<p>18S sequence-structure alignment (subset).</p>

The phylogeny of haptophytes, a diverse and ecologically significant group of microalgae, remains incompletely resolved despite extensive molecular studies. In this study, we apply a sequence-structure phylogenetic framework to the Haptophyta, utilizing ribosomal RNA (rRNA) small subunit (SSU) gene (18S; nearly complete sequences) and internal transcribed spacer 2 (ITS2) datasets. By integrating secondary structure information during sequence alignment and tree inference, we aim to enhance phylogenetic resolution and clarify evolutionary relationships within this lineage. Our taxon sampling reduced over 40,000 available 18S sequences to 396 representatives, alongside a compilation of 224 ITS2 sequences. Comparative modeling and homology-based structure prediction revealed both conserved and variable features in 18S and ITS2 secondary structures, with notable deviations in certain taxa. Maximum likelihood (ML) subset phylogenies based on 18S sequence-structure data showed the greatest congruence with established taxonomy, such as the division between calcifying and non-calcifying lineages. In contrast, ITS2 data presented alignment challenges due to high sequence variability, length differences, and limited taxon representation. Incorporating secondary structure information improved alignment quality and reduced phylogenetic artifacts, though ITS2 remained unsuitable for resolving deep relationships among haptophytes. Instead, ITS2 proved more valuable for distinguishing closely related species. While bootstrap support values were similar between sequence-only and sequence-structure approaches, the latter suggested alternative phylogenetic placements that better aligned with previous studies (using multiple markers or also some partial 18S sequences); for Hayaster perplexus in particular, these placements also better matched morphological data. Our results underscore the critical impact of taxon sampling and methodological choices on phylogenetic outcomes. Despite these challenges, the 18S sequence-structure ML tree offers a reliable depiction of haptophyte phylogeny, even though some backbone relationships remain weakly supported. Overall, this study highlights both the benefits and limitations of integrating RNA secondary structure into molecular phylogenetics and advances our understanding of haptophyte evolution.定鞭藻门（Haptophyta）是一类多样且具有重要生态意义的微藻类群，尽管已有大量分子生物学研究，但其系统发育关系仍未完全厘清。本研究针对定鞭藻门构建了序列-结构系统发育分析框架，采用核糖体RNA（ribosomal RNA, rRNA）小亚基（small subunit, SSU）基因（18S；近乎完整序列）与内部转录间隔区2（internal transcribed spacer 2, ITS2）数据集。本研究在序列比对与系统发育树推断过程中整合二级结构信息，旨在提升该类群的系统发育分辨率，厘清其内部演化关系。本研究的类群采样将4万余条可用的18S序列筛选精简至396条代表序列，并整合了224条ITS2序列。比较建模与基于同源性的结构预测结果显示，18S与ITS2的二级结构兼具保守与可变特征，部分类群存在显著的结构偏差。基于18S序列-结构数据的最大似然法（Maximum likelihood, ML）子集系统发育树，与已确立的分类学体系一致性最高，例如钙化与非钙化谱系的划分。与之相对，ITS2数据因序列变异性高、长度存在差异且类群代表性有限，面临较多序列比对难题。尽管整合二级结构信息可提升比对质量并减少系统发育推断偏差，但ITS2仍不适用于厘清定鞭藻门内的深层演化关系。相较而言，ITS2在区分近缘物种方面更具应用价值。尽管仅序列分析与序列-结构分析方法的自展支持率相近，但后者提出的替代系统发育定位结果，与此前采用多分子标记或部分18S序列的研究更为契合；尤其对于困惑海盘藻（Hayaster perplexus）而言，该定位结果也更匹配其形态学数据。本研究结果凸显了类群采样与方法选择对系统发育分析结果的关键影响。尽管存在上述挑战，基于18S序列-结构数据的ML系统发育树仍可可靠呈现定鞭藻门的系统发育关系，尽管部分主干演化关系的支持度仍较弱。总体而言，本研究既展现了将RNA二级结构整合入分子系统发育学的优势与局限，也推进了我们对定鞭藻门演化的认知。