MOLECULAR SYSTEMATICS OF RIVER DOLPHINS INFERRED FROM COMPLETE MITOCHONDRIAL CYTOCHROME-B GENE SEQUENCES

1,140 bp of the complete mitochondrial cytochrome- b gene sequences of baiji ( Lipotes vexillifer ), franciscana ( Pontoporia blainvillei ), and Ganges river dolphin ( Platanista gangetica gangetica ) were determined to address the systematic position and phylogeny of extant river dolphins with combination of homologous sequences of other cetaceans. The neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) phylogenetic analyses all identified the river dolphins into three lineages, i. e., Platanista, Lipotes , and Inia + Pontoporia . The Lipotes did not have sister relationship with either Platanista or Inia + Pontoporia , which strongly supported the referral of Lipotes to a separate family, i. e. , Lipotidae. There were very high sequence divergences between all river dolphin genera, suggesting a relatively longer period of separation time than those among other odontocete families.     

淡水豚类分子系统发生的研究

测定了白暨豚和恒河豚细胞色素ｂ基因３０７ｂｐ的ＤＮＡ序列,并与其它鲸类的相应序列合并,分析了淡水豚类（恒河豚、印河豚、白暨豚、亚河豚和弗西豚）的系统学位置和系统发生。淡水豚类不同属间的序列差异已达到或超过了其它齿鲸类科间的差异水平,因此它们分别归属于不同的科,即恒河豚科、白暨豚科、亚河豚科和弗西豚科。系统发生分析支持淡水豚类和海豚类之间具有下述的关系：（恒河豚科（（白暨豚科（亚河豚科,弗西豚科））海豚总科））。即淡水豚的４个科中,恒河豚科是最早分化的一支,其次是白暨豚科,然后是亚河豚科和弗西豚科的分化。白暨豚科和亚河豚科＋弗西豚科组成海豚总科的姊妹群,并与海豚总科一同组成海豚下目。恒河豚与其它淡水豚类间无直接的亲缘关系。淡水豚类是并系的。把恒河豚类独立为恒河豚总科是合理的。初步认为有理由把白暨豚类也作为一个总科级的支系。恒河豚和印河豚间的序列差异极小,两者可能只是同一个种的２个亚种。     

近年来我国关于白鱀豚Lipotes vexillifer的研究

白鱀豚是我国特有的珍贵水生哺乳动物,也是世界现存的4种淡水豚(拉河豚Pontoporia blainvillei,亚河豚Inia geoffrensis,恒河豚Platanista gangetica和白鱀豚)中存活头数最少的一种。近几年来,白鱀豚已为很多人所熟知,并像大熊猫一样,被视为我国的“国宝”。 对白鱀豚进行系统的研究工作,开始在中华人民共和国成立之后。特别是七十年代以来,我国的科学工作者对其进行了大量的调查研究。     

DISTRIBUTION AND ABUNDANCE OF THE AMAZON RIVER DOLPHIN (INIA GEOFFRENSIS) AND THE TUCUXI (SOTALIA FLUVIATILIS) IN THE UPPER AMAZON RIVER

A boat survey was conducted from 5 to 26 June 1993 to estimate the abundance of the Amazon river dolphin ( Inia geoffrensis ) and the tucuxi ( Sotalia fluviatilis ) along ca . 120 km of the Amazon River bordering Colombia, Peru, and Brazil. Two survey methods were used: line transects during 5 d and strip transects during 15 d. The line transects were used to estimate the abundance of both species in the main channels of the Amazon at distances greater than 200 m from river banks and islands, and strip transects were used to estimate abundance in the remainder of the habitat. A total of 29 sightings was obtained using line transects, including 8 of Inia , 15 of Sotalia , and 6 with both species present. The total number of sightings made while using strip transects was 143, including 78 of Inia , 51 of Sotalia , and 14 with both species present. The distributions of sightings with respect to distance from the nearest bank were not significantly different between the two species. Based on the results from the two methods, we estimate that there are 346 (CV = 0.12) Inia and 409 (CV = 0.13) Sotalia in the study area. Overall, the mean group size for Inia was 2.9 individuals and for Sotalia was 3.9 individuals. Inia density (dolphin/km²) was highest in tributaries (4.8), followed by areas around islands (2.7) and along main banks (2.0); while Sotalia density was highest in lakes (8.6), followed by areas along main banks (2.8) and around islands (2.0). These are among the highest densities measured to date for any cetacean.     

