Comprehensive Insights into Cnidarian Phylogenetics: cox3-Based Phylogeny, Mitochondrial Genome Variability, Introns, and Gene Rearrangements

doi:10.21203/rs.3.rs-4961541/v1

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Comprehensive Insights into Cnidarian Phylogenetics: cox3-Based Phylogeny, Mitochondrial Genome Variability, Introns, and Gene Rearrangements

https://doi.org/10.21203/rs.3.rs-4961541/v1

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Cnidarians, an ecologically and culturally significant group, possess mitochondrial genomes that exhibit several features deviating from conventional textbook descriptions. In this study, we analyzed mitochondrial genomes across various cnidarian orders, revealing significant variation in gene rearrangement patterns. Notably, we found that Anthozoa exhibits highly variable gene arrangements despite slow evolutionary rates, while Hydrozoa demonstrates relatively high levels of gene rearrangement coupled with faster molecular evolution. Our analysis of the cytochrome c oxidase subunit III (cox3) gene provides new insights into cnidarian phylogeny, supporting the monophyly of both Anthozoa and Medusozoa, which aligns more closely with nuclear data and the currently accepted taxonomic scheme. Within Medusozoa, our cox3-based phylogenetic tree reveals strong support for monophyly at the class level. Our reconstruction also suggests a novel topology wherein Staurozoa, Hydrozoa, and Scyphozoa form a sister group to Cubozoa. This finding is particularly significant given the frequent inconsistencies observed in previous mitochondrial gene studies. The cox3 gene's predominant distribution on the heavy(+) strand and limited intron presence in most cnidarian species potentially reduces the impact of strand bias on phylogenetic signal, making it a valuable marker for phylogenetic reconstruction.

However, our study also highlights the challenges inherent in using mitochondrial genes for deep phylogenetic reconstructions within Cnidaria. We observed incongruences between nuclear and mitochondrial phylogenies, as well as conflicts among different mitochondrial gene-based studies. These discrepancies underscore the complex nature of cnidarian evolution and the potential limitations of relying on a single type of genetic marker.

Cnidaria

cox3

gene rearrangements

mitochondrial genome variability

phylogeny

Cnidaria is a diverse phylum of basal metazoans comprising three major clades: Anthozoa, Medusozoa, and Endocnidozoa(Lang et al., 2009). The phylum includes approximately 12,789 recognized extant species, with an estimated 8,000 additional undescribed species(Appeltans et al., 2012). Anthozoa, which includes sea anemones, corals and sea pens, is characterized by the exclusive presence of the polyp life stage. Medusozoa encompasses all cnidarians that exhibit a medusa (jellyfish) stage in their life cycle, and is currently divided into four classes: Scyphozoa (true jellyfish), Cubozoa (box jellyfish), Staurozoa (stalked jellyfish), and Hydrozoa (hydroids). Endocnidozoa comprises two highly derived parasitic groups: Myxozoa and Polypodiozoa, both of which are microscopic and have undergone significant morphological simplification(Schierwater & DeSalle, 2021).

The evolutionary relationships within Cnidaria, particularly at the class level, have been challenging to resolve due to phenotypic plasticity and the limitations of morphology-based classifications. Recent molecular phylogenetic analyses have consistently supported the monophyly of Cnidaria and its position as a sister group to Bilateria(Zapata et al., 2015a). However, the relationships among major cnidarian clades remain contentious. Anthozoa is generally considered a well-defined monophyletic group, supported by extensive analyses of nuclear rRNA data(DeBiasse et al., 2024). In contrast, the phylogenetic relationships within Medusozoa are still debated, with various conflicting hypotheses proposed(Kayal et al., 2018). One prevailing hypothesis based on nuclear gene sequences suggests that Staurozoa is the sister group to all other medusozoans, potentially representing a key lineage for understanding early medusozoan evolution(Ling et al., 2023). An alternative hypothesis proposes that Staurozoa, Cubozoa, and Scyphozoa collectively form a sister group to Hydrozoa. This arrangement challenges traditional views and highlights the complexity of medusozoan phylogenetics(Collins et al., 2006a). Interestingly, some phylogenomic analyses based on mitochondrial protein-coding genes have suggested a different topology, with Staurozoa more closely related to Anthozoa than to other medusozoans(Feng et al., 2023). This finding further complicates our understanding of cnidarian phylogeny and underscores the potential for incongruence between nuclear and mitochondrial gene trees.

