Exploring Barbronia species diversity and phylogenetic relationship within Suborder Erpobdelliformes (Clitellata: Annelida)
Authors: Liu Yingkui , Fu Xinxin , Wang Yu , Liu Jing , Liu Yong , Li Chong , Dong Jiajia 
Published: May 2024

Background
Barbronia, a genus of freshwater macrophagous leeches, belongs to Erpobdelliformes (Salifidae: Clitellata: Annelida), and B. weberi, a well-known leech within this genus, has a worldwide distribution. However, the systematics of Barbronia have not yet been adequately investigated, primarily due to a few molecular markers, and only 20 Barbronia sequences available in the GenBank database. This gap significantly limits our understanding of the Barbronia species identification, as well as the phylogenetic placement of the genus Barbronia within Salifidae.

Methods
Next-generation sequencing (NGS) was used to simultaneously capture the entire mitochondrial genome and the full-length 18S/28S rDNA sequences. The species boundary of Barbronia species was estimated using bGMYC and bPTP methods, based on all available Barbronia COI sequences. Uncorrected COI p-distance was calculated in MEGA. A molecular data matrix consisting of four loci (COI, 12S, 18S, and 28S rDNA) for outgroups (three Haemopis leeches) and 49 erpobdellid leeches, representing eight genera within the Suborder Erpobdelliformes was aligned using MAFFT and LocARNA. This matrix was used to reconstruct the phylogenetic relationship of Barbronia via Bayesian inference (BI) and the maximum likelihood (ML) method.

Results
The full lengths of the mitochondrial genome, 18S and 28S rDNAs of B. cf. gwalagwalensis, are 14847 bp, 1876 bp 1876 bp, and 2863 bp, respectively. Both bGMYC and bPTP results based on COI data are generally congruent, suggesting that the previously proposed taxa (B. arcana, B. weberi formosana, and B. wuttkei or Erpobdella wuttkei) are synonyms of B. weberi. The specimens listed in the B. gwalagwalensis group, however, are split into at least two Primary Species Hypotheses (PSHs). The p-distance of the first PSH is less than 1.3% but increased to 4.5% when including the secondary PSH (i.e., B. cf. gwalagwalensis). In comparison, the interspecific p-distance between the B. weberi group and the B. gwalagwalensis group ranged from 6.4% to 8.7%, and the intraspecific p-distance within the B. weberi group is less than 0.8%. Considering the species delimitation results and the sufficient large p-distance, the specimen sampled in China is treated as B. cf. gwalagwalensis. The monophyly of the four Erpobdelliformes families Salifidae, Orobdellidae, Gastrostomobdellidae sensu stricto and Erpobdellidae is well supported in ML and BI analysis based on a data of four markers. Within the Salifidae, a well-supported Barbronia is closely related to a clade containing Odontobdella and Mimobdella, and these three genera are sister to a clade consisted of Salifa and Linta. According to the results of this study, the strategy of simultaneous obtaining both whole mitochondria and nuclear markers from extensively sampled Salifids species using NGS is expected to fathom both the species diversity of B. gwalagwalensis and the evolutionary relationship of Salifidae.