。铲鲟属(Scaphirhynchus)是大西洋鲟类的基部类群。研究也说明线粒体基因组数据在鲟形目鱼类系统与进化研究方面具有重要应用价值。     

Cytoarchitectonics and neuronal organization of the sturgeon telencephalon

Anatomy, cytoarchitecture and neuronal composition of the i everted-type telencephalon of various sturgeon species; ( Acipenser baeri Br., Acipenser stellatus Pall., Acipenser nudiventris Lov., Acipenser ruthenus L., Acipenser gueldenstaedtii Br., Huso huso L.) were studied by classical histological methods and by TV-imaging. Hemisphere cytoarchitecture was significantly variable across species and genus, while neurone composition proved to be rather conservative. Neurones were mainly of the undifferentiated, isodendritic type. The question of homology of structures between everted and inverted vertebrate telencephala is discussed, and our observations lead us to hypothesise that evolution of the unusual Actinopterygian (and acipenserid) telencephalon has been limited and is, in fact, an evolutionary "dead-end".     

Development of mesonephros in prolarvae of acipenserids (acipenseridae)

Development of the excretory system in early ontogenesis of acipenserids—the Russian sturgeon Acipenser gueldenstaedtii, the starred sturgeon A. stellatus, the sterlet A. ruthenus, and the great sturgeon Huso huso—is investigated histologically. In acipenserids, the mesonephros develops after hatching. At the 36th stage of development (after Detlaf et al., 1981), the mesonephros is a consecutively arranged group of spherical rudiments of placodes situated along the Wolffian duct. Morphological differentiation of mesonephros as the first generations of renal corpuscles, vascular glomeruli, and convoluted tubules is completed up to the 45th stage, i.e., to the moment of transition of prolarvae to active feeding.     

Evolution of ancient satellite DNAs in sturgeon genomes

This study characterizes a repetitive DNA family of sequences in sturgeon, the PstI satellite DNA. We have found a high degree of preservation for these sequences, which are present in all 13 species analyzed, including within the genera Acipenser, Huso, and Scaphirhynchus of the family Acipenseridae. This is one of the most ancient satellite DNAs found to date, because it has been estimated to be more than 100 million years old. Alternatively, to the current view that most satellite DNAs are species-specific or preserved in a few closely related species, the PstI family and other previously characterized sturgeon satellite DNA, the HindIII, represent the most fascinating exceptions to the rapid sequence change usually undergone by satellite DNAs. Here, we compare the evolutionary pattern of these two satellite DNA families, PstI and HindIII, which differ markedly in length, sequence, and nucleotide composition. We have found that, in contrast to the situation in most other living beings, a high degree of preservation, a slow sequence change rate and slowed concerted evolution, appears to be a general rule for sturgeon satellite DNAs. The possible causes for all these features are discussed in the light of the evolutionary specifics found within these ancient organisms.     

Molecular phylogeny of Acipenserinae and black caviar species identification.

The first phylogenetic tree for the Acipenserinae based on the combined sequence data for fragments of three mitochondrial genes, the cytochrome b (650 bp) gene, 12S (150 bp) and 16S (350 bp) rRNA genes is described. Three general conclusions are inferred from the tree: 1) Scaphirhynchus albus is the sister-species of all Acipenser and Huso species; 2) The two species of Huso are embedded within the genus Acipenser. Therefore, the genus Huso is not a separate taxonomic unit; and 3) There are three main clades within Acipenser: (a) A. sturio-A. oxyrinchus, (b) A. schrenckii-A. transmontanus, and (c) all Ponto-Caspian species plus A. dabryanus and A. brevirostrum. A method based on the identification of diagnostic nucleotide positions in the cytochrome b gene was developed and used to survey the United States and European caviar markets. The survey of 95 lots of caviar obtained at the American market suggests that in December 1995 and April 1996 17% of the designations made by caviar suppliers were mislabeled with respect to species identification. In December of 1996, this figure jumped to 32%. The main commercially harvested species (Huso huso, A. stellatus, A. gueldenstaedtii, A. schrenckii, and H. dauricus), as well as A. nudiventris, A. persicus, and A. baerii are threatened due to the increased demand of the international caviar market.     

Progressively developed myocardial apoptotic cell death during late phase of reperfusion

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins and infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flow, measured by colored microspheres during ischemia. Necrotic cell death (TTC staining) was significantly increased during R, starting at 27 ± 2% at 6 h R and increasing to 41 ± 2% at 24 h R. There was no further change at 48 (37 ± 3%) and 72 (36 ± 6%) h R, respectively. TUNEL positive cells (% total normal nuclei) in the peri-necrotic zone progressively increased from 6 (26 ± 2^*) to 24 (38 ± 1^*), 48 (48 ± 3^*) and 72 (59 ± 4^*) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compared with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 ± 0.8%^*) to 72 h R (78 ± 2%^*), and increase in expression of pro-apoptotic proteins including Bax from 6 (30 ± 3%^*) to 72 h R (66 ± 3%^*), and p53 from 6 (12 ± 1%^*) to 72 h R (91 ± 2%^*), respectively. Immunohistochemical staining revealed that infiltrated neutrophils (mm² myocardium) were significantly correlated with development of necrotic and apoptotic cell death from 6 to 24 h R, respectively (P < 0.05), while large macrophage infiltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apoptosis; 3) inflammatory cells may play a role in triggering cell death during R. ^* P < 0.05 vs. normal nuclei and tissue; P < 0.01 vs. 6 h R.     

