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. 2026 Mar 6;53(1):471. doi: 10.1007/s11033-026-11637-w

The complete mitogenome of Tylomelania sp. ‘Poso A’ (Caenogastropoda: Pachychilidae) from Lake Poso assembled from long-read sequencing data

Björn Stelbrink 1,2,✉, Carola Greve 3, Charlotte Gerheim 3, Walter Salzburger 2, Thomas von Rintelen 4
PMCID: PMC12966257  PMID: 41790322

Abstract

Background

Sulawesi’s ancient lakes represent biodiversity hotspots for various freshwater groups such as gastropods, shrimps, and crabs, as well as fishes. The less known Lake Poso in the northern part of the island is home to about 25 species belonging to the island-endemic freshwater gastropod genus Tylomelania within the family Pachychilidae. Here, we present the first complete mitogenome of a Tylomelania species from Lake Poso (Tylomelania sp. ‘Poso A’) assembled from long-read (PacBio) sequencing data.

Methods and Results

The mitogenome is 16,540 bp long and thus only minimally shorter than the mitogenome of its congener from Lake Towuti, T. sarasinorum (16,632 bp). Based on 13 protein-coding and 2 rRNA genes we place the species within a recently published mtDNA-based phylogeny and show its close relationship to T. carbo.

Conclusions

This is the first Tylomelania mitogenome to be generated using long-read sequencing data, and it is only the second complete mitogenome available for this species-rich radiation. Generating mitogenomes from such long-read data is only the first step towards creating the reference genome required for in-depth phylogenomic analyses.

Keywords: Indonesia, Sulawesi, Freshwater gastropods, PacBio, Molecular phylogeny

Introduction

The Indonesian island Sulawesi within the Malay Archipelago is a hotspot of biodiversity for various terrestrial, but also freshwater groups [1, 2]. The latter are particularly concentrated in the island’s ancient lakes and include morphologically and genetically diverse species flocks. These are monophyletic, species-rich, and endemic groups [3, 4], the most well-studied of which are gastropods, crabs, shrimps, and fish [5]. One of the most biodiverse and abundant freshwater invertebrate groups is the gastropod genus Tylomelania, which is endemic to the island and comprises about 50 mainly lacustrine species [5]. In recent years, evolutionary research has primarily focused on the well-studied Malili lake system. This freshwater feature was also the subject of an international deep-drilling endeavor, which revealed that Lake Towuti originated about 1 Ma [6]. In contrast, Lake Poso – located further north and not connected to the Malili lake system – is also very deep and species-rich (about 25 Tylomelania species, 3 gecarcinucid crabs, 8 Caridina shrimps, as well as 13 native and 17 non-native fishes; [5, 7, 8]), but its gastropod fauna is still insufficiently documented.

Much of the knowledge on species boundaries and phylogenetic relationships within Sulawesi’s freshwater species flocks is based on morphological and mitochondrial data (see, e.g. [5], for an overview). Despite the various advantages of mtDNA in taxonomy and systematics, several processes such as introgressive hybridization, incomplete lineage sorting, and horizontal gene transfer remain to be investigated (e.g. [9]). Moreover, young species groups, such as those inhabiting the ancient lakes of Sulawesi, may have evolved at a pace too quick to be detected for mitochondrial markers to resolve phylogenetically [5]. Therefore, genome-wide data is key to a better understanding of the relationships between these species and the role of introgression during the emergence of the different species flocks. For Tylomelania, genomic (short-read) data has been generated for the vast majority of species inhabiting the Malili lake system. However, this has not yet been exploited beyond the mitogenome level (see [10]).