Molecular identification of evolutionarily significant units in the Amazon River dolphin Inia sp. (Cetacea: Iniidae)

The Amazon river dolphin, genus Inia, is endemic to the major river basins of northern South America. No previous studies have focused on the genetic structure of this genus. In this work, 96 DNA samples from specimens of this genus were collected in the Orinoco basin (four rivers), the Putumayo River, a tributary of the Colombian Amazon and the Mamoré, and the Tijamuchí and Ipurupuru rivers in theBolivian Amazon. These samples were used to amplify a fragment of 400 bp of the mitochondrial DNA (mtDNA) control region. In addition, 38 of these samples were also used to sequence 600 bp of the mitochondrial cytochrome b gene. The analysis of the population structure subdivision with an analysis of molecular variance (AMOVA) revealed important aspects about the genetic structure of Inia groups fromthese three geographically separate regions. By comparing the control region DNA and cytochrome b sequences, distinct types of nonshared haplotypes were observed. The net genetic divergence of control region sequences was 6.53% between the Orinoco and Bolivian rivers, 5.32% between the Putumayo and Bolivian rivers, and 2.50% between the Orinoco and Putumayo rivers. For the cytochrome b gene, these values were 2.48%, 2.98%, and 0.06%, respectively. The nucleotide sequences were analyzed phylogenetically using several genetic distance matrices and applying neighbor-joining, maximum likelihood, and maximum parsimony procedures. The results support the proposal to subdivide the Inia genus into at least two evolutionarily significant units: one confined to the Bolivian river basin and the other widely distributed across the Amazon and Orinoco basins.     

淡水豚类线粒体DNA 12S rRNA基因的序列变异及其分子系统学研究

淡水豚类４个代表属「白暨豚（Ｌｉｐｏｔｅｓ）、恒河豚（Ｐｌａｔａｎｉｓｔａ）、弗西豚（Ｐｏｎｔｏｐｏｒｉａ）和亚河豚（Ｉｎｉａ）」ｍｔＤＮＡ １２Ｓ ｒＲＮＡ基因的序列差异水平,高于其他齿鲸类科间的差异,特别是远远高于海豚总科内的科间差异。研究结果支持它们应归属于不同的科,即白暨豚科（Ｌｉｐｏｔｉｉｄａｅ）、恒河豚科（Ｐｌａｔａｎｉｓｔｉｄａｅ）、弗西豚科（Ｐｏｎｔｏｐｏｒｉｄａｅ）和亚河豚科（Ｉ     

Purification and characterization of the major whey proteins from the milks of the bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris), and the beagle (Canis familiaris)

The major whey proteins of the milks of the dolphin, manatee, and beagle were purified by gel filtration and ion exchange chromatography and characterized and identified by molecular weight determination, amino acid analysis, N-terminal sequencing, and activity measurements. The major whey protein components from all three species were found to be monomeric beta-lactoglobulins. These proteins were all active in binding retinol. Dolphin milk contained two beta-lactoglobulins (designated 1 and 2) which showed a slight difference in molecular weight and considerably divergent N-terminal sequences, whereas the other milks only contained a single form of beta-lactoglobulin. alpha-Lactalbumins were purified from dolphin and dog milks and were active in promoting lactose synthesis by bovine galactosyltransferase. The dolphin protein had an N-terminal sequence more similar to ruminant alpha-lactalbumins than to those known from other species. Although alpha-lactalbumin activity has been detected in manatee milk at low levels, the corresponding protein was not isolated. In addition, dog milk was found to contain high levels of lysozyme (greater than 1.0 mg/ml), which were identified by activity and sequencing. The functional and evolutionary implications of these results are discussed.     

Evolution of river dolphins

The world's river dolphins (Inia, Pontoporia, Lipotes and Platanista) are among the least known and most endangered of all cetaceans. The four extant genera inhabit geographically disjunct river systems and exhibit highly modified morphologies, leading many cetologists to regard river dolphins as an unnatural group. Numerous arrangements have been proposed for their phylogenetic relationships to one another and to other odontocete cetaceans. These alternative views strongly affect the biogeographical and evolutionary implications raised by the important, although limited, fossil record of river dolphins. We present a hypothesis of river dolphin relationships based on phylogenetic analysis of three mitochondrial genes for 29 cetacean species, concluding that the four genera represent three separate, ancient branches in odontocete evolution. Our molecular phylogeny corresponds well with the first fossil appearances of the primary lineages of modern odontocetes. Integrating relevant events in Tertiary palaeoceanography, we develop a scenario for river dolphin evolution during the globally high sea levels of the Middle Miocene. We suggest that ancestors of the four extant river dolphin lineages colonized the shallow epicontintental seas that inundated the Amazon, Paraná, Yangtze and Indo-Gangetic river basins, subsequently remaining in these extensive waterways during their transition to freshwater with the Late Neogene trend of sea-level lowering.     

