Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish

Abstract

Body size is one of the most important attributes of a species, but the basic question of why and how each species reaches a different "right size" is still largely unknown. Herein, three phylogenetically closely related catfishes from Sisoridae, including one extraordinarily large-sized Bagarius yarrelli and two average-sized Glyptothorax macromaculatus and Oreoglanis setiger, were comparatively studied using RNA-Seq. Approximately 17,000 protein-coding genes were annotated for each of the three fishes, and 9509 genes were identified as high-confidence orthologous gene pairs. Comparative expressions uncovered a similar functional cluster about ribosome biogenesis was enriched in different tissues of the upregulated genes of Bagarius yarrelli. Moreover, differentially expressed genes and positively selected genes revealed that the glycolysis/pyruvate metabolism and cell cycle pathways have also greatly enhanced in this large-sized species. In total, 20 size-related candidate genes (including two growth modulators: the serine/threonine-protein kinases 3 (AKT3) and adaptor protein 1 (SH2B1), and a crucial pyruvate kinase (PKM2A)) were identified by multiplying comparative analyses along with gene functional screening, which would play major roles in enabling the large body size associated with Bagarius yarrelli and provide new insights into body size evolution. In conjunction with field observations and morphological comparisons, we hypothesize that habitat preferences promote size divergence of sisorids.

Keywords: Bagarius; body size; growth; pyruvate metabolic; ribosome.

Figure 1

Intergeneric phylogenetic relationships and body length differences in Sisoridae. (a) phylogenetic relationships recreated based on a previous study [13]; (b) the maximum body length recorded for each genus based on Fishbase (http://www.fishbase.org/search.php). The photographs present a typical appearance for each genus, among which Conta, Erethistes, Erethistoides, Gogangra, Nangra, Pseudolaguvia, and Sisor were provided by courtesy of Heok Hee Ng, Ayarnangra, Caelatoglanis, and Gagata by courtesy of Kamphol Udomritthiruj, Parachiloglanis by courtesy of Ryan Thoni, and the others were taken by the first author (Wansheng Jiang). The genera that include the species in this study are highlighted in blue.

Figure 2

Length frequency distributions and preliminary age estimations of Bagarius yarrelli (BY), Glyptothorax macromaculatus (GM), and Oreoglanis setiger (OS). (a) length frequency of the specimens deposited in the Kunming Natural History Museum of Zoology, Chinese Academy of Sciences; (b) age–length relationship of BY, GM, OS (solid line) and other eight catfish species (dotted line).

Figure 3

The front half body view of BY (a), GM (b), and OS (c). Arrows indicate the end of gill openings, and the numbers indicate the modified epidermal adhesive structures as follows: (1) thoracic adhesive apparatus; (2) adhesive sucker; and (3) adhesive unbranched rays.

Figure 4

Overview of the expression differentially expressed genes (DEGs) identified in BY relative to GM and OS species. (a) the principal component analysis of the overall gene expression among three tissues in three species; (b) a heatmap of the overlapped DEGs between any of the two in six DEG datasets. The six DEG datasets were obtained from the comparisons between BY vs. GM and BY vs. OS in three tissues; (c) Venn diagrams of upregulated BY DEGs when compared with GM and OS samples for all three types of tissues, with the overlapping genes showing no statistical transcriptional differences between GM and OS.

Figure 5

Fast-evolving genes (FEGs) enrichment and PKM2A sequence alignment and protein structure. (a) comparison of dN/dS ratios between BY and GM using gene ontology (GO) functional categories; highlighted red dots indicate GO terms with elevated evolutionary rates in BY that may be related to growth and proliferation, and blue dots indicate these GO terms with reduced evolutionary rates in BY; (b) sequence alignments of PKM2A & PKM2B between BY (red indicates positive selection sites), GM, OS, and zebrafish, with human PKM2 included; (c) the construction of the PKM2A protein of BY, GM, and OS, with human PKM2 used as a template (PDB id: 3srd). A possible hydrogen bond between H406 and G506, and an affinity enhancement between K433 and FBP (the red and yellow spheres) via the charge attraction of BY (in magenta) can be seen in the red cycle; no similar link was found between GM and OS (in cyan).

Figure 6

Flowchart that illustrating the comparative transcriptome analyses using RNA-Seq.

Figure 7

Schematic illustration of the main genetic clues revealed in this study that may be related to large BY body size when compared to GM and OS. Upregulated BY DEGs are shown in red (SH2B1, AKT3, PFKMA, and PKLR) and positively selected genes (PSGs) obtained from evolutionary analysis are shown in blue (PKM2A, HK2, TGFβ3, and CCNB2). The biological processes are denoted by red boxes (such as Ribosome biogenesis), with groups including many enriched GO terms or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are partially illustrated below the box. All of these clues could contribute to BY cell growth, proliferation, and increased biomass, thereby enabling the evolution of a much larger body size.