Human activities and climate change have changed the living environment of species, accelerated the rate of species extinction, and global biodiversity is facing a huge threat. The objective of this study is to determine the resource protection strategy of freshwater planarian by systematically exploring the population genetics and pedigree geography of the planarian population in the four mountain systems of Henan Province and Taihang Mountains.
Taking the Japanese planarian in the four mountain systems of Henan Province and Taihang Mountains as an example, DNA was extracted with the help of mitochondrial cytochrome c oxidase subunit I gene (COI), mitochondrial cytochrome b gene (Cytb), and nuclear gene internal transcribed spacer-1 (ITS-1), amplified by polymerase chain reaction and sequenced, and genetic diversity, genetic structure, and pedigree geographical pattern were analyzed by single-gene analysis and polygene joint analysis.
The corresponding length of Cytb, COI, and nuclear gene ITS-1 sequences in the four mountains of Henan Province is 308, 805, and 632 bp, respectively, and the nucleotide diversity and haplotype diversity are 0.00212 and 0.975, respectively. The gene length of ITS-1 and Cytb in Taihang Mountains is 668 and 315 bp, respectively, with a total length of 984 bp. There are 10 shared haplotypes and 36 unique haplotypes. The values of all nucleic acid diversity and haplotype diversity were 0.00156 and 0.965, respectively, and the genetic variation coefficients between populations and groups were 20.28% and 16.40%, respectively (P < 0.05). Different haplotypes of the four mountain systems in Henan Province and Taihang Mountains are scattered in different geographic populations, and there is no correlation between them and their geographic locations, which is consistent with the results of phylogeny.
The genetic diversity of Trionychia japonica population in the four mountain systems of Henan Province and Taihang Mountains shows high haploid diversity and low nucleotide diversity, and phylogenetic analysis has not found obvious pedigree geographical pattern.
In the current era of population growth, urbanization, rapid development of modern industry and agriculture, and increasingly serious environmental pollution, as well as in the context of global warming, drying up of water bodies, and accelerating tourism development, global biodiversity is facing a huge threat. In a large number of medical reports, planarian is used not only as a substitute model for screening chemical poisons, but also as a marker animal for monitoring water quality and as a research model for population genetics and pedigree geography. With the gradual improvement in new generation sequencing methods and molecular marker technology, the ideal model of planarian population genetics and systematic geography is becoming the focus of many medical researchers. Pedigree geography can investigate the genetic diversity and pedigree relationship of species from the perspective of genetics, providing reference for species protection [1,2,3]. The diffusion of individual organisms is an important factor affecting genetic differentiation among populations. In many different freshwater species, the genetic structure within their populations and the gene flow between populations have been studied. The evolutionary significance of gene flow is mainly reflected on the following two aspects: on the one hand, limited gene flow makes local adaptation and microevolutionary changes possible, thus increasing the fitness of local populations; on the other hand, when the population size is small, gene flow is a necessary condition to maintain gene variation. It can be observed that the degree of gene variation and differentiation between populations depends largely on the markers used. At present, pedigree geography is widely used to analyze the distribution pattern, migration, and diffusion of planarian. Mitochondrial genes and some nuclear gene fragments are now commonly used molecular markers for pedigree biogeography.
Maternal inheritance, simple structure, no gene recombination, fast evolution, large copy number, easy extraction, and other characteristics make mitochondrial genes an ideal marker for studying species pedigree geography and population genetic diversity [4,5]. Mitochondrial cytochrome c oxidase subunit I gene (COI) and mitochondrial cytochrome b gene (Cytb) are commonly used to study the genetic diversity of populations. When studying intraspecific and close interspecific relationships, the internal transcribed spacer-1 (ITS-1) of ribosome often has a high proportion of polymorphisms and information sites. Although there is no relevant report in China, studies in some European and American countries and regions have revealed the lineage differentiation model and possible lineage sources of different geographical populations to varying degrees. Henan Province is located in the middle and lower reaches of the Yellow River in the middle east of China and the south of the North China Plain, including the Taihang Mountains, the Taibei Mountains, the Tongbai Mountains, and the Funiu Mountains. Human activities and climate change have changed the living environment of species and accelerated the rate of species extinction. Global biodiversity is facing a huge threat. How to protect biodiversity has become a hot issue of general concern. Therefore, in order to prevent the extinction of species and the destruction of ecological integrity, the population genetics and phylogenetic geography of the four mountain systems in Henan Province and Taihang Mountains were investigated using genetic analysis methods to provide scientific guidance for the protection of freshwater planarian resources.