Isolation and characterization of twelve polymorphic microsatellite markers in bottlenose dolphins (Tursiops truncatus)

We developed polymerase chain reaction primers for 12 dinucleotide microsatellite loci in the bottlenose dolphin, Tursiops truncatus. Seven markers were obtained after hybridization screening, and five following random genome sequencing. Orthologous positions were computed for nine markers on the bovine genome and for seven on the human genome. The markers are distributed across chromosomes and found in different types of DNA regions. All 12 loci are polymorphic for Tursiops. Five loci were also polymorphic in the related species Stenella frontalis and the more distantly related river dolphin, Inia geoffrensis, indicating these markers will be informative across the Delphinidae and other cetacean taxa.     

Population,density estimates,and conservation of river dolphins (Inia and Sotalia) in the Amazon and Orinoco river basins

This study is part of an on‐going effort to evaluate and monitor river dolphin populations in South America. It comprises the largest initiative to estimate population size and densities of Inia and Sotalia dolphins using statistically robust and standardized methods. From May 2006 to August 2007, seven visual surveys were conducted in selected large rivers of Bolivia, Colombia, Brazil, Ecuador, Peru, and Venezuela in the Amazon and Orinoco river basins. Population sizes of Inia and Sotalia were estimated for different habitats (main river, tributary, lake, island, confluence, and channel). A total of 291 line and 890 strip transects were conducted, covering a distance of 2,704 linear kilometers. We observed 778 Inia geoffrensis, 1,323 Inia boliviensis, and 764 Sotalia fluviatilis. High‐density areas were identified (within 200 m from the river banks, confluences, and lakes) and we propose that these constitute critical habitat for river dolphins. High densities of river dolphins seem to coincide with well‐managed freshwater protected areas and should be considered as hot spots for river dolphins in South America.     

Microsatellite loci for population and parentage analysis in the Amazon River dolphin (Inia geoffrensis de Blainville, 1817)

We developed specific primers for microsatellite DNA regions for the Amazon River dolphin or boto Inia geoffrensis, for use in population and conservation genetic studies. We also tested their transferability for two other species, Pontoporia blainvillei (sister taxon of I. geoffrensis) and Sotalia guianensis. A total of 12 microsatellite loci were polymorphic for the boto. An additional 25 microsatellite loci previously isolated from other cetacean species were also tested in the boto. The 26 polymorphic microsatellite loci indicate they will be excellent markers for studies of population structure and kinship relations of the boto.     

中华白海豚3 个MHC 基因座位遗传变异的初步分析

主要组织相容性复合体(Major histocompatibility complex,MHC) 基因是由一组紧密连锁的基因组成,是哺乳动物免疫系统中最重要的组成部分。本文选择3 个MHC 基因座位的第二外元,即:MHC-I 类基因和II 类基因的DRA 和DQB 座位,初步调查濒危物种中华白海豚的遗传变异。共鉴定了2 个DRA、2 个DQB 和7 MHC-I等位基因。DRA 座位遗传变异非常低,而DQB 和MHC-I 座位具有相对较高水平的遗传变异。并且,在DQB 和MHC-I 基因座位的假定的抗原结合位点(Antigen binding sites,ABS),非同义替代明显大于同义替代,提示平衡选择(Balancing selection)维持这两个座位的多态性,而在DRA 座位上,并没有检测到平衡选择。系统发生分析表明中华白海豚的MHC 等位基因没有聚在一起,而是和其他的物种聚在一起,符合MHC 跨种进化(Transspecies evolution)的模式。     

Ultrastructure of enamel and dentine in extant dolphins (Cetacea: Delphinoidea and Inioidea)

