A new pheretimoid earthworm species, Metaphire liaoningensis sp. nov., was investigated in northeastern China. The taxonomy of M. liaoningensis sp. nov. using morphological characters and molecular data is reported, as well as its current geographical range in northeastern China. The new species, belonging to the M. houlleti species group, is characterized by having three pairs of spermathecal pores in 6/7/8/9, round and small pre-clitellar genital markings medio-ventral to the spermathecal pore opening, the first dorsal pore in 12/13, and the spermathecae ampulla large, nearly elliptical with a surface wrinkled and a duct long and stout. The molecular phylogenetic position of M. liaoningensis sp. nov. within the M. houlleti species group is performed by COI and mitogenomic data, respectively. The COI K2P genetic distance between the new species and the other species within the M. houlleti species group was at least 16%. This is the second updated report of a new pheretimoid species in northeastern China since the last decade.

Biodiversity, new species, Northeast China, taxonomy

It is well known that China is one of the countries in Asia having rich species diversity of earthworm fauna. To date, an estimated total of 640 species and subspecies of earthworms from nine families and 28 genera have been recorded in China (Huang et al. 2006; Aspe 2016; Jiang 2016; Shen et al. 2016, 2019; Zhao et al. 2017; Dong et al. 2018, 2019, 2024; Jiang and Qiu 2018; Jiang et al. 2018; Sun et al. 2018, 2021; Yuan et al. 2019; Han et al. 2024; Jin et al. 2024; Li et al. 2024); this number has nearly doubled in the last 16 years since Blakemore’s recorded lists (Blakemore 2007a, 2008). The Megascolecidae family accounts for more than 90% of earthworm species in China, with an estimated total of more than 600 species and subspecies recorded, making it the most dominant earthworm family in the country. Amynthas, with around 476 species and subspecies, and Metaphire, with around 135 species and subspecies, are the most speciose genera in this family, accounting for 89% of the total number of species in China.

Megascolecidae species showed an uneven distribution in China. Recent statistical data revealed larger occurrences in southern China, with more than 79 species recorded in each of the provinces of Guangxi, Hainan, Sichuan, and Taiwan, while only 13 species were recorded in the whole northern region (most are considered widespread species with higher adaptability). Although this phenomenon may be due to more favorable temperature and humidity in the southern regions, it may also be related to the insufficient sampling efforts invested in taxonomic studies in the northern part of the country (Jiang 2016). Moreover, Dong et al. (2024) have recently identified three new earthworm species in eastern and southern China, which includes one Metaphire species (M. changbaimontis Dong & Shen, 2024) located in the Changbai Mountain in Northeast China. This marked the first discovery of a pheretimoid species since the previous decades (Yu et al. 1992).

A recent investigation conducted in 2023 in Northeast China has led to the discovery of a new species belonging to the pheretimoid earthworm group of the genus Metaphire. Live specimens of these species share similar external characteristics to those of M. tschiliensis (Michaelsen, 1928) and M. guillelmi (Michaelsen, 1895) in terms of body size and pigmentation (updated taxonomic studies of these two species are ongoing). However, at present, the major range of occurrence of this new pheretimoid earthworm species is limited in northeastern China only to the province of Liaoning and east of the Inner Mongolia Autonomous Region.

This work therefore aims to report the taxonomy of M. liaoningensis sp. nov. using morphological characters and molecular data. The report of this new species will provide additional information on the earthworm diversity of Northeast China as well as updated information on the pheretimoid earthworm fauna in the country.

Earthworm specimens were collected in the summer of 2023, around the months of May and July, in Northeast China that includes the provinces of Heilongjiang, Jilin, and Liaoning and East of Inner Mongolia Autonomous Region. The collection sites chosen were mainly based on three habitat types, including forests, farmlands, and urban parks. Earthworms were collected through digging and hand sorting. Collections near the sites with surface castings were also taken into account. Earthworm specimens were then preserved in 100% ethanol in the field and stored at –20 °C in the laboratory. Out of over 200 pheretimoid earthworm individuals collected in this study, a total of 11 individuals of M. liaoningensis were morphologically and genetically identified (Table 1), while the rest of the pheretimoid earthworm species are reported separately in ongoing studies.

Earthworm external and internal morphological characters were examined using a stereomicroscope (ZEISS) and ZEN 3.3. pro software for image capturing and to aid in identifying and measuring small organs and other characters for morphological analysis. The generic diagnoses and taxonomic assignments follow Sims and Easton (1972) and Xiao (2019).