Phylogenetic studies of Cnidaria have employed various molecular markers, including nuclear ribosomal genes (e.g., 5S, 18S, 28S, ITS), mitochondrial genes (e.g., 12S, 16S, cox1, cox3), ultraconserved genomic elements (UCEs), and whole transcriptome data(Zapata et al., 2015b; Erickson et al., 2021; Quattrini et al., 2023). However, these approaches face challenges such as taxonomic sampling biases, exclusion of key lineages, high sequencing costs, and substantial computational requirements. Accurate phylogenetic reconstruction demands careful consideration of data quality, including the identification of orthologous genes that reliably reflect species relationships and the selection of markers less prone to systematic biases. Ideal molecular markers for phylogenetic analysis should meet several criteria: they should be orthologous, present in all taxa under study, under neutral selection, exhibit relatively constant character state frequencies and substitution rates across lineages, and contain both conserved and variable regions(Cruickshank, 2002). Mitochondrial genes generally fulfill many of these criteria and have been widely used in animal phylogenetics.

Cnidarian mitochondrial genomes exhibit unique features compared to those of other animals, including diverse genome structures and extensive gene rearrangements(Feng et al., 2023). This study aims to investigate these atypical characteristics of cnidarian mitochondrial genomes and evaluate their utility in resolving cnidarian phylogenetic relationships. By analyzing a comprehensive dataset of mitochondrial genomes across diverse cnidarian taxa, we will assess variations in genome structure, gene content, and gene order. This research will contribute to our understanding of mitochondrial genes evolution in cnidarians and provide insights into broader evolutionary processes in early-branching metazoan phyla. Furthermore, it will help assess the efficacy of mitochondrial genomes in resolving deep phylogenetic relationships within Cnidaria, potentially complementing nuclear gene-based phylogenies and contributing to a more comprehensive understanding of cnidarian evolution.

Data Retrieval

We retrieved 654 complete mitochondrial genome sequences representing all available cnidarian species from the National Center for Biotechnology Information (NCBI) GenBank database (accessed 31/05/2024). For species with multiple mitochondrial genome entries, we preferentially selected reference sequences to ensure dataset consistency. Our dataset includes representatives from all seven recognized cnidarian classes: Anthozoa, Hydrozoa, Scyphozoa, Staurozoa, Cubozoa, Myxozoa, and Polypodiozoa.

Genome Annotation

To ensure consistent and accurate annotation, we re-annotated all mitochondrial genomes using MITOS2(Bernt et al., 2013b), with the RefSeq63 database as the reference. This re-annotation process was crucial for standardizing gene identification and ensuring the reliability of subsequent analyses.

Phylogenetic Analysis

Maximum-Likelihood Phylogeny

We extracted protein-coding genes from the annotated mitochondrial genomes. Multiple sequence alignments for each gene were generated using MAFFT v 7.525(Katoh & Standley, 2013) with the L-INS-i algorithm, which is suitable for sequences with one conserved domain and long gaps. We inferred a maximum-likelihood (ML) phylogeny using IQ-TREE v2.1.2(Minh et al., 2020) with the following settings: -B 1000 -m MFP + MERGE. This approach incorporated 1,000 ultrafast bootstrap replicates to assess branch support and employed the ModelFinder Plus (MFP) algorithm to determine the best-fit model of sequence evolution. We selected Halisarca dujardini (GenBank accession: EU237483.1), a species from the phylum Porifera, as the outgroup for our phylogenetic analysis, following standard practice in metazoan phylogenetics(Kapli & Telford, 2020).

