南极鱼类多样性和适应性进化研究进展

南极地区是地球上唯一未被人类活动大量影响的地区, 其极端寒冷的环境为南极生物的进化提供了“温床”。过去三千万年间, 南极鱼亚目鱼类在南极海洋逐渐变冷的过程中快速进化, 从一个温暖海域的底栖祖先分化成南极海域最为多样化的鱼类类群。由于其在南极圈内和南极圈外的各种温度区间都有分布, 因而成为研究鱼类适应性进化和耐寒机制的良好生物模型。本文综述了有关南极海域鱼类区系组成与物种多样性现状, 南极鱼亚目鱼类适应低温的一系列特化的生物学性状及其关键的遗传进化机制。现有研究表明: 南极鱼类在几千万年零度以下低温环境的进化中发生了大量基因的大规模扩增和基因表达的改变, 如铁调素、卵壳蛋白和逆转座子等118个基因发生了显著的扩增。另外, 有些从南极鱼中获得的抗寒基因已经用于提高动植物低温抗性的研究并取得了良好的效果。在今后的几年中, 将会有多个南极鱼物种的全基因组得到破译, 在低温适应相关基因的功能和进化方面的研究也会更加深入, 这些研究将深入揭示低温压力下基因组的进化规律以及鱼类低温适应的分子机制。     

Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus, 1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of +1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a β-hairpin-like structure with two antiparallel β-sheets. This is the first report of an AMP from the coral fish Z. cornutus.     

Positive Darwinian selection operating on the immunoglobulin heavy chain of Antarctic fishes

The cooling of the Southern Ocean to the freezing point of seawater (-1.9 degrees C) over the past 25 million years played a dominant selective role in the evolution of the Antarctic fish fauna. During this period, the perciform suborder Notothenioidei, which is largely endemic to the Antarctic, diversified and developed numerous cold-adapted characters. In this report, we provide compelling evidence that the immunoglobulin heavy chain (IgH) of the notothenioid fishes has undergone adaptive selection. Two and four IgH clones were isolated, respectively, from spleen cDNA libraries prepared from the Antarctic icefish Chaenocephalus aceratus and the yellowbelly rockcod Notothenia coriiceps. The transmembrane region of the membrane form of the rockcod IgM heavy chain was located at the end of the second constant (C(H)) domain, in contrast to other teleost IgMs in which the transmembrane region is located at the end of the third constant domain. Phylogenetic analyses of C(H) regions revealed that rates of nonsynonymous nucleotide substitution were higher than rates of synonymous nucleotide substitution. Many of the nonsynonymous substitutions introduced charge changes, consistent with positive Darwinian selection acting to adapt the structure of the notothenioid immunoglobulins. The rates of nonsynonymous nucleotide substitutions were higher than the rates of synonymous nucleotide substitutions in complementarity determining regions of variable regions, suggesting that diversity at antigen binding sites is enhanced by genomic and/or somatic selection. Results of Southern blot hybridization experiments were consistent with a translocon type of IgH gene organization reminiscent of bony fishes and tetrapods.     

Phylogenetics of notothenioid fishes (Teleostei: Acanthomorpha): inferences from mitochondrial and nuclear gene sequences

Notothenioids represent an adaptive radiation of teleost fishes in the frigid and ice-laden waters of the Southern Ocean surrounding Antarctica. Phylogenetic hypotheses for this clade have resulted primarily from analyses of mtDNA gene sequences, and studies utilizing nuclear gene DNA sequence data have focused on particular sub-clades of notothenioid fishes. In this study, we provide the first phylogenetic analysis of notothenioids using both mtDNA and nuclear gene sequences for a comprehensive sampling of all major lineages in the clade. Maximum parsimony and Bayesian analyses of aligned mtDNA genes, an aligned nuclear gene (S7 ribosomal protein intron 1), and combined dataset containing the mtDNA and nuclear genes resulted in phylogenies that contained the previously identified Antarctic and High Antarctic Clades. There were areas of agreement and disagreement between different datasets and methods of phylogenetic analysis, and the phylogenies resulting from the nuclear encoded S7 ribosomal protein intron 1 sequences were considerably less resolved than those inferred from mtDNA gene sequences. However, we anticipate increased resolution of the notothenioid phylogeny from future analyses that sample DNA sequences from several nuclear genes.     

The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity

Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.     

Sex determination in Antarctic notothenioid fish: chromosomal clues and evolutionary hypotheses

In fish, the determination of sex can be controlled by genetic factors, environmental factors or a combination of both. The presence of heteromorphic sex-related chromosomes is widely acknowledged as strongly indicative of genetic control of sex determination (GSD) acting over other sex control systems. Heteromorphic sex-related chromosomes have been observed in a minority of teleosts (approximately 4 %). However, when looking at the fishes of the suborder Notothenioidei the frequency of sex-related chromosomes increases substantially, reaching 26.67 % of the cytogenetically studied species. Noteworthy, sex chromosomes were observed only in cold-adapted species which live in the Antarctic coastal waters, whereas morphologically differentiated sex chromosomes were never observed in the temperate non-Antarctic notothenioid families. Recent evidence suggests that the sex-linked chromosomes across the Antarctic notothenioid families may not share a common origin, but likely originated more than once during notothenioid evolutionary history, thus implying the presence of selection pressures operating toward fixation of GSD system. On the whole, the cytogenetic evidences suggest the Antarctic-specific fixation of differentiated heteromorphic sex-related chromosomes and of a prominent GSD across Antarctic notothenioids that may be an additional manifestation of notothenioid evolution in thermally stable cold environment.     

