Evolutionary trajectories of venomous fish: complete mitogenomes of Paracentropogon rubripinnis and Inimicus japonicus reshape Scorpaeniformes phylogeny

doi:10.21203/rs.3.rs-4960028/v1

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Evolutionary trajectories of venomous fish: complete mitogenomes of Paracentropogon rubripinnis and Inimicus japonicus reshape Scorpaeniformes phylogeny

https://doi.org/10.21203/rs.3.rs-4960028/v1

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The Scorpaeniformes order encompasses a diverse array of teleost fish, including commercially important and venomous species. Fish venoms offer significant pharmacological potential, but incomplete phylogenetic understanding has hindered research. Resolving relationships among venomous fish families is crucial for studying venom evolution and discovering novel bioactive compounds. To address these phylogenetic uncertainties, we generated and assembled the complete mitochondrial genomes of Paracentropogon rubripinnis (Tetrarogidae) and Inimicus japonicus (Synanceiidae), two representative venomous species. The circular mitogenomes, 16,465 bp and 16,676 bp in length, respectively, contain the typical vertebrate mitochondrial gene complement. Comparative analyses revealed a highly conserved gene order and orientation across Scorpaeniformes, with slight variations in the Notothenioidei outgroups. We identified novel conserved sequence blocks in the control regions and characterized structural features of protein-coding genes, tRNAs, and non-coding elements. Phylogenetic analyses using 13 mitochondrial protein-coding genes from 71 Scorpaeniformes and three outgroup species provided a higher-resolution phylogeny of the order, including 12 families and 31 genera. Our results support the monophyly of Tetrarogidae and Synanceiidae, placing them at the base of the Scorpaeniformes phylogeny. This study provides new insights into Scorpaeniformes evolution, particularly regarding venomous fish families, and lays a foundation for future research on fish venom evolution and applications.

Biological sciences/Computational biology and bioinformatics/Genome informatics

Biological sciences/Computational biology and bioinformatics/Phylogeny

Biological sciences/Computational biology and bioinformatics/Sequence annotation

Scorpaeniformes evolution

venomous fishes

Paracentropogon rubripinnis

Inimicus japonicus

complete mitogenomes

phylogenetic analysis

The Scorpaeniformes order is one of the largest and most morphologically diverse groups of ray-finned fish, comprising more than 1400 species classified into 38 families, including commercially important and venomous fish^1,2. Understanding the polyphyletic relationships within this order is crucial for effective conservation efforts, sustainable fisheries management, and potential pharmacological applications of venomous species. However, the complexity and diversity of Scorpaeniformes have presented significant challenges in revolving their evolutionary history, leading to unclear taxonomic boundaries and debates about the monophyly of the order^3–5. These uncertainties persist despite numerous studies employing diverse approaches such as morphological analyses, partial mitochondrial gene sequencing, and nuclear gene examinations^6,7. A prime example of this ongoing controversy is the unresolved phylogenetic relationships among families such as Synanceiidae, Tetrarogidae, Scorpaenidae, and Sebastidae. Previous studies using molecular sequence data or morphological features^4,7–13 have yielded two competing hypotheses: ((Synanceiidae + Tetrarogidae) + (Scorpaenidae + Sebastidae)) (Fig. 1A) and ((Synanceiidae + Tetrarogidae) + Scorpaenidae) + Sebastidae) (Fig. 1B).

Phylogenetic analyses face several challenges that can lead to discrepancies between gene and species trees. These discrepancies may arise from various factors, including incomplete lineage sorting, horizontal gene transfer, gene duplication and loss, and hybridization. Furthermore, sampling errors and biases in outgroup selections can significantly influence phylogenetic reconstruction¹⁴. Meanwhile, increasing the number of genes and sequence length can help mitigate some of these issues by providing more phylogenetic signals; however, it also introduces new challenges. Obtaining and analyzing large-scale genomic data, especially for large eukaryotic genomes, is often costly, time-consuming, and computationally intensive^15,16. Mitochondrial genomes have emerged as valuable tools for phylogenetic studies, including Scorpaeniformes phylogenetics^10,17–19. Mitochondrial genomes, which are relatively easier to obtain and analyze compared to nuclear genomes, typically range from 15 to 20 kb in fish and contain a compact informative set of genes: 13 protein-coding genes (PCGs), two ribosomal RNAs (12s rRNA and 16s rRNA), 22 transfer RNAs (tRNAs), and two non-coding regions (control region and origin of the light strand (O_L))—these are essential for transcription and replication²⁰. Moreover, mitogenomes possess unique characteristics that make them suitable for evolutionary and phylogenetic studies, including conserved gene content across species, relatively high mutation rate, maternal inheritance, and low levels of recombination. These features collectively contribute to the effectiveness of the mitochondrial genome as molecular markers in evolutionary biology and phylogenetics.

