Method for accurately identifying Yangtze river dolphin and East Asian dolphin based on dentin 87Sr / 86Sr ratio

Abstract

The invention discloses a method for accurately identifying a Yangtze river dolphin and an East Asian dolphin based on a dentin 87Sr / 86Sr ratio, which comprises the following steps: collecting a dolphin tooth sample, pretreating teeth, analyzing the dentin 87Sr / 86Sr ratio, measuring a tooth growth layer group, and reconstructing the 87Sr / 86Sr ratio change. The average values of different dentin 87Sr / 86Sr ratio change stages are compared with the Yangtze River main stream and ocean 87Sr / 86Sr ratios respectively, the habitat types of the dolphins in the forming period are judged, the dolphins are judged to be the Yangtze River dolphins or the East Asian dolphins according to the habitat types, and an important basis is provided for judging species of dead dolphin individuals at the Yangtze River estuary.

Description

Technical Field

[0001] The present invention belongs to the field of ecological technology and specifically relates to a dentin-based 87 Sr / 86 A method for accurately identifying Yangtze finless porpoises and East Asian finless porpoises using Sr ratio. Background Art

[0002] The finless porpoise (Neophocaena) is a small toothed whale that inhabits coastal waters and some rivers from the temperate waters of Japan in the east to the tropical waters of the Persian Gulf in the west. There has been much controversy about its taxonomic status and nomenclature. Based on morphological and genetic characteristics, the classification of the genus Neophocaena includes two species: the Indo-Pacific finless porpoise (Neophocaena phocaenoides) and the narrow-ridged finless porpoise (N.asiaeorientalis), the latter of which contains two subspecies (East Asian finless porpoise Nasunameri and Yangtze finless porpoise Naasiaeorientalis). In recent years, based on the whole genome sequencing of the Yangtze finless porpoise and the analysis of population genomics, it is believed that the Yangtze finless porpoise and the East Asian finless porpoise have significant and stable genetic differentiation, and the genetic differentiation has reached the interspecific level.

[0003] The Yangtze finless porpoise is a freshwater cetacean unique to China. It inhabits the middle and lower reaches of the Yangtze River in China and the two major lakes connected to the river (Dongting Lake and Poyang Lake). Since the Baiji dolphin (Lipotes vexillifer) was declared functionally extinct, the Yangtze finless porpoise has become the only cetacean in the Yangtze River freshwater ecosystem. In recent decades, the population of the Yangtze finless porpoise has declined significantly. It is estimated that the number of Yangtze finless porpoises had dropped to about 1,800 in 2006, and only 1,040 in 2012. The latest survey results show that the natural population of the Yangtze finless porpoise has rebounded for the first time in many years (2022), but it is only about 1,249. The East Asian finless porpoise inhabits the Taiwan Strait, the East China Sea and the Yellow Sea / Bohai Sea, the western, southern and southeastern coasts of South Korea, and the coastal areas of Japan. The species inhabits nearshore and shallow waters, including bays, mangrove swamps and estuaries, and is therefore extremely vulnerable to human activities such as shipping, fisheries, and shoreline engineering. Currently, the Yangtze finless porpoise and the East Asian finless porpoise are listed as critically endangered and endangered species respectively on the Red List of the International Union for Conservation of Nature (IUCN), highlighting the urgency of conservation work. In 2021, the Yangtze finless porpoise was upgraded to a national first-class key protected wild animal, and the need for conservation of the finless porpoise has further increased.