As a first step towards gaining a more comprehensive insight into the evolutionary history of this genus, we here selected a specimen from a Lake Poso endemic as this can serve as a useful (genome) reference for subsequent phylogenomic studies. The strategy was to use a lacustrine species from outside the Malili lake system (see Fig. 1) as an outgroup, which exhibits similar features due to presumably undergoing similar adaptation processes in a similar environment (i.e., parallel evolution; see [11] for examples). Moreover, the reference species was selected to be genetically close enough to the remaining lake species facilitating mapping and SNP discovery in a genome-wide context. For the first time, we were using long-read sequencing on this genus in order to create an accurate and contiguous reference genome. To assess and make use of these long and high-quality reads, we first generated a mitogenome, which is presented here. This represents an important genomic contribution to the understudied Lake Poso fauna and will also help to shed more light on the extent of mitochondrial introgression within the genus.

Fig. 1.

Fig. 1

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Example photo of Tylomelania sp. ‘Poso A’, taken in an aquarium (left panel). Photo by Chris Lukhaup (with permission). Middle panel shows Lake Poso (marked in red) and the Malili lake system (marked in orange) on Sulawesi including major drainages. Inset map (bottom-right) shows the island (marked in dark-grey) within the Indo-Australian Archipelago

Materials and methods

A single individual of Tylomelania sp. ‘Poso A’ (see [11]; NCBI: txid246313) from an aquarium stock (Fig. 1) was selected for the genomic analysis to ensure that high-molecular-weight DNA can be extracted for long-read sequencing. The species is morphologically very distinct from the remaining species endemic to Lake Poso given its shell size and sculpture, and its body colouration (see [11]). The specimen and its DNA, which have been used here, are stored in the Lab Centre for Biodiversity Genomics of the Senckenberg Research Institute under voucher number TBG_4316.

Genomic DNA was extracted from foot tissue using a slightly modified CTAB protocol as described by [12], which is suitable for polysaccharide-rich tissue such as those from molluscs. Long reads were generated on a PacBio Sequel IIe at the Radboudumc Genome Technology Center’s Sequencing Core Facility in Nijmegen, the Netherlands. A total of 5,807,967 polymerase reads were yielded from the PacBio run. HiFi reads (read quality > 0.99) were obtained using pbccs 6.4.0 (github.com/PacificBiosciences/ccs), demultiplexed with lima 2.9.0 (github.com/PacificBiosciences/barcoding) and cleared from PCR duplicates using pbmarkdup 1.0.3 (github.com/PacificBiosciences/pbmarkdup). The final dataset consisted of 2,994,657 reads for further analyses.

HiFi reads were used to assemble the mitogenome of the Tylomelania sp. ‘Poso A’ de novo. The MitoHiFi 2.0 [13] pipeline was used to assemble the mitogenome de novo using the published T. sarasinorum mitogenome [14] as the source (i.e., reference for mapping) and to annotate the final contig using the implemented software MitoFinder [15]. Differences between the two complete Tylomelania mitogenomes were visualized and assessed in Geneious Prime 2025.2.2 (geneious.com).

To place Tylomelania sp. ‘Poso A’ within the Tylomelania Lake Poso clade, we made use of a recently published mitogenome dataset including 13 CDS and 2 rRNAs [10]. Single-locus datasets were concatenated using AMAS [16] and subjected to IQ-TREE 2.2.0 [17, 18] for phylogenetic analysis. We reduced the published dataset [10] by only including T. carbo (GenBank acc. no. for the 15 mitochondrial markers available at: ncbi.nlm.nih.gov/nuccore/?term=tylomelania+190828_1 & *+192946_1), T. kuhli (*+191710_1 & *+192321_1), T. neritiformis (*+190814_4 & *+190814_6) from Poso River (two individuals each) and the newly sequenced specimen of Tylomelania sp. ‘Poso A’ (GenBank acc. no. PX289796). We further included the two riverine species Tylomelania sp. ‘Beau’ (*+190750_4 & *+191027a_2) as well as T. celebicola (*+190529_5 & *+190532_4; also two individuals each), of which the individual T. celebicola (2714) was used as outgroup to root the tree (see [10]).