On the origin and trigger of the notothenioid adaptive radiation

Adaptive radiation is usually triggered by ecological opportunity, arising through (i) the colonization of a new habitat by its progenitor; (ii) the extinction of competitors; or (iii) the emergence of an evolutionary key innovation in the ancestral lineage. Support for the key innovation hypothesis is scarce, however, even in textbook examples of adaptive radiation. Antifreeze glycoproteins (AFGPs) have been proposed as putative key innovation for the adaptive radiation of notothenioid fishes in the ice-cold waters of Antarctica. A crucial prerequisite for this assumption is the concurrence of the notothenioid radiation with the onset of Antarctic sea ice conditions. Here, we use a fossil-calibrated multi-marker phylogeny of nothothenioid and related acanthomorph fishes to date AFGP emergence and the notothenioid radiation. All time-constraints are cross-validated to assess their reliability resulting in six powerful calibration points. We find that the notothenioid radiation began near the Oligocene-Miocene transition, which coincides with the increasing presence of Antarctic sea ice. Divergence dates of notothenioids are thus consistent with the key innovation hypothesis of AFGP. Early notothenioid divergences are furthermore congruent with vicariant speciation and the breakup of Gondwana.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.     

Diversity and disparity through time in the adaptive radiation of Antarctic notothenioid fishes

According to theory, adaptive radiation is triggered by ecological opportunity that can arise through the colonization of new habitats, the extinction of antagonists or the origin of key innovations. In the course of an adaptive radiation, diversification and morphological evolution are expected to slow down after an initial phase of rapid adaptation to vacant ecological niches, followed by speciation. Such ‘early bursts’ of diversification are thought to occur because niche space becomes increasingly filled over time. The diversification of Antarctic notothenioid fishes into over 120 species has become one of the prime examples of adaptive radiation in the marine realm and has likely been triggered by an evolutionary key innovation in the form of the emergence of antifreeze glycoproteins. Here, we test, using a novel time‐calibrated phylogeny of 49 species and five traits that characterize notothenioid body size and shape as well as buoyancy adaptations and habitat preferences, whether the notothenioid adaptive radiation is compatible with an early burst scenario. Extensive Bayesian model comparison shows that phylogenetic age estimates are highly dependent on model choice and that models with unlinked gene trees are generally better supported and result in younger age estimates. We find strong evidence for elevated diversification rates in Antarctic notothenioids compared to outgroups, yet no sign of rate heterogeneity in the course of the radiation, except that the notothenioid family Artedidraconidae appears to show secondarily elevated diversification rates. We further observe an early burst in trophic morphology, suggesting that the notothenioid radiation proceeds in stages similar to other prominent examples of adaptive radiation.     

Hemoglobin from the antarctic fish Notothenia coriiceps neglecta. Amino acid sequence of the beta chain

1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

Disaptation and recovery in the evolution of Antarctic fishes

The radiation of notothenioid fishes provides an excellent system to explore issues of evolution and adaptation. Most studies emphasize adaptation to the extreme Antarctic environment; however, recent work provides cogent examples of disaptation or evolutionary loss of function. The nature and extent of regressive change is revealed by subsequent adaptive recovery. Ancestral notothenioids were benthic but some became secondarily pelagic through the retention of larval characters. Paedomorphosis has produced detrimental changes in lateral-line sensory systems that have been made good by compensatory adaptation. In the icefish family, compensatory adaptation has followed the loss of the oxygen-binding pigments haemoglobin and myoglobin.     

Ecomorphological disparity in an adaptive radiation: opercular bone shape and stable isotopes in Antarctic icefishes

To assess how ecological and morphological disparity is interrelated in the adaptive radiation of Antarctic notothenioid fish we used patterns of opercle bone evolution as a model to quantify shape disparity, phylogenetic patterns of shape evolution, and ecological correlates in the form of stable isotope values. Using a sample of 25 species including representatives from four major notothenioid clades, we show that opercle shape disparity is higher in the modern fauna than would be expected under the neutral evolution Brownian motion model. Phylogenetic comparative methods indicate that opercle shape data best fit a model of directional selection (Ornstein–Uhlenbeck) and are least supported by the “early burst” model of adaptive radiation. The main evolutionary axis of opercle shape change reflects movement from a broad and more symmetrically tapered opercle to one that narrows along the distal margin, but with only slight shape change on the proximal margin. We find a trend in opercle shape change along the benthic–pelagic axis, underlining the importance of this axis for diversification in the notothenioid radiation. A major impetus for the study of adaptive radiations is to uncover generalized patterns among different groups, and the evolutionary patterns in opercle shape among notothenioids are similar to those found among other adaptive radiations (three‐spined sticklebacks) promoting the utility of this approach for assessing ecomorphological interactions on a broad scale.     

Antifreeze glycoprotein levels in Antarctic notothenioid fishes inhabiting different thermal environments and the effect of warm acclimation

A quantification method was developed to determine the concentrations of the major antifreeze glycoprotein (AFGP) isoforms in the blood of Antarctic notothenioid fishes. Serum samples were precipitated with 2.5% TCA and the supernatant containing AFGPs were chromatographed on an HPLC size exclusion column and the concentrations of the major AFGP size classes were determined from the areas of the corresponding peaks in the elution profile. Eight species of Antarctic notothenioid fishes were examined and their blood AFGP concentrations varied from 5 to 35 mg/mL. All of these fishes synthesized both the large and small AFGPs, but maintained higher levels of small AFGPs than the large ones in their blood. The species inhabiting more severe water environments (lower temperature and presence of ice) had higher serum AFGP levels than those in milder environments. The cryopelagic Pagothenia borchgrevinki decreased their blood AFGP concentrations in response to warm acclimation, but to a much lower extent in comparison to the antifreeze-bearing fishes in the Northern Hemisphere. After being warm acclimated at +4 degrees C for 16 weeks, the serum concentrations of the small and large AFGPs were decreased by about 60% and 20%, respectively.