Among the diverse families within the Scorpaeniformes order, Tetrarogidae (also known as waspfish) and Synanceiidae (stonefish) are particularly interesting. Tetrarogidae comprises approximately 17 genera and 41 species, while Synanceiidae includes about nine genera and 36 species, and both are primarily distributed in the Indo-West Pacific. These bottom-dwelling fish are notorious for their venomous dorsal spines, which can inflict painful injuries on humans^21,22. The venom of these fish has garnered attention for its potential pharmacological and medical applications²³. Consequently, numerous studies have investigated the ecological distribution, morphological features, venom proteins, and evolutionary history of Scorpaeniformes fish^8,12,24–26. However, taxonomic studies based on molecular characteristics remain relatively scarce. To date, the complete mitochondrial genomes have only been sequenced for over 100 Scorpaeniformes species across 22 families and 57 genera. Notably, the family Tetrarogidae was absent from these mitogenomic studies. Paracentropogon rubripinnis (Tetrarogidae) and Inimicus japonicus (Synanceiidae) are two representative species of these families, inhabiting the coastal waters of southern Japan and the southern Korean peninsula in the Northwest Pacific. While two mitogenomes from the Synanceiidae family have recently been sequenced^11,27, no mitogenomic data are currently available for any Tetrarogidae species.

This study aimed to elucidate the phylogenetic positions of Tetrarogidae and Synanceiidae and their evolutionary relationships within the Scorpaeniformes order. Thus, we sequenced and assembled the complete circular mitochondrial genomes of P. rubripinnis and I. japonicus to achieve this. We comprehensively analyzed their nucleotide composition, gene content, genomic organization, and codon usage profiles of protein-coding genes (PCGs). Additionally, we examined the structure of non-coding elements, including tRNAs, and the replication origin and identified novel conserved sequenced blocks in the control region. To provide a broader phylogenetic context, we performed phylogenetic analyses using 71 Scorpaeniformes and three outgroup species, focusing on the phylogenetic positions of Tetrarogidae and Synanceiidae within the order.

Mitochondrial genome organization and structure

We sequenced and assembled the complete mitochondrial genomes of P. rubripinnis and I. japonicus using Illumina sequencing technology, yielding 59.8 and 33.2 Gb of raw data, respectively (Table S1). The resulting circular mitogenomes were 16,465 bp for P. rubripinnis and 16,676 bp for I. japonicus. Both mitogenomes exhibited the typical vertebrate mitochondrial gene composition, containing 13 protein-coding genes (COI–III, ND1–6, ND4L, Cytb, ATPase6, and ATPase8), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA genes (including two each for serine and leucine, and one for the other amino acids), and a control region (D-loop) (Fig. 2). Most genes (28 out of 37) were encoded on the heavy strand (H-strand). In comparison, the remaining nine genes (ND6 and eight tRNAs) were located on the light strand (L-strand) (Fig. 2 and Table 1). To assess the conservation of genes in the order across Scorpaeniformes, we compared the mitogenome structures of P. rubripinnis and I. japonicus with those of 71 other Scorpaeniformes species and three Notothenioidei species (Table 2). Our analysis revealed that the order and orientation of all 37 genes and the control region in P. rubripinnis and I. japonicus are identical to those of other sequenced Scorpaeniformes species^10,27–29. This high degree of conservation in gene arrangement appears to be a characteristic feature of Scorpaeniformes mitogenomes. Interestingly, we observed slight differences in the region between ND5 and the D-loop in the Notothenioidei species, highlighting a potential order-specific variation in mitogenome structures (Fig. S1).

Table 1

Organization constituents of mitogenomes of *P. rubripinnis* and *I. japonicus*.
Feature	Strand	P. rubripinnis			I. japonicus
Feature	Strand	Position	Spacer (+)/overlap (-)	Start/stop codon	Position	Spacer (+)/overlap (-)	Start/stop codon
tRNA-Phe (F)	+	1–69	0		1–69	0
12S rRNA	+	70–1021	0		70–1023	0
tRNA-Val (V)	+	1022–1093	0		1024–1095	0
16S rRNA	+	1094–2775	0		1096–2783	0
tRNA-Leu (L1)	+	2776–2849	0		2784–2857	0
ND1	+	2850–3824	3	ATG/TAA	2858–3832	2	ATG/TAA
tRNA-Ile (I)	+	3828–3897	-1		3835–3904	-1
tRNA-Gln (Q)	-	3897–3967	-1		3904–3974	-1
tRNA-Met (M)	+	3967–4035	0		3974–4043	0
ND2	+	4036–5081	0	ATG/TA*	4044–5089	0	ATG/TA*
tRNA-Trp (W)	+	5082–5152	1		5090–5161	1
tRNA-Ala (A)	-	5154–5222	1		5163–5231	1
tRNA-Asn (N)	-	5224–5296	0		5233–5305	0
OL	-	5297–5328	0		5306–5336	0
tRNA-Cys (C)	-	5329–5394	0		5337–5403	-1
tRNA-Tyr (Y)	-	5395–5464	1		5403–5472	1
COI	+	5466–7016	0	GTG/TAA	5474–7024	0	GTG/TAA
tRNA-Ser (S1)	-	7017–7087	3		7025–7095	3
tRNA-Asp (D)	+	7091–7163	7		7099–7171	9
COII	+	7171–7861	0	ATG/T*	7181–7871	0	ATG/T*
tRNA-Lys (K)	+	7862–7935	1		7872–7945	2
ATPase 8	+	7937–8104	-10	ATG/TAA	7948–8115	-10	ATG/TAA
ATPase 6	+	8095–8777	0	ATG/TA*	8106–8788	0	ATG/TA*
COIII	+	8778–9562	0	ATG/TA*	8789–9573	0	ATG/TA*
tRNA-Gly (G)	+	9563–9633	0		9574–9644	0
ND3	+	9634–9982	0	ATG/T*	9645–9993	0	ATG/T*
tRNA-Arg (R)	+	9983–10051	0		9994–10062	0
ND4L	+	10052–10348	-7	ATG/TAA	10063–10359	-7	ATG/TAA
ND4	+	10342–11722	0	ATG/T*	10353–11733	0	ATG/T*
tRNA-His (H)	+	11723–11791	0		11734–11802	0
tRNA-Ser (S2)	+	11792–11859	3		11803–11870	7
tRNA-Leu (L2)	+	11863–11935	0		11878–11950	0
ND5	+	11936–13774	-4	ATG/TAA	11951–13789	-4	ATG/TAA
ND6	-	13771–14292	0	ATG/TAA	13786–14307	0	ATG/TAA
tRNA-Glu (E)	-	14293–14361	5		14308–14376	4
Cytb	+	14367–15507	0	ATG/T*	14381–15521	0	ATG/T*
tRNA-Thr (T)	+	15508–15579	-1		15522–15593	-1
tRNA-Pro (P)	-	15579–15648	0		15593–15662	0
Control region	+	15649–16465	0		15663–16676	0
An asterisk indicates the incomplete stop codon.