Bayesian Inference

We conducted Bayesian inference (BI) using PhyloBayes MPI v1.7b(Lartillot et al., 2013). We implemented the site-heterogeneous mixture CAT + GTR model to account for variation in evolutionary processes across sites. We ran two independent Markov chain Monte Carlo (MCMC) chains for 50,000 cycles, sampling every 100 generations. Convergence was assessed using the maxdiff statistic, with a value < 0.3 considered acceptable.

Phylogenetic Tree Visualization

Phylogenetic trees were visualized using TreeViewer(Bianchini & Sánchez-Baracaldo, 2024), which are widely used tools for the interpretation and presentation of phylogenetic data.

Calculation of Branch Length

We calculated branch lengths of the maximum-likelihood tree using the 'adephylo' package v1.1-16 in R(Jombart et al., 2010). Specifically, we employed the sumDD method, which calculates the sum of direct descendants for all nodes along each path. This approach provides a comprehensive measure of evolutionary distances within the phylogenetic tree (Dray et al., 2023).

Gene Rearrangement Analysis

To quantify mitochondrial genome rearrangements, we calculated the rearrangement score (RS) for each mitochondrial genome using (Zhang et al., 2020). This method compares the gene order of each mitochondrial genome to a reference arrangement. For our analysis, we used the typical invertebrate mitochondrial gene arrangement as the reference: nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad5, nad4, nad4l, nad6, cob, nad1, rrns, rrnl. Due to the variability in tRNA gene content and arrangement within cnidarian mitochondrial genomes, we only considered protein-coding genes and rRNA genes for this analysis.

Statistical analyses

Statistical analyses were performed using Seaborn for visualization and Pearson correlation coefficients for assessing relationships between variables(Waskom et al., 2020).

Diversity of Cnidarian Mitochondrial Genomes

Cnidarians exhibit remarkable diversity in their mitochondrial genome structures, deviating significantly from the typical metazoan circular mitochondrial chromosome. Early phylogenetic hypotheses suggested that Anthozoa, characterized by predominantly circular mitochondrial genomes, is sister to Medusozoa, which generally possesses linear mitochondrial genomes(Bridge et al., 1992). This hypothesis has been supported by numerous subsequent studies(Schierwater & DeSalle, 2021). However, recent findings indicate that some anthozoans, such as Ceriantharia, also possess linear mitochondrial genomes, challenging the idea that linear mitochondrial genomes originated from a single common ancestor(Stampar et al., 2019). This discovery highlights the necessity of extensive sequencing across various taxa to better understand the evolutionary history of cnidarian mitochondrial genomes. Despite the considerable species diversity within Cnidaria, only 654 mitochondrial genomes are currently available in GenBank. This represents a small fraction of cnidarian diversity, including potential cryptic species (Supplementary Table S1).

Cnidaria is divided into seven major classes based on the National Center for Biotechnology Information (NCBI) Taxonomy: Anthozoa (sea anemones, corals, sea pens), Scyphozoa (true jellyfish), Cubozoa (box jellies), Hydrozoa (hydroids), Staurozoa (stalked jellyfish), and two highly derived parasitic classes, Myxozoa and Polypodiozoa(Schierwater & DeSalle, 2021). The abundance of species and the number of sequenced mitochondrial genomes vary greatly among these classes, with Anthozoa and Hydrozoa being the most well-represented in our dataset.

Class Anthozoa

Anthozoa, comprising approximately 7,500 extant species(Daly et al., 2007), inhabits diverse marine environments from shallow to deep waters across tropical, temperate, and polar regions. Despite their ecological significance, only about 400 complete mitochondrial genomes have been sequenced. Most anthozoan mitochondrial genomes are circular and encode 13 protein-coding genes and two ribosomal RNA genes. However, some tube anemones (Order Ceriantharia) possess multipartite linear mitochondrial genomes(Stampar et al., 2019), challenging previous assumptions about mitochondrial genome structure in this class.

