Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Effects of damming on population sustainability of Chinese sturgeon, Acipenser sinensis: evaluation of optimal conservation measures

The numbers of spawning sites for Chinese sturgeon have been drastically reduced since the construction of the Gezhouba Dam across the Yangtze River. This dam has blocked migration of Chinese sturgeon to their historic spawning ground causing a significant decline of the Chinese sturgeon population. We conducted a VORTEX population viability analysis to estimate the sustainability of the population and to quantify the efficiency of current and alternative conservation procedures. The model predicted the observed decline of Chinese sturgeon, resulting from the effect of the Gezhouba Dam. These simulations demonstrated the potential interest of two conservation measures: increasing spawning area and reducing predation on sturgeon eggs. The simulations also demonstrated that the actual restocking program is not sufficient to sustain sturgeon population as the artificial reproduction program induce the loss of more wild mature adults that the recruitment expected by the artificial reproduction.

Population status, threats and conservation of the Yangtze finless porpoise

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is currently limited to the middle and lower reaches of the Yangtze River from Yichang to Shanghai, China, and the adjoining Poyang and Dongting Lakes. Its population size has decreased remarkably during the last several decades due to the heavy impact of human activities, including overfishing of prey species, water development projects that cause attendant habitat loss and degradation, water pollution, and accidental deaths caused by harmful fishing gear and collisions with motorized vessels. It was estimated that the number of remaining individuals was down to approximately 1800 in 2006, a number that is decreasing at a rate as high as 5% per year. Three conservation measures - in situ and ex situ conservation and captive breeding have been applied to the protection of this unique porpoise since the early 1990s. Seven natural and two "semi-natural" reserves have so far been established. Since 1996, a small group of finless porpoises has been successfully reared in a facility at the Institute of Hydrobiology of the Chinese Academy of Sciences; three babies were born in captivity on July 5, 2005, June 2, 2007 and July 5, 2008. These are the first freshwater cetaceans ever born in captivity in the world. Several groups of these porpoises caught in the main stream of the Yangtze River, or rescued, have been introduced into the Tian'e-Zhou Semi-natural Reserve since 1990. These efforts have proven that, not only can these animals survive in the area, they are also to reproduce naturally and successfully. More than 30 calves had been born in the reserve since then, with one to three born each year. Taking deaths and transfers into account, there were approximately 30 individuals living in the reserve as of the end of 2007. Among eight mature females captured in April 2008, five were confirmed pregnant. This effort represents the first successful attempt at off-site protection of a cetacean species in the world, and establishes a solid base for conservation of the Yangtze finless porpoise. A lesson must be drawn from the tragedy of Chinese River Dolphin (Lipotes vexillifer), which has already been declared likely extinct. Strong, effective and appropriate protective measures must be carried out quickly to prevent the Yangtze finless porpoise from becoming a second Chinese River Dolphin, and save the biodiversity of the Yangtze River as a whole.

Abundance and conservation status of the Yangtze finless porpoise in the Yangtze River, China

middle and lower reaches of the Yangtze River, China. It is the only freshwater population of porpoises in the world and is currently listed as Endangered by IUCN. In November and December 2006 we used two boats and line transect methods to survey the entire current range of the population, except for two lakes (Poyang and Dongting). Sighting results were similar for both boats, so we pooled all data and analyzed them using two line transect models and a strip transect model. All models produced similar estimates of abundance (1111, 1225 and 1000). We then added independent estimates of the number of porpoises from the two lakes for a total estimate of approximately 1800 porpoises. Our findings indicate that the population continues to decline and that its distribution is becoming more fragmented. Our current estimate in the main river is slightly less than half the estimate from surveys between 1984 and 1991 (which was probably an underestimate). We also found an apparent gap in the distribution of porpoises between Yueyang and Shishou (similar to 150 km), where sightings had previously been common. Continued threats to Yangtze finless porpoises include bycatch in unregulated and unselective fishing, habitat degradation through dredging, pollution and noise, vessel strikes and water development. Immediate protective measures are urgently needed to ensure the persistence of finless porpoises in the Yangtze River. The survey design and analytical methods developed in this study might be appropriate for surveys of cetaceans in other river systems. (c) 2008 Elsevier Ltd. All rights reserved.