2 Experimental materials and research methods
2.1 Experimental materials
2.1.1 Sample collection
The population of Trionychia japonica from the four mountain systems of Taihang Mountain, Taibei Mountain, Tongbai Mountain, and Funiu Mountain in Henan Province was taken as sample data, with a total of 36 population types. Table 1 shows the locations of triangular planarian in the four mountain systems in Henan.
|Population code||Sampling location||Longitude and latitude||Altitude (m)||Collection date (year-month)|
|Qi||Qi County||35°43′ N/114°06′ E||206||2021-9|
|XW||Xiuwu County||35°28′ N/113°20′ E||490||2021-2|
|JY-1||Jiyuan City||35°15′ N/112°11′ E||791||2021-5|
|JY-2||Jiyuan City||32°51′ N/112°12′ E||532||2021-9|
|GS-1||Gushi County||31 °49′ N/115°44′ E||157||2021-8|
|GS-2||Gushi County||31947′ N/115*45′ E||211||2022-3|
|SC||Shangcheng County||31 °44′ N/115°28′ E||170||2021-8|
|Xin||Xinxian County||31°37′ N/114°49′ E||210||2021-9|
|XY-1||Xinyang City||31°49′ N/114°3′ E||180||2022-3|
|XY-2||Xinyang City||31°59′ N/113°51′ E||170||2022-1|
|TB-1||Tongbai County||32°21′ N/113°23′ E||160||2021-10|
|TB-2||Tongbai County||32°24′ N/113°16′ E||230||2021-7|
|WG||Wugang City||33°11′ N/113°36′ E||190||2021-8|
|SP||Suiping County||33°11′ N/113°40′ E||150||2021-6|
|FC||Fangcheng County||33°22′ N/1 12956′ E||300||2021-6|
|NZ||Nanzhao County||33°19′ N/112°1 7′ E||350||2021-6|
|NX||Neixiang County||33°22′ N/1 11°54′ E||430||2021-9|
|XiX||Xixia County||33°31′ N/1 11°38′ E||530||2021-7|
|LuanC||Luanchuan County||33°56′ N/1 11°43′ E||430||2021-8|
|LuS||Lushan County||33°46′ N/112°19′ E||190||2021-9|
Table 2 shows the location of triangular planarian in the Taihang Mountains. Each sample point has more than 20 samples, with an average of 22. The collected planarians were placed in a collecting bottle and fed in a 13°C incubator. They were fed with fresh beef liver homogenate once a week, and the water was changed every 2–3 days after feeding. The experiment required the living planarian to be hungry for more than 7 days.
