Genetic diversity and structure assessment of Macrobrachium nipponense populations: implications for the protection and management of genetic resources

– This article presents a study of D-loop sequences to characterize the genetic diversity of wild Macrobrachium nipponense populations in Yixing natural waters including two reservoirs (Hengshan reservoir, HS; Youche reservoir, YC), 3 brooks (Linjin Dang, LJD; Magong Dushan Dang, MDD; Yangshan Dang, YSD) and 3 shallow lakes (Dongjiu lake, DJ; Xijiu lake, XJ; Tuanjiu lake, XJ), and compared the genetic differentiation and population structure with wild populations of Taihu Lake (TH), Yangtze River (YZ), and the main local arti ﬁ cially bred varieties “ Taihu No. 2 ” (TH-2). A 747 bp D-loop sequence fragment was ampli ﬁ ed in 321 individuals and the results exhibited a higher content of A þ T (80.03%) than C þ G (19.97%). A total of 110 haplotypes were identi ﬁ ed. The h and p value proved the diversity of these populations was at the same level with high genetic diversity. TH-2 and YZ showed remarkable diversity, and XJ is even better. Fst estimates suggested that YZ and TH-2 were signi ﬁ cant differentiation with other Yixing populations ( P < 0.05). Three populations from shallow lake (DJ, XJ and TJ) displayed signi ﬁ cant differentiated with the left Yixing ones ( P < 0.05). The pairwise genetic distance, as well as haplotype network results, also suggested that all these 11 populations did not diverge at the species level ( < 15%). The P values of Tajima ’ s D and Fu Fs were relatively greater than 0.1 ( P > 0.1) and the nucleotide mismatch distribution analysis showed multiple peaks, giving a conclusion that the populations did not exhibited expansion. All these results suggested that TH-2 and YZ have remarkable diversity, and the germplasm resources and genetic diversity of M. nipponense in Yixing are very good and are suitable for original materials of breeding.


Introduction
Macrobrachium nipponense, commonly known as oriental river prawn or river shrimp, is an economically important indigenous prawn species in China, widely distributed in lakes, rivers, reservoirs, and other freshwater water areas throughout the country (Fu et al., 2004). Because of its fast growth, high adaptability, and strong reproductive capacity, this species has been continuously bred since the late 1950s, especially in the lower reaches of the Yangtze River and Taihu Lake basin.
However, due to natural and anthropogenic factors, the wild M. nipponense populations are currently surviving in smaller habitat areas; they are reducing and catches are declining (Fu et al., 2018). In 2001, the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences set up a scientific research team to carry out research on the improvement of prawn varieties. After years of efforts, two new prawn varieties were successfully cultivated: the hybrid prawn 'Taihu No. 1' and prawn 'Taihu No. 2'. Compared to the local wild prawn populations, individual specifications of both strains had been significantly improved. Its growth rate increased by more than 30% and the average yield per mu increased by 25% (mu, Chinese unit of landmeasurement that is commonly 666.7 square meters). the 'Taihu No. 2' showed better specifications than 'Taihu No.1'and has been cultured on a large scale and promoted in Jiangsu, Anhui, Zhejiang, Sichuan, Tianjin, Hubei, and other provinces (cities). However, the market is still short of supply. In 2019, the Chinese government implemented a comprehensive strategy to step up the conservation of the Yangtze River, and subsequently a fishing ban was imposed in the Taihu Lake basin. These measures are of great significance for the conservation of the wild M. nipponense germplasm resources and sustainable development of aquaculture.
In recent studies, a number of molecular markers, such as SSR (simple sequence repeats) and COI (cytochrome c oxidase subunit I), were applied to analyze wild M. nipponense populations in different areas of China (Chen et al., 2015(Chen et al., , 2017Ma et al., 2012;Qiao et al., 2013). Yixing city is located in the lower reaches of the Yangtze River in Jiangsu Province, on the west bank of Taihu Lake. The city's natural water system is very developed and diverse, and it includes a number of reservoirs, brooks, and shallow lakes that are connected with Taihu Lake. The excellent geographical environment is perfect for the survival of prawns, but to date no studies have been conducted on the genetic characteristics of the local wild prawn population in Yixing.
The mitochondrial DNA (mtDNA) D-loop sequence is widely used in population genetic analysis because it does not encode proteins and is not affected by selection (Liao et al., 2016;Maltsev et al., 2015). In the present study, wild M. nipponense populations were sampled from eight different natural water bodies in Yixing city, including reservoirs, brooks, and shallow lakes, and D-loop sequences were employed to examine their genetic diversity. Then, the genetic differentiation and structure of these populations were compared with those of wild populations from Taihu Lake, Yangtze River, and the main local artificial strain 'Taihu No. 2'. The results of this study may contribute to improving the germplasm resources of wild M. nipponense populations in China and provide reference data to assist in the formulation of policies for the species' breeding program.

