海洋生物分类与系统演化实验室知识库

舟形藻目隶属于硅藻门（Bacillariophyta）、硅藻纲（Bacillariophyceae），广泛分布于海水、半咸水、与淡水环境中，是硅藻中物种多样性最丰富的类群，在生态系统中扮演重要角色。舟形藻目（Naviculales）通常可划分为5个亚目、18个科，但目下阶元的系统发育关系仍然不明。其主要原因在于大多数研究仅依赖叶绿体或核糖体的部分基因片段，再加上采样不足和类群覆盖度较低，因此无法反映其确切的系统发育关系，由此阻碍了对舟形藻目演化生物学的研究。另外，舟形藻目是硅藻中出现时间最晚，却是物种多样性最高的类群，其中很可能存在快速辐射过程，但相关驱动因素及其环境适应机制还未见报道。

本研究针对上述问题，开展了舟形藻目物种全面的核基因取样，新获得83个舟形藻目物种的转录组数据，结合已发表数据，共得到100个高质量转录组数据（包括97个舟形藻目物种，3个外群物种），涵盖舟形藻目全部5个亚目及11个主要科级类群。通过对组装和注释后得到的蛋白质序列进行基因家族聚类分析，构建了两个直系同源基因数据集（S50_2525：2525个基因，50%覆盖度；S70_402：402个基因，70%覆盖度）。采用超矩阵串联模型与多物种溯源模型的方法对两个数据集进行系统发育分析和分歧时间估算，结合地质事件以及舟形藻目内部可能存在的全基因组复制事件（WGD），解析了舟形藻目多样化形成过程。此外，采用实验室模拟实验，探究了作为底栖类群代表的舟形藻目对海洋热浪的应答策略及分子机制。

主要研究结果如下：

（1）基于两个数据集构建的舟形藻目系统发育树具有较高的一致性，进一步证实了鞍型藻亚目（Sellaphorineae）的单系性，及其亚目下的鞍型藻科（Sellaphoraceae）与羽纹藻科（Pinnulariaceae）互为姐妹群的系统发育关系。明确了褐指藻科（Phaeodactylaceae）为双肋藻科（Amphipleuraceae）的姐妹群，伯克利藻科（Berkeleyaceae）为等半藻科（Diadesmidaceae）的姐妹群，普氏藻科（Proschkiniaceae）为辐节藻科（Stauroneidaceae）的姐妹群。

（2）分歧时间估算显示，舟形藻目与其姐妹群的分化发生在白垩纪早期（120.3 Ma），舟形藻目出现的时间约为116.6 Ma。在舟形藻目内部，舟形藻亚目（Naviculineae）与其姐妹群在109.1 Ma开始分化；长蓖藻亚目（Neidiineae）与褐指藻亚目（Phaeodactylineae）的共同祖先在100.3 Ma出现分化；双壁藻亚目（Diploneidineae）与舟形藻亚目+长蓖藻亚目+鞍型藻亚目在93.5 Ma开始分化；鞍型藻亚目与舟形藻亚目+长蓖藻亚目在87.5 Ma出现分化。

（3）通过系统发育学手段，确定了舟形藻目内7个未被报道的WGD，且都分布在舟形藻目内多样性较高的类群中，其中斜纹藻科（Pleurosigmataceae）经历2次WGD，舟形藻科（Naviculaceae）经历3次WGD，鞍形藻科经历1次WGD，双壁藻科（Diploneidaceae）经历1次WGD。结合分歧时间分析发现，这些WGD均分布在K-Pg界限、始新世地球温暖期与中中新世气候转变期，表明地质事件很可能是驱动舟形藻目发生WGD的重要因素。比较发生在不同时期的WGD保留基因的功能发现，重要的功能基因通常会在同时期所有的WGD中被保留，发生在K-Pg界限的斜纹藻科2个WGD中有57个共有基因，且包含3个转录因子（SBP、C3HC4、HSF），这些转录因子在斜纹藻物种适应当时低温、低光环境条件时发挥了重要作用；发生在中中新世气候转变期附近的2个WGD中共发现30个共有基因，其中4个（DnaJ、HSF、FAD、FAH）涉及在低温胁迫条件下维持藻细胞光合能力与细胞膜稳定性；发生在始新世温暖期的3个WGD有12个共有基因，其中3个（HSF、Pkinase、CRAL_TRIO）参与了藻细胞热胁迫应答以及抗氧化等相关通路。比较不同科内的4个WGD发现，复制事件后的保留基因在舟形藻目中具有明显的功能偏好性，集中在刺激应答、代谢过程、生物调节等6个主要的生物过程中，且有多个与大分子物质代谢物和定位有关，暗示大分子物质在舟形藻目快速辐射及应对环境变化过程中起着关键作用。