|Population code||Sampling location||Longitude and latitude||Altitude (m)||Collection date (year-month)|
|Qi||Qi County, Henan Province||35°43′ N/114°06′ E||206||2021-6|
|XW||Xiuwu County, Henan Province||35°28′ N/113°20′ E||490||2021-9|
|JY-1||Jiyuan City, Henan Province||35°15′ N112°11′E||791||2021-7|
|JY-2||Jiyuan City, Henan Province||35°25′ N112°12′ E||532||2021-8|
|XT-1||Xingtai City, Hebei Province||37°20′ N113°54′ E||1,055||2021-9|
|XT-2||Xingtai City, Hebei Province||37°05′ N113°47′ E||774||2022-3|
|Y||Yi County, Hebei Province||39°23′ N/115°15′E||433||2021-8|
|QY||Quyang County, Hebei Province||38°56′ N114°33′E||71||2021-9|
|LaiS||Laishui County, Hebei Province||39°39′ N/115°23′ E||430||2022-3|
|LingS||Lingshou County, Hebei Province||38°41′ N113°51′E||35||2021-9|
|PShan||Pingshan County, Hebei Province||38°15′ N113°43′ E||20||2021-2|
|FP||Fuping County, Hebei Province||38°55′ N/113°48′ E||840||2021-5|
|LQ||Lingqiu County, Shanxi Province||39°20′ N114°18′ E||1,391||2021-9|
|WT||Wutai County, Shanxi Province||38°54′ N113°38′ E||980||2021-8|
|Yu||Yu County, Shanxi Province||38°12′ N113°19′ E||970||2021-6|
|ZQ||Zuoquan County, Shanxi Province||37°00′ N113°29′ E||813||2021-6|
|PShun||Pingshun County, Shanxi Province||36°12′ N/113°36′ E||622||2021-7|
|HG||Huguan County, Shanxi Province||35°55′ N113°35′ E||575||2021-10|
|LC||Lingchuan County, Shanxi Province||35°32′ N113°26′ E||206||2021-8|
|YC||Yangcheng County, Shanxi Province||35°14′ N/112°26′E||490||2022-3|
Figure 1(a) and (b) shows the population distribution map of the Japanese planarian in the four mountain systems of Henan Province and Taihang Mountains, respectively.
2.1.2 Experimental reagents and instruments
The main reagents used in the experiment are Tris-balanced phenol, TAE buffer of Shanghai Baogui Biotechnology Co., Ltd, Merck KGaA Protease K, agarose Beijing Solabo Technology Co., Ltd, DNeasy Blood & Tissue Kit, 10× loading buffer, sodium dodecyl sulfate, Tris, 2× Taq Plus Master Mix, ethylenediaminetetraacetic acid, absolute ethanol, amyl alcohol, chloroform, gentamycin, and ethidium bromide. The main instruments used in the experiment are the SPH-250 thermostatic biochemical incubator of Shanghai Jinghong Experimental Equipment Co., Ltd, the electronic balance of Shanghai Tianping Instrument Technology Co., Ltd, the vertical high-pressure sterilizer of Beijing Wuzhou Dongfang Technology Development Co., Ltd, the electric blast drying oven of Shanghai Yiheng Scientific Instrument Co., Ltd, the polymerase chain reaction (PCR) instrument of Germany Biometra, PHS-25 precision pH meter of Shanghai Precision Scientific Instrument Co., Ltd, DYCP-31A agarose gel electrophoresis model of Beijing Dayi Biotechnology Co., Ltd, NanoDrop 2000 micro spectrophotometer of Thermo Company of the United States, and SE-93 automatic dual pure water distiller of Shanghai Yarong Biochemical Instrument Factory. The test process followed the manufacturer’s recommendations.
2.2 Research methods
2.2.1 Extraction of genomic deoxyribonucleic acid (DNA)
In the study, genomic DNA was extracted by the DNeasy Blood & Tissue Kit. First, 25 and 35 mL of absolute ethanol were added into AW1 and AW2 buffer solutions, respectively, and a single planarian with a length of about 1.5 cm was washed three times with double distilled water and placed in a 1.5 mL centrifuge tube. Then 20 μL of pyruvate kinase and 200 μL of Buffer ATL were added, and the centrifuge tube was placed in liquid nitrogen for 10 s. The planarian tissue was fully crushed and shaken in the centrifuge tube, and then 200 μL of Buffer ATL was added. The mixture was vortexed and 200 μL of absolute ethanol was added to the vortex again. Then the mixture was moved into a 2 mL collection tube and centrifuged for 1 min. The collection tube and the discard tube were discarded.