Ethics statement
The study was approved by the Animal Care and Use Ethics Committee in the Freshwater Fisheries Research Center (Wuxi, China).

Sample collection
Wild samples of M. nipponense were collected from eight different natural water bodies in Yixing city, consisting of two reservoirs (Hengshan reservoir, Youche reservoir), three brooks (Linjin Dang, Magong Dushan Dang, Yangshan Dang), and three shallow lakes (Dongjiu lake, Xijiu lake, Tuanjiu lake) from February to March 2021. Wild prawns from the Yangtze River in Zhenjiang city and Taihu Lake in Wuxi city were also sampled. Individuals from the main local artificially bred strain 'Taihu No. 2' were obtained from the Dapu scientific research test base of the Freshwater Fisheries Research Center at the Chinese Academy of Fishery Sciences in Yixing. The detailed information on the samples is included in Table 1 and the locations are shown in Figure S1. In total, 35 individual prawns were sampled for each population. Muscle tissue samples were stored in 95% ethanol.

Total DNA extraction, PCR amplification, and sequencing
About 50 mg of muscle was sampled from each individual and was used to extract total DNA following the protocol  . PCR products were detected by electrophoresis and fragments were sequenced with an ABI3730 automated sequencer (Invitrogen Biotechnology Co., Ltd, USA).

Data analysis
BioEdit version 7 (Hall, 1999) was used to edit and align the D-loop sequences. Nucleotide diversity (p) and haplotype diversity (h) were estimated using ARLEQUIN 3.5 (Excoffier and Lischer, 2010), and genetic distances were estimated in MEGA 7 (Kimura, 1980). The analysis of molecular variance model (AMOVA) was used to estimate genetic variation following 1000 permutations in ARLEQUIN 3.5. Pairwise genetic differentiations (F ST ) were calculated with 10,000 permutations in ARLEQUIN 3.5 and the false discovery rate (FDR) was implemented using the method of Benjamini and Hochberg (1995). The best model to approximate sequence evolution was estimated in MEGA 7 (Kumar et al., 2016;Tamura et al., 2011). Evolutionary analyses were conducted using the maximum likelihood method in MEGA 7 based on the Tamura-Nei model (Kumar et al., 2016). Bootstrap values were based on 1000 rapid bootstrap replicates. The median-joining method in POPART was used to build a haplotype network (Leigh and Bryant, 2015). The neutrality test values were calculated in ARLEQUIN 3.5 (Excoffier and Lischer, 2010;Fu, 1997;Tajima, 1989). The mismatch-distribution analysis was carried out in DnaSP v5 (Librado and Rozas, 2009).

Base composition and variation analysis of the D-Loop region sequences
The D-Loop region sequences of M. nipponense were amplified in 385 individuals from 11 populations. Through alignment analysis, incomplete and invalid sequences were removed and 720-bp-long sequences obtained from 321 individuals were retained for further analysis. The YZ and TH-2 populations had the most variable sites, while the HS and YC populations had the least variable ones (Tab. 2). The mean basic nucleotide composition in the obtained sequences was as follows: T = 36.28%, T, A = 43.75%, C = 10.75%, and G = 9.22%. The A þT and C þ G contents were estimated at 80.03% and 19.97%, respectively (Tab. S1).
A total of 110 haplotypes were observed from the 321 individuals examined (Table S2, accession number: OP972608-OP972717). We found no insertion/deletion (indel) polymorphisms. Among these 110 haplotypes, the least unique were detected in the HS population (Hap17, Hap19, and Hap21). The YZ population had the highest number (20) of unique haplotypes (Hap91-Hap110). The remaining haplotypes were shared by two or more populations. The additional details about distribution of haplotypes were listed in Table S2.