[0004] The Yangtze River Estuary is located at the confluence of the Yangtze River, the East China Sea and the Yellow Sea. It has extremely rich food biological resources and is one of the hotspots for Chinese finless porpoises. The distribution areas of the East Asian finless porpoise and the Yangtze finless porpoise are similar here. At the same time, the estuary area is greatly affected by factors such as tides and salinity changes. Therefore, the distribution ranges of the Yangtze finless porpoise and the East Asian finless porpoise in the Yangtze River Estuary may overlap in space. In recent years, with the in-depth development of conservation work, dead finless porpoises in the Yangtze River Estuary have been discovered and reported in a timely manner. Accurately identifying dead individuals as Yangtze finless porpoises or East Asian finless porpoises has become a prerequisite for conservation management and scientific research. There are significant differences in the habitats of the Yangtze River finless porpoise and the East Asian finless porpoise, but the boundaries of their distribution in the salt and fresh water confluence area of ​​the Yangtze River Estuary are not clear. In addition, dead finless porpoises may drift downstream, making it difficult to identify the species of dead finless porpoises found in this water area. The Yangtze finless porpoise and the East Asian finless porpoise are similar in morphology. Currently, they are mainly distinguished by morphological differences such as dorsal height, tubercle width, and tubercle number. However, since both Yangtze finless porpoise and the East Asian finless porpoise are national key protected wild animals, the number of samples that can be collected to establish morphological differentiation parameters is very small, and the above morphological characteristics are affected by the growth and development stage of the individual. Therefore, there is a lack of mature standards for morphological differentiation, and the identification results are highly subjective. At the same time, dead finless porpoises are usually found in varying degrees of decay, which will seriously affect the recognition of surface morphology. In addition, although high-throughput molecular sequencing technologies such as resequencing have shown that the East Asian finless porpoise and the Yangtze finless porpoise have significant genetic differentiation, there is currently a lack of molecular markers that can accurately and cost-effectively distinguish the two.

[0005] Teeth are of great value in the study of age, habitat reconstruction, and dietary changes of many marine and terrestrial mammals. Dental tissue is highly mineralized, with dentin as the main component of the tooth, covered by enamel, and the vascular pulp of the tooth surrounded by dentin. About 72% of the dentin mass is composed of inorganic mineral components (mainly hydroxyapatite), 10% is water, and various organic components account for about 20% of the dentin mass. Dentin begins to form during the fetal period of the animal, and then gradually grows toward the pulp cavity. During its accumulation, it reflects the changes in individual growth rates, thus forming a discernible growth layer group (i.e., annual rings). During the growth process, dentin records the elements and isotopes of the habitat it has experienced, and due to the metabolic inertness of highly mineralized tissues, the trace elements and isotopes in the teeth will not change once deposited, so teeth can record the elements and isotopes of the habitat throughout their lives. At present, studies have explored the use of tooth elements to distinguish the Yangtze River finless porpoise, which mainly lives in freshwater, from the East Asian finless porpoise, which mainly lives in the ocean.

[0006] However, since the elements are deposited into dentin through multiple biofilm barriers, the relationship between the final element content of dentin and the element content in the environment is difficult to quantify, and it is not yet possible to establish the corresponding element content standards for finless porpoises living in freshwater or seawater habitats. In addition, studies have shown that physiological factors in the growth and development of animals can also lead to changes in the content of elements such as Sr and Zn in mammalian teeth, which greatly interferes with the interpretation of habitat changes based on element changes, resulting in the objectivity of using tooth element content to study animal habitat conversion being limited. Therefore, it is urgent to develop methods to accurately identify Yangtze finless porpoises and East Asian finless porpoises. Summary of the invention

[0007] Technical problem to be solved: In view of the above technical problem, the present invention provides a dentin-based 87 Sr / 86 The method of accurately identifying Yangtze finless porpoise and East Asian finless porpoise by Sr ratio is different from that of Sr element content. In the formation process of biological materials, Sr isotope ( 87 Sr / 86 Sr) hardly undergoes fractionation and is not affected by physiological factors such as its growth and development, which directly reflects its source. 87 Sr / 86 Sr, therefore dentin 87 Sr / 86 Sr can represent the habitat experienced by animals. 87 Sr / 86 direct and complete record of Sr. Moreover, different habitats such as marine and freshwater have 87 Sr / 86 The significant difference in Sr ratios in the ocean 87 Sr / 86 The Sr ratio is relatively uniform, and the ocean 87 Sr / 86 The Sr ratio is about 0.7092, while in the middle and lower reaches of the Yangtze River in China, 87 Sr / 86 The Sr ratio is between 0.7100-0.7110. 87 Sr / 86 The Sr ratio is mainly affected by the geological factors of the watershed and remains stable in different years. 87 Sr / 86 The Sr ratio determines the habitat of individual finless porpoises. 87 Sr / 86 The Sr ratio is 0.7092, corresponding to the marine habitat life history. 87 Sr / 86 The Sr ratio of 0.7100-0.7110 corresponds to the freshwater life history of the Yangtze River, and thus it is judged to be a Yangtze finless porpoise or an East Asian finless porpoise.