2.2.2 Primer synthesis, PCR amplification, and sequencing
The molecular markers selected in the study were mitochondrial gene Cytb, COI, and nuclear gene ITS-1. Reference for amplification primer of ITS-1 can be found in previous studies [6,7]. The amplification primers of Cytb and COI were designed by referring to primers data. Table 3 shows the primers needed, which were synthesized by Shanghai Sangon Biotechnology Co., Ltd.
|Molecular marker||Primer sequence||Annealing temperature/°C||Amplification length/bp|
The most all volume of PCR reaction is 30 μL, and the amplification reaction system is 1 μL of DNA, 10 μmol/L of positive reaction primer, respectively 1 μL, 15 μL of TaqMix, 12 μL of sterilized double distilled water. The PCR procedure is as follows: pre-denaturation at 90°C for 5 min, denaturation at 94°C for 40 s, annealing at different primer temperatures for 30 s, and extension and re-extension at 72°C for 1 and 10 min, respectively. Detection and sequencing of PCR products are performed as follows: 0.2 g agarose and 1× TAE buffer solution are taken in a triangular flask, shaken evenly, placed in a microwave oven, and heated until bubbles appear on the page. It is then cooled to 50°C, and ethidium bromide dye is added. It is again cooled into agarose gel and placed in an electrophoresis tank. After electrophoresis is completed, the tailing and clarity of the detection strip are verified. When the obtained product band is the target band, the amplified product has to be returned to the company for sequencing.
2.3 Data processing and analysis methods
2.3.1 Sequence splicing and comparison
The sequencing results of PCR products are spliced and edited by DNAStar software, and the National Biotechnology Information Center determines whether they are target sequences. Cytb and COI genes can judge whether the spliced sequence can be translated into amino acid normally through the online software EMBOSS Transeq. If it can be translated normally, the sequence can be considered to be used normally; otherwise, the sequence will be detected again after reverse complementary operation through the program. After the sequence detection is confirmed to be correct, it is completely compared and cut using the MEGA 7.0 software in the fast format to obtain sequence fragments with the same length. The results after comparison are exported in the fast format.
2.3.2 Population haplotype distribution and genetic analysis
The haplotype was defined by the DNASP5.0 software, and the haplotype diversity and nucleotide diversity were calculated at the same time. Molecular variation analysis was completed using the Arlequin software, and differentiation coefficient F ST between populations was estimated. The neutral test was completed by the DNASP5.0 software to estimate Tajima’s D and Fu’s Fs values.
2.3.3 Haplotype phylogeny and network analysis
Phylogenetic tree analysis selected Dugesia ryukyuensis, a sister species of the Japanese planarian, Ryukyu, as an outgroup pair, based on the maximum parsimony (MP) method, Bayesian inference (BI), and maximum likelihood (ML) to construct MP trees, BI trees, and ML trees, respectively. Using the program jModeltest v2.1.7, the best model of nucleotide substitution for each phylogenetic tree was obtained according to the Akaike information standard. The haplotype network diagram was constructed by the median connection method in Network 10.2.0.0, and then the evolutionary relationship among haplotypes was analyzed. P < 0.05 and P < 0.001 were statistically significant and significant, respectively.
3.1 DNA extraction and PCR amplification results
Figure 2 shows the PCR amplification results of three molecular markers by agarose gel electrophoresis. The bands of the three populations are clear and free of tailing. The size of the bands is the same as the theory. It can be concluded that they are all target bands, and they can be sequenced in two directions later. The samples of Figure 2(a)–(c) are from QY, SC, and SP, respectively.
3.2 Genetic diversity of the population
A total of 116 mitochondrial gene Cytb, COI, and nuclear gene ITS-1 sequences were obtained from 20 planarian populations in the four mountains of Henan Province. The corresponding lengths of the three genes were 308, 805, and 632 bp, respectively, and the total gene length was 1,745 bp. The nucleotide diversity and haplotype diversity of 116 combined gene sequences were 0.00212 and 0.975, respectively, including 5 shared haplotypes (H1, H21, H55, H67, H69) and 82 exclusive haplotypes (H2–H20, H22–H54, H55–6–H66). Among them, H55 is the most widely distributed area, which is concentrated in six populations, namely JY-1, Lus, LuanC, XiX, NZ, and FC. The value of nucleic acid diversity was 0.00023–0.00314, and the values of NX and Lus were the highest and lowest, respectively. Except Qi, Lus, and LuanC, the haplotype diversity of the remaining 17 geographical regions was 0.900–1.000, and the specific results are shown in Table 4.