Table 2

A list of 74 complete mitogenomes in Scorpaeniformes.
Family	Species	Accession number	Size (bp)	Whole genome composition							PCGs
Family	Species	Accession number	Size (bp)	A%	T%	G%	C%	A + T%	AT skew	GC skew	AT skew	GC skew
Stichaeidae	Chirolophis japonicus	NC_028022	16521	25.50	28.55	18.31	27.64	54.05	-0.0564	-0.2030	-0.1632	-0.2344
Stichaeidae	Xiphister atropurpureus	NC_034669	16517	25.41	27.58	18.67	28.34	52.99	-0.0410	-0.2058	-0.1448	-0.2381
Anarhichadidae	Anarhichas denticulatus	NC_037606	16512	26.67	27.30	17.76	28.27	53.97	-0.0116	-0.2283	-0.1084	-0.2594
Anarhichadidae	Anarhichas lupus	NC_009773	16465	26.67	27.41	17.79	28.13	54.08	-0.0136	-0.2251	-0.1094	-0.2543
Anarhichadidae	Anarhichas minor	NC_037609	16507	26.70	27.21	17.77	28.33	53.90	-0.0094	-0.2291	-0.1070	-0.2587
Zoarcidae	Lycodes tanakae	NC_034649	16594	25.56	25.20	18.67	30.58	50.75	0.0071	-0.2418	-0.0953	-0.2703
Zoarcidae	Lycodes ygreknotatus	NC_034751	16486	26.29	25.32	17.96	30.43	51.61	0.0188	-0.2577	-0.0779	-0.2882
Gasterosteidae	Gasterosteus aculeatus	NC_041244	16543	27.03	28.37	17.24	27.36	55.40	-0.0243	-0.2269	-0.1157	-0.2647
Gasterosteidae	Gasterosteus wheatlandi	NC_011570	16538	27.69	28.72	16.87	26.72	56.41	-0.0183	-0.2260	-0.1040	-0.2630
Gasterosteidae	Pungitius hellenicus	NC_029471	16713	27.42	26.42	17.55	28.62	53.83	0.0186	-0.2398	-0.0804	-0.2751
Gasterosteidae	Pungitius kaibarae	NC_014893	16505	27.50	26.47	17.21	28.82	53.97	0.0191	-0.2523	-0.0726	-0.2961
Gasterosteidae	Pungitius laevis	NC_029473	16575	27.64	27.04	17.29	28.04	54.68	0.0109	-0.2372	-0.0868	-0.2717
Gasterosteidae	Pungitius platygaster	NC_029474	16566	27.82	26.57	17.21	28.41	54.38	0.0230	-0.2455	-0.0735	-0.2819
Gasterosteidae	Pungitius pungitius	NC_011571	16388	27.50	26.77	17.31	28.42	54.27	0.0135	-0.2431	-0.0838	-0.2754
Gasterosteidae	Pungitius sinensis	NC_014889	16581	27.50	26.90	17.34	28.26	54.40	0.0109	-0.2395	-0.0858	-0.2726
Gasterosteidae	Pungitius tymensis	NC_029472	16479	27.20	26.39	17.62	28.79	53.58	0.0152	-0.2407	-0.0801	-0.2802
Gasterosteidae	Culaea inconstans	NC_011577	16465	28.78	28.32	16.25	26.64	57.10	0.0081	-0.2422	-0.0972	-0.2755
Gasterosteidae	Apeltes quadracus	NC_011580	16472	27.60	27.54	16.74	28.12	55.14	0.0010	-0.2538	-0.0970	-0.2906
Gasterosteidae	Spinachia spinachia	NC_011582	16359	29.21	30.99	15.37	24.42	60.21	-0.0295	-0.2273	-0.1316	-0.2499
Cottidae	Comephorus baicalensis	NC_036148	16526	26.72	26.09	17.22	29.97	52.81	0.0118	-0.2702	-0.0865	-0.3081
Cottidae	Comephorus dybowskii	NC_036149	16527	26.73	26.19	17.20	29.88	52.92	0.0103	-0.2695	-0.0872	-0.3075
Cottidae	Cottus amblystomopsis	NC_035002	16528	25.91	26.09	17.80	30.20	52.00	-0.0034	-0.2583	-0.1051	-0.2903
Cottidae	Cottus asper	NC_036145	16511	27.21	26.29	16.86	29.64	53.50	0.0171	-0.2748	-0.0757	-0.3136
Cottidae	Cottus bairdii	NC_028277	16529	27.35	26.06	16.69	29.90	53.42	0.0241	-0.2836	-0.0740	-0.3193
Cottidae	Cottus czerskii	NC_025242	16534	26.40	26.07	17.47	30.07	52.47	0.0063	-0.2650	-0.0850	-0.3010
Cottidae	Cottus dzungaricus	NC_024739	16527	26.93	26.30	17.07	29.70	53.23	0.0119	-0.2701	-0.0824	-0.3135
Cottidae	Cottus hangiongensis	NC_014851	16594	25.49	25.88	18.22	30.41	51.37	-0.0075	-0.2506	-0.1137	-0.2840
Cottidae	Cottus perifretum	NC_036146	16523	26.97	26.13	17.08	29.81	53.11	0.0158	-0.2716	-0.0799	-0.3121
Cottidae	Cottus poecilopus	NC_014849	16560	25.69	25.74	18.18	30.39	51.43	-0.0011	-0.2513	-0.1040	-0.2822
Cottidae	Cottus reinii	NC_004404	16561	26.30	25.78	17.63	30.28	52.09	0.0100	-0.2640	-0.0919	-0.2999
Cottidae	Cottus rhenanus	NC_036147	16522	27.10	26.19	16.96	29.75	53.29	0.0171	-0.2738	-0.0785	-0.3144
Cottidae	Cottus szanaga	NC_032039	16518	26.47	26.20	17.42	29.91	52.66	0.0052	-0.2638	-0.0877	-0.2984
Cottidae	Cottus volki	NC_035001	16536	27.22	26.26	16.79	29.72	53.48	0.0179	-0.2780	-0.0730	-0.3161
Cottidae	Mesocottus haitej	NC_022181	16527	26.64	26.12	17.35	29.88	52.76	0.0099	-0.2653	-0.0854	-0.2995
Cottidae	Trachidermus fasciatus	NC_018770	16536	26.33	25.47	18.13	30.07	51.80	0.0167	-0.2478	-0.0815	-0.2750
Hexagrammidae	Hexagrammos agrammus	NC_021459	16512	26.88	26.16	17.24	29.72	53.04	0.0137	-0.2659	-0.0839	-0.3009