[0008] Technical solution: a dentin-based 87 Sr / 86 The method for accurately identifying Yangtze finless porpoises and East Asian finless porpoises by Sr ratio includes the following steps:

[0009] Step 1) Collect finless porpoise tooth samples: Collect the straight teeth in the middle of the left side of the lower jaw of the finless porpoise that died in the wild. If the individual is severely decayed, try to take teeth in the adjacent position. If there are too many missing teeth, try to choose teeth that are not severely worn;

[0010] Step 2) Tooth pretreatment: extract the tooth, soak it in a trypsin solution for 24 hours, use ultrasound to vibrate the tooth in the trypsin solution to remove the soft tissue attached to the tooth surface and the pulp cavity, and then use deionized water to repeatedly vibrate and clean the outer surface of the tooth and the inside of the pulp cavity; embed the cleaned and dried tooth with cold mounting resin, use a diamond saw blade of a small cutting machine to cut along the longest axis of the tooth coronal surface, and cut the tooth into two parts; use sandpaper to finely grind the cut part of the coronal surface, and then use a polishing cloth and a silica suspension to polish; after polishing, use deionized water to wash the tooth slices and dry them;

[0011] Step 3) Dentin 87 Sr / 86 Sr ratio analysis: Laser ablation coupled to a multi-collector inductively coupled plasma mass spectrometer was used to perform line scanning analysis of the dentin from the cusp to the pulp cavity to obtain the dentin ratio. 87 Sr / 86 Sr ratio;

[0012] Step 4) Measurement of tooth growth layer groups: Use a biological polarizing microscope to take a tooth image, distinguish the dentin growth layer groups, and use image measurement software to measure the growth layer groups of the teeth. 87 Sr / 86 The laser scanning line of Sr ratio analysis measures the width of the growth layer group. 87 Sr / 86 The change of Sr ratio is related to the growth time;

[0013] Step 5) Rebuild 87 Sr / 86 Sr ratio changes: Analysis of dentin from the enamel interface to the pulp cavity 87 Sr / 86 Sr ratio, analysis of dentin 87 Sr / 86 The change of Sr ratio and the location of the change; the different dentin 87 Sr / 86 The average values ​​of Sr ratio in the changing stages are respectively 87 Sr / 86The Sr ratios were compared to determine the habitat type of the finless porpoise during its formation, and then it was determined to be the Yangtze finless porpoise or the East Asian finless porpoise based on the habitat type.

[0014] Preferably, in step 3), laser ablation is used in conjunction with a multi-collector inductively coupled plasma mass spectrometer to perform dentin 87 Sr / 86 Sr ratio analysis was performed in medium resolution mode. The laser energy density in the laser ablation-connected multi-collector inductively coupled plasma mass spectrometry experiment was 6.5 J / cm 2 The spot diameter of the ablation was 64 μm, the frequency was 10 Hz, and the ablation speed was 5 μm / s. Before each laser ablation, the background intensity of each isotope was measured for 200 cycles, and the average value was used for blank background correction during sample analysis. The apatite standard (Durango) and modern marine shark teeth were measured at the beginning and end of each analysis and every 30 minutes during the analysis. 87 Sr / 86 Sr ratio to evaluate the finless porpoise tooth samples 87 Sr / 86 Accuracy and repeatability of Sr ratio.

[0015] Preferably, in step 3), by setting the Faraday cup configuration of the multi-receiver inductively coupled plasma mass spectrometer, signals at m / z 82, 83, 83.5, 84, 85, 86, 86.5, 87 and 88 are received. In the process of data analysis, the "peak zero point" method is used to correct Kr interference by subtracting the average signal of the blank background from each analysis cycle; the signal at m / z 82 is received to monitor 42 Ca 40 Ar / 42 Ca 40 Ca peak; by monitoring 85 Rb signal calculated from isotope ratio 87 Rb, and 87 Rb / 85 The instrument mass fractionation calibration was performed for Rb. The calibration process assumed that Rb and Sr have the same mass fractionation factor. 88 Sr and 86 Sr signal, according to 88 Sr / 86 The isotopic abundance ratio of Sr in nature is 8.375209. The mass fractionation factor is calculated using the exponential law. According to the mass fractionation factor and 87 Rb / 85 The natural isotopic abundance ratio of Rb is 0.38567, and the corrected 87 Rb / 85 Rb, thus obtaining the corrected 87Rb. Subtract the corrected 87 Rb, according to the correction 87 Rb / 85 Rb steps for deduction 87 After Rb 87 Sr / 86 The Sr ratio was corrected for instrument mass fractionation to obtain a measured value that was corrected for mass fractionation and interference.

