First Record of Ophidonais serpentina (Müller, 1773) (Oligochaeta: Naididae) in China: The Occurrence or Absence of Needles Are Intraspeciﬁc Differences

: A naidid oligochaete, Ophidonais serpentina (Müller, 1773) is redescribed based on specimens from the Xinkai River in Zhejiang Province, China. O. serpentina is very common in Europe and America. This study is the ﬁrst record of the species in China. By integrating the previously morphological descriptions related to O. serpentina in the world, it can be divided into three morphological groups: a group with dorsal chaetae starting from VI, a group without dorsal chaetae, and a group with an unstable starting position of the dorsal chaetae. By comparing the mitochondrial DNA (16S rDNA, COI), nuclear DNA (ITS2), and histones (H3) from the three groups, Bayesian inference and maximum likelihood phylogenetic analyses were performed based on the combined data set. Different analyses gave almost consistent phylogenetic trees. All of the genetic distances between the three groups were 0.00%. No genetic variation can be detected between the specimens regardless of the presence and starting position of dorsal chaetae. This result suggests that a single lineage of O. serpentina is widespread worldwide.


Introduction
Ophidonais serpentina (Müller, 1773) (Annelida: Clitellata: Naididae: Naidinae) [1] is the type species of the monotypic genus Ophidonais Gervais [2]. In phylogeny, O. serpentina seems to be the sister group to a clade consisting of Stylaria, Ripistes, and Arcteonais [3]. This species is a common species in Europe, America, and Africa. Timm suggested that this species may be widely distributed in the Holarctic realm [4]. In Asia, it has previously been reported in Iran, Japan, Korea and Siberia. This study is the first to find the species in China.
In terms of morphology, dorsal chaetae have been previously described as stout and straight, beginning from VI [4,5]. In North America, Kathman and Brinkhurst found that in some specimens, all of the dorsal chaetae had been shed [6]. Ohtaka also noted the absence of dorsal chaetae [7]. Our specimens, which were collected from Xinkai River, could be divided into two groups. Most of the individuals had no needles, while the rest had needles with an uncertain beginning segment. By integrating records from other regions of the world, there are three morphological groups in O. serpentina, namely the no needles group, the stable needles group, and the unstable needles group.
The aim of the present study was to compare the morphological characteristics of these new specimens with corresponding features of the previously examined specimens of O. serpentina. Through the inclusion of molecular data, we explored whether the differences in morphology were related to genetics or represent phenotypic plasticity.

Taxon Sampling and Collection of Specimens
The Xinkai River is a eutrophic urban river in Shaoxing City, Zhejiang Province, China (29 • 95 99.06 N, 120 • 47 57.55 E) and a coastal river near the East China Sea. Specimens of O. serpentina were collected from the surface of hydrophytes at the shore of Xinkai River. A total of 64 specimens were collected, as shown in Table 1. The conditions of the site were as follows: the sediment was composed of silt and blocks, the water temperature was 25 • C, the depth was 0.5 m, the pH was 7.29, the conductivity was 428.5 µS/cm, the total dissolved solids (TDS) was 290.23 mg/L, the oxidation-reduction potential (ORP) was 564.25 mV, and the dissolved oxygen (DO) was 5.97 mg/L. The specimens were preserved in 80-90% alcohol. The morphological characteristics were observed with a light microscope (LM) and a scanning electron microscope (SEM). The specimens used for SEM were acidified and dried naturally, then pasted onto the SEM copper platform with carbon conductive adhesive. After that, the specimens were sputtered with gold and placed under the SEM (Hitachi SU-8010) for observation and photography. The permanently preserved specimens were stained with borax carmine, separated by hydrochloric acid alcohol, gradient dehydrated by alcohol (70-99%), hyalinized with xylene, and sealed by Canadian gum. Drawings and measurement were based on preserved specimens. The specimens were deposited in the Institute of Hydrobiology, Chinese Academy of Sciences. In this study, six specimens were selected for the extraction of genes. The gene data of other related species were downloaded from the GenBank database. Rhyacodrilus coccineus and Rhyacodrilus falciformis were selected as outgroups (Table 2). Drawings were made with Adobe Photoshop CC 2019.   2.2. DNA Extraction, Amplification, and Sequencing DNA was extracted by using a TIANamp Genomic DNA Kit and following the manufacturer's manual. The conditions for gene amplification are specified in Table 2. The 1µL amplified product was extracted and detected by gel electrophoresis. If the target bands were found, the polymerase chain reaction (PCR) products were sent to Icongene Ltd. (Wuhan, China) for sanger sequencing. All of the primers used for sequencing are described in Table 3. Table 3. Primers and programs used for amplification and sequencing of fragments of the mitochondrial 16S and COI and nuclear ITS2 and H3 markers.

