Research Article
Research Article
Comparison of seven complete mitochondrial genomes from Lamprologus and Neolamprologus (Chordata, Teleostei, Perciformes) and the phylogenetic implications for Cichlidae
expand article infoJiachen Wang, Jingzhe Tai, Wenwen Zhang§, Ke He|, Hong Lan, Hongyi Liu
‡ Nanjing Forestry University, Nanjing, China
§ Institute of Environmental Sciences, Ministry of Ecology and Environment of China State Environmental Protection Scientific Observation and Research Station for Ecological Environment of Wuyi Mountains Research Center for Biodiversity Conservation and Biosafety, Nanjing, China
| Zhejiang Agriculture and Forestry University, Hangzhou, China
¶ Zhejiang Open University, Hangzhou, China
Open Access


In this study, mitochondrial genomes (mitogenomes) of seven cichlid species (Lamprologus kungweensis, L. meleagris, L. ornatipinnis, Neolamprologus brevis, N. caudopunctatus, N. leleupi, and N. similis) are characterized for the first time. The newly sequenced mitogenomes contained 37 typical genes [13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs) and 22 transfer RNA genes (tRNAs)]. The mitogenomes were 16,562 ~ 16,587 bp in length with an A + T composition of 52.1~58.8%. The cichlid mitogenomes had a comparable nucleotide composition, A + T content was higher than the G + C content. The AT-skews of most mitogenomes were inconspicuously positive and the GC-skews were negative, indicating higher occurrences of C than G. Most PCGs started with the conventional start codon, ATN. There was no essential difference in the codon usage patterns of these seven species. Using Ka/Ks, we found the fastest-evolving gene were atp8. But the results of p-distance indicated that the fastest-evolving gene was nad6. Phylogenetic analysis revealed that L. meleagris did not cluster with Lamprologus species, but with species from the genus Neolamprologus. The novel information obtained about these mitogenomes will contribute to elucidating the complex relationships among cichlid species.

Key words

Cichlidae, Lamprologus, mitogenome, Neolamprologus, phylogenetic analyses


Cichlids (Teleostei: Perciformes: Cichlidae) are widely distributed across the Neotropics, Africa, the Middle East, Madagascar, as well as southern India and Sri Lanka (Smith et al. 2008; López-Fernández et al. 2010). They stand out as one of the most species-diverse groups of acanthomorphs. Kullander (1998) divided the family Cichlidae into eight subfamilies: Astronotinae, Cichlasomatinae, Cichlinae, Etroplinae, Geophaginae, Heterochromidinae, Pseudocrenilabrinae, and Retroculinae. The ninth subfamily, the Ptychochrominae, was later recognized by Sparks and Smith (2004). Cichlids gained recognition as a prominent model species for the study of evolutionary biology due to the numerous species, diverse genetics, distinct evolutionary lineages, and significant ecological and morphological divergences (Kocher 2004; Schwarzer et al. 2015; Reis et al. 2016; Nam et al. 2021).

African cichlids (subfamily Pseudocrenilabrinae) boasted an abundant variety of more than 2000 species (Brawand et al. 2014; Astudillo-Clavijo et al. 2022). Biologists have long been fascinated by the diversity of cichlids in the East African cichlid radiation (EAR), which has promoted high levels of endemism in the Lakes Tanganyika, Malawi, and Victoria (Kornfield and Smith 2000). Lake Tanganyika is a deep tropical and large Rift Valley lake with an age of 9–12 million years (Irisarri et al. 2018). It has the most diverse species of cichlid fish in terms of morphology, ecology, and behavior, including several mouth-brooding and substrate-spawning lineages (Takahashi 2003; Salzburger 2009). The cichlid fauna of Lake Tanganyika is dominated by lamprologine cichlids, which colonized most lacustrine habitats, but most often inhabits the littoral zone (Sturmbauer et al. 2010). Although classified as a single tribe, lamprologine cichlids exhibit significant diversity in morphology, ecology, and behavior. Lamprologus kungweensis, Lamprologus meleagris, Lamprologus ornatipinnis, Neolamprologus brevis, Neolamprologus caudopunctatus, Neolamprologus leleupi, and Neolamprologus similis are among the smallest species within the lamprologine cichlids, small enough to live inside the empty shells of gastropod mollusks (Sturmbauer et al. 2010). These species are regarded as a highly valuable ornamental species in the aquatic trade industry due to their ease of maintenance and handling in aquariums (Nam et al. 2021).

The genera Lamprologus and Neolamprologus can be difficult to distinguish due to their similar morphology, ecology, and behavior. As discussed by Stiassny (1991), meristic and morphometric measurements, osteology, and dentition were insufficient to differentiate between the species, as many of these traits were homoplastic. Furthermore, there might be instances of ancient ancestral polymorphism, introgressive hybridization, or lack of diagnostic synapomorphic characters among certain species within these two genera, further complicating their classification (Sturmbauer et al. 2010; Gante et al. 2016). Therefore, additional method, like molecular analysis might be required for more accurate classification.