Class Hydrozoa

Hydrozoa includes approximately 3,800 species, with both marine and freshwater representatives(Appeltans et al., 2012). Hydrozoan mitochondrial genomes are typically linear. Of the 40 hydrozoan species with complete mitochondrial genomes in GenBank, 17 are annotated as circular, though these annotations may be spurious(Ahuja et al., 2024).

Class Scyphozoa

Scyphozoa comprises about 230 species, generally with linear mitochondrial genomes. Among the 20 scyphozoan species with complete mitochondrial genomes in GenBank, 12 are annotated as circular, which may also be spurious(Ahuja et al., 2024).

Class Cubozoa

Cubozoa, with approximately 47 described species, predominantly inhabit tropical and subtropical near-shore environments. The mitochondrial genome of Alatina moseri is highly fragmented, consisting of 18 genes on eight linear chromosomes, including 13 protein-coding genes, two ribosomal RNAs, and one transfer RNA(Smith et al., 2012).

Class Staurozoa

Staurozoa, consisting of about 50 species, are primarily found in cold, anti-tropical regions. The only staurozoan with a sequenced mitochondrial genome, Haliclystus antarcticus, has a single, circular mitochondrial genome(Li et al., 2016).

Class Myxozoa

Myxozoa, highly derived parasitic cnidarians, exhibit unusual mitochondrial genomes. For instance, Myxobolus squamalis has its mitochondrial genome partitioned into eight chromosomes, each with a large noncoding region(Yahalomi et al., 2017). These genomes lack tRNA genes, contain only five protein-coding genes, and exhibit high structural plasticity.

Class Polypodiozoa

Polypodiozoa is represented by Polypodium hydriforme, which has a large, single circular mitochondrial genome of 93,065 bp, the largest sequenced within Cnidaria(Novosolov et al., 2022).

The remarkable structural diversity of cnidarian mitochondrial genomes underscores the importance of extensive sequencing efforts across this phylum. Such efforts are crucial for accurately understanding the evolutionary relationships and genomic adaptations within Cnidaria.

Mitochondrial Genome Diversity in Cnidaria: Unique Genes and Intron Frequency

We analyzed 654 mitochondrial genomes from cnidarian species across seven classes: Anthozoa (566 species), Hydrozoa (59 species), Scyphozoa (18 species), Staurozoa (1 species), Cubozoa (1 species), Myxozoa (1 species), and Polypodiozoa (1 species). Our analysis revealed significant variability in mitochondrial genome structure and composition among cnidarians. Unlike vertebrate mitochondrial genomes, which typically encode 37 genes (13 protein-coding genes, 22 tRNAs, and two rRNAs) (Boore, 1999), cnidarian mitochondrial genomes encode between 16 and 20 genes. This variability is primarily due to differences in tRNA gene content, with most cnidarian species encoding only one or two tRNAs (tRNA-Met, tRNA-Trp), and the frequent absence of the atp8 gene (Supplementary Figure S1). Cnidarian mitochondrial genomes includes several unique protein-coding genes, such as lagli, gpI and gpII in Anthozoa, dpo in Cubozoa, Hydrozoa, and Scyphozoa (Supplementary Table S2). These genes likely play crucial roles in the adaptation of these organisms to their specific environments and may influence mitochondrial genome evolution in ways that are not yet fully understood.

Introns within mitochondrial genomes are another intriguing aspect of cnidarian mtDNA. Introns are relatively common in the cox1 and nad5 genes of various Anthozoa and Hydrozoa species. Notably, within the order Scleractinia (Class Anthozoa), introns are present in multiple genes, including cox1, cox2, cox3, lagli, nad1, nad2, nad3, nad4, nad4l, and nad5, making Scleractinia the group with the highest number of mitochondrial introns observed (Table 1). The presence of introns in mitochondrial genomes raises questions about their impact on gene substitution rates. Studies have shown that introns can alter evolutionary scenarios, potentially leading to diverse environmental adaptations (Chuang et al., 2017; Barrett et al., 2020). However, the exact impact on substitution rates can vary depending on the specific gene and the evolutionary pressures acting on it.

