Four new earthworm species of the genera Amynthas and Metaphire (Oligochaeta, Megascolecidae) from Hunan and Anhui provinces, China.

This paper describes four new species earthworms from Hunan and Anhui provinces, China, Amynthasxiangtanensis Qiu & Jin, sp. nov., Amynthastaoyuanensis Qiu & Jin, sp. nov., Amynthasxuanchengensis Jin & Li, sp. nov. and Metaphiredonganensis Jin & Jiang, sp. nov. Amynthasxiangtanensis sp. nov., and A.taoyuanensis sp. nov. belong to the Amynthascorticis group. Both have four pairs of intersegmental spermathecal pores in 5/6-8/9; male pores in segment XVIII, separated by 1/3 of body circumference, each on top of a slightly raised porophore, surrounded by several tiny genital papillae. Amynthastaoyuanensis sp. nov. prostate glands are degenerated. Amynthasxuanchengensis sp. nov. belongs to the Amynthasmorrisi group, it has two pairs of spermathecal pores in 5/6 and 6/7; male pores in XVIII, separated by 1/3 of body circumference, each on top of a slightly raised, circular porophore. Metaphiredonganensis sp. nov. belongs to the Metaphirehoulleti group. It has three pairs of spermathecal pores in 6/7-8/9; male pores in XVIII, separated by 1/3 of body circumference, each on the bottom center of the longitudinal copulatory chamber.

Roxarsone reduces earthworm-mediated nutrient cycling by suppressing aggregate formation and enzymic activity in soil with manure application.

The application of manure and earthworms are frequently used in fertilization practices to improve C, N, and P cycling in soil, which may be adversely affected by roxarsone (ROX), as an organoarsenical pollutant. To effectively address this issue, in this work, the interactive impacts of ROX and earthworm Eisenia foetida on the aggregate formation, input of organic carbon (OC), and changes in the available N and P following 56-day cultivation were systematically investigated. Compared to the control, earthworms increased the mean weight diameter (MWD) of the soil aggregates from 0.6 to 1.1 mm. Thereby, they activated soil enzymes including catalase (CAT), sucrase (SC), urease (UE), and neutral phosphatase (NP), with the soil's pH decreased to 7.1. Consequently, the values of OC, soluble nitrite (NO3-N), and Olsen-P content were respectively increased by 0.78-, 1.69-, and 0.87- folds in the E treatment (14.3 vs. 25.5 g/kg, 12.8 vs. 33.3 mg/kg, and 7.8 vs. 14.6 mg/kg). Although the changes in the R treatment were slight, ROX reduced the earthworm-mediated improvements of soil fertility during the application of the RE treatment compared to the E treatment, i.e., the values of MWD, OC, NO3-N, and Olsen-P were reduced to 0.9 mm, 20.4 g/kg, 25.4 mg/kg, and 11.6 mg/kg, respectively. From the well-fitted structural equation models, it was demonstrated that earthworms enhanced the aggregate formation and nutrient cycling of OC, NO3-N, and Olsen-P, which were inhibited by ROX. Overall, these adverse effects can be offset by earthworm addition, which can play the dual role of monitor and driver for the soil properties. Our work provides insightful strategies for ROX-bearing manure management.

Mitochondrial DNA evidence reflects high genetic divergence of Amynthas aspergillum (Oligochaeta: Megascolecidae) in southern China.

Amynthas aspergillum (Perrier, 1872), a natural resource used in traditional Chinese medicine (Guang-dilong) with high economic value, is widely distributed in forests and farmland habitats in the hilly areas of southern China. To investigate the extent of genetic differentiation and diversity in A. aspergillum, a population genetic structure study was performed on 157 samples from 75 locations in southern China using the mitochondrial genes COI, COII, 12S rRNA, 16S rRNA, and NDI. The results indicated that A. aspergillum had a high level of genetic diversity, and variation within populations was the main source of the total variation. Six deeply divergent mitochondrial clades (I-VI) were detected using both phylogenetic tree and haplotype network analyses. This finding was supported by the high Kimura two-parameter genetic distance and the pairwise fixation index value obtained based on the COI gene. No significant phylogeographic structures were observed. The widespread geographic distribution of clades II, IV, and VI suggested a recent demographic expansion based on multiple analysis results. These results include a high level of Hd and low π, star-shaped haplotype network structures with a high number of less frequent haplotypes, significantly negative neutrality test values, and a unimodal mismatch distribution pattern. The divergence time estimates and reconstruction of the ancestral area revealed that A. aspergillum originated in Guangxi Province and underwent initial intraspecific diversification in the early Pliocene to generate clade I. Then, it gradually dispersed eastward and rapidly differentiated into clades II-V during the Pleistocene. The Yunnan-Guizhou Plateau and Nanling and Wuyi Mountains might act as geographical barriers for the spread of A. aspergillum to the west and north.