[0016] Preferably, in step 5), CHANGEPOINT in the programming statistics software R is used to 87 Sr / 86 The original detection value data of Sr ratio were analyzed to obtain the individual 87 Sr / 86 The point where the Sr ratio changes significantly, thus obtaining 87 Sr / 86 The Sr ratio does not change significantly in the stable stage, and the stable stage is calculated. 87 Sr / 86 The average value of Sr ratio; the average value is compared with the mainstream of the Yangtze River and the ocean 87 Sr / 86 By comparing the Sr ratios, we can determine the habitat type of the finless porpoise during its formation period, and then infer the species of the finless porpoise based on the habitat type.

[0017] The Yangtze River is the best choice. 87 Sr / 86 The Sr ratio is 0.7100-0.7110, ocean 87 Sr / 86 The Sr ratio is 0.7092.

[0018] Beneficial effect: The present invention is based on animal biomineralization materials 87 Sr / 86 The Sr ratio can directly reflect the environment 87 Sr / 86 The characteristics of Sr ratios and the 87 Sr / 86 The Sr ratios were significantly different, and the finless porpoise teeth were used as the habitat of the finless porpoise. 87 Sr / 86The indicator of Sr ratio is used to invert the habitat of finless porpoises, and whether the finless porpoise lives in fresh water or ocean is inferred to be a Yangtze finless porpoise or an East Asian finless porpoise. The method of the present invention has the advantages of being accurate, not affected by physiological factors, and being able to establish numerical discrimination standards. It can be used to reconstruct the habitat experienced by finless porpoises and thus identify Yangtze finless porpoises and East Asian finless porpoises. It overcomes the shortcomings of traditional morphology that is difficult to identify and the appearance is easily affected by factors such as trauma and decay; it overcomes the shortcomings that the content composition of dentin elements is easily affected by factors such as growth and development, and it is difficult to establish an environmental corresponding content standard. It provides a basis for reconstructing the habitat of dead finless porpoises and thus distinguishing Yangtze finless porpoises and East Asian finless porpoises in the Yangtze River estuary. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A tooth from the left lower jaw of a Yangtze River porpoise;

[0020] Figure 2 This is a slice of a finless porpoise tooth. The continuous shallow groove from the tooth tip to the pulp cavity in the image is the trace left by laser ablation of dentin.

[0021] Figure 3 Dentin of a dead East Asian finless porpoise found on the east coast 87 Sr / 86 Sr ratio, the black curve is 87 Sr / 86 Sr ratio, the gray horizontal line is obtained by CHANGEPOINT analysis 87 Sr / 86 The gray horizontal line on the Y axis indicates the fragment with significant changes in Sr ratio. 87 Sr / 86 Average value of Sr ratio;

[0022] Figure 4 Dentin of a dead Yangtze River porpoise found in the lower reaches of the Yangtze River 87 Sr / 86 Sr ratio, the black curve is 87 Sr / 86 Sr ratio, not detected by CHANGEPOINT analysis 87 Sr / 86 The significant change of Sr ratio, the position of the gray horizontal line on the Y axis indicates the position of the fragment 87 Sr / 86 The average value of Sr ratio; the black line is the fitting value, the gray interval is the confidence interval, and the light blue background represents the mainstream of the Yangtze River. 87 Sr / 86 Sr ratio range, light red background indicates ocean 87 Sr / 86 Sr ratio, the x-axis represents the distance from the tooth cusp to the pulp cavity along the dentin growth axis. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0024] The finless porpoise tooth dentin used in the present invention is highly mineralized and metabolically inert. Once formed, it will not change. Moreover, the dentin begins to form in the fetal period of the animal, then gradually grows toward the pulp cavity, and forms discernible growth rings according to the changes in individual growth rates. In addition, the Sr isotopes ( 87 Sr / 86 Sr ratio) information. 87 Sr / 86 The Sr ratio is relatively uniform, and the contemporary ocean 87 Sr / 86 The Sr ratio is 0.70918, while in the middle and lower reaches of the Yangtze River in China, 87 Sr / 86 The Sr ratio is between 0.710 and 0.711. Therefore, the different habitats experienced by the Yangtze River porpoise and the East Asian porpoise will be reflected in their dentin 87 Sr / 86 For Sr ratio, dentin can be used 87 Sr / 86 The Sr ratio was used to determine the species.