Mitochondria are organelles found in most eukaryotic cells that play a critical role in energy production (Hebert et al. 2010). The mitochondrial genome (mitogenome) of acanthomorph fishes is usually a circular, double-stranded molecule that ranges from 16 to 23 kbp in size. It typically contains 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and one control region (CR) (Iwasaki et al. 2013). Mitogenomes have the characteristics of high evolutionary rate, matrilineal inheritance, low molecular weight, simple structure, and ease of amplification, which makes them a reliable marker for studying phylogenetics (Ye et al. 2022; Wang et al. 2023). Mitogenome components, such as nad2 or rrnL, are widely used for phylogenetic analyses (Sturmbauer et al. 2010; Schwarzer et al. 2015). Although partial mitochondrial sequences can offer some insights into evolutionary relationships, they are limited in their ability to provide a comprehensive understanding due to the absence of information such as gene rearrangement, genetic code changes, replication, and transcriptional regulation patterns. Therefore, complete mitogenome sequences can be more beneficial as they can provide improved resolution and sensitivity for investigating evolutionary relationships (Li et al. 2019; Fiteha et al. 2023; Wang et al. 2023).

In this study, we report the complete mitogenome organizations and characteristics of seven species (L. kungweensis, L. meleagris, L. ornatipinnis, N. brevis, N. caudopunctatus, N. leleupi, and N. similis). We also performed a phylogenetic analysis of the seven complete mitogenomes obtained in this study with the published complete cichlid mitogenomes. We hope that our study can enable better comprehension of cichlid biodiversity and expand genetic resources for future cichlid comparisons.

Materials and methods

Sample collection and DNA extraction

The seven species are commonly sold as ornamental fish and can be found in many pet markets. Specimens were obtained from the Qiqiaoweng pet market in Nanjing, Jiangsu province, China. The specimens were identified using morphological characteristics described in FishBase ( No fish were sacrificed during this study. The fish were reared at the Laboratory of Animal Molecular Evolution, Nanjing Forestry University. Total genomic DNA was extracted from each fin using a FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, Nanjing, China), and stored at –80 °C for future use.

Genome sequencing, assembly, and annotation

Seven complete mitogenomes were sequenced on an Illumina platform (Personalbio Nanjin, China) using total genomic DNA. The genomic DNA was used to generate an Illumina library with an insert size of 400 bp. The clean data were then assembled in Geneious Prime 2022 software, using Lamprologus signatus (MZ427900.1) as a template. The mitogenomes were assembled and manually revised using DNAstar v. 7.1 (Madison, WI, USA).

Conservative domains were detected using BLAST ( and MITOS WebServer ( (Bernt et al. 2013). Maps of the mitogenomes were constructed using CGView ( (Stothard and Wishart 2005). MEGA X was used for base composition analysis, relative synonymous codon usage (RSCU) analysis, pairwise relative genetic distance (p-distance) calculation, as well as non-synonymous (Ka) and synonymous substitutions (Ks) analysis (Fay and Wu 2003; Kumar et al. 2016). Composition skew values were calculated using the following formulas: “AT-skew = (A − T) / (A + T) GC-skew = (G − C) / (G + C)” (Perna and Kocher 1995).

Phylogenetic analysis

Phylogenetic analysis was conducted using the sequences of 13 PCGs and two rRNA genes from the complete mitogenomes of 105 species, including seven species from this study (Suppl. material 1). Channa andrao and Hyphessobrycon sweglesi were selected as outgroups, while the remaining specimens belonged to the Cichlidae family. Phylogenetic analysis was conducted using maximum likelihood (ML) and Bayesian inference (BI) methods with PhyloSuite v. 1.2.3 software package (Zhang et al. 2020; Xiang et al. 2023). All genes were aligned using MAFFT v. 7.313, and the best-fit substitution model and partitioning scheme were determined using ModelFinder. ML phylogenies were inferred using IQ-TREE with the Edge-linked partition model for 5000 ultrafast bootstraps (Minh et al. 2013; Nguyen et al. 2015). BI phylogenies were inferred using MrBayes v. 3.2.7a with a partition model (Ronquist et al. 2012). The analysis consisted of two parallel runs with 2,000,000 generations each, and the initial 25% of sampled data was discarded as burn-in. The trees were visualized and edited using iTOL v. 6 (Letunic and Bork 2021).

Results and discussion

Genome organization and composition

Seven complete mitogenomes covering two genera were obtained. L. kungweensis (16,587 bp), L. meleagris (16,582 bp), L. ornatipinnis (16,585 bp), N. brevis (16,586 bp), N. caudopunctatus (16,586 bp), and N. similis (16,580 bp) had similar lengths, while N. leleupi had the shortest length at 16,562 bp (Fig. 1) (accession numbers: OP805601.1, OP805600.1, OQ076695.1, OP930818.1, OP930816.1, OP930817.1, and OP930815.1). The seven mitogenomes possessed the typical gene composition found in most bony fish, including 13 PCGs, 22 tRNAs, two rRNAs, and a CR. Among these genes, 12 PCGs, 14 tRNA genes, and two rRNA genes, were located on the major strand (H-strand), while the remaining eight tRNA genes and a PCG were encoded on the minor strand (L-strand). The gene order of these mitogenomes was identical to that of previously published species L. signatus (MZ427900.1) and Neolamprologus brichardi (AP006014.1) (Nam et al. 2021). Seventeen intergenic regions of the same length were observed between the mitochondrial regions of species L. kungweensis, L. meleagris, L. ornatipinnis, N. brevis, with lengths ranging from 10 bp (between atp8 and atp6) to 35 bp (between trnN and trnC). However, N. caudopunctatus exhibited a 24 bp intergenic region between trnV and rrnL, and N. similis displayed a 38 bp intergenic region at the same location. The trnC and trnY of N. similis overlapped by 1 bp, whereas there was no overlap in this region in the other six species. In addition, N. leleupi had one more intergenic region (24 bp between cox1 and trnS2) than other species (Table 1).