Population differentiation of Procypris rabaudi (Tchang) in the upper reaches of the Yangtze River revealed by mitochondrial DNA and RAPD markers

Procypris rabaudi (Tchang) is a cyprinid fish endermic to middle and upper reaches of the Yangtze River. Besides in main stream and large tributaries, there exists an early matured, small-sized ecological type in a small tributary, Tang River. In this study, mitochondrial DNA cytochrome h (cyt b) gene sequence analysis and randomly amplified polymorphic DNA (RAPD) analysis were performed to investigate the differentiation of the Tang River population from the Mudong reach population of the Yangtze River, with the purpose of conservation and exploitation of this fish. In the 1140 bps of cyt b gene sequence surveyed, 20 sites were found polymorphic, which defined 23 haplotypes. Among them, four haplotypes accounted for 54.4% of all individuals, while population-specific haplotypes occurred in low frequencies. Analysis of molecular variation on cyt b data revealed no significant partition existing between Tang River population and Mudong reach population. Analyses of 132 RAPD loci suggested that genetic variation between populations was significant, though values of different F-ST were not very high. The results revealed low genetic diversity and the beginning of population differentiation, suggesting that Tang River population should be designated as a separate Management Unit.

Molecular phylogeography of endangered sharp-snouted pitviper (Deinagkistrodon acutus; Reptilia, Viperidae) in Mainland China

Using phylogenetic and population genetic approaches, the present study reports the phylogeographic structure of the sharp-snouted pitviper (Deinagkistrodon acutus), a threatened snake species with commercial and medicinal importance in China. The entire mitochondrial ND2 gene (NADH dehydrogenase subunit 2) sequences of 86 individuals of D. acutus from 14 localities across its range in China were determined. Based on the results of phylogenetic analyses, distribution of diagnostic sites, haplotype network, and AMOVA hierarchical analysis, an cast-west division of the whole D. acutus population could be observed. Geographically, a line formed by a lake, river, and mountain chain (the Poyang Lake, Gan River to the southern end of the Wuyi Mountains), results in vicariance and approximately vertically splits the range into two and the whole population into two main lineages (western and eastern). The bifurcating tree suggested generally west to east dispersal trend. The data fit the isolation by distance (IBD) model well. Star-like clusters in haplotype network, significantly negative values of Fs statistics, and unimodal mismatch distributions all suggest recent demographic expansions in four areas. The results show that isolation, dispersal, bottleneck, and expansion jointly constitute the history of D. acutus. In a haplotype network, the excessive predominance of central haplotypes, few medium-frequency haplotypes, predominance (73.1 %) of the singletons among the derived haplotypes, most of which are connected to the central haplotype by only one mutational step, unsymmetrical campanulate unimodal curve of mismatch distributions and leftwards shift of the peaks, all suggest that the whole D. acutus population is a young population with low genetic diversity. Based on the data, the first priority for conservation action should be given to the Huangshan unit. (c) 2007 Elsevier Inc. All rights reserved.

Microsatellite diversity and population genetic structure of redfin culter (Culter erythropterus) in fragmented lakes of the Yangtze River

Redfin culter (Culter erythropterus) is a small lethic freshwater fish and widely distributed in the adjacent lakes of the Yangtze River of China. Five microsatellite loci were applied to investigate the genetic variation and population structure of redfin culter from seven lakes in the middle-and-lower reaches of the Yangtze River. The gene diversity was high among the populations (H > 0.9), the average number of alleles among seven populations was low with a range from 2.00 to 3.87. The mean observed (H-O) and expected (H-E) heterozygosity ranged from 0.111 to 0.419 and from 0.162 to 0.750, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found in 50% of the total locus-population combination tests in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percentage of variance among and within these populations were 6.18 and 93.82, respectively. The Fst values (0.062, P < 0.001) among studied populations indicated that there were significant genetic differentiations among redfin culture populations from the scattered lakes with different connections to the Yangtze River. These results are useful for the evaluation and conservation of small freshwater fishes. The factors that may be involved in low intra-population polymorphism and the pattern of the population genetic structure of redfin culter from the Yangtze River were discussed.