Population genetic structure analysis
The genetic diversity parameters obtained are presented in Table 2 Based on the AMOVA, 16.46% of the total genetic variation was attributed to genetic differences among populations and 83.54% to variation within populations (Tab. 3). The F ST value was high (F ST = 0.165) and significant (P-value < 0.000). The population pairwise F ST results after FDR testing are shown in Table 4. The F ST values between populations varied from À0.0111 (between the HS and TH populations) to 0.469 (between the YZ and TJ populations). The results indicated that the genetic differentiation between YZ and other populations was statistically highly significant (P < 0.05). The TH population was significantly differentiated from all the other populations (P < 0.05) except for MDD (P > 0.05). TH-2 was significantly differentiated from the XJ, TJ, TH, and YZ populations (P < 0.05). Among the Yixing populations, TJ was genetically differentiated from HS, YC, LJD, MDD, and YSD (P < 0.05); XJ was genetically differentiated from HS, YC, LJD, and MDD (P < 0.05); and DJ was genetically differentiated from HS and YC (P < 0.05). No obvious differentiation was detected in the remaining pairwise combinations of populations (P > 0.05).
The genetic distance among the 11 populations ranged from 0.011 to 0.044 (Tab. 5). The largest value was observed between YZ and TH-2 (0.044), which was higher than the values among the other nine populations (all below 0.20). Moreover, the genetic distance between YZ and the other nine populations was above 0.032, and that between TH-2 and the other nine populations was between 0.028 and 0.031. The genetic distance between the TJ and XJ populations was the smallest (0.011). The clustering analysis results (Fig. 1) showed the eight Yixing populations were grouped with TH and TH-2 in a main branch, while the YZ population clustered separately into a single branch. The haplotype network obtained from the comparison of all sequences displayed no Notes: Pairwise F ST , below the diagonal; significance of corresponding P-values (above the diagonal) based on pairwise differences in the concatenated mtDNA sequences. *: P < 0.05 as per Benjamini & Hochberg (1995) false discovery rate correction.  (Fig. 2). Haplotypes did not cluster according to the classification of populations, which showed that individuals of different groups interlaced with each other to form complex clusters.

Population dynamics analysis
The neutrality tests showed that most Tajima's D values were negative except in HS, YC, and TH-2, while Fu's Fs values were negative in LJD, DJ, XJ, TJ, and YZ. Overall, the P values were relatively greater than 0.1 (P > 0.1), which indicated that the neutrality tests values were not significant (Tab. S3). The mismatch distribution analysis showed multiple peaks, suggesting that the population size remained relatively stable and there was a demographic equilibrium in all populations (Fig. 3).