The presence of unique protein-coding genes and introns in cnidarian mitochondrial genomes underscores the evolutionary plasticity of these genomes and their potential role in environmental adaptation. Further research is needed to unravel the mechanisms by which these elements influence mitochondrial function, genome evolution, and species diversification in Cnidaria.

Relationships among the Cnidarian Classes

Phylogenetic Relationships within Cnidaria: Historical Context and Recent Insights

The elucidation of phylogenetic relationships within Cnidaria has evolved significantly over recent decades, driven by advancements in molecular techniques and the expansion of genetic data. Early studies, such as Bridge et al. integrated 18S and 16S rRNA sequences with morphological characters to investigate class-level relationships within Cnidaria(Bridge et al., 1995). Subsequent research utilized multi-locus datasets, including nuclear ribosomal genes (e.g., 5S, 18S, 28S, ITS) and mitochondrial genes (e.g., 12S, 16S, cox1, cox3), as well as complete mitochondrial genome sequences(Park et al., 2012; Kayal et al., 2013; Feng et al., 2023). The advent of phylogenomics marked a significant advancement in cnidarian systematics. Studies began to utilize data from hundreds or thousands of loci obtained from transcriptome sequences and target-capture sequencing approaches (Zapata et al., 2015b; Kayal et al., 2017, 2018; Quattrini et al., 2018; Quek et al., 2020; McFadden et al., 2021). Despite the high resolution afforded by these methods, phylogenetic relationships among and within cnidarian lineages often differed across studies, particularly concerning the major lineages within Medusozoa.

Cox3 Gene Analysis and Phylogenetic Insights

The maximum likelihood and Bayesian trees based on the cox3 gene exhibit nearly identical topologies, with the main difference being that the Bayesian trees show less resolution at internal branches compared to those generated by the maximum likelihood method. Our recent phylogenetic analysis provides robust support for two reciprocally monophyletic clades within Cnidaria: Anthozoa and Medusozoa (Fig. 1). This aligns with the widely accepted view of these groups as relatively independent evolutionary branches within Cnidaria. Within Medusozoa, our cox3-based phylogenetic tree reveals strong support for monophyly at the class level. Our reconstruction suggests a novel topology wherein Staurozoa, Hydrozoa, and Scyphozoa form a sister group to Cubozoa. This contrasts with previous studies based on complete mitochondrial genomes, where Staurozoa appeared more closely related to Anthozoa(Feng et al., 2023), and with studies suggesting paraphyly of both Anthozoa and Scyphozoa(Park et al., 2012; Kayal et al., 2013). The cox3 gene, with its conserved and variable regions, has proven to be a reliable marker for resolving phylogenetic relationships at both high and intermediate taxonomic levels within Cnidaria. Our results corroborate the monophyly of Anthozoa and Medusozoa while providing a refined view of the internal branching order within Medusozoa.

Gene Rearrangements in the Phylum Cnidaria

Gene rearrangements in mitochondrial genomes refer to the alteration of the order of genes within the genome. These rearrangements can be used as phylogenetic markers because they often occur infrequently and can provide insights into the evolutionary history of the organism. In this study, we used the typical invertebrate mitochondrial gene arrangement as a reference to investigate gene rearrangements within the phylum Cnidaria(Supplementary Table S3).

Rearrangement Scores Across Cnidarian Orders

Our analysis revealed significant variation in mitochondrial gene rearrangements among different orders within Anthozoa:

Order Corallimorpharia and Order Scleractinia exhibited rearrangement scores of 28 and 30, respectively, indicating the closest gene arrangements to the typical invertebrate arrangement among Anthozoan mitochondrial genomes.

Order Malacalcyonacea showed a wide range of rearrangement scores (15, 17, 19, 22, 23, 24, 25, 26, 27), indicating at least nine distinct gene arrangement types.