（4）不同生活型硅藻类群对于海洋热浪的响应策略与应答机制存在显著差异。作为底栖硅藻的代表类群，舟形藻目物种Navicula avium选择下调TCA循环和核苷酸合成过程，同时上调脂肪酸和氨基酸合成代谢通路来储存更多的大分子（脂质、蛋白质），这些大分子物质可作为碳和氮的储存库，维持N. avium在高温胁迫条件下基本的代谢活动，并帮助其在环境适宜时快速复苏。这一新发现表明WGD后保留基因是决定舟形藻目应答策略的重要因素。与N. avium相比，浮游硅藻Pseudo-nitzschia multiseries采取了不同的应答策略。它通过上调玉米素与不饱和脂肪酸合成代谢通路，并下调物质与能量代谢（糖酵解、TCA循环、碳与氮的吸收）来快速增加细胞数量，从而增加自身扩散至适宜环境的机会。另一方面，兼性硅藻Paralia guyana展现了更强的生理调节能力来维持自身稳态，包括激活叶黄素循环、合成更多抗氧化物质以及与热应答有关蛋白。

总之，本研究利用转录组数据对舟形藻目进行系统发育分析，理清了高阶元之间的亲缘关系与演化历程，明确了驱动舟形藻目物种快速辐射的重要因素以及面对极端环境时的应答策略与分子机制。这些成果为更深入研究物种起源和多样化过程提供了理论依据，为了解白垩纪以来舟形藻目的快速辐射提供新的线索，为预测全球气候变化背景下硅藻的进化方向提供了重要的参考。

Naviculales belongs to Bacillariophyta, Bacillariophyceae. It is widely distributed in marine, brackish, and freshwater environments, and is the most diverse group of diatoms, playing important roles in ecosystems. Naviculales is usually divided into 5 suborders and 18 families, but the phylogenetic relationships within the order are still unclear. This is mainly due to most studies relying on only partial gene fragments from chloroplasts or ribosomes, combined with limited sampling and low taxon coverage, which cannot reflect the exact phylogenetic relationships and hinders the study of the evolutionary biology of Naviculales. Additionally, Naviculales is the most recently evolved and species-rich group in diatoms, likely undergoing a rapid radiation process. However, the driving factors and mechanisms of environmental adaptation in this group have not been reported.

To address these aforementioned issues, this study conducted comprehensive sampling of nuclear genes in Naviculales. We newly obtained transcriptomic data for 83 species of Naviculales and combined them with published data to obtain a total of 100 high-quality transcriptomes (including 97 species of Naviculales and 3 outgroup species), covering all 5 suborders and 11 major families of Naviculales. We performed gene family clustering analysis on the protein sequences assembled and annotated, and constructed two datasets of orthologous genes (S50_2525: 2525 genes, 50% coverage; S70_402: 402 genes, 70% coverage). Using the supermatrix concatenation model and the multispecies coalescent model, we conducted phylogenetic analysis and divergence time estimation on the two datasets. By integrating geological events and possible whole-genome duplication events (WGD) within Naviculales, we elucidated the process of diversification in Naviculales. Additionally, using laboratory simulation experiments, we investigated the response strategies and molecular mechanisms of Naviculales, as a representative benthic group, to marine heatwaves.

The main results of this study are as follows:

(1) The phylogenetic trees constructed based on the two datasets showed high consistency, further confirming the monophyly of Sellaphorineae and the sister relationship between the Sellaphoraceae and the Pinnulariaceae within Sellaphorineae. It was clarified that the Phaeodactylaceae is the sister group to the Amphipleuraceae, the Berkeleyaceae is the sister group to the Diadesmidaceae, and the Stauroneidaceae is the sister group to the Proschkiniaceae.