Discussion
Within the context of the Yangtze River conservation and Taihu Lake fishing ban, the protection and appropriate use of the elite local germplasm resources are of great significance for the sustainable and efficient development of the prawn aquaculture industry. M. nipponense is an excellent local prawn species and its farming has become an important way to increase agricultural efficiency and the income of farmers in China (Fu et al., 2018). Therefore, it is very important to identify local populations with superior qualities and protect them to ensure the sustainable development of the M. nipponense seed industry, breeding, and aquaculture.
Some reports have been published on the genetic diversity of M. nipponense in China, mainly based on SSR and COI markers. SSR was employed in the genetic diversity analysis of populations in the Yellow River and Qiandao Lake, and showed that the wild populations had a high genetic diversity (Ma et al., 2012;Qiao et al., 2013). Mitochondrial DNA COI and 16SrRNA fragments were used in a study on the genetic structure of a wild population from Taiwan and showed that population expansion occurred recently (Chen et al., 2015(Chen et al., , 2017. In the present study, the abundant variable sites and haplotypes detected suggested that the D-loop was an effective molecular marker to detect genetic differences in M. nipponense populations. The mean basic nucleotide configurations obtained indicated that the content of AþT was higher than that of CþG. This was similarly observed in other shrimp and crab species, and is consistent with the fact that commonly the GC combination is relatively scarce while AT content is high in the mtDNA base composition of arthropods (Beati et al., 2013).
High genetic diversity means high adaptive survival potential and high evolutionary potential, which are advantageous for conservation and the utilization of germplasm resources. The p and h values are good indicators of genetic variation in a population (Jiang et al., 2019). All the11 populations examined in this study showed h > 0.7 and p > 0.009 proving that they are highly genetically diverse. The TH-2, YZ, and XJ populations showed remarkable diversity, especially the latter. The diversity of reservoir populations was slightly lower than that of other populations, the h of YC and HS populations were the lowest (ranged from 0.7438 to 0.8519). All the results suggested that the germplasm resources and genetic diversity of M. nipponense in Yixing are of excellent quality and the species is suitable for breeding.
The pairwise F ST value was considerably lower than the genetic differentiation index of the eight populations in China (Zhang et al., 2022). This might be due to frequent interaction between these populations given the relatively short geographical distance separating them. The genetic distance ranged from 0.011 to 0.044, indicating that the differences among the 11 populations were considerably smaller than those at the species level (<15%) for Macrobrachium spp. (Zhang et al., 2009). The YZ and TH-2 populations showed a high genetic differentiation from the other Yixing populations due to geographical isolation and parental origin differences. According to the distribution analysis of sampling sites, the brooks, lakes, and reservoirs of Yixing are connected with Taihu lake by trenches, which results in frequent water exchange. The haplotype network results also revealed the same inconspicuous differences in populations.
Population genetics studies have suggested that the main causes of population differentiation are genetic drift and natural selection, and that the process is also influenced by population history dynamics (Chen et al., 2005). The neutrality and mismatch distribution tests have been used to measure the historical evolution of populations. The populations have undergone expansion in the past, as shown by the Tajima's D value deviating significantly from the neutral test and the nucleoside acid mismatch curve presenting a single-peak distribution (Fratini et al., 2005). In this study, the P values (Tajima's D and Fu Fs) were relatively greater than 0.1 Fig. 2. The median-joining network based on haplotype frequencies of M. nipponense. The circle represents haplotype; circle size is proportional to the number of individuals in the haplotype. Each line in the network represents a single mutational change, and the branches are scaled to the number of polymorphic sites between each haplotype. Each black circle represents one missing haplotype.
(P > 0.1) and the nucleotide mismatch distribution analysis showed multiple peaks, indicating that the populations did not undergo expansion.

Conclusions
In this study, D-loop sequences were employed to examine the genetic diversity of eight wild M. nipponense populations in Yixing's natural waters, and their genetic differentiation and population structure were compared with those of wild populations of TH, YZ, and the main local strain TH-2. Our results showed that the TH-2 and YZ populations have a remarkable genetic diversity; the germplasm resources of M. nipponense in Yixing are in excellent condition and populations show a high genetic diversity, suggesting this species is suitable for breeding.

Author contributions
Hongtuo Fu and Hui Qiao designed the research; Yiwei, Xiong, Sufei Jiang collect samples and performed the study; Lijuan Zhang and Wenyi Zhang analyzed the data;, Jisheng Wang, Shubo Jin, Yongsheng Gong and Yan Wu contributed reagents, materials and tools; Sufei Jiang and Hui Qiao drafted the manuscript; Hongtuo Fu revised the manuscript; all authors approved the final version.

Supplementary Material
Table S1. The nucleotide composition from 11 M. nipponense populations. Table S2. Distribution of haplotypes of 11 M. nipponense populations. Table S3. Neutrality tests for11 M. nipponense populations Figure S1. A location map for the 11 inferred populations of M. nipponense.