Endangered migratory sturgeons of the lower Danube River and its delta

Historically, five acipenserid species migrated from the Black Sea into the Danube River: beluga Huso huso, Russian sturgeon Acipenser gueldenstaedtii, stellate sturgeon A. stellatus, ship sturgeon A. nudiventris, and perhaps European Atlantic sturgeon A. sturio. The freshwater sterlet A. ruthenus thrived in the Danube and its tributaries. Presently, only three anadromous species occur in the Romanian part of the Danube, Huso huso, A. gueldenstaedtii, and A. stellatus, while A. ruthenus lives in the Danube and its tributaries. Extreme depletion in the number of sturgeons was caused by many, primarily anthropogenic, factors which affected the Danube and the Black Sea shelf during recent last decades. Measures necessary for saving anadromous sturgeon species in the lower Danube are recommended.     

Development of the muscles associated with the mandibular and hyoid arches in the Siberian sturgeon,Acipenser baerii (Acipenseriformes: Acipenseridae)

The skeleton of the jaws and neurocranium of sturgeons (Acipenseridae) are connected only through the hyoid arch. This arrangement allows considerable protrusion and retraction of the jaws and is highly specialized among ray‐finned fishes (Actinopterygii). To better understand the unique morphology and the evolution of the jaw apparatus in Acipenseridae, we investigated the development of the muscles of the mandibular and hyoid arches of the Siberian sturgeon, Acipenser baerii. We used a combination of antibody staining and formalin‐induced fluorescence of tissues imaged with confocal microscopy and subsequent three‐dimensional reconstruction. These data were analyzed to address the identity of previously controversial and newly discovered muscle portions. Our results indicate that the anlagen of the muscles in A. baerii develop similarly to those of other actinopterygians, although they differ by not differentiating into distinct muscles. This is exemplified by the subpartitioning of the m. adductor mandibulae as well as the massive m. protractor hyomandibulae, for which we found a previously undescribed portion in each. The importance of paedomorphosis for the evolution of Acipenseriformes has been discussed before and our results indicate that the muscles of the mandibular and the hyoid may be another example for heterochronic evolution.     

Germination of conidia of poppy powdery mildew (Erysiphe polygoni DC)

Mode of germination and effect of various substrates, temperature, humidities, light and darkness on germination of poppy powdery mildew conidia have been studied.Conidia germinated on all substrates tested. Highest germination (89.41 %) was on agar 2 %. Germination was equally good on dry slide and in water. Generally one, rarely two germ tubes emerged from each conidium from the corner or occasionally from other places. Germination started after two hours of incubation and was almost over in 5 hours. Germination energy was 86.2.Maximum temperature for germination was 32.5 C. Optimum lied near 20 C.Light and darkness did not affect germination.Conidia germinated at all relative humidities tested including 0 and 100 %.Department of Plant Pathology, Agricultural Experiment Station, University of Udaipur, Udaipur, India.Based on a part of Ph.D. thesis of the senior author.     

Studies on the fungus flora of three grains in Egypt

In wheat, corn and sorghum grains twenty-six genera and seventy-seven species including sixteen ofAspergillus and twenty-one ofPenicillium were identified.In grain samples adjusted to various moisture levels (up to 18.8 % on a dry-weight basis) and stored for 4 months at 8, 20 and 30 C seed-borne fungi were monthly identified and counted, and the germinability of the grains was tested. In the control samples (moisture content 7.1–8.2 %) temperature had no effect on the fungal counts and on the precentage germination. With the rise of temperature and moisture content the fungal counts markedly increased and the germinability declined.The list of fungi dominant in one or more of the experiments, included eight species ofAspergillus, six ofPenicillium, two ofFusarium and one each ofAlternaria andCurvularia. The order of dominance of these fungi varied according to the type of grain, the length of storage and the treatment.A. niger was the dominant organism in the control samples of the three grains. It could grow successfully at high moisture contents but above 15 %, it was usually overgrown by other fungi. In wheatP. citrinum andA. sydowii, in cornP. citrinum andA. terreus, and in sorghumA. terreus andA. niger were the dominant fungi at moisture contents above 15 % after four months storage at 30 C. When stored at 8 CPenicillium species tended to predominate over other fungi in grains with high moisture contents.In surface-sterilized grains adjusted to 15 % moisture content, inoculated with six dominant fungi separately and incubated at 30 C,A. niger, P. citrinum andP. variabile caused severe deterioration in the three grains;A. ochraceus in wheat only andF. moniliforme in wheat and corn;C. spicifer was slightly injurious to the three grains.     