Three new species of the genus Amynthas (Oligochaeta: Megascolecidae) from Guangxi Zhuang Autonomous Region, China.

Three new earthworm species of the genus Amynthas Kinberg, 1867 were found in Guangxi Zhuang Autonomous Region, China: Amynthas huapingensis sp. nov., Amynthas retentus sp. nov. and Amynthas linguiensis sp. nov. Amynthas huapingensis sp. nov. belongs to Amynthas corticis-group characterized by having four pairs of spermathecal pores in 5/68/9; Amynthas retentus sp. nov., without spermathecal pores, belongs to Amynthas illotus-group; Amynthas huapingensis sp. nov. and Amynthas retentus sp. nov. have similar characteristics of male porophores medially surrounded by 23 genital papillae in a pulvinate pad within the male pore area. Amynthas linguiensis sp. nov. has variable spermathecal pores, three pairs in 6/78/9 or four pairs in 5/68/9. And its male pores each on the center of a large elliptical glandular porophore which extends from 1/4xvii to 1/4xix. The morphological comparison and COI gene data of these three new species and their similar species are given in this paper.

Biotransformation of roxarsone by earthworms and subsequent risk of soil arsenic release: The role of gut bacteria.

The organoarsenical feed additive roxarsone (ROX) is a ubiquitous threat due to the unpredictable levels of arsenic (As) released by soil bacteria. The earthworms representing soil fauna communities provide hotspots for As biotransformation genes (ABGs). Nonetheless, the role of gut bacteria in this regard is unclear. In this study, the changes in As speciation, bacterial ABGs, and communities were analyzed in a ROX-contaminated soil (50 mg/kg As in ROX form) containing the earthworm Eisenia feotida. (RE vs. R treatment). After 56 d, earthworms reduced the levels of both ROX and total As by 59 % and 17 %, respectively. The available As content was 10 % lower in the RE than in R treatment. Under ROX stress, the total ABG abundance was upregulated in both earthworm gut and soil, with synergistic effects observed following RE treatment. Besides, the enrichment of arsM and arsB genes in earthworm gut suggested that gut bacteria may facilitate As removal by enhancing As methylation and transport function in soil. However, the bacteria carrying ABGs were not associated with the ABG abundance in earthworm gut indicating the unique strategies of earthworm gut bacteria compared with soil bacteria due to different microenvironments. Based on a well-fit structural equation model (P = 0.120), we concluded that gut bacteria indirectly contribute to ROX transformation and As detoxification by modifying soil ABGs. The positive findings of earthworm-induced ROX transformation shed light on the role of As biomonitoring and bioremediation in organoarsenical-contaminated environments.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as "Guang Dilong".

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

Dynamics of bacterial community in the foregut and hindgut of earthworms with the nutrition supplied by kitchen waste during vermicomposting.

Earthworm gut microbiota is vital in degrading bio-waste during vermicomposting. However, microbial dynamics in earthworm gut during this process are unclear. Thus, the aim is to firstly report the bacterial dynamics in both foregut and hindgut of earthworms over a 28 days' timeframe of vermicomposting by Eisenia foetida with the nutrition supplied by kitchen waste. Results showed that except the changing of the bacterial diversity, composition and structure, dynamics of the foregut and hindgut bacteria also differed during vermicomposting which related to the changes of nutrient provision. Day 3 was a turning point. The abundant bacteria of the top 20 % genera nearly did not overlap between the foregut and hindgut. In the end of vermicomposting, a remarkable stable bacterial structure appeared in the hindgut compared to somewhat muddled one in the foregut. Understanding the dynamics of earthworm gut microbiota enables the improvements to regulate the efficiency of organic waste vermicomposting.