|Population code||Number of samples||Number of haplotypes||Haplotype||Haplotype diversity||Nucleotide diversity||Tajima’s D||Fu’s Fs|
|GS-1||6||5||H1, H2, H3, H4, H5||1.000||0.00204||0.372||0.307|
|GS-2||5||4||H1, H6, H7, H8, H9, H10||1.000||0.00257||0.414||0.272|
|SC||6||6||H11, H12, H13, H14||1.000||0.00092||−1.284**||−1.362*|
|Xin||9||9||H15, H16, H17, H18, H19, H20, H21, H22, H23||1.000||0.00287||−0.408||−0.079|
|XY-1||5||5||H24, H25, H26, H27, H28||1.000||0.00315||0.185||0.129|
|XY-2||5||5||H21, H29, H30, H31||1.000||0.00122||0.657||0.665|
|TB-1||6||4||H32, H33, H34, H35, H36||I.000||0.00179||0.510||0.650|
|TB-2||4||4||H37, H38, H39, H40||0.952||0.00301||−0.871||−0.948|
|WG||5||5||H41, H42, H463, H44, H45||0.644||0.00090||1.335||1.509|
|SP||4||3||H46, H47, H48, H49, H50, H51, H52, H53, H54||0.933||0.00295||0.899||1.325|
|FC||9||9||H55, H56, H57||1.000||0.00267||−0.874||−0.940*|
|NZ||5||5||H55, H58, H59, H60, H61||0.900||0.00253||0.074||0.171|
|NX||5||5||H62, H63, H64, H65, H66||1.000||0.00279||−0.395||−0.324|
|XiX||10||4||H55, H67, H68, H69, H70||0.933||0.00292||−1.78||−0.465|
|LuanC||7||5||H55, H67, H69, H71||0.900||0.00121||0.635||1.579|
|Qi||4||6||H73, H74, H75||0.978||0.00150||−0.446||−0.410|
|XW||6||3||H76, H77, H78, H79, H80, H81||1.000||0.00058||0.591||1.009|
|JY-1||6||4||H55, H67, H82, H83||1.000||0.00278||1.031||1.345|
|JY-2||4||4||H84, H85, H86, H87||1.000||0.00024||−0.854||−0.905|
Note: * refers to P < 0.05 and ** refers to P < 0.001.
A total of 120 mitochondrial Cytb and nuclear gene ITS-1 sequences and 116 COI sequences were obtained from 20 populations in Taihang Mountains. According to the joint analysis of ITS-1–Cytb, the gene length of ITS-1 and Cytb is 668 and 315 bp, respectively, with a total length of 984 bp. There are 10 shared haplotypes and 36 unique haplotypes, respectively. The values of all nucleic acid diversity and haplotype diversity were 0.00156 and 0.965, respectively. Table 5 refers to the specific results of ITS-1–Cytb sequence in Taihang Mountains. The length of COI sequence is 789 bp, including 24 unique haplotypes and 8 shared haplotypes. The values of all nucleic acid diversity and haplotype diversity were 0.920 and 0.00082, respectively. Range of nucleic acid diversity is 0-00112, XT-2, Yu, Qi, FP are the highest and lowest respectively.