Figure 1. 

The gene maps of the seven newly sequenced mitogenomes. Different gene types are shown in different colors.

Table 1.

Features of the mitogenomes of L. kungweensis, L. meleagris, L. ornatipinnis, N. brevis, N. caudopunctatus, N. leleupi, and N. similis.

Gene Position Size (bp) Intergenic Nucleotides Codon Strand
From To Start Stop
trnF 1/1/1/1/1/1/1 69/69/69/69/69/69/69 69/69/69/69/69/69/69 0/0/0/0/0/0/0 H
rrnS 70/70/70/70/70/70/70 1013/1012/1013/1012/1012/1013/1015 944/943/944/943/943/944/946 0/0/0/0/0/0/0 H
trnV 1014/1013/1014/1013/1013/1014/1016 1085/1084/1085/1084/1084/1085/1087 72/72/72/72/72/72/72 0/0/0/0/0/0/0 H
rrnL 1108/1107/1108/1107/1109/1108/1126 2776/2776/2776/2777/2778/2775/2777 1669/1670/1669/1671/1670/1668/1652 22/22/22/22/24/22/38 H
trnL2 2777/2777/2777/2778/2779/2776/2778 2850/2850/2850/2851/2852/2849/2851 74/74/74/74/74/74/74 0/0/0/0/0/0/0 H
nad1 2851/2851/2851/2852/2853/2850/2852 3825/3825/3825/3826/3827/3824/3826 975/975/975/975/975/975/975 0/0/0/0/0/0/0 ATG/ATG/ATG/ATG/ATG/ATG/ATG TAG/TAA/TAG/TAG/TAG/TAG/TAG H
trnI 3829/3829/3829/3830/3831/3828/3830 3898/3898/3898/3899/3900/3897/3899 70/70/70/70/70/70/70 3/3/3/3/3/3/3 H
trnQ 3898/3898/3898/3899/3900/3897/3899 3968/3968/3968/3969/3970/3967/3969 71/71/71/71/71/71/71 -1/-1/-1/-1/-1/-1/-1 L
trnM 3968/3968/3968/3969/3970/3967/3969 4036/4036/4036/4037/4038/4035/4037 69/69/69/69/69/69/69 -1/-1/-1/-1/-1/-1/-1 H
nad2 4037/4037/4037/4038/4039/4036/4038 5082/5082/5082/5083/5084/5081/5083 1046/1046/1046/1046/1046/1046/1046 0/0/0/0/0/0/0 ATG/ATG/ATG/ATG/ATG/ATG/ATG TA/TA/TA/TA/TA/TA/TA H
trnW 5083/5083/5083/5084/5085/5082/5084 5154/5154/5154/5155/5156/5153/5155 72/72/72/72/72/72/72 0/0/0/0/0/0/0 H
trnA 5156/5156/5156/5157/5158/5155/5157 5224/5224/5224/5225/5226/5223/5225 69/69/69/69/69/69/69 1/1/1/1/1/1/1 L
trnN 5226/5226/5226/5227/5228/5225/5227 5298/5298/5298/5299/5300/5297/5299 73/73/73/73/73/73/73 1/1/1/1/1/1/1 L
trnC 5334/5334/5334/5335/5336/5333/5335 5399/5399/5399/5400/5400/5398/5400 66/66/66/66/65/66/66 35/35/35/35/35/35/35 L
trnY 5400/5400/5400/5401/5401/5399/5400 5469/5469/5469/5470/5470/5468/5469 70/70/70/70/70/70/70 0/0/0/0/0/0/-1 L
cox1 5471/5471/5471/5472/5472/5470/5471 7066/7066/7066/7067/7067/7020/7066 1596/1596/1596/1596/1596/1551/1596 1/1/1/1/1/1/1 GTG/GTG/GTG/GTG/GTG/GTG/GTG TAA/TAA/TAA/TAA/TAA/TAA/TAA H
trnS2 7067/7067/7067/7068/7068/7045/7067 7137/7137/7137/7138/7138/7115/7137 71/71/71/71/71/71/71 0/0/0/0/0/24/0 L
trnD 7141/7141/7141/7142/7142/7119/7141 7213/7213/7213/7214/7214/7191/7213 73/73/73/73/73/73/73 3/3/3/3/3/3/3 H
cox2 7219/7219/7219/7220/7220/7197/7219 7909/7909/7909/7910/7910/7887/7909 691/691/691/691/691/691/691 5/5/5/5/5/5/5 ATG/ATG/ATG/ATG/ATG/ATG/ATG T/T/T/T/T/T/T H
trnK 7910/7910/7910/7911/7911/7888/7910 7983/7983/7983/7984/7984/7961/7983 74/74/74/74/74/74/74 0/0/0/0/0/0/0 H
atp8 7985/7985/7985/7986/7986/7963/7985 8152/8152/8152/8153/8153/8130/8152 168/168/168/168/168/168/168 1/1/1/1/1/1/1 ATG/ATG/ATG/ATG/ATG/ATG/ATG TAA/TAA/TAA/TAA/TAA/TAA/TAA H