[0025] Example 1

[0026] A method based on accurate determination of dentin 87 Sr / 86 The method of identifying Yangtze River finless porpoise and East Asian finless porpoise by Sr ratio, the specific steps are as follows:

[0027] Step 1) Tooth collection: The second tooth on the left side of the lower jaw was extracted from the mouths of dead finless porpoises found in Chongming, Lianyungang and Hangzhou Bay. For individuals with severe decay and missing teeth, other teeth available were used for analysis;

[0028] Step 2) Tooth pretreatment: The extracted teeth were soaked in a trypsin solution (2000 BAEE U / mg) (Sigma-Aldrich, Seelze, Germany) for 24 h, and then ultrasonically vibrated in the trypsin solution for 10 min to clean the soft tissue and attachments on the tooth root and remove the residual nerve tissue in the pulp cavity; then the teeth were ultrasonically cleaned 3 times with deionized water, and dried after cleaning (the cleaned teeth were as follows Figure 1The tooth was embedded with cold mounting resin; the tooth was cut into two parts along the longest axis of the coronal surface using a diamond saw blade of a small cutting machine (Struers, Copenhagen, Denmark); the coronal surface of one half was finely ground using sandpaper and then polished with a polishing cloth and a silica suspension; the tooth sections were washed with deionized water and dried;

[0029] Step 3) Dentin 87 Sr / 86 Sr ratio analysis: Laser ablation coupled to a multi-collector inductively coupled plasma mass spectrometer (LA-MC-ICP-MS) was used to perform line scanning analysis from the interface between enamel and dentin at the cusp to the pulp cavity to obtain the dentin ratio. 87 Sr / 86 The instrument system included a RESOlution SE 193 nm laser ablation system (Australian Scientific Instruments, ACT, Australia) and a Neptune plus multi-collector inductively coupled plasma mass spectrometer (Thermo Scientific, Dreieich, Germany). The laser energy density was 6.5 J / cm 2 , the spot diameter of the ablation was 64μm, the frequency was 10Hz, and the ablation speed was 5μm / s. Before the analysis, the laser ablation NIST SRM610 glass standard was used to debug the peak shape of the multi-collector inductively coupled plasma mass spectrometer to ensure that the peak shape reached an acceptable level; the laser ablation apatite international standard Durango was used to debug the instrument signal and stability to ensure that the instrument sensitivity and stability were within an acceptable range; before each laser ablation, the background intensity (V) of each isotope was measured for 200 cycles, and the average value was used for blank background correction during sample analysis. By measuring the apatite standard (Durango) and modern marine shark teeth (collected offshore) at the beginning and end of each analysis and every 30 minutes during the analysis, the background intensity (V) of each isotope was measured for 200 cycles, and the average value was used for blank background correction during sample analysis. 87 Sr / 86 Sr ratio to evaluate the finless porpoise tooth samples 87 Sr / 86 Accuracy and repeatability of Sr ratio.

[0030] By setting up the Faraday cup configuration of the multi-collector ICP-MS, signals at m / z 82, 83, 83.5, 84, 85, 86, 86.5, 87, and 88 were received. During data analysis, the “peak zero” method was used to correct for Kr interference by subtracting the average signal of the blank background from each analysis cycle; the signal at m / z 82 was received to monitor 42 Ca 40 Ar / 42 Ca 40 Ca peak; by monitoring 85 Rb signal calculated from isotope ratio 87 Rb, and 87 Rb / 85 The instrument mass fractionation calibration was performed for Rb. The calibration process assumed that Rb and Sr have the same mass fractionation factor and first used the measured 88 Sr and 86 Sr signal, according to 88 Sr / 86 The isotopic abundance ratio of Sr in nature is 8.375209. The mass fractionation factor is calculated using the exponential law. According to the mass fractionation factor and 87 Rb / 85 The natural isotopic abundance ratio of Rb is 0.38567, and the corrected 87 Rb / 85 Rb, thus obtaining the corrected 87 Rb. Subtract the corrected 87 Rb, according to the correction 87 Rb / 85 Rb steps for deduction 87 After Rb 87 Sr / 86 The Sr ratio was corrected for instrumental mass fractionation to obtain the measured value after mass fractionation correction and interference correction. Data processing and correction were performed using the open source LA-MC-ICP-MS data fast offline reduction program "SrDR". The analysis mode of MC-ICP-MS was medium resolution static mode. The mass numbers measured by the mass spectrometer during the analysis and the corresponding isotopes to be measured are shown in Table 1.