Alignments and Phylogenetic Analysis
We used BioEdit to check the original sequence, and then used SeqMan (DNASTAR) to connect the sequence when the sequence showed as single peak. We aligned the complete sequences into NCBI and did BLAST online. The newly acquired and downloaded sequences from the GenBank database were stored in FASTA format for further analysis. Then, we chose PhyloSuite [24] to analyze the data. Four sequences in batches were aligned by using the '-auto' strategy and normal alignment mode in MAFFT [25] and aggregated into a sequence matrix. The best model for the BIC criterion was selected by a model finder [26]. The optimal model was chosen by BIC: GTR + F + G4. Under the conditions of the GTR + G + F model (2 parallel runs, 1 × 10 7 generations), Bayes 3.2.6 [27] was used to infer the occurrence of the Bayesian inference system, in which 25% of the initial sample data were discarded as burn-in. Maximum likelihood phylogenies were inferred using IQ-TREE [28] under the GTR + G4 + F model for 1000 standard bootstraps. MEGA X was used to calculate the genetic distance between two pairs, and a p-distance model was selected to complete the calculation [29]. We used the Interactive Tree of Life (https://itol.embl.de accessed on 2 March 2022) tool [30] and Adobe Illustrator 2021 to manipulate and combine the phylogenetic trees.

Taxonomy and Morphology
Ophidonais serpentina (Müller, 1773) See Speber (1948) for synonymies [5]. Description of Chinese specimens: Length 4-30 mm, segments 27-87. The living individual is pale and has pigment stripes on its anterior segments, wrapped by a thin crust of foreign particles ( Figure 1D). Coelomocytes are abundantly present ( Figure 1C). Obtuse prostomium, eyes present ( Figure 1B). Stomach slowly widening in VIII-IX. Unable to swim. Description of Chinese specimens: Length 4-30 mm, segments 27-87. The living individual is pale and has pigment stripes on its anterior segments, wrapped by a thin crust of foreign particles ( Figure 1D). Coelomocytes are abundantly present ( Figure 1C). Obtuse prostomium, eyes present ( Figure 1B). Stomach slowly widening in VIII-IX. Unable to swim.  Needles are absent or beginning at posterior segments, often absent ( Figures 2B and 3C-E). Needles are stout and straight, length 62-140 µm, width 5 µm, 1 per bundle, 2-3 equal pointed distal end, indistinct under the light microscope, proximal blunt, nodulus proximal 1/3-1/4, and indistinct. Ventral chaetae are 3-5 per bundle, with those of II longer than the rest, having a length 150-178 µm and width 5 µm; other ventral chaetae have length 95-125 µm and width 5 µm. The distal teeth of ventral chaetae are longer than the proximal teeth in anterior segments and equal in the following segments ( Figures 2D-F and 3A,B). Nodulus median or proximal, nodulus distal gradually in the following segments. proximal 1/3-1/4, and indistinct. Ventral chaetae are 3-5 per bundle, with those of II longer than the rest, having a length 150-178 μm and width 5 μm; other ventral chaetae have length 95-125 μm and width 5 μm. The distal teeth of ventral chaetae are longer than the proximal teeth in anterior segments and equal in the following segments ( Figures 2D-F  and 3A-B). Nodulus median or proximal, nodulus distal gradually in the following segments.  Specimens deposited. IHB ZJ20210628a-b, two whole-mounted specimens, immature, no needles; IHB ZJ20210628c-d, two whole-mounted specimens, immature, with needles. Six specimens were used for extracting DNA and two specimens were used for electron microscopy. The rest of the specimens were dipped in 80-90% alcohol, and preserved in the Institute of Hydrobiology, Chinese Academy of Sciences.