atp6 8143/8143/8143/8144/8144/8121/8143 8826/8826/8826/8827/8827/8804/8826 684/684/684/684/684/684/684 -10/-10/-10/-10/-10/-10/-10 ATG/ATG/ATG/ATG/ATG/ATG/ATG TAA/TAA/TAA/TAA/TAA/TAA/TAA H
cox3 8826/8826/8826/8827/8827/8804/8826 9609/9609/9609/9610/9610/9587/9609 784/784/784/784/784/784/784 -1/-1/-1/-1/-1/-1/-1 ATG/ATG/ATG/ATG/ATG/ATG/ATG T/T/T/T/T/T/T H
trnG 9610/9610/9610/9611/9611/9588/9610 9681/9681/9681/9682/9682/9659/9681 72/72/72/72/72/72/72 0/0/0/0/0/0/0 H
nad3 9682/9682/9682/9683/9683/9660/9682 10030/10030/10030/10031/10031/10008/10030 349/349/349/349/349/349/349 0/0/0/0/0/0/0 ATG/ATG/ATG/ATG/ATG/ATG/ATG T/T/T/T/T/T/T H
trnR 10031/10031/10031/10032/10032/10009/10031 10099/10099/10099/10100/10100/10077/10099 69/69/69/69/69/69/69 0/0/0/0/0/0/0 H
nad4l 10100/10100/10100/10101/10101/10078/10100 10396/10396/10396/10397/10397/10374/10396 297/297/297/297/297/297/297 0/0/0/0/0/0/0 ATG/ATG/ATG/ATG/ATG/ATG/ATG TAA/TAA/TAA/TAA/TAA/TAA/TAA H
nad4 10390/10390/10390/10391/10391/10368/10390 11770/11770/11770/11771/11771/11748/11770 1381/1381/1381/1381/1381/1381/1381 -7/-7/-7/-7/-7/-7/-7 ATG/ATG/ATG/ATG/ATG/ATG/ATG T/T/T/T/T/T/T H
trnH 11771/11771/11771/11772/11772/11749/11771 11839/11839/11839/11840/11840/11817/11839 69/69/69/69/69/69/69 0/0/0/0/0/0/0 H
trnS1 11840/11840/11840/11841/11841/11818/11840 11906/11905/11906/11907/11907/11884/11906 67/66/67/67/67/67/67 0/0/0/0/0/0/0 H
trnL1 11911/11910/11911/11912/11912/11889/11911 11983/11982/11983/11984/11984/11961/11983 73/73/73/73/73/73/73 4/4/4/4/4/4/4 H
nad5 11984/11983/11984/11985/11985/11962/11984 13822/13821/13822/13823/13823/13800/13822 1839/1839/1839/1839/1839/1839/1839 0/0/0/0/0/0/0 ATG/ATG/ATG/ATG/ATG/ATG/ATG TAA/TAA/TAA/TAA/TAA/TAA/TAA H
nad6 13819/13818/13819/13820/13820/13797/13819 14340/14339/14340/14341/14341/14318/14340 522/522/522/522/522/522/522 -4/-4/-4/-4/-4/-4/-4 ATG/ATG/ATG/ATG/ATG/ATG/ATG TAA/TAA/TAA/TAA/TAA/TAA/TAA L
trnE 14341/14340/14341/14342/14342/14319/14341 14409/14408/14409/14410/14410/14387/14409 69/69/69/69/69/69/69 0/0/0/0/0/0/0 L
cytb 14414/14413/14414/14415/14415/14392/14414 15554/15553/15554/15555/15555/15532/15554 1141/1141/1141/1141/1141/1141/1141 4/4/4/4/4/4/4 ATG/ATG/ATG/ATG/ATG/ATG/ATG T/T/T/T/T/T/T H
trnT 15555/15554/15555/15556/15556/15533/15555 15626/15625/15626/15627/15627/15604/15626 72/72/72/72/72/72/72 0/0/0/0/0/0/0 H
trnP 15627/15626/15627/15628/15628/15605/15627 15696/15695/15696/15697/15696/15673/15696 70/70/70/70/69/69/70 0/0/0/0/0/0/0 L
CR 15697/15696/15697/15698/15697/15674/15697 16587/16582/16585/16586/16586/16562/16580 891/887/889/889/890/889/884 0/0/0/0/0/0/0

Nucleotide composition

The nucleotide composition of the seven newly sequenced Lamprologus and Neolamprologus mitogenomes were biased toward A and T (Table 2). The AT-skews exhibited inconspicuously positive values, while all GC-skews were markedly negative. The analysis revealed a clear preference for the utilization of C, along with a minor inclination towards A, across the entire genome (Table 2).