Hexagrammidae	Hexagrammos lagocephalus	NC_026888	16505	26.98	26.29	17.26	29.48	53.27	0.0130	-0.2615	-0.0838	-0.3002
Hexagrammidae	Hexagrammos otakii	NC_028630	16513	26.90	25.90	17.33	29.87	52.80	0.0189	-0.2656	-0.0728	-0.3067
Hexagrammidae	Pleurogrammus azonus	NC_023129	16591	26.94	27.06	17.22	28.77	54.01	-0.0022	-0.2512	-0.0994	-0.2858
Hexagrammidae	Pleurogrammus monopterygius	NC_023475	16575	27.05	27.12	17.15	28.68	54.17	-0.0012	-0.2517	-0.0956	-0.2913
Hexagrammidae	Ophiodon elongatus	NC_026887	16528	26.67	25.64	17.53	30.16	52.31	0.0198	-0.2647	-0.0679	-0.3057
Anoplopomatidae	Anoplopoma fimbria	NC_018119	16507	26.04	26.03	18.34	29.59	52.07	0.0001	-0.2348	-0.0999	-0.2702
Anoplopomatidae	Erilepis zonifer	NC_026889	16500	26.68	26.48	17.80	29.04	53.16	0.0039	-0.2399	-0.0967	-0.2721
Triglidae	Lepidotrigla microptera	NC_034944	16610	26.53	25.05	17.15	31.28	51.58	0.0286	-0.2918	-0.0668	-0.3326
Triglidae	Chelidonichthys kumu	NC_035059	16495	26.63	25.20	17.04	31.13	51.83	0.0277	-0.2925	-0.0641	-0.3351
Scorpaenidae	Pterois miles	NC_024746	16497	27.40	25.60	18.26	28.74	53.00	0.0340	-0.2229	-0.0504	-0.2418
Scorpaenidae	Pterois volitans	NC_025290	16500	27.55	25.58	18.02	28.85	53.13	0.0372	-0.2312	-0.0474	-0.2483
Scorpaenidae	Scorpaenopsis cirrosa	NC_027735	16966	27.91	26.35	17.71	28.02	54.27	0.0288	-0.2254	-0.0602	-0.2448
Sebastidae	Sebastes aleutianus	NC_039779	16976	27.47	26.94	17.44	28.15	54.41	0.0097	-0.2349	-0.0840	-0.2645
Sebastidae	Sebastes fasciatus	NC_036048	16399	27.36	27.06	17.64	27.93	54.42	0.0055	-0.2258	-0.0864	-0.2550
Sebastidae	Sebastes hubbsi	NC_027440	16453	27.86	26.66	17.20	28.28	54.52	0.0221	-0.2436	-0.0672	-0.2714
Sebastidae	Sebastes inermis	NC_023456	16504	27.77	26.76	17.13	28.34	54.53	0.0186	-0.2466	-0.0716	-0.2735
Sebastidae	Sebastes koreanus	NC_023265	16499	27.95	26.63	17.06	28.37	54.57	0.0242	-0.2491	-0.0657	-0.2808
Sebastidae	Sebastes longispinis	NC_026100	16445	27.91	26.66	17.12	28.31	54.57	0.0230	-0.2462	-0.0701	-0.2713
Sebastidae	Sebastes minor	NC_027444	16408	27.79	27.25	17.33	27.63	55.04	0.0099	-0.2292	-0.0817	-0.2609
Sebastidae	Sebastes nigrocinctus	NC_039778	16893	28.10	26.57	16.84	28.49	54.67	0.0280	-0.2570	-0.0665	-0.2854
Sebastidae	Sebastes oblongus	NC_024549	16396	27.91	26.41	16.96	28.72	54.32	0.0276	-0.2574	-0.0597	-0.2842
Sebastidae	Sebastes owstoni	NC_026191	16465	27.71	26.57	17.30	28.41	54.28	0.0210	-0.2430	-0.0749	-0.2723
Sebastidae	Sebastes rubrivinctus	NC_039777	16891	28.29	26.70	16.68	28.33	54.99	0.0290	-0.2589	-0.0671	-0.2839
Sebastidae	Sebastes schlegelii	NC_005450	16525	27.47	26.34	17.45	28.74	53.80	0.0210	-0.2444	-0.0705	-0.2772
Sebastidae	Sebastes steindachneri	NC_027445	16450	27.36	27.09	17.54	28.01	54.46	0.0049	-0.2298	-0.0875	-0.2587
Sebastidae	Sebastes taczanowskii	NC_027439	16452	27.71	26.47	17.29	28.53	54.18	0.0229	-0.2452	-0.0710	-0.2748
Sebastidae	Sebastes thompsoni	NC_027447	16405	27.98	26.77	17.04	28.21	54.75	0.0222	-0.2468	-0.0678	-0.2781
Sebastidae	Sebastes trivittatus	NC_027446	16409	27.86	26.67	17.09	28.37	54.54	0.0218	-0.2480	-0.0692	-0.2789
Sebastidae	Sebastes vulpes	NC_027438	16462	27.71	26.55	17.14	28.61	54.25	0.0214	-0.2506	-0.0708	-0.2808
Sebastidae	Sebastiscus marmoratus	NC_013812	17301	28.69	26.67	16.51	28.14	55.36	0.0364	-0.2605	-0.0613	-0.2877
Sebastidae	Helicolenus avius	NC_020349	16651	27.88	26.36	17.08	28.68	54.24	0.0280	-0.2535	-0.0632	-0.2842
Sebastidae	Helicolenus hilgendorfi	NC_003195	16728	27.82	26.35	17.19	28.65	54.17	0.0270	-0.2500	-0.0649	-0.2827
Synanceiidae	Synanceia verrucosa	NC_026989	16506	31.01	28.34	15.06	25.60	59.35	0.0451	-0.2593	-0.0288	-0.2883
Bathydraconidae	Parachaenichthys charcoti	NC_026578	18202	25.81	25.30	17.87	31.02	51.11	0.0100	-0.2691	-0.0882	-0.2761
Channichthyidae	Chionodraco hamatus	NC_029737	17457	26.38	26.00	17.44	30.18	52.38	0.0074	-0.2674	-0.0846	-0.2689
Nototheniidae	Pagothenia borchgrevinki	NC_030320	17299	25.11	29.46	20.45	24.98	54.57	-0.0797	-0.0996	-0.1905	-0.0950
Tetrarogidae	Paracentropogon rubripinnis	MT506029	16465	28.77	29.11	16.34	25.78	57.88	-0.0059	-0.2242	-0.0867	-0.2491
Synanceiidae	Inimicus japonicus	MT506040	16674	29.57	29.09	15.95	25.39	58.66	0.0083	-0.2285	-0.0663	-0.2602

