Enhancing inbreeding estimation and global conservation insights through chromosome-level assemblies of the Chinese and Malayan pangolin

Abstract

A high-quality reference genome coupled with resequencing data is a promising strategy to address issues in conservation genomics. This has greatly enhanced the development of conservation plans for endangered species. Pangolins are fascinating animals with a variety of unique features. Unfortunately, they are the most trafficked wild animal in the world. In this study, we assembled a chromosome-scale genome with HiFi long reads and Hi-C short reads for the Chinese and Malayan pangolin and provided two new representative reference genomes for the pangolin species. We found a great improvement in the evaluation of genetic diversity and inbreeding based on these high-quality genomes and obtained different results for the detection of genome-wide extinction risks compared with genomes assembled using short reads. Moderate inbreeding and genetic diversity were reverified in these two pangolin species, except for one Malayan pangolin population with high inbreeding and low genetic diversity. Moreover, we identified a much higher inbreeding level (FROH = 0.54) in the Chinese pangolin individual from Taiwan Province compared with that from Mainland China, but more than 99.6% runs of homozygosity (ROH) fragments were restricted to less than 1 Mb, indicating that the high FROH in Taiwan Chinese pangolins may have accumulated from historical inbreeding events. Furthermore, our study is the first to detect relatively mild genetic purging in pangolin populations. These two high-quality reference genomes will provide valuable genetic resources for future studies and contribute to the protection and conservation of pangolins.

Keywords: Chinese pangolin; Malayan pangolin; conservation genomics; genetic purging; inbreeding.

Figure 1:

Introduction to the species distribution and chromosome synteny of the Chinese and Malayan pangolins. (A) The distribution area and sampling sites of the Chinese and Malayan pangolins in this study. The circles represent sampling sites of the Chinese pangolins reported by Wang et al. [27]. Samples without detailed locations are not shown on the map. (B) The chromosome-scale synteny analysis between the Malayan pangolin and Chinese pangolin genomes.

Figure 2:

Comparison of the genome-wide genetic diversity and inbreeding estimated based on the LG (long-read HiFi assembled genome) and SG (short-read assembled genome). (A) Comparison of genome-wide π calculated based on the LG and SG in the Chinese and Malayan pangolin genomes. (B) Comparison of F_ROH calculated based on LG and SG in the Malayan pangolin genomes. (C) Comparison of F_ROH calculated based on LG and SG in the Chinese pangolin genomes. NS: P ≥ 0.5, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3:

Genome-wide inbreeding estimated by ROH in the Chinese and Malayan pangolin populations. (A) The length distribution of ROH across the genome in the Chinese pangolin population. (B) The length distribution of ROH across the genome in the Malayan pangolin population. (C) Genome-wide heterozygosity and inbreeding estimates (F_ROH) for all five pangolin populations. (D) Comparison of the averaged F_ROH in the MjavA and MjavB populations of the Malayan pangolins. (E) Comparison of the averaged F_ROH in the MpenA, MpenB, and MpenC populations of Chinese pangolins.

Figure 4:

Mutational load in the Chinese and Malayan pangolin populations. (A) Total number of individual-level LoF mutations across the Chinese pangolin populations. (B) Total number of individual-level LoF mutations across the Malayan pangolin populations. (C) The ratio of homozygous LoF mutations in the Chinese pangolin populations was calculated by the following formula: 2 × homozygous sites/(2 × homozygous sites + heterozygous site). (D) The ratio of homozygous LoF mutations in the Malayan pangolin populations was calculated using the same formula as that for the Chinese pangolin. (E) Relative mutational load in the Chinese pangolin populations (top 0.1% of GERP scores). (F) Relative mutational load in the Malayan pangolin populations (top 0.1% of GERP scores). The LoF here means loss-of-function mutations.

Figure 5:

The SFS and genetic signals of genetic purging in pangolin populations. (A) SFS for putatively damaging (LoF and missense mutations) and neutral mutations (intergenic variants) in the MpenA, MpenB, and MpenC populations. (B) SFS for putatively damaging and neutral mutations in the MjavA and MjavB populations. (C) Dot plot showing the occurrence of LoF mutations in the two Malayan pangolin populations calculated as the ratio of the number of the mutational load to synonymous mutations in the ROH regions (ROHf) or non-ROH (non-ROHf) regions across the genome. Each point signifies the LoF frequency of an individual. The large dots represent the average LoF frequency for the population, while the lines indicate the standard deviation range around the mean. (D) The ratio of ROHf to non-ROHf for the LoF in the two Malayan pangolin populations. The box represents the interquartile range (IQR), stretching from the first quartile (Q1) to the third quartile (Q3). The line that bisects the box indicates the median value. The whiskers extended from the box to show the variability of the data, typically reaching to the minimum and maximum values that fall within 1.5 times of the IQR from Q1 and Q3, respectively. (E) Dot plot showing the occurrence of LoF mutations in the three Chinese pangolin populations calculated as that in (C). (F) The ratio of ROHf to non-ROHf for the LoF in the three Chinese pangolin populations.