Abstract：
Coral reef ecosystems are the most productive and biodiverse marine ecosystems, with their productivity levels highly dependent on the symbiotic dinoflagellates belonging to the family Symbiodiniaceae. As a unique life history strategy, resting cyst production is of great significance in the ecology of many dinoflagellate species, those HABs-causing species in particular, however, there has been no confirmative evidence for the resting cyst production in any species of the family Symbiodiniaceae. Based on morphological and life history observations of cultures in the laboratory and morpho-molecular detections of cysts from the marine sediments via fluorescence in situ hybridization (FISH), cyst photography, and subsequent singe-cyst PCR sequencing, here we provide evidences for the asexual production of resting cysts by Effrenium voratum, the free-living, red tide-forming, and the type species of the genus Effrenium in Symbiodiniaceae. The evidences from the marine sediments were obtained through a sequential detections: Firstly, E. voratum amplicon sequence variants (ASVs) were detected in the cyst assemblages that were concentrated with the sodium polytungstate (SPT) method from the sediments collected from different regions of China Seas by high-throughput next generation sequencing (NGS); Secondly, the presence of E. voratum in the sediments was detected by PCR using the species-specific primers for the DNA directly extracted from sediment; Thirdly, E. voratum cysts were confirmed by a combined approach of FISH using the species-specific probes, light microscopic (LM) photography of the FISH-positive cysts, and a subsequent single-cyst PCR sequencing for the FISH-positive and photographed cysts. The evidences from the laboratory-reared clonal cultures of E. voratum include that: 1) numerous cysts formed in the two clonal cultures and exhibited a spherical shape, a smooth surface, absence of ornaments, and a large red accumulation body; 2) cysts could maintain morphologically intact for a storage of two weeks to six months at 4 °C in darkness and of which 76-92 % successfully germinated through an internal development processes within a time period of 3-21 days after being transferred back to the normal culturing conditions; 3) two or four germlings were released from each cyst through the cryptopylic archeopyle in all cysts with continuous observations of germination processes; and 4) while neither sexual mating of gametes nor planozygote (cells with two longitudinal flagella) were observed, the haploidy of cysts was proven with flow cytometric measurements and direct LM measurements of fluorescence from cells stained with either propidium iodide (PI) or DAPI, which together suggest that the cysts were formed asexually. All evidences led to a conclusion that E. voratum is capable of producing asexual resting cysts, although its sexuality cannot be completely excluded, which guarantees a more intensive investigation. This work fills a gap in the knowledge about the life cycle, particularly the potential of resting cyst formation, of the species in Symbiodiniaceae, a group of dinoflagellates having unique life forms and vital significance in the ecology of coral reefs, and may provide novel insights into understanding the recovery mechanisms of coral reefs destructed by the global climate change and suggest various forms of resting cysts in the cyst assemblages of dinoflagellates observed in the field sediments, including HABs-causing species.
摘要：
珊瑚礁生态系统是最具生产力和生物多样性的海洋生态系统，它们的生产力水平高度依赖于共生菌科的共生鞭毛藻。作为一种独特的生活史策略，静息囊肿的产生在许多鞭毛藻物种的生态学中具有重要意义，特别是那些引起HAB的物种，然而,没有确凿的证据表明共生菌科的任何物种都会产生静止的囊肿。根据实验室培养物的形态和生活史观察以及通过荧光原位杂交（FISH）对海洋沉积物中囊肿的形态分子检测，囊肿摄影,以及随后的单囊PCR测序，在这里，我们提供了Effreniumvoratum无性产生静息囊肿的证据，自由生活，赤潮形成,以及共生菌科中Effrenium属的类型种。来自海洋沉积物的证据是通过顺序检测获得的：首先，通过高通量下一代测序（NGS），从从中国海洋不同地区收集的沉积物中，用聚钨酸钠（SPT）方法浓缩的囊肿组合中检测到E.voratum扩增子序列变异体（ASV）；其次，使用直接从沉积物中提取的DNA的物种特异性引物，通过PCR检测沉积物中E.voratum的存在；第三，通过使用物种特异性探针的FISH组合方法确认了E.voratum囊肿，FISH阳性囊肿的光学显微镜（LM）摄影，以及随后对FISH阳性囊肿和拍照囊肿进行单囊肿PCR测序。来自实验室培养的E.voratum克隆培养物的证据包括：1）在两种克隆培养物中形成了许多囊肿，并呈球形，光滑的表面，没有装饰品，和大的红色积聚体；2）囊肿在黑暗中在4°C下可以保持形态完整，保存两周至六个月，其中76-92％的囊肿在转移回正常培养条件后的3-21天内通过内部发育过程成功发芽；3）在所有囊肿中，有两个或四个胚芽通过隐藻从每个囊肿中释放，同时没有连续观察到长子的发芽过程（也没有观察到有两个性子）通过流式细胞术测量和直接LM测量用碘化丙啶（PI）或DAPI染色的细胞的荧光，证明了囊肿的单倍体。这表明囊肿是无性系形成的。所有证据都得出了这样的结论，即沃鲁坦能够产生无性静息囊肿，虽然它的性不能完全排除，这保证了更深入的调查。这项工作填补了有关生命周期知识的空白，特别是静息囊肿形成的可能性，共生虫科的物种，一群在珊瑚礁生态中具有独特生命形式和重要意义的鞭毛藻，并可能提供新的见解，以了解全球气候变化破坏的珊瑚礁的恢复机制，并建议在野外沉积物中观察到的鞭毛藻囊肿组合中的各种形式的静息囊肿，包括引起HAB的物种。