Three new widely distributed and polymorphic species of Amynthas earthworms (Oligochaeta, Clitellata, Megascolecidae) from South China.

Three new earthworm species are described from South China, namely Amynthas rusticanus sp. nov., Amynthas scaberulus sp. nov., and Amynthas yuanjiangensis sp. nov. They are widely distributed in South China, and be polymorphic in reproductive organs. Of three new species, A. rusticanus sp. nov. is a small species with three pairs of spermathecal pores in 5/6-7/8 or two pairs of spermathecal pores in 5/6-6/7; it has developed prostate glands in XVI-XX. A. scaberulus sp. nov. is a medium-sized species with two pairs of spermathecal pores in 7/8-8/9; the lower edge of each spermathecal pore forms a tiny papilla, and the prostate glands are either degenerated or developed. A. yuanjiangensis sp. nov. is a medium-sized species with four pairs of spermathecal pores in 5/6-8/9 and has degenerated or developed prostate glands. Barcode data for each species (partial sequence of mitochondrial cytochrome c oxidase subunit I) are provided as well.

High-quality genome assembly of Metaphire vulgaris.

Earthworms enrich the soil and protect the health of their ecological environment. Previous studies on these invertebrates determined their protein content, hormone secretions, medicinal value, and ecological habits, but their whole genomic sequence remains incomplete. We performed whole genome sequencing of Metaphire vulgaris (Chen, 1930), which belongs to the genus Metaphire of the family Megascolecidae. The genome assembly was 729 Mb, with a N50 contig size of 4.2 Mb. In total, 559 contigs were anchored to 41 chromosomes according to the results of Hi-C (High-throughput Chromosome Conformation Capture) technology, which was confirmed by karyological analysis. A comparison of the genomic sequences and genes indicated that there was a whole-genome duplication in M. vulgaris followed by several chromosome fusion events. Hox genes and lumbrokinase genes were identified as partial clusters surrounding the genome. Our high-quality genome assembly of M. vulgaris will provide valuable information for gene function and evolutionary studies in earthworms.

Population Genetic Structure Reveals Two Lineages of Amynthas triastriatus (Oligochaeta: Megascolecidae) in China, with Notes on a New Subspecies of Amynthas triastriatus.

Amynthas triastriatus (Oligochaete: Megascolecidae) is a widely distributed endemic species in Southern China. To shed light on the population genetic diversity and to elucidate the population differentiation and dispersal of A.triastriatus, a population genetic structure study was undertaken based on samples from 35 locations collected from 2010 to 2016. Two exclusive lineages within A. triastriatus-lineage A and lineage B-were revealed. Lineage A was mainly distributed at high altitudes while lineage B was mainly distributed at low altitudes in Southeast China. The genetic diversity indices indicated that the populations of A. triastriatus had a strong genetic structure and distinct dispersal histories underlying the haplogroups observed in this study. Combined with morphological differences, these results indicated a new cryptic subspecies of A. triastriatus. Lineage A was almost degenerated to parthenogenesis and lineage B had a trend to parthenogenesis, which suggested that parthenogenesis could be an internal factor that influenced the differentiation and dispersal of A. triastriatus. The divergence time estimates showed that A.triastriatus originated around Guangxi and Guangdong provinces and generated into two main lineages 2.97 Ma (95%: 2.17-3.15 Ma) at the time of Quaternary glaciation (2.58 Ma), which suggested that the Quaternary glaciation may have been one of main factors that promoted the colonization of A.triastriatus.

Three new species and one subspecies of the Amynthas corticis-group from Guangxi Zhuang Autonomous Region, China (Oligochaeta, Megascolecidae).

