On the identity and phylogenetic position of Dero indica (Clitellata: Naididae)

The identity and validity of the freshwater worm Dero indica (Clitellata: Naididae; Naidinae) has been debated, and it has been suggested that it is likely to be identical with D. digitata. In this study we combine a newly generated COI sequence of D. indica with available sequences from GenBank, to estimate the phylogeny of Dero using both Bayesian Inference and Maximum Likelihood. The trees show that D. indica is well separated from D. digitata, instead it is closest to D. vaga, but with low support. Furthermore, the analyses confirm the close relationship between Dero and Branchiodrilus found in previous studies, and indicates the presence of cryptic species in D. furcata and D. digitata.


Introduction
The identity of the naidid worm Dero indica Naidu, 1962; pp 110-112 (Clitellata: Naididae: Naidinae) has historically been questioned. In the original description Naidu (1962) described it as being closest to Dero digitata and D. zeylanica, and in their global compendium on aquatic Oligochaeta, Brinkhurst and Jamieson (1971) regarded D. indica as most likely being identical to D. digitata. The ambiguity regarding D. indica was partly resolved by Naveed (2012) by studying several live and preserved specimens of D. indica, and found morphological differences. The two species of Dero differ from each other in the number of dorsal chaetae: D. digitata is characterized as having one hair and one needle chaeta, D. indica as having two hair and two needle chaetae. However, so far, no genetic data from D. indica has been available, and the phylogenetic position is still unknown. Aulophorus Schmarda, 1861 is often treated as a junior synonym to Dero and are often synonomised, also in this study. Please note that Naidu (1962;pp 137-139) also descibed Aulophorus indicus, which becomes a junior homonym to D. indica when the two genera are treated as synonyms.
The aim of this study is to test if Dero indica is genetically distinct from D. digitata and other congenerics, as well as placing it phylogenetically. To obtain this, phylogenies were estimated, with both Bayesian Inference and Maximum Likelihood, on a dataset consisting of newly obtained COI (Cytochrome c oxidase subunit I) sequence of D. indica combined with available sequences of Dero spp. from GenBank.

Material and methods
A specimen of Dero indica was collected at Neithavayal Pond (Minjur), Tamil Nadu, India, 13 o 16′43.1904″ N, 80 o 15′ 11.7252″ E and identified using a combination of the monograph by Brinkhurst and Jamieson (1971), the work of Naidu (2005) on Indian aquatic Oligochaeta and the guide by Timm (2009). The specimen was preserved in ethanol. The DNA extraction and amplification were performed at the Biozone Lab, Chennai, following a phenol chloroform protocol (Pachamuthu et al. 2000). The standard barcoding gene COI Sivabalan Srinivasan and Svante Martinsson contributed equally to this work.
(Cytochrome c oxidase subunit I) was amplified with the primer pair LCO1490 and HCO2198 (Folmer et al. 1994), using the following PCR-program: initial denaturation at 94°C for 3 min followed with 32 cycles with 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min 20 s, and finishing with the final extension at 72°C for 7 min. The obtained COI sequence of D. indica was deposited in GenBank (accession no. MK302407).
The new COI sequence of D. indica was combined with all COI sequences of Dero available in GenBank (downloaded 2019-09-02), as well as a set of other representatives of Naidinae as outgroups (see Table 1 for details), in total 27 sequences, the sequences were aligned using MAFFT v7.017 (Katoh et al. 2002) as implemented in Geneious 8.1.9 (Biomatters Ltd., Auckland, New Zealand), using default settings.
Phylogenies were estimated using both Bayesian Inference in MrBayes v.3.2.6 (Ronquist et al. 2012) and Maximum likelihood (ML) using PhyML 3.0 (Guindon et al. 2010), as implemented at the Montpellier Bioinformatics platform (http://www.atgc-montpellier.fr/). For the Bayesian analysis the alignment was partitioned according to codon position, partitions were unlinked. Rate variation across sites was set to gamma distribution with a proportion of invariable sites; model jumping was implemented to integrate over substitution model space. The analyses ran for 20 million generations sampling every 10,000 generations, the first 25% were discarded as burn-in, and a majority-rule consensus tree was constructed. For the ML analysis the Smart Model Selection (Lefort et al. 2017) with Bayesian Information criterion was used for automatic model selection; Subtree Pruning and Regrafting were used for tree improvement. Branch support was calculated with the SH-like (Shimodaira-Hasegawa test-like) approximative likelihood ratio test (aLRT) (Anisimova and Gascuel 2006). The trees were rooted according to the result in Erséus et al. (2017). All trees were drawn in FigTree 1.4.2 (Rambaut 2014) and further edited in Adobe Illustrator.

Results
The newly generated sequence of Dero indica is 507 base pairs (bp) long, and the COI alignment is 658 bp long, whereof 272 are variable. The Bayesian phylogenetic estimation resulted in mainly well resolved tree (Fig. 1a). However, Dero is not recovered as monophyletic, instead Dero and Branchiodrilus are found in a well-supported, but unresolved clade. This clade is a polytomy, consisting of a well-supported Branchiodrilus, and three groups of Dero. One of them consists of D. indica and D. vaga, but the sister relationship between them are unsupported. The other two groups of Dero are well supported. The first group consists of D. borellii, D. furcata, and D. superrenus, D. furcata forms two groups, and D. borellii is very close to one of them. The second group well-supported group consists of D. digitata, D. obtusa, and unidentified D. sp. the two D. sp. are both found together with one D. digitata, whereas the other D. digitata forms a separate group.
The ML phylogenetic estimation (Fig. 1b) is in most aspect similar to the Bayesian tree, Dero and Branchiodrilus form a weakly supported clade, and in contrast to the Bayesian analysis, both genera are recovered as monophyletic, the monophyly of Dero is weakly supported, whereas Branchiodrilus is strongly supported. The same three groups of Dero found recovered in the Bayesian tree are also recovered here, with the same internal relationships as in the Bayesian analysis. The support for the sister-group relationship between D indica and D. vaga is weak.

Discussion
Both phylogenetic analyses (Fig. 1) indicated a sister-group relationship between D. indica and D. vaga but, the support for this relationship is weak in both trees. However, we can conclude that D. indica is not closely related to D. digitata, as they well-separated in the trees, as well as differing morphologically (Naveed 2012), also summarised in the Introduction. We only have representatives of seven of the about 35-40 described species of Dero, and it is likely that D. indica is closer to some of the species not included in this study. More species need to be sequenced to find the exact position of D. indica.
Of the included species, two have deep divergences, indicating that there could be cryptic species involved. Furthermore, D. borellii is very close to one of the lineages of D. furcata, indicating that they could be the same species. To properly test this, more genetic markers are needed.
There are very few molecular studies on the clitellate fauna of India (Chakma et al. in press;Martin et al. 2018; see also Lalthanzara et al. 2018) and there are currently only seven records of COI sequences from the family Naididae from India in GenBank (search performed 2019-09-13). There is great potential for the use molecular methods in the exploration of the clitellate fauna of India, and hopefully, there will be more studies in the future, characterising the clitellate fauna of India.
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