A comprehensive analysis of the structural and genomic features of P. rubripinnis and I. japonicus mitogenomes revealed several distinctive characteristics. Subsequently, we checked the intergenic regions, overlapping genes, nucleotide composition, and skewness of two mitogenomes. Intergenic spacer sequences were identified in nine areas, totaling 25 bp in P. rubripinnis and 30 bp in I. japonicus, with lengths ranging from 1 to 9 bp (Table 1). Concurrently, we observed gene overlaps in both species: six regions in P. rubripinnis (24 bp total) and seven in I. japonicus, involving various genes such as tRNAs, ATPase, ND, and Cytb. Nucleotide composition analysis showed a high A+T content in both mitogenomes (57.88% in P. rubripinnis and 58.66% in I. japonicus; Table 2), notably exceeding the average found in the Scorpaeniformes species (53.97 ± 1.7%). This elevated A+T content aligns with previous findings in S. verrucosa (59.35%), another member of the Synanceiidae family²⁷.

Further examination of nucleotide bias through AT and GC skew analyses revealed interesting patterns. The AT skew was slightly negative for P. rubripinnis (− 0.0059) but positive for I. japonicus (0.0083), reflecting the broader trend in Scorpaeniformes, where 57 of 71 species exhibited positive AT skew values (average 0.0112 ± 0.017). Notably, Triglidae, Scorpaenidae, Sebastidae, and Synanceiidae fish demonstrated high AT skew (family average > 0.02), contrasting with the low AT skew (< − 0.01) observed in Stichaeidae and Anarhichadidae. The GC skew values were consistently negative across all Scorpaeniformes fishes, indicating a higher content of Cs than Gs, with P. rubripinnis and I. japonicus showing values of − 0.2242 and − 0.2285, respectively. These values are less negative than the Scorpaeniformes average (− 0.2487 ± 0.018), aligning with an increased GC skew observed in families such as Stichaeidae, Anarhichadidae, Gasterosteidae, Anoplopomatidae, Scorpaenidae and Tetrarogidae, in contrast to the lower GC skew seen in Cottidae, Hexagrammidae, and Triglidae. These findings collectively highlight the unique genomic features of P. rubripinnis and I. japonicus within the Scorpaeniformes mitochondrial genome evolution.

