# Molecular Phylogenetics of Seven Cyprinidae Distant Hybrid Lineages: Genetic Variation, 2nNCRC Convergent Evolution, and Germplasm Implications

## TL;DR

This study explores how distant hybridization in Cyprinidae fish leads to genetic variation and speciation, using molecular markers to trace lineage relationships and support aquaculture breeding.

## Contribution

The study introduces a nine-gene integrative marker system for analyzing Cyprinidae hybrid lineages and reveals convergent evolution in 2nNCRC hybrids.

## Key findings

- Hybrid lineages clustered intermediately between parental species with maternal affinity.
- 2nNCRC hybrids showed convergent evolution toward Carassius auratus, not their parents.
- Nine genes serve as pivotal markers for parentage tracing and understanding hybrid stabilization.

## Abstract

Distant hybridization fuels trait innovation and speciation, but the stabilization mechanism of Cyprinidae interspecific distant hybrid lineages remains unclear. We analyzed 7 such hybrid lineages and their parents using 4 mitochondrial genes (Cytb, COI, 16S rRNA, D-loop) and 5 nuclear genes (EGR2b, IRBP2, RAG1, RAG2, RH2), with 41 Cyprinidae species (85 samples) from GenBank supplementing the dataset. The hybrids exhibited variation patterns analogous to other Cyprinidae species; Maximum Likelihood (ML) and Bayesian Inference (BI) trees displayed congruent, well-supported topologies. Most hybrids clustered intermediately between their parental species with maternal affinity, except 2nNCRC (derived from distant hybridization between Cyprinus carpio and Megalobrama amblycephala), which showed convergent evolution toward Carassius auratus. These nine genes and the integrated marker system advance research on cytonuclear coadaptation and support parentage tracing, germplasm conservation, and hybrid breeding—laying a foundation for studies on hybrid speciation and the development of elite aquaculture germplasms.

Distant hybridization is key to trait innovation and speciation, with Cyprinidae hybrid phylogeny helping to clarify diversification mechanisms. Yet, a major gap persists in Cyprinidae studies: the stabilization mechanisms of interspecific distant hybrid lineages. To address this, we systematically analyzed the molecular phylogeny of seven Cyprinidae distant hybrid lineages and their parental species, using an integrative genetic framework encompassing four mitochondrial genes (Cytb, COI, 16S rRNA, D-loop) and five nuclear genes (EGR2b, IRBP2, RAG1, RAG2, RH2). Homologous sequences of 41 representative Cyprinidae species (85 samples) were retrieved from GenBank to supplement the dataset. Phylogenies were reconstructed from concatenated sequences, complemented by haplotype networks. Intra-/interspecific divergence was quantified using two mitochondrial genes (COI, Cytb) and two nuclear (RAG1, RH2). The results showed that these hybrid lineages exhibited variation patterns analogous to other Cyprinidae species. Both ML and BI trees reconstructed exhibited congruent topologies with high support (bootstrap/BPP > 80%), resolving genus/species-level relationships. While most hybrids clustered intermediately between their parental species, they typically displayed maternal affinity. A notable exception was the 2nNCRC (a homodiploid hybrid from Cyprinus carpio ♀ × Megalobrama amblycephala ♂), which displayed convergent evolution toward Carassius auratus. COI-based K2P genetic distance analysis revealed 2nNCRC had a much closer relationship with C. auratus (0.0119) than with its parents (0.1249 to C. carpio, 0.1552 to M. amblycephala). These nine genes elucidate the genetic relationships between Cyprinid hybrid lineages and progenitors, serving as pivotal molecular markers for parentage tracing and genetic dissection of distant hybridization mechanisms. The integrated mitochondrial–nuclear marker system in this study advances understanding of cytonuclear coadaptation and the stabilization of interspecific distant hybrid lineages in Cyprinidae. Specifically, it provides a precise tool for parentage tracing, Cyprinid germplasm conservation, and targeted regulation of hybrid breeding—laying a foundation for exploring hybrid speciation and developing elite aquaculture germplasms.