Total genomic DNA was extracted from the posterior part of the specimen using the TIANamp Genomic DNA Kit (China). Four mitogenomic data were obtained from M. liaoningensis sp. nov., M. tschiliensis, and M. guillelmi using high-throughput sequences with a paired-end 150 bp strategy on the platform of DNBseq in BGI Genomics (China). Clean data was filtered from the raw data following the steps of Zhao et al. (2022). The whole mitogenomic sequence was assembled by MitoZ v2.4 (Meng et al. 2019) and annotated by the online tool MITOS2 (https://usegalaxy.org/root?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fiuc%2Fmitos2%2Fmitos2%2F2.1.3%20galaxy0) and checked manually by comparing the published mitogenomes of Metaphire. The four mitogenomic sequences and annotation information were submitted to GenBank (accession numbers see Table 1).

Mitogenomic phylogenetic analyses based on the two datasets of 13 protein-coding genes (PCGs), including the 3^rd site of codon (named Site123) and excluding the 3^rd site of codon (named Site12), were performed using Bayesian inference (BI) and maximum likelihood (ML) methods with a concatenated and partitioned strategies. The best DNA substitute model was chosen using jModelTest 2.1 (Darriba et al. 2012), and GTR + I + G was the best model based on the Akaike information criterion. BI was performed by MrBayes 3.2.7a (Ronquist et al. 2012); two million generations were run to make the average standard deviation of split frequencies less than 0.01. ML analysis was performed in RAxML 8.0 (Stamatakis 2014) using the default rapid hill-climbing algorithm and the GTRGAMMA model to search for the best tree, and the clade support was assessed using 1000 rapid bootstrap replicates. Perionyx excavatus was set as the outgroup in both BI and ML analyses.

The former region of the cytochrome c oxidase subunit I (COI) was amplified using the primers LCO1490 (5-GGTCAACAAATCATAAAGATATTGG-3) (Folmer et al. 1994) and COIE (5-TATACTTCTGGGTGTCCGAAGAATCA-3) (Bely and Wray 2004) through polymerase chain reaction (PCR). The mixture (total volume 25 μl) contained 1 μl DNA and 17.25 μl sterile dH₂O, 2.0 μl of dNTP, 2.5 μl of buffer, 0.25 μl Easy Taq Polymerase (TransGen Biotech Co., LTD, Beijing, China), 1.0 μl of former primer LCO1490, and 1.0 μl of reverse primer COIE. The cycling profile was as follows: initial denaturation for 5 min at 95 °C; then denaturation for 30 sec at 95 °C, annealing for 30 sec at 45 °C, and extension for 30 sec at 72 °C for 35 cycles; and final extension for 5 min at 72 °C. Positive PCR products were sent to Tianyi Huiyuan Biotechnology Co., Ltd. (Beijing, China) for Sanger sequencing. Fasta sequences of COI were submitted to GenBank (accession numbers see Table 1). COI sequences of other pheretimoid species included in the analysis were downloaded from GenBank (accession numbers see Table 1). Genetic distances were calculated using the Kimura 2 parameter (K2P) model (Kimura 1980) performed in MEGA 5.0 (Tamura et al. 2011).

The discovery of M. liaoningensis sp. nov. in Northeast China is the second report of a new pheretimoid species in the region in the past decades. There has been an issue regarding an uneven distribution of pheretimoid earthworms in China, with less record in the northern part compared to the south. However, through the recent investigation and earthworm survey conducted last year, the discovery of M. liaoningensis sp. nov. and other pheretimoid earthworms in the region (on-going work) will lead to substantial information on recent earthworm fauna diversity in Northeast China and in the country as well. Still, more fieldwork in the future will be performed in northern China to further investigate the geographical range of M. liaoningensis sp. nov. and to broaden the pheretimoid earthworm survey in the region in aiming to discover newer endemic pheretimoid species in northern China.

We thank Robert J. Blakemore for improving the manuscript. This research was funded by the Fundamental Research Funds for the Universities in Hebei Province (JYT202401, JYQ202402), the China National Tobacco Corporation of Science and Technology Major Projects (110202201018 [LS-02]), the “One Belt and One Road” international scholarship from the Ministry of Science and Technology of China (DL2023130001L), and the President’s International Fellowship Initiative from the Chinese Academy of Sciences (2024VCA0009), and the Program of Central Guidance Fund for Local Science and Technology Development (246Z2907G).