Table 2.

Base compositions of the complete genomes, PCGs, rRNAs, tRNAs, and CRs of the seven newly sequenced mitogenomes.

Species Whole genome AT - skew GC - skew PCGs tRNAs rRNAs CR
Size AT Size AT Size AT Size AT Size AT
(bp) (%) (bp) (%) (bp) (%) (bp) (%) (bp) (%)
Lamprologus kungweensis 16,587 54.1 0.002 -0.300 11,466 53.5 1,554 54.7 2,613 54.1 891 62.9
Lamprologus meleagris 16,582 55.1 0.002 -0.300 11,466 54.7 1,553 55.8 2,613 54.1 887 63.6
Lamprologus ornatipinnis 16,585 53.9 0.006 -0.304 11,466 53.2 1,554 55.4 2,613 53.5 889 62.7
Neolamprologus brevis 16,586 53.6 0.011 -0.311 11,466 53.0 1,554 54.9 2,614 53.0 889 63.8
Neolamprologus caudopunctatus 16,586 53.9 0.002 -0.299 11,466 53.2 1,552 55.4 2,613 53.2 890 63.0
Neolamprologus leleupi 16,562 53.7 0.017 -0.318 11,421 53.0 1,553 54.7 2,612 53.0 889 62.5
Neolamprologus similis 16,580 54.1 0.010 -0.311 11,466 53.5 1,554 54.9 2,598 53.9 884 63.0

To determine the nucleotide composition of Cichlidae, the A + T content, AT-skew, G + C content, and GC-skew of 103 complete mitogenomes (including 8 subfamilies Astronotinae, Cichlasomatinae, Cichlinae, Etroplinae, Geophaginae, Pseudocrenilabrinae, Ptychochrominae, and Retroculinae of the family Cichlidae) were calculated. The H-strand in the mitogenomes of 103 cichlid species showed a similar preference for A and T nucleotides. The 103 Cichlidae mitogenomes had a comparable nucleotide composition, A + T content (52.1 ~ 58.8%) were higher than the G + C content (41.1 ~ 47.8%) (Fig. 2). The GC-skew were negative (–0.351 ~ –0.221), indicating a higher occurrence of C than G except for Andinoacara rivulatus (–0.019), Pelvicachromis pulcher (–0.005), and Etroplus canarensis (–0.002). The AT-skew were inconspicuously positive (0.002 ~ 0.076), indicating a small difference in the content of A and T in the mitogenomes. This phenomenon is also observed in other published Teleostei genomes (Liu et al. 2020; Ruan et al. 2020; Xu et al. 2021a, 2021b; Wang et al. 2023). The A nucleotide composition is commonly used to indicate gene direction and replication orientation during transcription and replication (Wei et al. 2010a, 2010b).

Figure 2. 

A + T content vs AT-skew and G + C content vs GC-skew in the 103 mitogenomes of family Cichlidae. Values are calculated on H-strands for full-length mitogenomes.

Protein-coding genes

In the seven newly sequenced mitogenomes, PCG nad6 was on the L-strand, while other PCGs were on the H-strand. The average A + T content of the PCGs ranged from 53.0% (N. leleupi and N. brevis) to 54.7% (L. meleagris). Six of them had the same 13 PCGs length of 11,466 bp, while the remaining species, N. leleupi, had a slightly shorter length of 11,421bp. The reason for this difference was that the cox1 gene in N. leleupi had a mutation causing a premature stop codon compared to other species, resulting in a reduction of 45 base pairs in length (Tables 1, 2).

Most of the PCGs in the seven newly sequenced mitogenomes began with the start codon ATG, except for cox1, which started with GTG. Most PCGs terminated with the codon TAA or incomplete codon (TA− / T−−), with the exception of nad1, which ended with TAG (Table 2). The cichlid species are relatively conservative in their use of start codons, and their preferences are generally consistent with those of the seven newly sequenced species with the only exception of the occurrence of a rare start codon ATC in the cox1 and nad3. All the Cichlids share the stop codons with TAA, TAG, AGA, and incomplete codons (TA− / T−−) (Fig. 3).

Figure 3. 

Start codon and stop codon usage for the mitochondrial genome protein-coding genes of 103 cichlid species.

RSCU was calculated to identify the predominant synonymous codon (Grantham et al. 1980). The comparative analysis based on RSCU of all PCG codons showed that the codon usage patterns of these seven species were similar (Fig. 4). Genes encoding Ile and Leu2 had high frequency, while those encoding Cys, Met, and Ser1 were infrequent.

Figure 4. 

The codon distribution and RSCU of the mitogenomes of the seven newly sequenced mitogenomes.

