Vertebrates from aquatic to terrestrial is a leap in the evolutionary history of vertebrates, and this process requires various physiological and morphological innovations in the respiratory system, motor system, nervous system and other aspects in order to achieve the transformation from aquatic to terrestrial.
After a long period of paleontological and vertebrate zoology studies, it is known that the most recent fish relative of the tetrapods that are still alive today is the lungfish. Echinoss, lungfish, and tetrapods form the suborder of finfish, which, along with the suborder of rayfin fish in which common species of fish are found, is collectively known as teleost fish.
However, what is the basis for genetic innovation for various evolutionary changes, from the ancestors of bony fish to the ancestors of finfish to terrestrial vertebrates, has been a major scientific question that remains unresolved in the scientific community.
Dissecting the genomes of lungfish and early ray-finned fish, "living fossil" fish, is key to solving this major problem. With the exception of the African Echinoss (Speartail) genome, which was resolved in 2013, the genetic code of other species has not yet been resolved and systematic studies are lacking.
Lungfish, in particular, have the largest known genome of vertebrates (more than 40 Gb, the human genome is about 3 Gb), and scientists have long wanted to analyze their genomes, but have not been successful.
Lungfish Source: alchetron
On February 4, 2021, Cell published online titles :"Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes" and "African lungfish genome sheds light on the vertebrate water-to-land." transition" of two research papers (Figures 1 and 2).
Two companion studies analyzed the genomes of five species of the original spoke-fin fish Senegalese polyfin fish, spoon-sturgeon, bowfin fish and alligator eel, as well as African lungfish in living meatfin fish that are closestly related to tetrapods.
The two papers cross-integrate the disciplines of genomics, evolutionary biology, ichthyology, paleontology, computational biology and experimental biology, revealing the mystery of the genetic basis of the aquatic to terrestrial transition of vertebrates from different perspectives and at different evolutionary nodes.
<h1>An elucidation of the genome of primitive spokefin fish found that the genetic basis for the landing has emerged in the ancestors of teleost fish</h1>
There are many obstacles to be overcome in the evolution of vertebrates from aquatic to terrestrial, two important of which are how to support the body to move in the absence of buoyancy in the water body, and how to breathe oxygen in the air.
Primitive rayfin fish such as multifin fish, sturgeon, bowfin fish, and garnets, although far related to the landing "advance team", retain some of the characteristics associated with overcoming the above-mentioned water-to-land obstacles. Multifin fish, in particular, have primitive lungs for breathing air, in which polyfin fish can suck in air through the spray holes in the back of the water with very low dissolved oxygen, and even survive from the water for a period of time.
In addition, multifin fish are also similar to empty spiny fish, with muscle and endoskeletal support of the pectoral fin handle, which can crawl underwater. The unique biological properties and evolutionary status of these primitive fish have attracted many scientists to study them.
The first study analyzed the genomes of high-quality chromosomal-level polyfin fish and the genomes of three other primitive rayfin fish, and based on these genomic information, through comparative analysis and experimental verification, revealed the genetic premise that has evolved in the original rayfin fish that has supported the further evolution of meatfin fish into quadrupeds.
Genetic basis for regulating the motor flexibility of the limbs
Comparisons of the skeletons of current and extinct species already know that the "big arms" (humerus) of quadrupeds, including humans, are homologous to the posterior basal fin bones of the pectoral fins of ancient fish, which are lost in the hyperbony fish of the spokes (teleost, which includes most common fish such as eels, carps, and sea bass).
After obtaining the genome sequences of primitive spokefin fish multifin fish, spoon-sturgeon, bowfin fish and alligator eel, by comparing it with the genomes of various jawed vertebrates, the researchers found that enhancers that regulate limb development in many tetrapods already existed in primitive spokefin fish.
One of the ultra-conserved enhancers can even be traced back to cartilaginous fish, an enhancer that regulates the expression of the downstream Osr2 gene in synovial joints. Osr2 is associated with the formation of synovial joints and increases flexibility in limb movements.
Experiments on pectoral fin regeneration in multifin fish and in situ expression analysis confirmed that the gene was mainly expressed at the junction of the posterior basal fin bone and the fin strip, which corresponded to the synovial joint structure in tetrapod species. This enhancer is lost in the genome of eukeletal fish, and in response, eubony fish also lose the posterior basal fin bone and the synovial joints that connect.