Longitudinal and cross sections of teeth from 17 species of the Recent dolphins (Delphinoidea and Inioidea) were examined under scanning electron microscope to study the arrangement and ultrastructure of dental tissues with reference to phylogenetic and functional constraints. For most species, enamel had a simple bi-layered structure of radial enamel and an outer layer of prismless enamel. The outer prismless layer varied from 5 to 30 % of enamel thickness. The enamel of Burmeister’s porpoise (Phocoena spinipinnis) was entirely prismless. The prisms had an open sheath; tubules and tuft-like structures were common at the enamel-dentine junction. Cetacean dentine was characterized by irregularly distributed dentinal tubules in a relatively homogenous dentinal matrix. Radial enamel was observed in all Delphinoidea and in the franciscana (Pontoporia blainvillei), whereas the Amazon river dolphin (Inia geoffrensis) had prisms organized in Hunter–Schreger bands. HSB in enamel are regarded as a device for resisting propagation of cracks. These may occur due to increased functional demands, possibly related to the hardness of the species diet. Simplification in tooth shape and reduced biomechanical demands plausibly explain the primitive radial organization among delphinoids and Pontoporia. The HSB structure in the Amazon river dolphin, similar to those of extinct archaeocetes, seems to have secondary functional implications. However, the distribution of HSB in more-basal odontocetes is too poorly known to judge whether the HSB of Inia are a retained plesiomorphic feature or convergence.     

Potentially infanticidal behavior in the Amazon river dolphin (<Emphasis Type="Italic">Inia geoffrensis</Emphasis>)

Infanticide by males is a phenomenon common in species in which the reproductive output of large numbers of females can be monopolized by a small number of males. It is thought to increase a male’s fitness, at the expense of the fitness of the infant’s parents, by bringing females into season more quickly. Infanticide by males has been recorded in just three cetacean species. We report aggressive behavior suggestive of infanticide in a fourth, the Amazon river dolphin (Inia geoffrensis). We observed and photographed a series of attacks on a neonate Amazon river dolphin by a large male, with apparent protective behavior by the mother. Although infanticide was not confirmed, the forceful, aggressive behaviors were highly suggestive of infanticidal behavior and represent another important data point for comparative studies of infanticide in mammals. Amazon river dolphins may have a polygynous, polyandrous, or promiscuous mating system, the latter two of which are not the norm in species in which the reproductive output of large numbers of females are monopolized by a small number of males. However, sexual dimorphism, high rates of aggression by males, socio-sexual object-carrying displays by males, and a long interbirth interval suggest that successful male Amazon river dolphins may be able to monopolize a large proportion of mating opportunities, and it is plausible that male dolphins can improve their reproductive success by bringing females into estrous sooner by killing the offspring of other males.     

Patterns of Adaptive and Neutral Diversity Identify the Xiaoxiangling Mountains as a Refuge for the Giant Panda

Genetic variation plays a significant role in maintaining the evolutionary potential of a species. Comparing the patterns of adaptive and neutral diversity in extant populations is useful for understanding the local adaptations of a species. In this study, we determined the fine-scale genetic structure of 6 extant populations of the giant panda (Ailuropoda melanoleuca) using mtDNA and DNA fingerprints, and then overlaid adaptive variations in 6 functional Aime-MHC class II genes (DRA, DRB3, DQA1, DQA2, DQB1, and DQB2) on this framework. We found that: (1) analysis of the mtDNA and DNA fingerprint-based networks of the 6 populations identified the independent evolutionary histories of the 2 panda subspecies; (2) the basal (ancestral) branches of the fingerprint-based Sichuan-derived network all originated from the smallest Xiaoxiangling (XXL) population, suggesting the status of a glacial refuge in XXL; (3) the MHC variations among the tested populations showed that the XXL population exhibited extraordinary high levels of MHC diversity in allelic richness, which is consistent with the diversity characteristics of a glacial refuge; (4) the phylogenetic tree showed that the basal clades of giant panda DQB sequences were all occupied by XXL-specific sequences, providing evidence for the ancestor-resembling traits of XXL. Finally, we found that the giant panda had many more DQ alleles than DR alleles (33∶13), contrary to other mammals, and that the XXL refuge showed special characteristics in the DQB loci, with 7 DQB members of 9 XXL-unique alleles. Thus, this study identified XXL as a glacial refuge, specifically harboring the most number of primitive DQB alleles.     