Phylogenetic relationships of the North American sturgeons (order Acipenseriformes) based on mitochondrial DNA sequences

The evolutionary relationships of the extant species within the order Acipenseriformes are not well understood. Nucleotide sequences of four mitochondrial genes (12S rRNA, COII, tRNA(Phe), and tRNA(Asp) genes) in North American sturgeon and paddlefish were examined to reconstruct a phylogeny. Analysis of the combined gene sequences suggests a basal placement of the paddlefish with regard to the sturgeons. Nucleotide sequences of all four genes for the three Scaphirhynchus species were identical. The position of Scaphirhynchus based on our data was uncertain. Within the genus Acipenser, the two Acipenser oxyrinchus subspecies were very similar in sequence and found to be basal to the remaining Acipenser species examined. Based on our data, Acipenser transmontanus and Acipenser medirostris were sister taxa, as were Acipenser fulvescens and Acipenser brevirostrum. Comparison of our results with hypotheses of sturgeon relationships proposed by previous authors is presented. The sequence data presented here are phylogenetically useful and provide a solid foundation of genetic information for the North American Acipenseriformes that can be expanded to include Eurasian species to provide a global picture of sturgeon evolution.     

Morphological and functional development of characters associated with settlement in the yellowfin goby, <Emphasis Type="Italic">Acanthogobius flavimanus</Emphasis>

Morphological and functional development of characters from pelagic larval to benthic juvenile periods in the yellowfin goby, Acanthogobius flavimanus, were examined on the basis of 275 specimens (9.4–25.9mm in standard length) collected in shallow water in the Tama River estuary, central Japan. Judging from the development of osteological features, late pelagic juveniles possessed fully developed swimming and feeding abilities. Rapid changes in various characters, including the formation of a loop-shaped gut, increases in condition factor and teeth number, broader upper-field view capability, and extended scaly area and pigmentation development, occurred during the transition from late pelagic to early benthic periods. Most of the changes were associated with structural and functional aspects enabling more efficient use of benthic resources or the avoidance of predators in the benthic habitat.     

The skull of the Chinese sturgeon,Acipenser sinensis (Acipenseridae)

The Chinese sturgeon, Acipenser sinensis, is a large member of Acipenseridae now found only in the Yangtze River and the Yellow and East China seas. The goal of this paper was to describe the skull of A. sinensis in the context of recent anatomical and systematic studies of sturgeons. Five specimens (354–670 mm standard length) were prepared as skeletons. The left and right parietals and frontals are broadly separated by a median fontanelle. The lateral‐most lateral extrascapular variably supports the confluence of the supratemporal, occipital and trunk lateral lines. There is no distinct ventral supraorbital process as found in other sturgeons. The anterodorsal portion of the snout is unique among Acipenseridae by having a single large anamestic dorsal rostral bone instead of a series of separate dorsal rostral bones. There are 0–2 lateral rostral bones on each side positioned anterior to but not in contact with the horizontal arm of the jugal. The dorsal surface of the neurocranium lacks a pineal opening, and its anterior tip is sharply pointed in the smaller specimens examined and gently curved in larger specimens. The anteromedial arm of the palatopterygoid is broad relative to other acipenserids. These new morphological data are discussed and compared among Acipenseridae.     

Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNA ND4L -ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DMA (mtDNA) ND4L and partial A7D4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNA ND4L -ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

Immunoglobulin heavy chain constant region of five Acipenseridae: cDNA sequence and evolutionary relationship

The immunoglobulin M (IgM) heavy chain constant region genes of Russian sturgeon (Acipenser gueldenstaedtii), Sterlte sturgeon (Acipenser ruthenus), Amur sturgeon (Acipenser schrenckii), Chinese sturgeon (Acipenser sinensis) and Great sturgeon (Huso huso) were cloned and analyzed with molecular biology and bioinformatics methods. We cloned IGH nucleic acid sequences by RT-PCR using the specific primer, then determined the characteristics and functions of the amino acid sequences. The gene contains four constant region domain-encoding exons (CH1-4), of which CH4 sub-regions were the most conserved in IgM heavy chain constant region domain and had the highest identity within all the experimental species. According to the analysis of the phylogenetic tree, the variation expectation value (K(aa)), and species differentiation time (T) in the CH4 sub-region, we found that Chinese sturgeon and the other five sturgeon form one whole bifurcation of the tree, while, among the five left, Amur sturgeon and Huso sturgeon, Russian sturgeon and Siberian sturgeon (data from GenBank), Sterlte itself forms three other bifurcations. This result can clearly explain the relations of taxonomic status, geographical distribution and evolution among the species studied.