This result reveals that genetic innovations in the prototype of synovial joints have emerged in the ancestors of bony fish, while bony fish secondarily lost their corresponding functions.
Origin of air breathing and lungs
Olfactory receptors sense stimuli from chemical molecules in the environment and convert them into olfactory nerve impulse information.
In-depth analysis of comparative genomics found that there were two types of olfactory receptors in the olfactory receptors of these ancient fish, in addition to the olfactory receptors that fish possessed to detect water-soluble molecules, but also the olfactory receptors capable of detecting air molecules. This is consistent with their ability to breathe air.
In addition, through the clustering and phylogenetic relationship analysis of multi-organ expression profiles, it was found that the expression profiles of lung and fish maw were the closest, and some lung-specific expression genes had also appeared in the ancestors of fish, implying that the molecular basis of "protopulmonary" formation already existed in the ancestors of teleost fish.
Transcriptome analysis of multiple species showed that genes highly expressed in the lungs of primitive spokefin fish were significantly enriched in angiogenesis pathways, which explained why the lungs or bladder surfaces of these primitive spokefin fish were densely packed with blood vessels, helping to spread and transport oxygen in the lungs.
These results genetically validate Darwin's hypothesis that the lung and the fish maw are homologous organs, and also clearly show that the fish maw of our common bonefish is derived from the early lung evolution of vertebrates.
Evolution of the cardiac system
The co-evolution of the heart and respiratory system plays an important role in the evolution of vertebrates. The respiratory system provides oxygen for the maintenance of normal heart function while relying on the heart to transport the blood that carries oxygen throughout the body.
From one atrium and one ventricle in fish to two atriums and two ventricles in people, the structure of the heart tends to be perfected and the function becomes more complex.
The arterial cone is located in the upper part of the outflow channel of the heart and borders the right ventricle, acting as an auxiliary organ for cardiac activity, which prevents blood reflux as well as balances ventricular blood pressure.
The arterial cone is also present in primitive spokefin fish and earlier cartilaginous fish.
Through genomic collinear analysis, it was found that genes related to the heart system retained a very conservative collinear relationship between humans and polyfin fish, suggesting that these genes also retained a fairly conservative regulatory mechanism. The study also found for the first time a conservative regulatory original that regulates the Hand2 gene.
The researchers targeted deletion of this regulatory element in the mouse genome and found that newly born mutant mice in early embryonic development, due to decreased expression of the Right Ventricle Hand2 gene, resulting in cardiac hypoplasia and congenital death.
It has previously been reported that functional mutations in the Hand2 gene can lead to the occurrence of Tetralogy of Farroy,a congenital heart defect disorder. The results will contribute to human research into heart developmental defects.
summary
This study not only reveals the homologous relationships of many vital organs of vertebrates at the molecular level, but also the molecular genetic mechanisms that regulate these organs and related functions.
Based on the comparative analysis of the evolutionary process of species, it is also proposed for the first time that the genetic regulation mechanism of organs and physiological functions related to the terrestrial adaptation of tetrapods has begun to take shape in their bony fish ancestors.
In particular, the ancient genes and regulatory elements that regulate air respiration function, skeletal motility, and cardiopulmonary system development provide an important basis for genetic innovation for the subsequent leap forward in the evolution of tetrapods in the subsequent landfall of finfish.
Fig. 1 Primitive spokefin fish already possessed a large number of key genomic elements associated with terrestrial properties
The corresponding authors of the study are Professor Zhang Guojie (Shenzhen Huada Gene Research Institute/Kunming Institute of Zoology, Chinese Academy of Sciences/University of Copenhagen), Researcher He Shunping (Institute of Hydrology, Chinese Academy of Sciences), Researcher Zhu Min (Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences) and Professor Wang Wen (Northwestern Polytechnical University/Kunming Institute of Zoology, Chinese Academy of Sciences), the first authors are Bi Xupeng (Institute of Hydrobiology, Chinese Academy of Sciences/Shenzhen Huada Institute of Genetics), Wang Kun (Northwestern Polytechnical University), Yang Liandong (Institute of Hydrobiology, Chinese Academy of Sciences), Pan Hailin (Shenzhen Huada Genetics Research Institute), Jiang Haifeng (Institute of Aquatic Sciences, Chinese Academy of Sciences) and Wei Qiwei (Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences).