Genetic structure and low-genetic diversity suggesting the necessity for conservation of the Chinese Longsnout catfish, Leiocassis longirostris (Pisces : Bagriidae)

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Ex situ conservation status of an endangered Yangtze finless porpoise population (Neophocaena phocaenoides asiaeorientalis) as measured from microsatellites and mtDNA diversity

The Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis, is all endangered small cetacean that occurs only in the middle and lower reaches of the Yangtze River of China. The establishment of a breeding population of the porpoise in Tian-e-Zhou Baiji National Natural Reserve represents the first attempt at ex situ conservation efforts for a cetacean species. With the goal of effective protection, management, and monitoring of this preserved population, we examined its genetic diversity using 930 bp of mtDNA control region sequences and 13 polymorphic microsatellite loci. A very low level of genetic variation (h = 0.6010 +/- 0.0029 s.d.; pi = 0.0007 +/- 0.0000002 s.d.) in the mtDNA control region sequences and a moderate genetic diversity (Ho = 0.5740 +/- 0.2575 s.d.) in the microsatellites were detected in the population. It is necessary to introduce more individuals with representative genetic variations into the reserve ill order to foml a larger and healthier group structure for long-term survival of the population. Successful establishment of the Yangtze finless porpoise population in the Reserve also provides a useful model for an ex situ conservation programme for other rare and endangered species. (c) 2005 International Council for the Exploration ofthe Sea. Published by Elsevier Ltd. All rights reserved.

Molecular phylogenetics of Gymnocypris (Teleostei : Cyprinidae) in Lake Qinghai and adjacent drainages

149 complete mitochondrial DNA (mtDNA) cytochrome b (Cyt b) genes (1140 bp) of Gymnocypris przewalskii, Gymnocypris eckloni and Gymnocyptis scolistomus from the Lake Qinghai, Yellow River and Qaidam Basin were sequenced and analyzed. Consistent dendrogram indicated that the samples collected from the same species do not constitute a separate monophyletic group and all the samples were grouped into three highly divergent lineages (A, B and C). Among them, Lineage A contained all samples of G. przewalskii from the Lake Qinghai and partial samples of the G. eckloni from the Yellow River. Lineage B contained the remaining samples of G. eckloni from the Yellow River. Lineage C was composed of a monophyletic group by G. eckloni from the Qaidam Basin. Analysis of molecular variance (AMOVA) indicated that most of genetic variations were detected within these three mtDNA lineages (93.12%), suggesting that there are three different lineages of Gymnocypris in this region. Our Cyt b sequence data showed that G. przewalskii was not a polytypic species, and G. scolistomus was neither an independent species nor a subspecies of G. eckloni. The divergent mtDNA lineages of G. eckloni from the Yellow River suggested that gene flow between the different populations was restricted to a certain extent by several gorges on the upper reach of the Yellow River. Lineage B of G. eckloni might be the genetic effect from the ancestor which was incorporated with the endemic schizothoracine fishes when the headward erosion of the Yellow River reached to its current headwaters of late. The G. eckloni from Basin Qaidam was a monophyletic group (lineage C) and F-st values within G. eckloni from the Yellow River were higher than 0.98, suggesting that the gene flow has been interrupted for a long time and the G. eckloni from Basin Qaidam might have been evolved into different species by ecology segregation. The correlation between the rakers number of Gymnocypris and population genetic variation was not significant. All Gymnocypris populations exhibited a low nucleotide diversity (pi = 0.00096-0.00485). Therefore the Gymnocyptis populations from Basin Qaidam could have experienced severe bottleneck effect in history. Our result suggested Gym-nocypris populations of Basin Qaidam should give a high priority in conservation programs.