Three new species and one subspecies of the genus Amynthas are described from Guangxi Zhuang Autonomous Region, China: The new species are: Amynthas maximus Qiu & Dong, sp. nov. and Amynthas tortuosus Qiu & Dong, sp. nov., and Amynthas shengtangmontis Dong & Jiang, sp. nov., the subspecies is Amynthas shengtangmontis minusculus subsp. nov. All have four pairs of spermathecal pores in 5/6-8/9, which indicates that they should belong to the corticis-group. Their morphological characteristics are compared to other similar species in the corticis-group from China and other Asian countries, such as Amynthas pulvinus Sun & Jiang, 2013, Amynthas homosetus (Chen, 1938), Amynthas corticis (Kinberg, 1867), Amynthas dorsualis Sun & Qiu, 2013, and Amynthas carnosus (Goto & Hatai, 1899). In addition, the results presented are confirmed by the pairwise comparison of COI barcode sequences. The pairwise distances between each new species and the other eighteen corticis-group species are greater than 14.7% on average. Furthermore, the pairwise distance between A. shengtangmontis shengtangmontis and A. shengtangmontis minusculus is 10.7-11.4%.

Three new species of earthworms (Oligochaeta: Megascolecidae) from Yunnan Province, China.

Earthworm specimens collected from Yunnan Province, China were found to represent three new species of Megascolecidae: Amynthas demptus sp. nov., Amynthas lacustris sp. nov. and Metaphire reclusa sp. nov. A. demptus belongs to Amynthas hawayanus-group and has three pairs of spermathecal pores in 5/6-7/8. A. lacustris belongs to Amynthas morrisi-group and has two pairs of spermathecal pores in 5/6-6/7. M. reclusa has no spermathecal pores. A. demptus has no male pores. The male pores of A. lacustris are about 1/3 body circumference ventrally apart, with a presetal and a postsetal papilla medial to each pore, surrounded with two circular folds. The male pores of M. reclusa are in copulatory chambers at XVIII, with openings of copulatory chambers about 1/3 body circumference ventrally apart. In this paper, the morphological characters of the three new species are described. We also sequenced COI and 12S gene fragments of the holotypes and selected paratype specimens to provide molecular data.

Two new species of earthworms belonging to the genus Amynthas (Oligochaeta: Megascolecidae) from Guangxi Province, China.

Two new species of earthworms belonging to the genus Amynthas are described from Guangxi Province, China: Amynthas crassitubus sp. nov. and Amynthas stabilis sp. nov. from Shiwan Mountain Nature Reserve. The two species have three pairs of spermathecal pores in 6/7-8/9, and belong to the sieboldi-group. COI barcode sequences support their status as new species.

Three new earthworm species of the tokioensis-group in the genus Amynthas (Oligochaeta: Megascolecidae) from Guangxi Province, China.

Three new species of the genus Amynthas are described from Guangxi Province, China. They are named A. dissimilis sp. nov., A. anteporus sp. nov. and A. marsupiformis sp. nov. All of them have two pairs of spermathecal pores in 6/7-7/8, and belong to the tokioensis-group. Their morphological characteristics are compared to similar species in tokioensis-group from China and other Asian countries. In addition, the mitochondrial COI and 12S-tRNA-Val-16S sequence of three new species were determined, then pairwise distances between species were calculated. Both morphological and molecular evidences could easily distinguish the new species from earthworms previously reported in the tokioensis-group.

Four new Amynthas and Metaphire earthworm species from nine provinces in southern China.

Four new species belonging to genera Amyntha and Metaphire were discovered from an extensive area in southern China, covering the provinces Sichuan, Yunnan, Guizhou, Guangdong, Guangxi, Fujian, Hunan, Jiangxi and Anhui. The species are named Amynthas dispersus Sun Qiu, sp. nov., Amynthas shanghangensis Sun Qiu, sp. nov., Amynthas dentiformis Sun Jiang, sp. nov. and Metaphire sanmingensis Sun Jiang, sp. nov. The first two new species have four pairs of intersegmental spermathecal pores in 5/6-8/9. A. dispersus has elliptical and glandular male pore porophores raised on a low pad-like area surrounded by two to three shallow skin folds, the genital papillae are variable in the spermathecal pore and male pore regions, and prostate glands are vestigial or rudimentary. Amynthas shanghangensis has male porophores surrounded by three papillae, each prostate gland accompanied by an accessory gland, and the distal ½-⅓ of the spermathecal diverticulum dilated into a rod-shaped seminal chamber. Amynthas dentiformis has two pairs of spermathecal pores in 7/8-8/9, male pores always surrounded by two papillae, and small sacs on the dorsal and ventral margins of the intestinal caecae; the prostate gland occasionally has stalked accessory glands. M. sanmingensis is in the Metaphire houlleti-group, and has secondary male pores opening to copulatory pouches, rod-shaped spermathecal seminal chamber, as well as variable genital papillae in spermathecal and male pores regions. The GenBank accession numbers of DNA barcode data are attached under the description of each species.