(2) Divergence time estimation suggests that the divergence between the Naviculales and its sister group occurred in the early Cretaceous period (120.3 Ma), with the emergence of Naviculales dating to approximately 116.6 Ma. Within Naviculales, the suborder Naviculineae began diverging with its sister group around 109.1 Ma, while the common ancestor of the suborders Neidiineae and Phaeodactylineae diverged around 100.3 Ma. The divergence between Diploneidineae and the clade containing Naviculineae, Neidiineae, and Sellaphorineae occurred around 93.5 Ma. Finally, the divergence between the suborder Sellaphorineae and the clade containing Naviculineae and Neidiineae occurred around 87.5 Ma.

(3) Through phylogenetic analysis, seven previously unreported WGDs were identified within Naviculales, all of which are distributed among lineages with high diversity within the order. Specifically, the Pleurosigmataceae experienced two WGDs, the Naviculaceae experienced three WGDs, the Sellaphoraceae experienced one WGD, and the Diploneidaceae experienced one WGD. Combined with divergence time analysis, these WGDs were found to be distributed around the Cretaceous-Paleogene boundary, the Eocene thermal maximum, and the mid-Miocene climate transition, suggesting that geological events were likely important drivers of WGDs in Naviculales. Comparative studies of gene retention after WGDs at different times revealed that important functional genes are usually retained in all WGDs occurring at the same time period. For example, in the two WGDs occurring around the Cretaceous-Paleogene boundary in the Pleurosigmataceae, 57 common genes were found, including three transcription factors (SBP, C3HC4, HSF), which played important roles in adapting Pleurosigmataceae species to low-temperature and low-light environments at that time. In the two WGDs occurring around the mid-Miocene climate transition, 30 common genes were found, including four (DnaJ, HSF, FAD, FAH) involved in maintaining algal cell photosynthetic capacity and cell membrane stability under low-temperature stress conditions. In the three WGDs occurring during the Eocene thermal maximum, 12 common genes were found, including three (HSF, Pkinase, CRAL_TRIO) involved in algal cell heat stress response and antioxidant-related pathways. Comparing the four WGDs within different families revealed that retained genes after duplication events in Naviculales show a clear functional bias, concentrating in six major biological processes, including response to stimulus, metabolic processes, and biological regulation. Many of these genes are related to macromolecule metabolism and localization, indicating that macromolecules play a key role in the rapid radiation and environmental adaptation processes of Naviculales.

(4) Different growth form diatom taxa exhibit significant differences in their response strategies and mechanisms to marine heatwaves. As the representative group of benthic diatoms, the Naviculales species Navicula avium downregulates the tricarboxylic acid (TCA) cycle and nucleotide synthesis processes while upregulating pathways involved in lipid and amino acid synthesis to store more macromolecules such as lipids and proteins. These synthesized macromolecules serve as carbon and nitrogen reserves, helping the species maintain basic metabolic activities under high-temperature stress conditions and facilitating rapid recovery when environmental conditions become favorable. This result further confirms that the retention of genes related to macromolecule metabolism after WGD plays a crucial role in the survival of Naviculales during extreme environmental changes on Earth. In comparison, the euplanktonic species Pseudo-nitzschia multiseries, when faced with heatwaves, upregulates pathways involved in carotenoid and unsaturated fatty acid synthesis while downregulating pathways related to substance and energy metabolism (glycolysis, TCA cycle, carbon fixation, nitrogen assimilation) to rapidly increase cell numbers, thereby increasing its chances of spreading to suitable environments. The tychoplanktonic species Paralia guyana, on the other hand, demonstrates stronger physiological regulatory abilities to maintain its homeostasis, including activating the xanthophyll cycle and synthesizing more antioxidant substances and heat-resistant proteins.

In summary, this study conducted a phylogenetic analysis of the order Naviculales using transcriptome data, clarifying the relationships and evolutionary history among higher taxonomic ranks. It identified the key factors driving the rapid radiation of Naviculales species and the response strategies and molecular mechanisms in extreme environments. These findings provide a theoretical basis for further research on species origins and diversification processes, offer new clues for understanding the rapid radiation of Naviculales since the Cretaceous, and provide important references for predicting the evolutionary direction of diatoms under the background of global climate change.