Aquatic resource conservation - The first Yangtze finless porpoise successfully born in captivity

Background. As the sole freshwater subspecies of finless porpoise (Neophocaena phocaenoides), the Yangtze finless porpoise (N. p. asiaeorientalis) lives only in the middle and lower reaches of the Yangtze River and its appended Poyang and Dongting Lakes. As a result of human activity on the river, including over and illegal fishing, pollution, transportation and dam construction, the population of Yangtze finless porpoises has been steadily and rapidly decreasing during the past several decades, which leads the animal to be endangered. Methods. For saving this unique animal from extinction, three corresponding measures, in situ conservation, ex situ conservation, and intensifying breeding research in captivity, were proposed and have been implemented since the 1980s. Results. After successfully rearing the animals in captivity for almost nine years, the first Yangtze finless porpoise was successfully born in captivity on July 5, 2005. The calf is male, with a body length of 69 cm. This is the first freshwater cetacean ever born in captivity. Conclusion. The successful birth of this calf confirms that it is possible to breed the Yangtze finless porpoise in captivity. Furthermore, this will greatly benefit the conservation efforts, and also greatly bolster our on-going efforts to study the reproductive biology of these animals. Recommendation. More studies and efforts are expected to establish a sustainable, captive colony of the Yangtze finless porpoise, which will not only greatly expand our knowledge about the reproduction biology of this animal, but also help to redeem the wild population through a careful yearly 'soft releasing' process.

Phylogenetic relationships among Trichodinidae (Ciliophora : Peritricha) derived from the characteristic values of denticles

The phylogenetic relationships among trichodinids remain obscure. As an important diagnostic marker, the morphology of the denticles in the adhesive disc as well as the adoral spiral has been widely used in generic discrimination and species identification of trichodinids. We studied the characters of denticles of the ten genera of Trichodinidae and the sole genus Urceolaria of Urceolariidae by using a quantitative method. The characteristic values were used to generate Manhattan distance, on which the dendrogram was based to construct with the Unweighted Paired Group Method using the Arithmetic mean (UPGMA). The investigations show that all the genera of the family Trichodinidae were clearly separate from the outgroup Urceolaria, and within the Trichodinidae: (i) Dipartiella grouped with Trichodinella and Tripartiella and lay in the closest position to the outgroup with a low dissimilarity, suggesting Dipartiella might be the most primitive genus in the family; (ii) Hemitrichodina clustered in a single clad and lay in the farthest position to the outgroup with the highest dissimilarity, indicating that it might be the most advanced genus; and (iii) the other 6 genera, Trichodina, Paratrichodina, Semitrichodina, Vauchomia, Pallitrichodina and Trichodoxa clustered in a big clad with very low dissimilarity, showing that they are closely related to each other. We discuss the evolutionary trend of the denticle and conclude that the denticles of the adhesive disc should be an apomorphic feature of the trichodinids and their changes could reflect the evolutionary tendencies of these ciliates.

Population genetic structure of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis): Implications for management and conservation

Understanding the population genetic structure is a prerequisite for conservation of a species. The degree of genetic variability characteristic of the mitochondrial DNA control region has been widely exploited in studies of population genetic structure and can be useful in identifying meaningful population subdivisions. To estimate the genetic profile of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), an endangered freshwater population endemic to China, the complete mtDNA control region was examined in 39 individuals belonging to seven different stocks inhabiting the middle and lower reaches of the Yangtze River. Very low genetic diversity was found (nucleotide diversity 0.0011 +/- 0.0002 and haplotypic diversity 0.65 +/- 0.05). The mtDNA genetic pattern of the Yangtze population appears to indicate a founder event in its evolutionary history and to support the marine origin for this population. Analyses by F-st and Phi(st) yielded statistically significant population genetic structure (F-st = 0.44, P < 0.05; phi(st) = 0.36, P < 0.05). These results may have significant implications for the management and conservation of the Yangtze finless porpoise in the future.