Two partition files were tested (15 partitions for the 13 protein-coding genes + 2 rRNAs vs. 41 partitions (codon partitioning for the protein-coding genes) with MFP+MERGE to identify best-fit substitution models and partition schemes. Nodal support was estimated using 1,000 ultrafast bootstrap (UFBoot) replicates and 10,000 replicates of the SH-like approximate likelihood ratio test (SH-aLRT; [19]). Nodes with > 95% for UFBoot and > 80% for SH-aLRT are considered well-supported [19].

Results

A total of 5,312 reads were mapped against the source with a mean and maximum coverage of 1,376.7 and 3,384, respectively (Fig. 2, inset). This is considerably higher than the mean coverage of 812.9 obtained from the short-read sequencing data analysed for the 78 Tylomelania species in [10]. More importantly, the present mitogenome is contiguous, whereas this only applied to 25 out of 78 mitogenomes in [10]. The assembled mitogenome is 16,540 bp long (Fig. 2) and thus only slightly shorter than that of T. sarasinorum (16,632 bp; [14]). Base composition was markedly biased towards a high A + T content (65.2%; A = 29.2%, T = 36.0%), whereas the G + C content was considerably low (34.8%; G = 18.6%, C = 16.2%). These ratios were very similar to those in T. sarasinorum (A + T = 65.2%, G + C = 34.9%). A single, longer repeat of 23 and 43 bp (TA motif) was identified within the D-loop/control region in the present and the T. sarasinorum mitogenome, respectively.

Fig. 2.

Fig. 2

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Annotated mitogenome of Tylomelania sp. ‘Poso A’ visualized in Geneious Prime 2025.2.1 (see Table 1 for more details on the features). Inset represents the output obtained from MitoHiFi showing the coverage depth along the assembled mitogenome (mean depth = 1,376.7, max depth = 3,384)

After comparing both mitogenomes, the final annotation of the present mitogenome was modified: (1) the range of 16 S-rRNA was extended from 5,432 to 6,814 bp to 5,432–6,816 bp to start at the same position as the one of T. sarasinorum; (2) the range of atp8 was extended from 9,240 to 9,392 bp to 9,240–9,410 bp until a stop codon (TAA) was reached; (3) the range of nd5 was extended from 15,274 to 16,518 bp to 14,799–16,520 bp until a fragment size was reached that was observed in other Tylomelania species [10] and that began with a starting codon (ATG); moreover, a ‘GA’ [coverage = 773] insert was added after 15,310 bp that was missing in the consensus sequence and thus caused the presence of multiple stop codons across the gene; and (4) the range of atp8 was extended from 9240 to 9392 bp to 9240–9410 bp until a stop codon (TAA) was reached.

On the heavy strand, gene order was: tRNAPhe (68 bp), tRNASer2 (67 bp), nd2 (1,074 bp), tRNAAsp (67 bp), atp8 (171 bp), atp6 (696 bp), tRNAIle (70 bp), nd3 (354 bp), cox1 (1,533 bp), cox2 (690 bp), tRNASer1 (67 bp), nd4l (291 bp), nd4 (1,368 bp), tRNAHis (68 bp), and nd5 (1,206 bp). Gene arrangement on the light strand was: tRNACys (63 bp), tRNAAla (68 bp), tRNAAsn (68 bp), tRNATrp (66 bp), tRNAGlu (64 bp), tRNATyr (65 bp), tRNAArg (70 bp), tRNAGln (67 bp), tRNALys (68 bp), cox3 (780 bp), tRNAMet (66 bp), cob (1,140 bp), nd6 (552 bp), tRNAPro (64 bp), nd1 (933 bp), tRNALeu1 (68 bp), tRNALeu2 (70 bp), 16 S-rRNA (1,385 bp), tRNAVal (70 bp), tRNAGly (66 bp), tRNAThr (72 bp), and 12 S-rRNA (900 bp). Two main overlaps were observed between cob and nd6 (47 bp) as well as nd4 and nd4l (7 bp). All PCGs started with an ATG codon and stopped with a TAA codon except for nd1, nd2, and nd3 (TAG stop codon). The complete features of the mitogenome with the position of each gene are provided in Table 1.