Order Zoantharia had rearrangement scores of 22, 24, 25, 26, with at least four gene arrangement types.

Order Scleralcyonacea presented rearrangement scores of 13, 19, 26, 27, 28, 30, suggesting at least six gene arrangement types.

Order Antipatharia exhibited rearrangement scores of 26 and 28, indicating at least two gene arrangement types.

Order Actiniaria showed rearrangement scores of 24 and 26, with at least two gene arrangement types.

Order Octocorallia demonstrated high levels of gene rearrangement with scores of 13, 23, and 30, reflecting severe gene rearrangement within this group.

For Class Hydrozoa, the analysis was complicated by the presence of genes on several linear chromosomes, making direct comparisons to the typical invertebrate arrangement challenging. By extracting single-chromosome mitochondrial genomes for comparison, we found that Hydrozoa exhibited rearrangement scores ranging from 21 to 28, indicating significant gene rearrangement among species.

Correlation Between Gene Rearrangement and Substitution Rates

The correlation between gene rearrangement and substitution rates in mitochondrial genomes is a critical area of study, especially in understanding the evolutionary dynamics of different cnidarian classes. In Anthozoans, the remarkably slow rate of mitochondrial genome evolution—50 to 100 times slower than in most other animals—presents a unique challenge for examining this correlation(Shearer et al., 2002). The shallow, nearly equidistant branch lengths observed in phylogenetic trees of Anthozoans indicate a level of evolutionary stasis that obscures the potential relationships between gene rearrangement and substitution rates. This evolutionary pattern, while fascinating, complicates efforts to draw meaningful conclusions about how gene rearrangement might influence substitution rates in these organisms.

In contrast, Hydrozoa offers a more promising avenue for investigating the correlation between gene rearrangement and substitution rates. The variability in branch lengths observed among different Hydrozoan species suggests a more dynamic evolutionary process, with some lineages experiencing rapid evolutionary changes while others remain more conserved. This diversity in evolutionary rates provides a valuable opportunity to explore how gene rearrangements in mitochondrial genomes might be linked to changes in substitution rates. To examine the correlation between gene rearrangement and substitution rates in Hydrozoa, we compared mitochondrial gene order shuffling with branch lengths(substitution rates) in phylogenetic trees. Consistent with findings in bilaterian examples(Bernt et al., 2013a), we observed a strong correlation between complete mitochondrial genome shuffling and long branches, indicative of high substitution rates(Fig. 2).

Our study highlights the significant variability in mitochondrial gene rearrangements across different cnidarian orders and classes. Anthozoans exhibit highly variable gene arrangements and slow evolutionary rates, while Hydrozoa show relatively high levels of gene rearrangement and faster molecular evolution. These findings underscore the importance of considering gene rearrangement patterns in phylogenetic studies and provide valuable insights into the evolutionary dynamics of Cnidaria.

Challenges of Using Mitochondrial Genes in Phylogenetic Analysis

The phylogenetic relationships within Cnidaria have been a subject of ongoing debate, with different genetic markers yielding conflicting topologies. Our study, focusing on the cox3 mitochondrial gene, provides new insights while highlighting the challenges inherent in using mitochondrial genes for deep phylogenetic reconstructions within this phylum.

