Naididae, Ehrenberg, 1831

Mercan, Deniz, Horenkamp, Austin, Aydemir, Merve Nur, Aydemir, Habeş Bilal, Neubig, Kurt, Arslan, Naime & Anderson, Frank E., 2025, Comparative mitogenomics of Clitellata reveals mitogenome organization can affect the mode of phylogeny, Turkish Journal of Zoology 49 (4), pp. 197-206 : 199-200

3.1. Mitogenome characterization of Naididae

The P. hammoniensis mitogenome assembly was 14,881 bp long and was completely annotated. The partial mitogenomes of St. lacustris , C. diaphanus , C. diaphanus sp. B, Sl. appendiculata , and St. fossularis were 14,813; 15,724; 15,694; 15,699, and 15,719 bp long, respectively. All genes are dispersed and transcribed in the heavy chain, like other annelids ( Oceguera-Figueroa et al., 2016; Weigert et al., 2016) ( Figure 1 ; Table S2). The mitogenome sequences of P. hammoniensis , St. fossularis , St. lacustris , C. diaphanus , C. diaphanus sp. B, and Sl. appendiculata can be accessed from NCBI under accession numbers OQ654101, PP909790, PP909791, PP909792, PP909793 and PP893275, respectively.

As seen in other invertebrates, these mitogenomes showed an A+T bias of 67.8– 70.9%). Several adjacent genes overlapped from two (trnY-trnG, trnM-rrnS, trnAtrnS2 for all species) to 29 (trnQ- nd6, nd2-cox1 for C. diaphanus ) nucleotides (Table S2). In the mitogenomes, the overall lengths of PCGs, tRNAs, and rRNAs were 11928, 1389, and 1982 bp, respectively. Conserved atp6-atp8 overlap was missing in this mitogenome, and accelerated evolution seems to lead to both differentiation of gene order and accumulation of intergenic regions. The conserved transcriptional sequence WHWGHTW identifiedinEcdysozoawasdetectedinthe P. hammoniensis mitogenome, 14 bp upstream of the trnT gene ( Aydemir et al., 2023).

All PCGs were found to start with the standard ATG (methionine) codon, except for nd6 (ATT) in P. hammoniensis ; nd1 (GTG) in St. lacustris ; nd2 (CTT), nd1 (GTG), and nd3 (ATT) in St. fossularis ; nd2 (TTT), nd4L (ATA), and nd1 (GTG) in Sl. appendiculata ; nd3 (ATA) and nd1 (GTG) in C. diaphanus and nd2 (ATC), nd1 (GTG), nd3 (ATA), and atp8 (TTA) in C. diaphanus sp. B. While cox1, cox3, cytB, atp6, nd5, nd4L, and nd4 genes had complete TAA triplets as stop codons, cox2, atp8, nd6, nd1, nd3, and nd2 genes had incomplete T-stop codons that are completed via polyadenylation processes. Like the total mitogenome, all PCGs had negative AT- and negative GC-skew values. From the ORF Finder analysis, the P. hammoniensis mitogenome is capable of coding 780 different ORFs that start with any sense codon and are longer than 30 nucleotides (Supplementary file S1). Interestingly, 445 of these 780 ORFs are potentially encoded in the light chain, which does not code any known mitochondrial gene. Based on BLAST searches, 39 of 780 potential ORFs aligned with protein sequences in the nonredundant protein database (Supplementary file S2). Thirteen of the 39 were formal mitochondrial PCGs (atp6, atp8, cox1, cox2, cox3, cytB, nd1, nd2, nd3, nd4, nd4L, nd5, nd6) and five of the 39 were different framed forms of cox1 and cox2 sequences. Eighteen of the 39 aligned amino acid sequences were potentially coded in the light chain, eight of which seemed to be an alternative form of some nd genes (nd1, nd3, nd4 and nd5) in different organisms (Supplementary file S2). Similarly, 726, 727, 757, 770 and 736 potential ORF sequences were detected in the St. fossularis , St. lacustris , C. diaphanus , C. diaphanus sp. B, and Sl. appendiculata mitogenomes, respectively.

The average length of tRNAs was 63.13 bp and varied from 56 bp in trnT to 69 bp in trnQ (Table S2). All tRNAs constructed had a conserved clover-leaf structure except trnS1 and trnT, both of which had a DHU-replacement ( Figure S1 ). A reduced secondary structure pattern is common in invertebrates for trnS1, but trnT reduction is unique to P. hammoniensis in Clitellata. trnE, trnP, trnS1, and trnS2 tRNAs had negative AT- and negative GC- skew values. Unlike the total mitogenome and PCGs, tRNA genes tended to have at least one positive skewness value (Table S2).

The rrnS and rrnL genes were 737 and 1245 bp long, respectively, and they had positive AT- and negative GCskew values (Table S2). Both rRNA genes had conserved putative secondary structures ( Figures 2a and 2b ). The putative secondary structures of rrnS and rrnL genes comprised three domains with 30 helices and five domains (domain III is absent in invertebrates) with 49 helices, respectively.

Noncoding regions of the mitogenome of P. hammoniensis were dispersed between 11 consecutive gene clusters and had a total length of 472 bp, varying from 2 (trnH-trnR and rrnS-trnV) to 208 (trnR- nd5) bp long. Intergenic regions did not exhibit significant matches in the BLAST search with low query coverage values (e> 0.01). In Clitellata, the putative control region is generally located in the atp6-nd5 gene cluster (trnW- atp6 -trnHtrnR- nd5 -trnF), and the P. hammoniensis mitogenome had 39-bp-long total intergenic sequences in this region (Table S2). In the nd5 -trnF intergenic region, transcription polarity was dramatically changed. This region probably has the replication origin that can construct a stable secondary structure with significant ΔG value (−13.60 kcal/mol) ( Figure S2 ).

Figures

Figure 1.a.Mitogenome organizationofP. hammoniensis drawn using OGDraw (Greiner et al., 2019). All genes are encoded in heavy (J-) strand. Gaps between genes indicate IGSs. b. Different gene order patterns in Naididae mitogenomes. Two tRNAgenes (trnF and trnP) were rearranged with TDRL events in Naididae (between Tubificinae and Naidinae) mitogenomes. St. fossularis has the same mitochondrial gene order pattern with other Naidinae species sequenced in this study (St. lacustris, C. diaphanus, C. diaphanus sp. B, and Sl. appendiculata).

Figure 2. The putative secondary structures of (a) rrnS and (b) rrnL of P. hammoniensis. Structures were predicted and helixes were numbered with reference W. laevis secondary structures (Ye et al., 2015). Roman numbers refer to domain names.Dashes indicate Watson-Crick base pairing and dots indicate G-U base pairing.