Table 1.

Feature table of the mitogenome of Tylomelania sp. ‘Poso A’

Start (bp) End (bp) Type Gene Product
1 68 tRNA tRNA-Phe
809 747 tRNA tRNA-Cys
964 897 tRNA tRNA-Ala
1039 972 tRNA tRNA-Asn
1124 1059 tRNA tRNA-Trp
1257 1194 tRNA tRNA-Glu
1354 1290 tRNA tRNA-Tyr
1457 1388 tRNA tRNA-Arg
1594 1528 tRNA tRNA-Gln
1669 1602 tRNA tRNA-Lys
2533 1754 CDS cox3 Cytochrome c oxidase subunit 3
2601 2536 tRNA tRNA-Met
3773 2634 CDS cob Cytochrome b
4278 3727 CDS nd6 NADH dehydrogenase subunit 6
4346 4283 tRNA tRNA-Pro
5288 4356 CDS nd1 NADH dehydrogenase subunit 1
5356 5289 tRNA tRNA-Leu
5438 5369 tRNA tRNA-Leu
6816 5432 rRNA 16 S-rRNA
6868 6799 tRNA tRNA-Val
6968 6903 tRNA tRNA-Gly
7050 6979 tRNA tRNA-Thr
7948 7049 rRNA 12 S-rRNA
8033 8099 tRNA tRNA-Ser
8100 9173 CDS nd2 NADH dehydrogenase subunit 2
9172 9238 tRNA tRNA-Asp
9240 9410 CDS atp8 ATP synthase F0 subunit 8
9432 10,127 CDS atp6 ATP synthase F0 subunit 6
10,135 10,204 tRNA tRNA-Ile
10,205 10,558 CDS nd3 NADH dehydrogenase subunit 3
10,635 12,167 CDS cox1 cytochrome c oxidase subunit 1
12,238 12,927 CDS cox2 cytochrome c oxidase subunit 2
12,940 13,006 tRNA tRNA-Ser
13,024 13,314 CDS nd4l NADH dehydrogenase subunit 4 L
13,308 14,675 CDS nd4 NADH dehydrogenase subunit 4
14,731 14,798 tRNA tRNA-His
14,799 16,520 CDS nd5 NADH dehydrogenase subunit 5
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MFP-MERGE favoured three and five partitions for the non-codon partitioning and the codon-partitioning scheme, respectively. As there was no difference between these two phylogenetic analyses in terms of topology and branch lengths, only the non-codon partitioning tree is shown (Fig. 3). Accordingly, Tylomelania sp. ‘Poso A’ clustered within the highly-supported clade P (Lake Poso; see also [10]) and is closely related to one of the two specimens of T. carbo from the lake’s east shore (Fig. 3). As in the previous study [10], the two individuals of each of these Lake Poso/Poso River species do not cluster together, but are scattered within the Poso clade.

Fig. 3.

Fig. 3

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Phylogenetic relationships between Tylomelania from Lake Poso and the Poso River species (marked in orange) including Tylomelania sp. ‘Poso A’ (marked in pink) as well as selected riverine species (marked in dark-blue; see also [10] for a more comprehensive dataset). Numbers on branches indicate nodal support (left: SH-aLRT, right: UFBoot). Shells of Lake Poso species in apertural view are reproduced from [11] (scale bar = 10 mm)