A well-documented issue in Cnidarian phylogenetics is the incongruence between nuclear and mitochondrial gene trees, often attributed to factors such as substitution saturation, introgression, and the unique properties of mitochondrial genomes (Pratlong et al., 2017; Quattrini et al., 2023). While numerous analyses of nuclear rRNA data support the monophyly of Anthozoa(Zapata et al., 2015b; DeBiasse et al., 2024), our review of previous studies shows that mitochondrial DNA data often suggest a paraphyletic Anthozoa, with octocorals forming a sister group to medusozoans(Kayal & Lavrov, 2008; Lavrov et al., 2008; Park et al., 2012). Interestingly, our analysis based on the cox3 gene aligns more closely with the nuclear data and the currently accepted taxonomic scheme, supporting the monophyly of both Anthozoa and Medusozoa. This result is particularly noteworthy given the frequent inconsistencies observed in mitochondrial gene studies. The restoration of a monophyletic Scyphozoa in our cox3-based phylogeny further demonstrates the potential of this gene to resolve relationships that have been contentious in previous mitochondrial studies(Kayal et al., 2013). The selection of cox3 for our study was deliberate, taking into account the unique characteristics of Cnidarian mitochondrial genomes. The predominant distribution of cox3 on the heavy strand (+) in most Cnidarian species, with only a few exceptions in certain Anthozoan orders, potentially reduces the impact of strand bias on phylogenetic signal. Additionally, the limited presence of introns in cox3 across Cnidaria further justifies its use as a phylogenetic marker.

However, the inconsistencies observed between different mitochondrial gene studies in Cnidaria are cause for concern. The varying phylogenetic performance of different mitochondrial genes, influenced by factors such as gene rearrangements, strand bias, and the presence of introns, makes the selection of appropriate markers a critical consideration in Cnidarian phylogenetics. Our study underscores the need for careful evaluation of mitochondrial genes used in phylogenetic reconstructions within this phylum(Collins et al., 2006b; Feng et al., 2023). These discrepancies raise important questions about the evolutionary history of Cnidaria and the methodological approaches used to reconstruct it. The incongruence between mitochondrial and nuclear gene trees is not unique to Cnidaria and has been observed in various taxa(Cameron, 2014; Quattrini et al., 2023). However, the fact that different mitochondrial gene-based studies also yield conflicting results is particularly concerning and warrants further investigation.

Future Directions and Implications

While our cox3-based phylogeny provides support for some traditional groupings within Cnidaria, the overall picture of Cnidarian phylogenetics remains complex. Future studies should consider: A multi-faceted approach incorporating a wide range of both mitochondrial and nuclear markers, as well as morphological and developmental data. More sophisticated phylogenetic methods that can account for the unique characteristics of Cnidarian mitochondrial genomes. Cautious interpretation of phylogenetic results, especially when dealing with ancient lineages like Cnidaria.

The challenges highlighted in our study underscore the importance of a nuanced understanding of both the strengths and limitations of different genetic markers in advancing our knowledge of Cnidarian phylogeny. As we continue to unravel the evolutionary history of this diverse phylum, integrating multiple lines of evidence and employing advanced analytical techniques will be crucial for resolving longstanding phylogenetic questions.

Author Contributions

Lingming Kong wrote the paper; Ying Gao contributed to data collection and performed the data analysis; Ying Gao helped with data analysis and figure modification.

Funding

This research work was supported by the Shandong Provincial Natural Science Foundation, China (ZR2019PC027).

Conflicts of Interest

The authors declare no conflict of interest.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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Comprehensive Insights into Cnidarian Phylogenetics: cox3-Based Phylogeny, Mitochondrial Genome Variability, Introns, and Gene Rearrangements

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Abstract

Figures

Introduction

Materials and Methods

Data Retrieval

Genome Annotation

Maximum-Likelihood Phylogeny

Bayesian Inference

Phylogenetic Tree Visualization

Calculation of Branch Length

Gene Rearrangement Analysis

Results and Discussion

Diversity of Cnidarian Mitochondrial Genomes

Mitochondrial Genome Diversity in Cnidaria: Unique Genes and Intron Frequency

Relationships among the Cnidarian Classes

Phylogenetic Relationships within Cnidaria: Historical Context and Recent Insights

Cox3 Gene Analysis and Phylogenetic Insights

Gene Rearrangements in the Phylum Cnidaria

Rearrangement Scores Across Cnidarian Orders

Correlation Between Gene Rearrangement and Substitution Rates

Challenges of Using Mitochondrial Genes in Phylogenetic Analysis

Future Directions and Implications

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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