[0031] Table 1 Mass numbers and corresponding isotopes measured by MC-ICP-MS

[0032]

[0033] Step 4) Tooth growth layer group measurement: Use a biological polarizing microscope (Leica, Solms, Germany) to take tooth images (such as Figure 2 ), distinguish the dentin growth layer groups, and use image measurement software to 87 Sr / 86 The laser scanning line of Sr ratio analysis measures the width of the growth layer group. 87 Sr / 86 The change of Sr ratio is related to the growth time;

[0034] Step 5) Rebuild 87 Sr / 86Sr ratio changes: Changepoint Statistics was used to analyze the changes in dentin from the enamel interface to the pulp cavity. 87 Sr / 86 Sr ratio, evaluation of dentin 87 Sr / 86 Whether the Sr ratio has a significant change and where the change occurs; the different dentins divided by Changepoint Statistics analysis 87 Sr / 86 The average values ​​of Sr ratio in the Changjiang mainstream are 87 Sr / 86 Sr ratio 0.7100-0.7110, ocean 87 Sr / 86 The Sr ratio of 0.7092 was compared to determine the habitat type of the finless porpoise during its formation, and thus the species of the finless porpoise was inferred based on the habitat type.

[0035] This example includes tooth samples of a dead finless porpoise found on the coast and a dead finless porpoise found in the lower reaches of the Yangtze River. The results of Sr isotope analysis are as follows: Figure 3-Figure 4 As shown in the figure, the horizontal axis represents the distance from the tooth tip to the pulp cavity along the dentin growth axis; the vertical axis represents the life history 87 Sr / 86 Changes in the Sr ratio are related to changes in the living water environment.

[0036] like Figure 3 Shown is the dentin of a dead finless porpoise found along the coast 87 Sr / 86 The Sr ratio fluctuates very little and is always around 0.70918. This result is consistent with the measured value in the contemporary ocean (0.70918), indicating that the finless porpoise lives in 87 Sr / 86 The Sr ratio is stable in the ocean, indicating that it is an East Asian finless porpoise. Figure 4 Shown are the teeth of a dead finless porpoise found in the lower reaches of the Yangtze River 87 Sr / 86 The change in the Sr ratio is not large, basically within the range of 0.7100-0.7110. This result is close to the measured value of water samples in the main stream of the Yangtze River, indicating that these individuals live in the freshwater environment of the Yangtze River, indicating that they should be Yangtze finless porpoises.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dentin-based 87 Sr / 86 The method for accurately identifying Yangtze River finless porpoises and East Asian finless porpoises by using Sr ratio is characterized in that: The following steps are involved: Step 1) Collect finless porpoise tooth samples: Collect the straight teeth in the middle of the left side of the lower jaw of dead finless porpoises in the wild. If the individual is severely decayed, try to take teeth in nearby positions. If there are too many missing teeth, try to choose teeth that are not severely worn; Step 2) Tooth pretreatment: Extract the tooth and soak it in trypsin solution for 24 hours. Then use ultrasound to vibrate the tooth in the trypsin solution to remove the soft tissue attached to the tooth surface and the pulp cavity. Then use deionized water to repeatedly vibrate and clean the outer surface of the tooth and the inside of the pulp cavity. Use cold mounting resin to embed the cleaned and dried tooth. Use a diamond saw blade of a small cutting machine to cut along the longest axis of the tooth coronal surface to cut the tooth into two parts. Use sandpaper to finely grind the cut coronal surface, and then use a polishing cloth and silica suspension to polish. After polishing, use deionized water to wash the tooth slices and dry them. Step 3) Dentin 87 Sr / 86 Sr ratio analysis: Laser ablation coupled to a multi-collector inductively coupled plasma mass spectrometer was used to perform line scanning analysis of the dentin from the cusp to the pulp cavity to obtain the dentin ratio. 87 Sr / 86 Sr ratio; Step 4) Measurement of tooth growth layer groups: Use a biological polarizing microscope to take a tooth image, identify the dentin growth layer groups, and use image measurement software to 87 Sr / 86 The laser scanning line of Sr ratio analysis measures the width of the growth layer group. 87 Sr / 86 The change of Sr ratio is related to the growth time; Step 5) Rebuild 87 Sr / 86 Sr ratio changes: Analysis of dentin from the enamel interface to the pulp cavity 87 Sr / 86 Sr ratio, analysis of dentin 87 Sr / 86 The change of Sr ratio and the location of the change; the different dentin 87 Sr / 86 The average values ​​of Sr ratio in the changing stages are respectively 87 Sr / 86 The Sr ratios were compared to determine the habitat type of the finless porpoise during its formation, and then it was determined to be the Yangtze finless porpoise or the East Asian finless porpoise based on the habitat type.