Protein-coding genes

The total length of 13 protein-coding genes (PCGs) was 11,428 bp, encoding 3800 codons in both P. rubripinnis and I. japonicus mitogenomes. Most PCGs utilized ATG as the start codon, except for COI, which possessed GTG, an accepted canonical mitochondrial start codon in vertebrates^30–32 (Table 1). For termination, six PCGs (ND1, COI, ATPase8, ND4L, ND5, and ND6) contained the TAA stop codon, four (COII, ND3, ND4, and Cytb) used the incomplete T stop codon, and three (ND2, ATPase6, and COIII) used the incomplete TA stop codon. This pattern of stop codon usage was identical to that of S. verrucosa, another member of the Synanceiidae subfamily²⁷. The incomplete stop codons are likely completed to TAA by post-transcriptional polyadenylation³³. The A+T content of the PCGs was 58.08% in P. rubripinnis and 58.43% in I. japonicus, while the A + T content at the third codon positions was 67.8% and 67.6%, respectively. Analysis of the relative synonymous codon usage (RSCU) revealed a preference for NNA and NNT codons over NNC and NNG (Fig. 3), consistent with the observed A and T bias at the third codon positions, which is typical in metazoan mitochondria^19,36. Most PCGs showed negative AT skew values, indicating a higher A and T content, except for ATPase8 in P. rubripinnis and ND2 and ATPase8 in I. japonicus (Fig. 4). GC skew values were negative for most PCGs except for ND6, indicating a higher C content than G. ND6 exhibited the highest GC skew and lowest AT skew in both species. These skew patterns are consistent with those observed in other Scorpaeniformes mitogenomes^10,18.

Transfer RNA genes and ribosomal RNA genes

The mitogenomes of P. rubripinnis and I. japonicus each contained 22 tRNA genes, including two for leucin and serine. Fourteen tRNA genes were located on the plus strand and eight on the minus strand (Table 1). In P. rubripinnis, tRNA gene lengths ranged from 66 bp (tRNA-Cys) to 74 bp (tRNA-Leu1 and tRNA-Lys), and in I. japonicus they ranged from 67 bp (tRNA-Cys) to 74 bp (tRNA-Leu). Secondary structure predictions revealed that 21 tRNA genes displayed canonical cloverleaf structures, while tRNA-Ser2 lacked a dihydrouridine (DHU) stem in both species (Fig. 5). This tRNA-Ser2 feature is consistent with observations in other vertebrate mitogenomes, including Scorpaenifromes^10,18,37. All amino acid acceptor stems in the tRNA genes were conserved at 7 bp, including non-Watson–Crick base pairs. Unmatched base pairs, exclusively T–G base pairs, were present in stem regions, a common phenomenon that can be resolved by post-transcriptional editing³⁸. The concatenated sequence of all tRNA genes showed positive AT skew (0.0289 in P. rubripinnis; 0.0166 in I. japonicus) and GC skew (0.0523 in P. rubripinnis; 0.0783 in I. japonicus), indicating a bias towards A and G nucleotides.

The 12S and 16S rRNA genes were located between tRNA-Phe and tRNA-Leu1, separated by tRNA-Val. In P. rubripinnis, the 12S gene was 952 bp long with 55.15% A + T content, while the 16S rRNA gene was 1682 bp with 58.32% A + T content. In I. japonicus, the 12S rRNA gene was 954 bp (56.71% A + T), and the 16S rRNA gene was 1688 bp (57.94% A + T).

Non-coding regions

The mitogenomes of P. rubripinnis and I. japonicus contained two major non-coding regions: the origin of light-strand replication (O_L) and the control region (CR). These regions contain regulatory sequences essential for mitochondrial transcription and replication initiation³⁹. The O_L was located between tRNA-Asn and tRNA-Cys, with lengths of 32 bp in P. rubripinnis and 31 bp in I. japonicus. Both formed hairpin secondary structures (Fig. 6A), consistent with typical vertebrate O_L characteristics. However, the I. japonicus O_L exhibited an atypical structure with relatively lower predicted scores, warranting further investigation into its structural significance.

The CR was positioned between tRNA-Pro and tRNA-Phe. In P. rubripinnis, it was 817 bp long with 61.57% A + T content and negative AT (-0.0099) and GC (-0.0764) skews. The I. japonicus CR was 1014 bp long with 64.99% A + T content and negative AT (-0.0137) and GC (-0.1324) skews. Multiple sequence alignment and conserved sequence analysis of the CRs from 12 Scorpaeniformes species revealed the presence of six previously described conserved sequence blocks (CSBs)⁴⁰ in both P. rubripinnis and I. japonicus: CSB-1, -2, -3, -D, -E, and -F (Fig. 6B and Table S2). Additionally, we identified two novel CSBs (Region-1 and Region-2) located upstream of the CSB-F in Scorpaeniformes. While Region-2 was highly conserved across Scorpaeniformes, Region-1 was absent in P. rubripinnis and I. japonicus. These novel CSBs may have functional roles similar to previously known CSBs in Scorpaeniformes.