<h1>The elucidation of the lungfish genome and its major implications for the study of tetrapod landings</h1>
The second study on aquatic to terrestrial evolution of vertebrates reported the largest genome ever resolved, with a size of more than 40 billion pairs of bases (40 Gb), more than 10 times the size of the human genome (3 Gb).
In order to analyze this high-complexity giant genome with high quality, the project has spawned two three-generation sequencing assembly software NextDenovo and wtdbg.2.0, which once again highlights China's cutting-edge influence in the field of genomics research.
The final lungfish assembly result coincided with the predicted size, and the completeness was as high as 99% of the chromosomal mounting rate. The genome contains more than 95% of the intact genes of vertebrates.
In contrast, the Australian lungfish genome assembly size of 34 Gb and only 67% complete vertebrate intact genes published in Nature in January 2021, and the assembly size of the Salamander genome published in Nature in February 2018 was 32 Gb, with a completion rate of only 70%.
Therefore, the African lungfish genome reported in this work is not only the largest genome at present, but also the first complete and high-quality super genome, marking that China's scientific and technological personnel have reached the international top level in the analysis of super large genomes.
Amplification of lungfish genomes
Although the lungfish genome is more than 10 times that of humans, the number of genes is similar to that of humans, so lungfish have a large number of ultra-long genes, the longest gene is 18 Mb long, and the longest human gene is 2.8 Mb.
The study found that the expression levels of these ultra-long genes in lungfish are similar to those of short genes and homologous genes of other species, suggesting that lungfish have evolved a mechanism for efficiently transcribe these ultra-long genes.
The researchers found that the lungfish genome is so large large because of the continuous insertion of transposons into the evolutionary branches of lungfish for hundreds of millions of years.
The reason why the lungfish genome can tolerate a large number of transposons to replicate the accumulation of "junk DNA" may be because the ability to inhibit the expression of transposons in germline cells has evolved at the same time, because the specific zinc finger protein gene family composed of the two functional domains of KRAB and zf-C2H2 is greatly expanded in the lungfish genome, and its expression level in the reproductive glands is also greatly improved.
In addition, the length growth rate of the ultra-long gene is also lower than that of the short gene, suggesting that the growth rate of gene length in lungfish is also controlled by natural selection. These results solve the mystery of the lungfish's oversized genome.
The respiratory system evolved further
For organisms that breathe air directly, the surface tension caused by the liquid (blood) -gas (air) interface on the alveoli must be solved, and the lung surfactant has a very critical role in solving the surface tension of the alveoli.
Pulmonary surfactants are mainly composed of proteins and lipids, and the proteins in them are decisive for the physiological properties of pulmonary surfactants.
In land vertebrates, there are four lung surfactant proteins, namely SP-A, SP-B, SP-C and SP-D.
Studies of primitive rayfin fish, lungfish and tetrapods found that these breath-related genes have undergone three steps of evolution, SP-B is the oldest protein, which has appeared from the ancestors of teleost fish, which is confirmed by the initial air respiration ability of the ancestors of the bony fish found in the first study; SP-C is a new gene that appeared from the ancestors of the meat fin fish, representing the enhancement of the breathing ability of the ancestors of the meat fin fish, and SP-A and SP-D are new genes that appeared from the ancestors of tetrapods. Marks the maturation of breathing capacity in terrestrial vertebrates.
In addition, the slc34a2 gene plays a key role in recycling phospholipids of pulmonary surfactants.
The study found that this gene is highly expressed in the lungs of lungfish and tetrapods, while low expression in the lungs of rayfin fish. Previous studies have shown that this gene is mainly expressed in zebrafish in the digestive system to improve the utilization of phosphorus. Therefore, this gene may have been recruited by the lungs in the ancestors of finfish, which is one of the mechanisms for further enhancement of their respiratory function.
Terrestrial kinematic systems
In the evolution from vertebrate aquatic to terrestrial, the change in the motor system encompasses multiple layers, the most notable of which is the formation of the five fingers, which is also the iconic phenotype of quadrupeds.