Evolutionary analyses

The selection pressure was analyzed by calculating the ratio of Ka/Ks across Lamprologus and Neolamprologus for each aligned PCG (Fig. 5) (Yang and Nielsen 2002). It was found that atp8 showed the largest Ka/Ks value among the 13 PCGs, which suggested more amino acid variety in the biomolecule. This suggests that the atp8 gene might have evolved faster than other PCGs due to slight selection pressure (Hassanin et al. 2005). The faster evolution of the atp8 gene could result in greater amino acid diversity, indicating its potential as an effective marker for population classification. The Ka/Ks values for all PCGs were lower than 1, suggesting that purifying selection was likely the main driver of mitochondrial PCG evolution (Hurst et al. 2002).

Figure 5. 

Ka/Ks values for the 13 PCGs. Pale pink box plots, five species of gnus Neolamprologus; orange box plots, four species of Lamprologus; blue box plots, nine species of Lamprologus and Neolamprologus. The band inside the box represents the median; upper and lower hinges correspond to the 25th and 75th percentiles; circles, to outliers.

Besides the Ka/Ks analysis, an assessment of the degree of divergence in Lamprologus and Neolamprologus was conducted by analyzing the overall p-distance between nucleotides of 13 PCGs + two rRNA genes (Fig. 6). The results of p-distance indicated that the fastest-evolving gene was nad6, which was inconsistent with the results of Ka/Ks value. However, the difference in this gene might be not comparable with the selection since this force is acting in a contemporary period.

Figure 6. 

Genetic p-distances for nucleotide sequences for 13 PCGs and 2 rRNAs. Pale pink box plots, five species of Neolamprologus; orange box plots, four species of Lamprologus; blue box plots, 9 species of genera Lamprologus and Neolamprologus.

Ribosomal RNA genes, transfer RNA genes, and control regions

The size of the rrnS genes were between 943 bp (L. meleagris, N. brevis, and N. caudopunctatus) and 946 bp (N. similis), while the size of the rrnL genes in seven species ranged between 1,652 bp (N. similis) to 1,671 bp (N. brevis) (Table 1). The two rRNA genes located between trnF and trnL2, with trnV separating them. The A + T content of rRNAs ranged from 53.0% ~ 54.1% (Table 2).

The sizes of the tRNA genes ranged from 66 bp (trnY of N. caudopunctatus) to 74 bp (trnK). The combined length of the 22 tRNA genes varied between 1,552 bp (N. caudopunctatus) and 1,554 bp (L. kungweensis, L. ornatipinnis, and N. similis). The A + T contents of tRNA genes ranged from 54.7% to 55.8% among the seven species analyzed in this study (Table 2).

As with other fish mitogenomes, the CRs were discovered to exist between trnF and trnP in all seven species. The sizes of the CRs ranged from 884 bp (N. similis) to 891 bp (L. kungweensis). The A + T contents of PCGs, tRNAs, and rRNAs sequences were found to be similar to that of the entire mitogenomes, whereas CR sequences had a higher A + T content (62.5% ~ 63.8%) (Table 2).

Phylogenetic analysis

To elucidate the phylogenetic inter-relationships within the family Cichlidae and genera Lamprologus and Neolamprologus, concatenated nucleotide sequences of 13 PCGs + two rRNAs from 103 cichlid species were obtained. Additionally, Channa andrao, and Hyphessobrycon sweglesi from two other families were used as outgroups. It was found that BI and ML analysis generated the same topology structure on most nodes (Fig. 7).

Figure 7. 

13 PCGs-based phylogenetic tree of 103 cichlid species and two outgroups. Numbers at nodes represent the posterior probability and bootstrap values for BI and ML analysis, respectively. “-” indicates this clade not supported by BI or ML analysis.

Specifically, the seven complete mitogenomes covered two genera in this study have good clustering in phylogenetic trees, and within the family Cichlidae, the subfamily Etroplinae and Ptychochrominae were monophyletic across analyses. They diverged with species in other subfamilies early in the evolutionary history of cichlid fishes. This result was similar to a previous molecular phylogenetic study (Astudillo-Clavijo et al. 2022). Thirty-one species from subfamily Astronotinae, Cichlasomatinae, Cichlinae, Geophaginae, and Retroculinae were clustered into one branch, indicating these five subfamilies were closely related. Moreover, 67 Pseudocrenilabrinae species also formed a monophyletic clade. Pseudocrenilabrinae tribes and their interrelationships were for the most part well supported as reported by Astudillo-Clavijo et al. (2022). Due to the addition of seven newly sequenced mitogenomes, three pairs of sisters (N. brichardi + N. leleupi, N. caudopunctatus + L. meleagris, and L. signatus + L. kungweensis) were newly identified, as shown in Fig. 7. Lamprologus meleagris did not cluster with Lamprologus species, but with species from the genus Neolamprologus. Previous studies have identified such taxonomic issues in the genera Lamprologus and Neolamprologus (Schelly et al. 2006; Sturmbauer et al. 2010). Sturmbauer et al. (2010) think a viable way might be to re-assign the genus name Lamprologus to most Neolamprologus species. Our results also support this scenario. However, the species from the genera Lamprologus and Neolamprologus used in this study were limited, making it impossible to perform a more detailed analysis. Therefore, to better understand the relationships between members of these two genera, it will be beneficial to include more species in future studies.