RIVERINE HABITAT PREFERENCES OF BOTOS (INIA GEOFFRENSIS) AND TUCUXIS (SOTALIA FLUVIATILIS) IN THE CENTRAL AMAZON

The distribution and density of the Amazon's two contrasting endemic dolphins–boto, or Amazon river dolphin, Inia geoffrensis , and tucuxi, Sotalia fluviatilis –were examined on two adjoining large rivers in western Brazil. Using a 17-m river boat as a sightings platform, strip transects were used to cover areas within 150 m of the river margin and line transects were used in all other areas. Highest densities of both dolphins occurred near the margin, and lowest in the center of rivers. Seven different habitats were identified along river margins. The boto and the tucuxi differed in some elements of habitat choice, but they shared a preference for areas with diminished current and where two channels joined. Neither species favored the two most common edge types in this region of the Amazon-mud banks and flooded forest margins. Overall, the most preferred habitat type was the least common, and known as "meeting of the waters." In these areas a channel of sediment-rich white water meets one carrying acidic black water; the resultant mixing produces particularly productive, and obviously attractive, conditions for dolphins. These results demonstrate that Amazonian dolphins selectively occur in areas known to be favored for gill net deployment by local fishermen, and may explain why entanglement is apparently a common cause of mortality.     

Gene duplications and sequence polymorphism of bovine class II DQB genes

The genetic diversity of bovine class II DQB genes was investigated by polymerase chain reaction amplification and DNA sequencing. The first domain exon was amplified from genomic DNA samples representing 14 class II haplotypes, defined by restriction fragment length polymorphism (RFLP) analysis. The presence of a polymorphism in the copy number of DQB genes was confirmed since two DQB sequences were isolated from certain haplotypes. Four subtypes of bovine DQB genes were found. DQB1 is the major type and was found in almost all haplotypes. DQB2 is very similar to DQB1 but was found only in the duplicated haplotypes DQ9 to 12. DQB3 and DQB4 are two quite divergent genes only present in certain duplicated haplotypes. The bovine DQB complexity thus resembles that in the human DRB region. Bovine DQB genes were found to be highly polymorphic as ten DQB1 alleles and four DQB2 alleles were identified. The observed sequence polymorphism correlated well with previously defined DQB RFLPs. Bovine and human DQB alleles show striking similarities at the amino acid level. In contrast, the frequency of silent substitutions is much higher in comparisons of DQB alleles between species than within species ruling out the possibility that any of the contemporary DQB alleles have been maintained since the divergence of humans and cattle. The frequency of silent substitutions between DQB alleles was markedly lower in cattle than in humans, in agreement with a previous comparison of human and bovine DRB alleles.     

苏铁nrDNA ITS区的序列多态性:不完全致同进化的证据

本研究对苏铁(Cycas revoluta) nrDNA ITS进行克隆测序, 并以cDNA ITS为参照, 比较分析获得的序列的碱基变异、GC含量、5.8S二级结构的稳定性和5.8S保守基序的有无以及系统发育关系。结果发现苏铁nrDNA ITS存在较高的基因组内多样性, 同时, 这些分化的nrDNA ITS拷贝中包含有假基因的存在, 而且假基因与功能拷贝之间已经形成了较大的遗传分化, 这暗示假基因起源有较长历史。苏铁核仁组织区不仅多达16个, 而且分布在13条染色体上, 这可能是其nrDNA ITS致同进化不完全的主要原因。     

Vocalizations of Amazon River Dolphins,Inia geoffrensis: Insights into the Evolutionary Origins of Delphinid Whistles

Oceanic dolphins (Odontoceti: Delphinidae) produce tonal whistles, the structure and function of which have been fairly well characterized. Less is known about the evolutionary origins of delphinid whistles, including basic information about vocal structure in sister taxa such as the Platanistidae river dolphins. Here we characterize vocalizations of the Amazon River dolphin (Inia geoffrensis), for which whistles have been reported but not well documented. We studied Inia at the Mamirauá Sustainable Development Reserve in central Brazilian Amazônia. During 480 5‐min blocks (over 5 weeks) we monitored and recorded vocalizations, noted group size and activity, and tallied frequencies of breathing and pre‐diving surfaces. Overall, Inia vocal output correlated positively with pre‐diving surfaces, suggesting that vocalizations are associated with feeding. Acoustic analyses revealed Inia vocalizations to be structurally distinct from typical delphinid whistles, including those of the delphinid Sotalia fluviatilis recorded at our field site. These data support the hypothesis that whistles are a recently derived vocalization unique to the Delphinidae.