|Population code||Number of samples||Number of haplotypes||Haplotype||Haplotype diversity||Nucleotide diversity||Tajima’s D||Fu’s Fs|
|LaiS||5||3||H1, H5, H6||0.833||0.00093||−1.214||−1.251|
|Yi||7||2||H2, H3, H4||0.803||0.00130||−0.744||−0.593|
|LingS||12||5||H2, H5, H9, H10, H11||0.600||0.00032||−0.920||−1.109|
|XT-1||4||4||H14, H15, H16, H17||0.476||0.00185||0.598||0.965|
|XT-2||7||3||H9, H18, H19, H20||0.700||0.00194||0.050||0.461|
|Qi||4||4||H5, H6, H7, H8||0.714||0.00061||−1.748**||−2.023**|
|XW||9||8||H21, H22, H23, H24, H25, H26, H27, H28, H29||0.607||0.00001||0.864||1.002|
|JY-1||4||5||H30, H31, H32, H33||0.833||0.00183||0.578||0.925|
|JY-2||4||3||H16, H34, H35||0.700||0.00147||−0.874||0.926|
|LQ||8||4||H5, H6, H11||1.000||0.00127||0.675||0.687|
|ZQ||11||4||H8, H30, H37, H38||0.658||0.00146||−2.248**||−3.146**|
|PShun||6||4||H16, H39, H40, H41, H42||1.000||0.00104||−0.865||−0.902|
|LC||7||3||H6, H45, H46||0.833||0.00161||−1.273*||−1.387**|
Note: * refers to P < 0.05 and ** refers to P < 0.001.
3.3 Population genetic structure and genetic differentiation
According to the geographical region, the four mountain systems in Henan Province are divided into three species groups: the north, the middle, and the south. The molecular variation analysis shows that the F ST value is 0.368, and the difference between populations is statistically significant (P < 0.05). The genetic variation coefficients between populations and groups were 20.28% and 16.40%, respectively. The range of F ST value of ITS-1–Cytb–COI was −0.095–0.9972, and 190 pairs of population showed differences (P < 0.05). The variability between Lus and Qi populations and between Qi and JY-2 populations was the largest and smallest, respectively, with F ST values of 0.997 and 0.095 (P < 0.05). Table 6 refers to the F ST values of ITS-1–Cytb–COI of the four mountain systems in Henan.
|XW||0.544*||0.324*||0.118||0.401||0.516*||0. 140||0388 *||0.142||0.361*||0.360*||0.264*||0.178||0.273*||0.156||0.178||0.375*||0.527*||0.000||—||—|
Note: * refers to P < 0.05.
The F ST value of COI in Taihang Mountains was 0.480, with significant statistical significance (P < 0.01). According to its geographical location, it can be divided into two subgroups, namely, north and south subgroups. The genetic variation between groups and within populations was 49.25 and 52.31%, respectively. Table 7 refers to the F ST values of ITS-1–Cytb in Taihang Mountains. The F ST value in this area was 0.404, and the difference was statistically significant (P < 0.01). The genetic variation among groups, between populations, and within populations was 3.94, 36.54 and 59.52%, respectively. The range of F ST value of ITS-1–Cytb was −0.292–0.926190, among which 118 pairs showed differences (P < 0.05). XT-1 and JY-1 and PShan and HG had the smallest and largest genetic variation, respectively.
Note: * refers to P < 0.05.
3.4 Haploid phylogeny and haplotype network analysis results
The haplotype MP tree and BI number were reconstructed from the combined sequences of three genes in the four mountains of Henan Province, and the topological structures of the two systems were consistent. A total of 87 haplotypes formed 6 haplotype groups, mainly concentrated in 1–3 lines of a total of 5 lines. Branch 1 includes 28 haplotypes of 10 geographic populations; Clade 2 includes 17 haplotypes of 12 geographic populations; clade 3 includes 29 haplotypes of 17 geographic populations. Figure 3 refers to the haplotype network of the four mountain systems in Henan. Since they are scattered in different geographic populations, they should show a correlation with the geographic location.
The haplotype MP tree and BI number were reconstructed from the COI–Cytb joint sequence in Taihang Mountains, and the topological structures of the two systems were consistent. A total of 46 haplotypes formed 3 haplotype groups, mainly concentrated in 1 line of a total of 3 lines. Figure 4 refers to the haplotype network diagram of Taihang Mountains. The results are consistent with those of phylogeny.
Because of its unique biological characteristics, experiments on planarian are applicable to experimental animals in many fields. It has a strong regenerative capacity and is praised as an “immortal animal” by the biological community. Asian countries often use planarian japonica as a model animal to complete stem cell regeneration, development, and other research. European and American countries usually use Mediterranean planarian as a model animal for relevant research in these fields. A planarian can grow or regress to its own according to its own nutritional status and has advantages in building a good model of autophagy . In addition, planarian has extremely high sensitivity to harmful substances and low indoor-feeding cost, so it is also considered as an ideal model animal in toxicology research.