Previously, there have been a lot of studies on the appearance of five fingers, and the most widely accepted view is that in fish, hoxa11 and hoxa13 are expressed together at the ends of the limbs, and the traits of fins appear, while in tetrapods, hoxa13 inhibits the expression of hoxa11 at the ends of the limbs, and the traits of the five fingers appear.
However, previous studies have not been able to find the genetic regulatory mechanism behind this expression change.
This study found that hoxa11 has a new regulatory element of tetrapod origin around 200 bp upstream, which may be the key element used by hoxa13 to control the spatiotemporal expression of hoxa11.
Interestingly, this element also showed large variation in snakes and birds, and may be closely related to the violent variation of the limbs of snakes and birds, indicating that its function has great research value.
In addition, the study also found multiple genetic changes associated with arm formation such as the presence of radius and the enhancement of motor neuron capacity, including the gradual loss of the actinotrichia proteins encoding genes that affect the development of the radius in lungfish and tetrapods, and the deletion of quadruped-specific large fragments of the Hox13b gene that controls hindlimb development. The Hoxc10 gene upstream, which controls the development of motor neurons in the lumbar spine, has a specific new origin regulatory element in tetrapods.
These results reveal previously unknown processes and mechanisms of genetic innovation in the origin and evolution of limbs and land mobility with five fingers.
Increased anxiolytic abilities
During the aquatic terrestrial transitions in vertebrates, the most significant changes in the brain are in the amygdala region.
The amygdala is an important organ responsible for emotional processing. In tetrapods, the amygdala began to have a zoning structure and more complex links. Previous studies have also shown that the amygdala of lungfish may also be more similar to that of quadrupeds.
This study found that two new genes, Nps and Npsr, appeared in the ancestors of lungfish and tetrapods, encoding neuropeptide S and its receptors, respectively. These two genes are known to be expressed in the amygdala and are important genes responsible for anti-anxiety.
In addition, the study also found that multiple genes associated with the amygdala showed large amino acid changes in lungfish and tetrapod ancestors. These results suggest that the ancestors of lungfish and tetrapods may have had greater anti-anxiety abilities, and this enhancement of this ability may have some positive effect on vertebrate landings.
Figure 2 The African lungfish genome provides an important bridge for understanding the aquatic terrestrial transitions of vertebrates
This study shows that the evolution of vertebrates from aquatic to terrestrial animals exhibits a three-step evolution.
In the case of air respiration, for example, the molecular basis of air respiration and air smell has emerged in the common ancestor of teleost fish, while bony fish have lost this property and belong to a more specialized taxa.
Subsequently, more breath-related genes and functional elements appeared in the ancestors of finfish, which further enhanced their air-breathing ability.
Eventually, quadrupeds evolved more genes and functional elements to be able to breathe air and successfully get rid of the shackles of water.
The analysis of genetic innovations related to various terrestrial adaptation traits shows that three types of genetic innovations, such as the origin of new genes, the emergence of new regulatory elements, and the change of gene-coded amino acids, have played a role in the evolution of vertebrate aquatic to terrestrial animals.
The corresponding authors of this work are Professor Wang Wen (Kunming Institute of Zoology, Chinese Academy of Sciences/Northwestern Polytechnical University), Professor He Shunping (Institute of Hydrobiology, Chinese Academy of Sciences), Professor Qiu Qiang (Northwestern Polytechnical University), and Researcher Zhao Wenming (Beijing Institute of Genomics, Chinese Academy of Sciences), and the first authors are Wang Kun (Northwestern Polytechnical University), Wang Jun (South China Agricultural University), Zhu Chenglong (Northwestern Polytechnical University), Yang Liandong (Institute of Fisheries, Chinese Academy of Sciences), Ren Yandong (Northwestern Polytechnical University), Ruan Jue (Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences), Fan Guangyi (Qingdao Huada Gene Research Institute) and Hu Jiang (Wuhan Hope Group Biotechnology Co., Ltd.).
Original link:
https://www.cell.com/cell/fulltext/S0092-8674(21)00089-1
https://www.cell.com/cell/fulltext/S0092-8674(21)00090-8
Source: WeChat public account "BioArt", the official website of the Kunming Institute of Zoology, Chinese Academy of Sciences