Subsequently, two other I. japonicus mitogenome sequences were reported (accession numbers: MT604162 and MT375601)^41,42. We compared our I. japonicus sequence with the available MT604162 sequence and observed a 99.84% identity. While general mitogenomic features were consistent across studies, our analysis provides additional insights into tRNA and O_L secondary structures, PCG codon usage, and novel Scorpaeniformes-specific CSBs.

Phylogenetic analysis

To elucidate the relationships among Scorpaeniformes families and reconstruct a higher-resolution interrelationship of the Scorpaeniformes species, we collected 13 mitochondrial PCGs from 71 Scorpaeniformes and three Notothenioidei species (outgroups), including 12 families and 31 genera (Table 2). Phylogenetic trees were constructed using Bayesian inference (Fig. 7A) and maximum-likelihood (Fig. 7B) methods, resulting in highly congruent topologies with strong posterior probabilities and bootstrap values. Our analysis revealed that Tetrarogidae and Synanceiidae, including P. rubripinnis and I. japonicus, respectively, formed a monophyletic clade that occupied the most basal position within the Scorpaeniformes phylogeny. This finding builds upon previous studies, which identified monophyly between these families yet could not determine their exact phylogenetic location^7,8. A recent study using mitochondrial PCGs placed Synanceiidae at a basal position within Scorpaeniformes, albeit without including Tetrarogidae¹¹. Our analysis, which incorporates both families, confirms and extends these findings, providing a more comprehensive view of their phylogenetic placement.

The Scorpaenidae family, which includes most marine venomous fish with 26 genera and 223 species, has been subject to different phylogenetic interpretations⁸. Our analysis incorporates newly sequenced mitochondrial genomes from Tetrarogidae and Synanceiidae, supporting the phylogenetic relationship depicted in Fig. 1A rather than Fig. 1B. This result is particularly significant as it includes, for the first time, mitochondrial genome data from Tetrarogidae. While the overall topology of our phylogenetic tree is consistent with some previous studies, including these new data provides a higher resolution of the interrelationships between Scorpaeniformes, especially regarding the phylogenetic positions of Tetrarogidae and Synanceiidae.

To further investigate these basal relationships within Scorpaeniformes, we expanded our analysis to include 13 mitochondrial PCGs from two Platycephalidae species, as a recent study suggested that Platycephalidae and Synanceiidae might occupy basal positions in the Scorpaeniformes phylogeny¹⁸. Our results showed that the phylogenetic position of Platycepahlidae is sensitive to the choice of outgroup. When using Notothenioidei as outgroups, Platycephalidae appeared in the most basal position within Scorpaeniformes (Fig. S2A). However, when Perciformes species were used as outgroups, Platycephalidae clustered with Tetrarogidae and Synanceiidae, forming a clade not at the tree base (Fig. S2B). Notably, the bootstrap values for many of the deeper nodes in both trees were relatively low (below 70), indicating uncertainty in these relationships.

Our comprehensive analysis of the complete mitochondrial genomes of P. rubripinnis and I. japonicus, representing the Tetrarogidae and Synanceiidae subfamilies, respectively, has yielded several significant findings. The mitogenomes exhibit the typical vertebrate mitochondrial structure, with 13 PCGs, 2 rRNAs, 22 tRNAs, and 2 non-coding regions. Comparative genomic analyses revealed highly conserved gene orders and orientations across Scorpaeniformes, distinguishing them from the outgroup—the Notothenioidei species. We confirmed previously identified CSBs in the control region and discovered two novel Scorpaeniformes-specific CSBs, expanding our understanding of mitochondrial regulatory elements within this order.

Phylogenetic analyses incorporating our newly sequenced mitogenomes and those of 71 other Scorpaeniformes species provided a higher-resolution phylogeny of the order. Our results strongly support the monophyly of Tetrarogidae and Synanceiidae, positioning them at the base of the Scorpaeniformes phylogeny. This placement offers new insights into venom evolution in these fish and their relationships to other Scorpaeniformes families.

These mitogenomic data and phylogenetic findings in this study significantly advance our understanding of Scorpaeniformes evolution. Our results provide a robust framework for future investigations into biodiversity, biogeography, and adaptation in the order. These insights have potential implications for the classification and taxonomy of Scorpaeniformes, particularly in refining the placement of venomous fish families within the phylogenetic tree. By demonstrating the value of mitogenomic data in resolving complex phylogenetic relationships, our study underscores the importance of comprehensive genomic sampling across diverse fish lineages. As we expand our genomic database, we can further refine our understanding of teleost evolution, potentially uncovering new evolutionary patterns and mechanisms. Therefore, this work not only contributes to the field of ichthyology but also provides a foundation for future research in areas such as venom evolution and potential pharmacological applications, highlighting the broader impact of phylogenetic studies on basic and applied sciences.

Sample collection and DNA extraction

P. rubripinnis and I. japonicus specimens were collected from Geomun-do (34.1 N, 127.18 E), Yeosu, Republic of Korea. Total genomic DNA was extracted from 100 mg of muscle tissues using the cetyltrimethylammonium bromide (CTAB)-based method. Briefly, tissue samples were incubated in CTAB buffer (2% cetyltrimethylammonium bromide, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl, and pH 8.0) at 56°C for 1 hour. Then, DNA was isolated using phenol/chloroform extraction followed by ethanol precipitation. The extracted DNA was resuspended in TE buffer and stored at -20°C until further use. All experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-YB-2023-363). All methods were carried out in accordance with relevant guidelines and regulations, including the ARRIVE guidelines for reporting animal research.