It is believed that a clear and separate band of gene DNA of planarian was obtained by electrophoresis detection, indicating that the DNA extraction method with DNeasy Blood & Tissue Kit has high purity and integrity, with ideal extraction performance. However, researchers believe that the improved phenol chloroform method has a good effect on DNA extraction. The research results also found that the corresponding length of the three genes, Cytb, COI, and nuclear gene ITS-1 sequences in the four mountains of Henan Province, were 308, 805, and 632 bp, respectively, and the nucleotide diversity and haplotype diversity were 0.00212 and 0.975 respectively. The gene length of ITS-1 and Cytb in Taihang Mountains is 668 and 315 bp, respectively, with a total length of 984 bp. There are 10 shared haplotypes and 36 unique haplotypes, respectively. The population of the four mountain systems and Taihang Mountains in Henan Province may have experienced a genetic bottleneck, and then the population grew rapidly, but the population expansion period was short, and the accumulation of nucleotide variation was insufficient. A few domestic scholars have studied the population genetics and pedigree geography of species of Mediterranean planarian, namely, the model species for regeneration and development research. Leria et al. analyzed the genetic variation and population parameters of 11 populations of Mediterranean planarian based on mitochondrial gene COI, YB, and nuclear gene N13. The results show that the Mediterranean planarian forms an obvious pedigree pattern in its entire distribution area, namely, three branches in the west, middle, and southeast, and no haplotype is shared among the branches, thus confirming the hypothesis that the current distribution pattern of this species is formed by paleogeological events, and also suggesting the ancient Mediterranean planarian species [9,10,11]. Contrary to the local distribution of Mediterranean planarian, Dugesia sicula is the only worldwide species of the genus Tricerata in the Mediterranean region, and most of its populations are asexual populations that undergo fission reproduction. Researchers such as Harrath et al. evaluated 58 D the COI haplotype and nucleotide diversity of icula population, showed that the haplotype did not show geographical distribution [12,13,14]. The single-gene and joint-gene analysis results of the Japanese triangle planarian in the Taihang Mountains showed that the haplotype diversity of Yu population is 0. The reason may be that the terrain of this area is unique. Yu is located in the southwest of Zhangjiakou City, Hebei Province, at the junction of Mount Hengshan, Mount Taihang, and Mount Yanshan. Due to the development of the Taihang Mountain’s scenic area in recent years, human interference may be of serious concern, limiting the migration ability of the Japanese triangle planarian. The genetic diversity of the population is low [15,16,17]. In the study of the population of Japanese planarian in the four mountain systems of Henan Province, the haplotype phylogenetic tree based on the joint sequence of mitochondrial genes Cytb, COI, and nuclear gene 1ITS-I was basically consistent with the haplotype network diagram [18,19,20]. Similarly, in the study of the population of the Japanese planarian in the Taihang Mountains, the haplotype network diagram based on the COI single-gene sequence and the ITS-I–Cytb joint character sequence showed a genetic structure similar to the corresponding haplotype phylogenetic tree. The haplotype network relationship can directly see the evolutionary relationship between haplotypes. The haplotypes of each population of the four mountain systems in Henan Province and the Taihang Mountains are scattered among different geographic populations, and the haplotype topology does not show the distance relationship corresponding to the geographic location of the population. It shows that there is no obvious pedigree geographical pattern of planarian in the four mountain systems and different geographic populations in Taihang Mountains of Henan Province [21,22,23].