Discussion

Generating long-read sequences and a reliable mitogenome are the first steps towards a Tylomelania reference genome to be used in subsequent phylogenomic projects. Conducting (phylo)genomic studies on molluscs frequently poses challenges, for example due to the complexity of extracting high-purity, high-molecular-weight genomic DNA in the presence of high levels of mucopolysaccharides (e.g. [20–22]). Mollusc-specific CTAB-based isolation methods mitigate some of these issues and are widely used for traditional PCR and Sanger sequencing, but also for short-read sequencing approaches. Nevertheless, mucopolysaccharides may also affect long-read sequencing by interfering with the chemistry or blocking the pores used for sequencing [21]. We cannot evaluate this potential effect here, but the PacBio sequencing conducted appeared to be overall successful as reflected in the contiguous, highly covered, and well-annotated mitogenome. This contiguity is probably the main advantage of long reads for the mitogenome assembly, which could not be achieved for the majority of species analysed in [10], despite the high number of short reads and considerable mean coverage. Moreover, long reads help to better identify gene duplications, along with structural variants and tandem repeats in non-coding regions (e.g. [23]), which however were not observed here. A broader comparison is needed to validate possible structural differences and the accuracy of gene annotations. This should highlight the advantages and disadvantages of short-read and long-read approaches for mitogenome assembly.

In the present molecular phylogeny, the species from Lake Poso and the Poso River form a highly supported clade as in [10]. Discussing the closer relationship between T. carbo and Tylomelania sp. ‘Poso A’ remains difficult, as the former species has not yet been studied in a phylogenetic context, and the latter clustered within an unresolved clade [24]. Moreover, none of these species were recovered reciprocally monophyletic. Despite the limited dataset and the scattered distribution of the specimens across the lake, we assume that mitochondrial introgression could also play a significant role in Lake Poso as already discussed for the Malili lake system [10, 11].

Conclusion

The non-monophyly of these morphospecies and several others within the Malili lakes (see also [10, 24]) highlights the need for other, non-mitochondrial, markers in future studies to postulate a more accurate species tree hypothesis (see also discussion in [10]).

Such future phylogenomic studies will probably become increasingly collection-based given the threats to these fascinating ancient lakes. Habitat loss, increasing pollution, and particularly the introduction of the invasive fish species – such as the ‘flowerhorn’ cichlid (a man-made hybrid complex; see [25] for details) – to Lake Matano around 2005 [25–27] caused a decline in endemic biodiversity in recent years. In Lake Poso, where the present Tylomelania species occurs naturally, the situation is probably not much better. The fact that the fish fauna today consists mainly of non-native species that may feed on the endemic fauna should be a cause for concern [8]. Initiatives to protect these species are thus urgently needed.

Acknowledgements

Analyses were performed at sciCORE (scicore.unibas.ch) scientific computing center at University of Basel.

Author contributions

Björn Stelbrink: Conceptualization, Data curation, Formal analysis, Visualization, Writing – original draft. Carola Greve: Data curation, Investigation, Writing – review & editing. Charlotte Gerheim: Data curation, Investigation, Writing – review & editing. Walter Salzburger: Conceptualization, Resources, Writing – review & editing. Thomas von Rintelen: Conceptualization, Funding acquisition, Resources, Writing – review & editing, Supervision.

Funding

Open Access funding enabled and organized by Projekt DEAL. Sequencing was funded by the Center for Integrative Biodiversity Discovery (Museum für Naturkunde Berlin). BS was supported by a research grant from the German Science Foundation (DFG, STE 2460/8–1). CG was supported by the program ‘LOEWE – Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz’ of Hesse’s Ministry of Higher Education, Research, and the Arts (LOEWE/1/10/519/03/03.001(0014)/52).

Data availability

The mitogenome is deposited at NCBI’s GenBank under accession number PX289796. GenBank accession numbers for the 15 mitochondrial markers of the remaining species analysed can be found at: ncbi.nlm.nih.gov/nuccore/?term=tylomelania+190828\_1,*+192946\_1,*+191710\_1,*+192321\_1,*+190814\_4,*+190814\_6,*+190750\_4,*+191027a\_2, *+190529\_5, and *+190532\_4.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The mitogenome is deposited at NCBI’s GenBank under accession number PX289796. GenBank accession numbers for the 15 mitochondrial markers of the remaining species analysed can be found at: ncbi.nlm.nih.gov/nuccore/?term=tylomelania+190828\_1,*+192946\_1,*+191710\_1,*+192321\_1,*+190814\_4,*+190814\_6,*+190750\_4,*+191027a\_2, *+190529\_5, and *+190532\_4.

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