第1章 绪论... 1

1.1 硅藻概述... 1

1.1.1 硅藻简介... 1

1.1.2 硅藻的起源... 3

1.1.3 硅藻的分类学研究... 3

1.2 舟形藻目概述... 8

1.2.1 舟形藻目分类学研究... 8

1.2.2 舟形藻目系统发育研究... 11

1.2.3 舟形藻目快速辐射的驱动因素... 14

1.2.4 舟形藻目环境适应性研究... 15

1.3 转录组概述... 16

1.3.1 转录组学及其在藻类研究中应用... 16

1.3.2 系统发育转录组学及其在藻类研究中的应用... 17

1.4 研究目的和意义及科学问题... 18

1.4.1 研究目的... 18

1.4.2 研究意义... 19

1.4.3 拟解决的科学问题... 19

第2章 基于转录组的舟形藻目系统发育重建... 21

2.1 引言... 21

2.2 材料与方法... 22

2.2.1 样品采集与藻株分离... 22

2.2.2 标本制备与形态学鉴定... 23

2.2.3 RNA提取... 24

2.2.4 转录组测序、质控及组装... 25

2.2.5 基因功能注释... 27

2.2.6 直系同源基因筛选... 27

2.2.7 系统发育树的构建与拓扑结构验证... 30

2.2.8 分歧时间估算... 31

2.2.9 多样化速率计算... 32

2.3 研究结果... 32

2.3.1 转录组组装结果... 32

2.3.2 直系同源基因的筛选与数据集构建... 35

2.3.3 舟形藻目系统发育分析... 35

2.3.4 舟形藻目分歧时间分析... 40

2.3.5 舟形藻目多样性速率与地质事件的联系... 43

2.4 讨论... 44

2.4.1 舟形藻目下各亚目间的系统发育关系... 44

2.4.2 舟形藻亚目下各类群系统发育关系... 45

2.4.3 鞍型藻亚目下各类群系统发育关系... 46

2.4.4 褐指藻亚目系统发育关系... 47

2.4.5 舟形藻目起源及目下各类群演化过程... 48

2.5 小结... 49

第3章 舟形藻目全基因组复制事件... 50

3.1 引言... 50

3.1.1 全基因组复制事件研究概述... 50

3.1.2 全基因组复制事件的意义... 51

3.1.3 全基因组事件研究方法... 52

3.2 材料与方法... 53

3.2.1 基于系统发育的WGD推断... 53

3.2.2 WGD基因功能注释与GO富集... 53

3.3 研究结果... 54

3.3.1 舟形藻目内WGD分析... 54

3.3.2 WGD后保留基因GO富集分析... 56

3.3.3 不同类群间WGD后保留基因功能分析... 58

3.3.4 不同时期WGD后保留基因功能分析... 59

3.4 讨论... 63

3.4.1 地质事件是导致舟形藻目发生WGD的重要因素... 63

3.4.2 WGD在舟形藻目环境适应中扮演重要角色... 64

3.4.3 不同时期WGD保留基因拥有不同的功能偏好性... 65

3.5 小结... 67

第4章 基于转录组探究舟形藻对海洋热浪的适应机制... 69

4.1 引言... 69

4.2 材料与方法... 70

4.2.1 藻种选择... 70

4.2.2 海洋热浪模拟实验... 70

4.2.3 藻细胞密度与叶绿素荧光参数测定... 70

4.2.4 RNA提取与纯化... 71

4.2.5 测序、质控与组装... 71

4.2.6 转录组分析... 71

4.3 研究结果... 72

4.3.1 热浪对不同硅藻细胞生长与光合作用的影响... 72

4.3.2 热浪条件下不同硅藻差异基因数量与GO富集分析... 74

4.3.3 热浪对不同硅藻特定代谢通路的影响... 78

4.3.4 热浪条件下不同硅藻抗逆基因的变化... 83

4.4 讨论... 84

4.4.1 不同硅藻对海洋热浪的应对策略与分子机制存在显著差异... 84

4.4.2 WGD后保留基因是决定舟形藻目应答策略的重要因素... 86

4.5 小结... 88

第5章 结论与展望... 90

5.1 结论... 90

5.2 创新点... 91

5.3 展望... 91

参考文献... 93

附录... 109

致谢... 154

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 156