Phylogenetic evaluation of Amynthas earthworms from South China reveals the initial ancestral state of spermathecae.

Our knowledge of the phylogeny of the earthworm genus Amynthas under the family Megascolecidae, which is comprised of a huge number of species, is very limited compared to the better-known and much smaller family Lumbricidae. In order to investigate the phylogenetic relationships among the species within the genus Amynthas, which is the largest genus of the Megascolecidae family, nuclear and mitochondrial DNA sequences of 77 species, including 76 in-group Amynthas species collected from South China and 1 out-group species, were analyzed. A 5402bp segments composed of whole nuclear 18S rDNA and the mitochondrial genes COI, COII, ND1, 12S, and 16S was assembled from 77 species. Maximum Likelihood and Bayesian analyses of the concatenated sequences were performed. The results revealed evolution of two geographically independent lineages, both showing the ancestral state of two pairs of spermatheca (Sp.p 7/8/9). We found the species groups described by Sims and Easton (1972) to be non-monophyletic, and the origin of the parthenogenetic species group to likely be a quadthecal ancestor. These results provide modest evidence in support of an Indochinese peninsula origin of the Chinese Amynthas species and divergence of the genus once it had spread to mainland China. The findings of this study are consistent with a divergence scenario that resulted in at least one branch spreading to the Southeast of China and another branch spreading to the Southwest of China, but further research is required to confirm this interpretation of the Amynthas phylogeny.

Fifteen new earthworm mitogenomes shed new light on phylogeny within the Pheretima complex.

The Pheretima complex within the Megascolecidae family is a major earthworm group. Recently, the systematic status of the Pheretima complex based on morphology was challenged by molecular studies. In this study, we carry out the first comparative mitogenomic study in oligochaetes. The mitogenomes of 15 earthworm species were sequenced and compared with other 9 available earthworm mitogenomes, with the main aim to explore their phylogenetic relationships and test different analytical approaches on phylogeny reconstruction. The general earthworm mitogenomic features revealed to be conservative: all genes encoded on the same strand, all the protein coding loci shared the same initiation codon (ATG), and tRNA genes showed conserved structures. The Drawida japonica mitogenome displayed the highest A + T content, reversed AT/GC-skews and the highest genetic diversity. Genetic distances among protein coding genes displayed their maximum and minimum interspecific values in the ATP8 and CO1 genes, respectively. The 22 tRNAs showed variable substitution patterns between the considered earthworm mitogenomes. The inclusion of rRNAs positively increased phylogenetic support. Furthermore, we tested different trimming tools for alignment improvement. Our analyses rejected reciprocal monophyly among Amynthas and Metaphire and indicated that the two genera should be systematically classified into one.

Complete mitochondrial genome of an Amynthas earthworm, Amynthas aspergillus (Oligochaeta: Megascolecidae).

We have determined the mitochondrial genome of the first Amynthas earthworm, Amynthas aspergillus (Perrier, 1872), which is a natural medical resource in Chinese traditional medicine. Its mitogenome is 15,115 bp in length containing 37 genes with the same contents and order as other sequenced earthworms. All genes are encoded by the same strand, all 13 PCGs use ATG as start codon. The content of A + T is 63.04% for A. aspergillus (33.41% A, 29.63% T, 14.56% G and 22.41% C). The complete mitochondrial genomes of A. aspergillus would be useful for the reconstruction of Oligochaeta polygenetic relationships.

Complete mitochondrial genome of a Pheretimoid earthworm Metaphire vulgaris (Oligochaeta: Megascolecidae).

We have determined the mitochondrial genome of the first Pheretimoid earthworm, Metaphire vulgaris (Chen, 1930). This mitogenome is 15,061 bp in length containing 37 genes typical of other annelid. All genes are encoded by the same strand, ATP8 is not adjacent to ATP6, all 13 PCGs use ATG as a start codon. These features are consistent with first determined earthworm Lumbricus terrestris, but unusual among animal mtDNAs.