Phylogenetic Analyses
We combined 119 nucleotide sequences into a dataset (3603bp) for phylogenetic analysis. The trees based on the combined dataset were largely consistent in the maximum likelihood (ML) and Bayesian inference (BI) analysis. Both trees were shown in an equidistant version (Figure 4), and the support values were given as BI posterior probabilities (pp) and ML bootstrap support (bs). In the selected specimens, Ophidonais serpentina 1-3 were the individuals without dorsal chaetae, and O. serpentina 4-6 were those with dorsal chaetae. O. serpentina A-C were downloaded from GenBank. Two phylogenetic trees highly supported that all of the O. serpentina specimens were recovered as a monophyletic clade (pp 1.00, bs 100). In maximum likelihood analysis, O. serpentina was recovered as sister to the clade comprising four species: Piguetiella blanci, Specaria josinae, Stylaria lacustris, and Stylaria fossularis (bs 54). In Bayesian inference analysis, O. serpentina was sister to the clade comprising Slavina appendiculata and Vejodovskyella comata (pp 0.66). In both trees, all seven species formed a single clade (pp 0.99, bs 89). Specimens deposited. IHB ZJ20210628a-b, two whole-mounted specimens, immature, no needles; IHB ZJ20210628c-d, two whole-mounted specimens, immature, with needles. Six specimens were used for extracting DNA and two specimens were used for electron microscopy. The rest of the specimens were dipped in 80-90% alcohol, and preserved in the Institute of Hydrobiology, Chinese Academy of Sciences.

Phylogenetic Analyses
We combined 119 nucleotide sequences into a dataset (3603bp) for phylogenetic analysis. The trees based on the combined dataset were largely consistent in the maximum likelihood (ML) and Bayesian inference (BI) analysis. Both trees were shown in an equidistant version (Figure 4), and the support values were given as BI posterior probabilities (pp) and ML bootstrap support (bs). In the selected specimens, Ophidonais serpentina 1-3 were the individuals without dorsal chaetae, and O. serpentina 4-6 were those with dorsal chaetae. O. serpentina A-C were downloaded from GenBank. Two phylogenetic trees highly supported that all of the O. serpentina specimens were recovered as a monophyletic clade (pp 1.00, bs 100

Pairwise Genetic Distances
The uncorrected p-distance for 16S rDNA genes in the Ophidonais showed barcoding gaps were between 1.7 and 4.6%, and the COI genes were between 11.4 and 15.8%. The pdistances between the three morphological groups were 0.00% (Table 4).

Pairwise Genetic Distances
The uncorrected p-distance for 16S rDNA genes in the Ophidonais showed barcoding gaps were between 1.7 and 4.6%, and the COI genes were between 11.4 and 15.8%. The p-distances between the three morphological groups were 0.00% (Table 4).

Morphological Characters of Ophidonais Serpentina
Of the 64 specimens, only 11 individuals had dorsal chaetae which appeared on one side of the body and with one on each segment. So far, the descriptions of this species in the published records were mostly consistent with Sperber (1948) and Timm (2009), without mentioning the lack of dorsal chaetae. This phenomenon has been shown in reports from North America [6] and Japan [31] and was also found in specimens collected in China. Additionally, some scholars found cilia in the papillae [32] but they were absent in our specimens and the arrangement of papillae was irregular. More detailed descriptions of the differences between the species from different regions of the world are listed in Table 5.

Intraspecies Analysis
The results of pairwise distance and phylogenetic analysis showed that Ophidonais serpentina was related closely to Vejdovskyella comata (pp 89, bs 0.66, mean distance 2.7% (16S), 13.8% (COI)), which was consistent with Bely and Wray's conclusion. Additionally, we found that Piguetiella blanci (pp 0.99, bs 54, mean distance 2.8% (16S), 12.1% (COI)) was also closely related to O. serpentina, which was not mentioned in Bely's study. All the results showed that the three groups (the absent dorsal chaetae group, the stable dorsal chaetae group and the unstable dorsal chaetae group) from a single monophyly. Whether the needles existed or not, and whether the starting position of the needles was stable or not, there was no genetic variation that was detected between the specimens. This