Scheel and other researchers used low-diffusion terrestrial planarian C. Bergi to conduct another comprehensive pedigree geography study. They found that most of the sampled areas have high genetic diversity, and the pedigree of common ancestors can be traced back to the Pleistocene. The values of all nucleic acid diversity and haplotype diversity were 0.00156 and 0.965, respectively. The genetic variation coefficients between populations and groups were 20.28% and 16.40%, respectively. The range of F ST value of ITS-1–Cytb–COI is −0.095–0.99712, and 190 pairs of the population showed differences. The F ST value of ITS-1–Cytb in Taihang Mountains is 0.404, and the difference is statistically significant. The genetic variation between groups, between populations, and within populations is 3.94, 36.54, and 59.52%, respectively. The range of F ST value of ITS-1–Cytb is −0.292–0.926190, and 118 pairs of the population showed differences. The haplotype–network relationship can directly see the evolutionary relationship between haplotypes. Different haplotypes of the four mountain systems in Henan Province and the Taihang Mountains are scattered in different geographic populations, and there is no correlation between the geographic location of and. This result is consistent with the result of phylogeny [24,25]. Species diffusion, geographical isolation, and recent human activities will affect the population structure of species, while geological and agricultural morphological changes such as mountains, plateaus, and basins, bottleneck effects, and other factors will also affect the genetic structure or population of species [26,27,28]. From Dabie Mountain to Tongbai Mountain and then to Funiu Mountain in Henan Province, from southeast to northwest, there are several mountains and rivers, forming a barrier of special geographical environment. Aquatic organizations, especially freshwater aquatic organizations, there are often obvious geographical barriers between different water systems and different geographic populations, and the relatively significant genetic differentiation between populations is usually related to different degrees of isolation, because these isolation will restrict the gene exchange between populations. The Dabie Mountains, Tongbai Mountains, and Funiu Mountains in Henan Province have complex landforms, and the geology has gone through multiple crustal movements and long-term denudation and accumulation, which may cause significant genetic differentiation among populations. Since the Cenozoic, the Taihang Mountains have also experienced multiple geological movements, forming a variety of highly heterogeneous features such as terrain, soil, climate, and vegetation. The unique and complex terrain has also produced isolation effect, which may hinder the genetic exchange between different geographic populations in Taihang Mountains, thus increasing the genetic variation among different populations and promoting the high differentiation of population genes. In addition, the genetic variation of species with strong diffusion ability among different geographic populations is small [29,30]. As a benthic animal, the Japanese planarian moves slowly in the freshwater environment where it lives, which makes it impossible to spread far away from the mouth. Therefore, the weak diffusion ability may be another reason for the great genetic differentiation of the Japanese planarian in the Dabie Mountains, Tongbai Mountains, Funiu Mountains, and Taihang Mountains of Henan Province. The four mountain systems in Henan and Taihang Mountains may be experiencing population decline, although not very significant. Regional climate type, terrain, natural environment, human activities, and other factors will affect the species diversity and population richness of a region. As a species of freshwater planarian, the Japanese planarian mainly lives in freshwater environments such as springs, streams, and lakes. In recent years, with the rapid development of mountain tourism, the freshwater environment where the planarian lives has been polluted or artificially destroyed. In addition, the drying up of water bodies in the context of global warming may be another direct reason for the decline of the population of Japanese planarian in Henan and Taihang Mountains. At the same time, the genetic diversity, population structure, and population history of Rhododendron taihang and the population analysis based on mitochondrial nuclear sequence: the research report on the reason for the decline of Rhododendron taihang in China pointed out the relationship between species and gene evolution, which further demonstrated that gene analysis contributed to the way and type of species change.
The genetic characteristics of the population of Triticollis japonicus in the four mountain systems of Henan Province and the Taihang Mountains show high haploid diversity and low nucleotide diversity. Triticollis japonicus may have experienced rapid population growth, but the expansion history is short and the accumulation of nucleotide variation is insufficient. Most populations have significant genetic differentiation, but phylogenetic analysis has not found obvious pedigree geographical pattern. This neutral experiment showed that population decline of the planarian might occur in the four mountain systems of Henan and Taihang Mountains. Relevant responsible departments need to take effective measures to reduce human interference and environmental pollution and strengthen the protection of Trionychia japonica populations.
Funding information: There is no funding for this article.
Conflict of interest: The authors declare that they have no competing interest.
Data availability statement: The data used to support the findings of this study are available from the corresponding author upon request.
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