Sequencing, mitochondrial genome assembly, and annotation

Genomic DNA libraries with 550 bp insert were constructed for P. rubripinnis and I. japonicus using the Illumina standard protocol (San Diego, USA). Paired-end sequencing generated 2 × 101 bp reads for P. rubripinnis, and 2 × 151 bp reads for I. japonicus. Raw reads were preprocessed using Trimmomatic (v0.36)⁴³, to remove adapter sequences, poly-N sequences, and low-quality bases. The parameters were set as follows: ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 for the 101 bp library and MINLEN:50 for the 151 bp library. Mitochondrial genome assembly was performed using two different approaches: Norgal (v1.0.0)⁴⁴ with default parameters and NOVOPlasty (v2.7.2)⁴⁵. For NoVOPlasty, we used COI coding sequences of each species (GeneBank: KU216091.1 for P. rubripinnis; KU199176.1 for I. japonicus) as seed sequences with parameters set to ‘Type = mito, K-mer = 39, Genome range = 12,000–22,000’. The assembled genomes from both methods showed high concordance, with only a 1 bp difference in length and > 99.9% sequence identity. We selected the NOVOPlasty assembly for subsequent analyses. Mitochondrial genome annotation was performed using MitoAnnotator⁴⁶ to generate a gene map and identify genome features.

Bioinformatics analysis

The 13 protein-coding genes (PCGs) of P. rubripinnis and I. japonicus were extracted based on their annotation information, and relative synonymous codon usage (RSCU) scores were calculated using CAIcal⁴⁷. Secondary tRNA gene structures were predicted using tRNAscan-SE⁴⁸ with default parameters and visualized using PseudoViewer⁴⁹. To analyze nucleotide composition bias, AT and GC skew values were calculated using the following formulas: AT skew = (A – T)/(A + T) and GC skew = (G – C)/(G + C)⁵⁰.

To analyze the origin of light-strand replication (O_L), the sense strand sequence forming the regular O_L, located between tRNA-Asn (N) and tRNA-Cys (C), was extracted. The secondary O_L structure was predicted using RNAstructure software⁵¹. To examine the control region, we aligned the control regions from mitogenomes of 12 species using the MUSCLE algorithm implemented in MEGA X software⁵² with default settings. Conserved sequence blocks within the control region were identified using DnaSP (v.6.12.03)⁵³ with a window length of 8 and a conservation score of 6. Regions with a homozygosity score above 0.9 were designated as conserved sequence blocks.

Phylogenetic analysis

Phylogenetic relationships were investigated using mitogenomes of 71 Scorpaeniformes species and 3 Notothenioidei species, available in the MitoFish database (Table 2). The mitogenomes of the 3 Notothenioidei species (Parachaenichthys charcoti, Chionodraco hamatus, and Pagothenia borchgrevinki) were assigned as outgroups. Nucleotide sequences of the 13 protein-coding genes (PCGs) were extracted from the mitochondrial genome sequences using their annotation information. Multiple sequence alignment was performed using MAFFT (v7.407)⁵⁴ with an --auto parameter. The resulting alignments were quality-checked and formatted using trimAl (v1.2rev59)⁵⁵. A supermatrix was generated by concatenating the alignments of all 13 PCGs using phyutility (v2.7.1)⁵⁶ for phylogenetic tree reconstruction. Bayesian inference analysis was conducted using MrBayes⁵⁷ and implemented in the TOPALi (v2.5) platform⁵⁸. The GTR + I + G model was applied with 300,000 generations. For the maximum-likelihood (ML) analysis, the best-fit model was determined using ModelFinder⁵⁹. Then, the ML tree was inferred based on the concatenated alignment using IQTREE (v1.6.12)⁶⁰ with 1000 bootstrap replicates and the GTR + F + R6 model.

Competing interests

The authors declare that they have no competing interests.

Author Contribution

S.-G.L. designed the study and performed the experiments. S.-G.L. and S.K. conducted the mitogenome analyses. S.-G.L., S.K., and C.P. wrote the manuscript. C.P. supervised and contributed to the project coordination. All authors reviewed the manuscript.

Acknowledgments

This work was supported by research grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022R1A2C1010731 to C.P.).

Data Availability

Whole genome sequencing data of P. rubripinnis and I. japonicus were deposited to the NCBI database with the following SRA accession numbers: SRR11802645 and SRR11802644, and BioProject accession numbers: PRJNA632998 and PRJNA633000, respectively. The complete mitogenomes of P. rubripinnis and I. japonicus are available with GenBank accession numbers MT506029 and MT506030, respectively.

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Evolutionary trajectories of venomous fish: complete mitogenomes of Paracentropogon rubripinnis and Inimicus japonicus reshape Scorpaeniformes phylogeny

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Version 1

Abstract

Figures

Introduction

Results

Mitochondrial genome organization and structure

Protein-coding genes

Transfer RNA genes and ribosomal RNA genes

Non-coding regions

Phylogenetic analysis

Discussion

Materials and Methods

Sample collection and DNA extraction

Sequencing, mitochondrial genome assembly, and annotation

Bioinformatics analysis

Phylogenetic analysis

Declarations

Competing interests

Author Contribution

Acknowledgments

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1