In conclusion, our study increased the database of mitogenome in Cichlidae, and showed that mitogenome sequences are efficient molecular markers for studying the phylogenetic relationships within Cichlidae. However, there is a lack of analyses in nuclear genes. In the future study, we will further improve these deficiencies.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.


This research was funded by the Innovation and Entrepreneurship Training Program for College Students of China (202210298070Z), the biodiversity investigation, observation and assessment program (2019–2023) of Ministry of Ecology and Environment of China (2110404), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author contributions

Conceptualization: WZ, HL. Data curation: JW. Formal analysis: JT, JW. Funding acquisition: WZ, JW, HL. Methodology: HL. Project administration: HL, WZ. Resources: WZ, HL, KH. Software: JT, JW. Supervision: HL, KH. Validation: HL. Visualization: JW, JT. Writing - original draft: JW.

Author ORCIDs

Jiachen Wang

Jingzhe Tai

Wenwen Zhang

Ke He

Hong Lan

Hongyi Liu

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.


  • Astudillo-Clavijo V, Stiassny MLJ, Ilves KL, Musilova Z, Salzburger W, Lopez-Fernandez H (2022) Exon-based phylogenomics and the relationships of African Cichlid fishes: Tackling the challenges of reconstructing phylogenies with repeated rapid radiations. Systematic Biology 72(1): 134–149.
  • Bernt M, Donath A, Jühling F, Externbrink F, Florentz C, Fritzsch G, Pütz J, Middendorf M, Stadler PF (2013) MITOS: Improved de novo metazoan mitochondrial genome annotation. Molecular Phylogenetics and Evolution 69(2): 313–319.
  • Brawand D, Wagner CE, Li YI, Malinsky M, Keller I, Fan SH, Simakov O, Ng AY, Lim ZW, Bezault E, Turner-Maier J, Johnson J, Alcazar R, Noh HJ, Russell P, Aken B, Alfoldi J, Amemiya C, Azzouzi N, Baroiller JF, Barloy-Hubler F, Berlin A, Bloomquist R, Carleton KL, Conte MA, D’Cotta H, Eshel O, Gaffney L, Galibert F, Gante HF, Gnerre S, Greuter L, Guyon R, Haddad NS, Haerty W, Harris RM, Hofmann HA, Hourlier T, Hulata G, Jaffe DB, Lara M, Lee AP, MacCallum I, Mwaiko S, Nikaido M, Nishihara H, Ozouf-Costaz C, Penman DJ, Przybylski D, Rakotomanga M, Renn SCP, Ribeiro FJ, Ron M, Salzburger W, Sanchez-Pulido L, Santos ME, Searle S, Sharpe T, Swofford R, Tan FJ, Williams L, Young S, Yin SY, Okada N, Kocher TD, Miska EA, Lander ES, Venkatesh B, Fernald RD, Meyer A, Ponting CP, Streelman JT, Lindblad-Toh K, Seehausen O, Di Palma F (2014) The genomic substrate for adaptive radiation in African cichlid fish. Nature 513(7518): 375–381.
  • Sturmbauer C, Salzburger W, Duftner N, Schelly R, Koblmüller S (2010) Evolutionary history of the Lake Tanganyika cichlid tribe Lamprologini (Teleostei: Perciformes) derived from mitochondrial and nuclear DNA data. Molecular Phylogenetics and Evolution 57(1): 266–284.
  • Fiteha YG, Rashed MA, Ali RAM, Magdy M (2023) Characterization and phylogenetic analysis of the complete mitochondrial genome of Mango tilapia (Sarotherodon galilaeus: Cichlidae). Molecular Biology Reports 50(4): 3945–3950.
  • Gante HF, Matschiner M, Malmstrom M, Jakobsen KS, Jentoft S, Salzburger W (2016) Genomics of speciation and introgression in Princess cichlid fishes from Lake Tanganyika. Molecular Ecology 25(24): 6143–6161.
  • Hassanin A, Léger N, Deutsch J (2005) Evidence for multiple reversals of asymmetric mutational constraints during the evolution of the mitochondrial genome of metazoa, and consequences for phylogenetic inferences. Systematic Biology 54(2): 277–298.
  • Hebert SL, Lanza IR, Nair KS (2010) Mitochondrial DNA alterations and reduced mitochondrial function in aging. Mechanisms of Ageing and Development 131: 451–462.
  • Irisarri I, Singh P, Koblmuller S, Torres-Dowdall J, Henning F, Franchini P, Fischer C, Lemmon AR, Lemmon EM, Thallinger GG, Sturmbauer C, Meyer A (2018) Phylogenomics uncovers early hybridization and adaptive loci shaping the radiation of Lake Tanganyika cichlid fishes. Nature Communications 9(1): 3159.
  • Iwasaki W, Fukunaga T, Isagozawa R, Yamada K, Maeda Y, Satoh TP, Sado T, Mabuchi K, Takeshima H, Miya M, Nishida M (2013) Mitofish and Mitoannotator: A mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Molecular Biology and Evolution 30(11): 2531–2540.
  • Kocher TD (2004) Adaptive evolution and explosive speciation: The cichlid fish model. Nature Reviews. Genetics 5(4): 288–298.