2. A dentin-based method according to claim 1 87 Sr / 86 The method for accurately identifying Yangtze River finless porpoise and East Asian finless porpoise by Sr ratio is characterized by: In step 3), laser ablation coupled to a multi-collector inductively coupled plasma mass spectrometer was used to analyze the dentin 87 Sr / 86 Sr ratio analysis was performed using medium resolution mode. Laser energy density of 6.5 J / cm in laser ablation-connected multi-collector inductively coupled plasma mass spectrometry experiment 2 The spot diameter of the ablation was 64 μm, the frequency was 10 Hz, and the ablation speed was 5 μm / s. Before each laser ablation, the background intensity of each isotope was measured, and 200 cycles were measured. The average value was used for blank background correction during sample analysis. The apatite standard and modern marine shark teeth were measured at the beginning and end of each analysis and every 30 minutes during the analysis. 87 Sr / 86 Sr ratio to evaluate the finless porpoise tooth samples 87 Sr / 86 Accuracy and repeatability of Sr ratio.

3. A dentin-based method according to claim 1 87 Sr / 86 The method for accurately identifying Yangtze River finless porpoise and East Asian finless porpoise by Sr ratio is characterized by: In step 3), the Faraday cup configuration of the multi-collector ICP-MS was set to receive signals at m / z 82, 83, 83.5, 84, 85, 86, 86.5, 87, and 88. During data analysis, the "peak zero" method was used to correct for Kr interference by subtracting the average signal of the blank background from each analysis cycle; the signal at m / z 82 was received to monitor 42 Ca 40 Ar / 42 Ca 40 Ca peak; by monitoring 85 Rb signal calculated from isotope ratio 87 Rb, and 87 Rb / 85 The instrument mass fractionation calibration was performed for Rb. The calibration process assumed that Rb and Sr have the same mass fractionation factor. 88 Sr and 86 Sr signal, according to 88 Sr / 86 The isotopic abundance ratio of Sr in nature is 8.375209. The mass fractionation factor is calculated using the exponential law. According to the mass fractionation factor and 87 Rb / 85 The natural isotopic abundance ratio of Rb is 0.38567, and the corrected 87 Rb / 85 Rb, thus obtaining the corrected 87 Rb. Subtract the corrected 87 Rb, according to the correction 87 Rb / 85 Rb steps for deduction 87 After Rb 87 Sr / 86 The Sr ratio was corrected for instrument mass fractionation to obtain a measured value that was corrected for mass fractionation and interference.

4. A dentin-based method according to claim 1 87 Sr / 86 The method for accurately identifying Yangtze River finless porpoise and East Asian finless porpoise by Sr ratio is characterized by: In step 5), the CHANGEPOINT function in the statistical programming software R is used to 87 Sr / 86 The original detection value data of Sr ratio were analyzed to obtain the individual 87 Sr / 86 The point where the Sr ratio changes significantly, thus obtaining 87 Sr / 86 The Sr ratio does not change significantly in the stable stage, and the stable stage is calculated. 87 Sr / 86 The average value of Sr ratio; the average value is compared with the mainstream of the Yangtze River and the ocean 87 Sr / 86 By comparing the Sr ratios, we can determine the habitat type of the finless porpoise during its formation period, and then infer the species of the finless porpoise based on the habitat type.

5. The dentin-based method according to claim 1 87 Sr / 86 The method for accurately identifying Yangtze River finless porpoise and East Asian finless porpoise by Sr ratio is characterized by: Yangtze River mainstream 87 Sr / 86 The Sr ratio is 0.7100-0.7110, ocean 87 Sr / 86 The Sr ratio is 0.7092.

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