International Journal of Marine Science, 2025, Vol.15, No.2, 107-117 http://www.aquapublisher.com/index.php/ijms 109 2.3 Disputes on non-single-system sources and system classification It should be pointed out that "algae" is not a single-line group, and its members come from different phylogenetic origins. Based on the theory of endosymbiosis, experts propose that algae are divided into prokaryotic algae (i.e., cyanobacteria) and eukaryotic algae, and are further grouped according to the pigment membrane structure (Keeling, 2010). Among eukaryotic algae, primary chloroplasts (surrounded by two membranes) are only found in red algae, green algae and gray algae; while chloroplasts surrounded by more membranes appear in a series of variopoid groups (such as Cryptophyta, Mixoplankton, Prhinophyla, etc.) as well as some phyla and dinoflagellate phyla. Different scholars have different interpretations of these relationships, such as the theory of stained biosynthesis once assumed a single-original endosymbiotic event of red algae ( Stiller et al., 2014 ), while others advocated multiple independent secondary or tertiary endosymbiotic events. In summary, algae taxonomy is controversial in terms of endosynthetic events and genealogical definition, suggesting the need for a re-evaluation of traditional classification in a comprehensive molecular, morphological and ecological evidence. 3 Molecular Evidence of Algae Phylogenetic Development 3.1 Comparative analysis of nuclear genes and chloroplast genes In algae phylogenetic research, nuclear genes and chloroplast genes are widely used. Classic nuclear genes include the small subunit rRNA gene (18S rDNA) and the ITS region, while commonly used chloroplast genes such as rbcL, rbcS, psaA/psbA, etc. Both have their own characteristics: nuclear genes usually evolve slowly and are suitable for higher-order groups; chloroplast genes have moderate relative evolution speed and are commonly inherited by single parents, which is suitable for studying close relationships. In practical applications, people often compare the topological differences between nuclear gene trees and chloroplast gene trees to test system relationships (Fang et al., 2017). In recent years, the application of omics data has become mainstream, such as the entire chloroplast genome contains a large number of genomic loci, which can significantly improve resolution; some studies have pointed out that compared with traditional small number of gene markers, chloroplast genome and nuclear genome data provide more genetic information, especially suitable for the problems of related taxa or classification disorder (Fučíková et al., 2016). For example, joint analysis of the intact chloroplast genome reveals a more stable kinship hint than single gene analysis in certain green algae and cryptoalgae studies. 3.2 Multigenome joint analysis and molecular phylogenetic tree construction Single-gene markers often find it difficult to fully reflect complex evolutionary history, so multigene joint analysis (supermatrix analysis) has become a necessary means. Studies have shown that short-sequence markers such as single rDNA or conservative protein-encoded genes are difficult to meet the needs of algae identification and phylogenetic analysis, and the use of multi-marker combination is an inevitable choice. Usually, researchers will select multiple nuclear genes to be selected with chloroplast genes or intranuclear insertion subregions, etc., to align and jointly build a phylogenetic tree (Lemieux et al., 2015). In the modern era of high-throughput sequencing, we will further use strategies such as chloroplast or mitochondrial genome sequences, multigenome splicing data (genome skimming) and haplotype networks to conduct comprehensive analysis of complex lineages. These multigenome joint analyses can sometimes unravel the problems of instability or insufficient branching support in single gene trees, providing more credible evidence for kinship among algae taxa. For example, by combining Bayesian and maximum likelihood analysis of nuclear and chloroplast genes, a relatively stable phylogenetic tree can be constructed among multiple algae phyla to provide a basis for discussing the origins of different groups. 3.3 Application of genomics and transcriptomics in algae lineage research With the development of omics technology, genome-wide and transcriptome data have played an important role in the study of algae lineage evolution. Large-scale data sets such as chloroplast whole genome, mitochondrial whole genome, nuclear genome, and RNA-Seq transcriptome are used to build high-resolution phylogenetic trees (Figure 1) (Mekvipad and Satjarak, 2019). The chloroplast genome is particularly important, which contains hundreds to thousands of genes, providing more loci for phylogenetic analysis than a single or a few molecular markers. In addition, comparative genomics methods can identify conserved evolutionary genomic features, such
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