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Despite the ecological function of earthworms being of undeniable importance, little is known about their population genetic diversity, dispersal, and gene flow patterns. Here, we applied 769,957 single-nucleotide polymorphisms (SNPs) gathered by restriction site-associated DNA sequencing (RAD-seq) to unearth the genetic variability of populations of the earthworm Amynthas_YN2017 sp. collected from Western Yunnan, China. The neighbor-joining (NJ) tree method revealed that 30 individuals strongly clustered into three lineages (G2, G3, and G5 lineages) that coincided with the geographic distribution proximity of the sampling locations. There was a low level of genetic differentiation among these lineages with an average genetic differentiation (FST) value ranging from 0.080 to 0.135. Clustering analysis demonstrated a clear population genetic structure with a low level of admixture. There was only one migration edge in the population from the G3 lineage to the G2 lineage. Therefore, we presume that a reduction in gene flow caused by geographic isolation influences the population genetic differentiation of Amynthas_YN2017 sp. Additionally, the presence of river (Nujiang River) as geographical barrier is likely to have limited the gene flow. Overall, the large number of SNPs gathered by RAD-seq reveals a significant population genetic structure over a restricted geographic scale, highlighting that the RAD-seq approach could be used to explore the population phylogenetic relationships and population genetic diversity of earthworms.

Enrofloxacin (EF) is widely used in veterinary medicine while the bulk accumulation of EF residues and their metabolites have been raised more and more public concerns nowadays. The aim of this study was to explore the acute and sub-acute toxicity of EF on the earthworms (Eisenia fetida) in an artificial soil substrate. The 50% lethal concentration (LC50) of EF to earthworms at day 14 after exposure to different concentration of EF (0, 2.5, 5, 10, 15 and 25 g EF per kg soil dry weight) was calculated to examine the acute toxicity of EF. Sub-acute toxicity test was conducted through detecting the effects of EF on growth, reproduction, avoidance response and respiration on earthworms. The LC50 of EF at day 14 to earthworms was 11.01 g kg−1, which was far higher than the environmentally relevant concentration of EF residues. The inhibition of EF on the growth and reproduction of earthworms was shown in a dose-dependent manner and the high concentration of EF (above 1 g kg−1 on growth rate; above 0.05 g kg−1 on juvenile amounts) obviously disturbed the growth and reproduction of earthworms. Earthworms exposed to ≥0.25 g kg−1 of EF displayed avoidance response while the soil only spiked with ≥2 g kg−1 of EF showed decreased habitat function. The respiration of earthworms was inhibited when exposed to 2.0 g kg−1 of EF for more than 14 days or 1.0 g kg−1 of EF for more than 28 days. It is concluded that EF could cause acute and sub-acute toxicity to earthworms (E. fetida) at relatively high concentration. The long-term exposure of EF to earthworms and the test conducting on natural soil substrates specific to that region should be further studied.

The Hengduan Mountains (HMs), China, is a biodiversity hotspot that uplifted mainly between Late Miocene and Late Pliocene, the uplift movement might have a significant impact on the evolution of earthworms. Here, we investigated the species richness and the distribution of the earthworms in the HMs. A total of 170 species/subspecies belonging to four families and 12 genera were preliminary identified, species number in Megascolecidae account for more than 88%. Molecular phylogeny and divergence times analysis based on concatenated genes suggest that the divergence time of Moniligastridae (Outgroup) and Lumbricidae (Group 1) in the HMs was 215.84 million years ago (MYA). The divergence of first two clades in Megascolecidae (M405 and Group 2) can be date to around 44.42 MYA (95% HDP: 38.14–52.62 MYA). Dispersal of Megascolecidae from outside of the HMs to this region coincides with Indian-Eurasian collision between Eocene and Oligocene. The reconstruction of ancestral-area reveals the clades in Megascolecidae (Group 2–8) may have experienced different evolutionary histories following their divergence time during Cenozoic, which suggests that paleogeographical events might promote the dispersal and diversification of earthworm in the HMs. Unearthing the biogeographical pattern of earthworms provide insight into the evolution of soil invertebrate fauna in the temperate northern hemisphere and can be used for tracing the complex paleogeographical history of the HMs.