  • Kullander S (1998) A phylogeny and classification of the South American Cichlidae (Teleostei: Perciformes). Phylogeny and Classification of Neotropical Fishes, 461–498.
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874.
  • Letunic I, Bork P (2021) Interactive Tree of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Research 49(W1): 293–296.
  • Li R, Wang G, Wen ZY, Zou YC, Qin CJ, Luo Y, Wang J, Chen GH (2019) Complete mitochondrial genome of a kind of snakehead fish Channa siamensis and its phylogenetic consideration. Genes & Genomics 41(2): 147–157.
  • Liu HY, Sun CH, Zhu Y, Li YD, Wei YS, Ruan HH (2020) Mitochondrial genomes of four American characins and phylogenetic relationships within the family Characidae (Teleostei: Characiformes). Gene 762: 145041.
  • López-Fernández H, Winemiller KO, Honeycutt RL (2010) Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Perciformes: Cichlidae: Cichlinae). Molecular Phylogenetics and Evolution 55(3): 1070–1086.
  • Minh BQ, Nguyen MA, von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution 30(5): 1188–1195.
  • Nam SE, Eom HJ, Park HS, Rhee JS (2021) The complete mitochondrial genome of Lamprologus signatus (Perciformes: Cichlidae). Mitochondrial DNA, Part B, Resources 6(12): 3487–3489.
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274.
  • Perna NT, Kocher TD (1995) Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. Journal of Molecular Evolution 41(3): 353–358.
  • Reis RE, Albert JS, Di Dario F, Mincarone MMM, Petry PL, Rocha LAR (2016) Fish biodiversity and conservation in South America. Journal of Fish Biology 89(1): 1–36.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542.
  • Ruan HT, Li M, Li ZH, Huang JJ, Chen WY, Sun JJ, Liu L, Zou KS (2020) Comparative analysis of complete mitochondrial genomes of three Gerres fishes (Perciformes: Gerreidae) and primary exploration of their evolution history. International Journal of Molecular Sciences 21(5): 1874.
  • Schelly R, Salzburger W, Koblmüller S, Duftner N, Sturmbauer C (2006) Phylogenetic relationships of the lamprologine cichlid genus Lepidiolamprologus (Teleostei: Perciformes) based on mitochondrial and nuclear sequences, suggesting introgressive hybridization. Molecular Phylogenetics and Evolution 38(2): 426–438.
  • Schwarzer J, Lamboj A, Langen K, Misof B, Schliewen UK (2015) Phylogeny and age of chromidotilapiine cichlids (Teleostei: Cichlidae). Hydrobiologia 748(1): 185–199.
  • Stiassny MLJ (1991) Atavisms, phylogenetic character reversals, and the origin of evolutionary novelties. Netherlands Journal of Zoology 42(2–3): 260–276.
  • Sturmbauer C, Salzburger W, Duftner N, Schelly R, Koblmuller S (2010) Evolutionary history of the Lake Tanganyika cichlid tribe Lamprologini (Teleostei: Perciformes) derived from mitochondrial and nuclear DNA data. Molecular Phylogenetics and Evolution 57(1): 266–284.
  • Wang JC, Xu W, Liu YY, Bai YW, Liu HY (2023) Comparative mitochondrial genomics and phylogenetics for species of the snakehead genus Channa Scopoli, 1777 (Perciformes: Channidae). Gene 857: 147186.
  • Wei SJ, Shi M, Sharkey MJ, van Achterberg C, Chen XX (2010b) Comparative mitogenomics of Braconidae (Insecta: Hymenoptera) and the phylogenetic utility of mitochondrial genomes with special reference to Holometabolous insects. BMC Genomics 11(1): 1–16.
  • Xiang CY, Gao FL, Jakovlić I, Lei HP, Hu Y, Zhang H, Zou H, Wang GT, Zhang D (2023) Using PhyloSuite for molecular phylogeny and tree‐based analyses. iMeta 2(1): e87.
  • Xu W, Ding JY, Lin SP, Xu RF, Liu HY (2021a) Comparative mitogenomes of three species in Moenkhausia: Rare irregular gene rearrangement within Characidae. International Journal of Biological Macromolecules 183: 1079–1086.
  • Xu W, Lin SP, Liu HY (2021b) Mitochondrial genomes of five Hyphessobrycon tetras and their phylogenetic implications. Ecology and Evolution 11(18): 12754–12764.
  • Ye WT, Wang JC, Zhao XY, Liu HY, Zhu S (2022) Mitochondrial Genomes of two Lycosa spiders (Araneae, Lycosidae): Genome Description and Phylogenetic Implications. Diversity (Basel) 14(7): 538.
  • Zhang D, Gao FL, Jakovlic I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355.

Supplementary material

Supplementary material 1 

Summary of the mitochondrial genomes used for phylogenetic analysis

Jiachen Wang, Jingzhe Tai, Wenwen Zhang, Ke He, Hong Lan, Hongyi Liu

Data type: xlsx

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (14.99 kb)
login to comment