Comparative repeatome analysis of Pyrgomorphidae and Acrididae (Orthoptera: Caelifera) revealed the contribution of repetitive DNA in genome gigantism

Abstract

Eukaryotic genomes are often rich in DNA repetitive elements, involving both transposable elements (TE) and tandemly repeated satellite DNA. Grasshopper species, known for their large genome sizes, comprising relatively a high proportion of genomic repeats. This study aimed to identify and perform a comparative analysis of DNA repetitive content in eight grasshopper species from the Pyrgomorphidae and Acrididae families. We utilized unassembled low-coverage Illumina paired-end short reads in the RepeatExplorer2 pipeline to identify genomic repeats, and RepeatMasker to estimate their abundance and divergence activity. Flow cytometry estimated genome sizes, ranging from 1C = 7.670 pg to 18.612 pg, with Aularches miliaris (18.612 pg) being the second largest insect genome reported to date. The repeat content ranged from 51% to 74%, with a mean value of 64.26% of the total genome. The major identified repeat elements included LINE, Ty3_Gypsy, Penelope, Ty1-copia, Helitron, Maverick, and satellite repeats, with LINE elements being the most abundant, constituting 24% to 54% of the total repetitive content in Apalacris varicornis and A. miliaris, respectively. The positive correlation of repetitive content and TEs with genome size suggests that their expansion has contributed to the large genome sizes observed. Satellite DNA analysis identified 65 satDNA families across the eight species. Additionally, phylogenetic analysis of TE protein domains revealed that consensus sequences from the same domain cluster together, suggesting domain-specific evolutionary pathways for TEs in the genome. This study reveals new dynamics into the role of repetitive DNA in genome gigantism as well as other evolutionary mechanisms in the Pyrgomorphidae and Acrididae families of Orthoptera.

Fig 1. Genome repeat proportion and composition of DNA repetitive content Pyrgomorphidae and Acrididae.

(A) Phylogenetic analysis based on 13 mitochondrial protein coding genes (PCGs) and 2 rRNA genes, the initial split into two clades separating both families with Pyrgomorphidae species on upper and Acrididae on lower branch. The color pattern indicates the bootstrap support. (B) Genome size across the species in Gb. (C) Singlets and repeats proportions across the species. (D) Repeat content composition of species with LINE, Ty3_gypsy and satellite repeats being abundant across the genomes.

Fig 2. The correlation analysis between repeat content and TE content with genome size.

Blue line shows strongly positive correlation between repeat content and genome size and green line indicates the strong positive correlation between TE content and genome size.

Fig 3. Comparative repeatome analysis visualization of Pyrgomorphidae and Acrididae.

(A) The size of each cluster (number of reads) is represented in the top bar plot. (B) The scaled rectangles represent the proportion of individual repeats in each species. (C) The scaled rectangles are re-calculated based on genome size, representing the actual number of reads in the genome. The size of each peak shows the total read numbers in the respective cluster. The top clusters labeled as “shared” denote repeats that showed similar repeat landscape. “Genus-Spec” refers to repeats that are specific to a particular genus. “AMI/YCO/AVA/PIN/PPU/PSA-Spec” are sets of species-specific groups containing unique repeats.

Fig 4. TE genetic divergence landscapes across the species.

(A) TE divergence landscapes of family Pyrgomorphidae with x-axis indicating the Kimura divergence percentage and y-axis shows the proportion of genome occupied by each TE. (B) TE divergence landscapes of family Acrididae.

Fig 5. Heatmaps of percentage abundance, divergence and copy numbers of 65 satDNA families across the species.

(A) The abundance percentage of 65 satDNA families across the genomes with PsaSat01 showing the highest proportion in P. sauteri.. (B) The divergence percentage satDNA families in the eight species. (C) The copy numbers of satDNA repeats occupied by each genome, represented in millions.

Fig 6. Satellitome divergence landscape in Pyrgomorphidae and Acrididae.

(A) The satellite DNA divergence landscapes of family Pyrgomorphidae with y-axis showing the genome proportion occupied by different satDNA sequences and x-axis indicates the Kimura divergence%, which measures the divergence of satDNA from their respective consensus sequences based on the Kimura distance. Lower Kimura substitution levels indicate less divergence and therefore more recent copies, while higher levels suggest older, more diverged sequences. (B) The satellite DNA divergence landscapes of family Acrididae.

Fig 7. Phylogenetic tree of protein-domain based transposable element (TE) sequences.

The maximum likelihood phylogenetic tree illustrates the distinct clustering of various TEs based on their protein-domain consensus sequences, represented by the distinctive star colors. The phylogenetic tree uses a gradient color scheme to represent bootstrap values, where red indicates the highest support (100), transitioning through purple, blue, green, yellow, and orange (31) as bootstrap values decrease.