Electronic supplementary information: Independent and adaptive evolution of phenotypic novelties is driven by coral symbiosis in barnacle larvae

CORAL BARNACLE COLLECTIONS Live corals of Leptastrea purpurea and Psammocora profundacella bearing burrowing and adult females of Berndtia barnacles were collected by SCUBA diving on coral reefs in the vicinity of the Northeast (NE) coast of Taiwan. Adult specimens on approximately 3 cm2 host colonies were transferred live to the lab and kept in 1-liter beakers containing aerated, filtered seawater with a 10L:14D cycle under LED lamps. The seawater was changed daily, and the tanks changed regularly. All tanks were checked daily for the release naupliar larvae (which precede the invasive cyprid larva). Once released, the nauplius larvae were concentrated using a pointed light source and then transferred using glass pipettes to sterilized aerated petri dishes. The larval material of the acorn coral barnacle Darwiniella angularis (Thoracica: Pyrgomatidae; inhabiting Cyphastrea chalcidicum) originate from Liu et al. (37). Larval culture and imaging Hatched nauplius larvae of Berndtia from the field-collected females were cultured in autoclaved seawater in petri dishes at ~26°C. As the larvae of Berndtia are lecithotrophic (non-feeding; Video 2) and no food was supplied during the culture. Seawater was changed at daily intervals until the nauplii metamorphosed to the cyprid stage (seen by the emergence of two compound eyes laterally to the nauplius eye; Fig 3A). To assess the larval swimming behaviors and gross structure in culture, both nauplii and cyprids were video recorded and photographed alive in either an Olympus SZX7 stereo microscope (SM) or a Zeiss AX10 light microscope (LM) fitted with differential interference contrast (DIC) optics. All larvae investigated originated from mixed broods of various females. To gain higher morphological resolution of the larval structure, >50 nauplii (all instars) and >100 cyprids were fixed and prepared for LM and scanning electron microscopy (SEM) following the guidelines in (8, 29). Digital editing of images was done using Corel PHOTO-PAINT X8 and the photo plates were assembled in Corel DRAW X8. The videos were edited in ACDSee Photo Studio. Larval experiments To track how the Berndtia cypris larvae explore and invade their hosts experimentally, we released between six and 50 live cyprids from different broods in 10-litre transparent, rectangular aquarium tank containing host colonies. All tanks were aerated by air pumps and sterile glass pipettes. The filtered seawater was changed daily. The cyprids of all three species are non-feeding. The condition of the cyprids were checked every 10-15 minutes during daytime under a stereomicroscope (SM) after exposure to the hosts. The SM was fitted with a digital Lumix G8 camera. Live videos were recorded at 15-minute intervals for 4-30 minutes from about 06:30am-11:00pm but were on several occasions followed for 24 hours. This was repeated for 32 days for all three species. To document larval and juvenile metamorphosis, observations and videos were made every 30 minutes after larval invasion. COLLECETION, REARING AND IMAGING OF OTHER BARNACLE LARVAE We collected ascothoracid (Baccalaureus sp.; Video 1; Fig 6B), burrowing (Acrothoracica; Video 1) and acorn (Balanomorphasp; Video 1) cypris larvae by deploying light traps in Gongguan Harbor on the NE coast of Green Island, Taiwan. The plankton was sorted in Petri dishes under an Olympus SZX7 dissection microscope, and 10 ascothoracid, one burrowing and one acorn live cypris larvae were recorded in an Olympus IX70 light microscope. Five specimens of the ascothoracid cypris larvae were processed for SEM as described above. The material of Trypetesa lampas (Acrothoracica: Trypetesidae; Video 1), Peltogaster paguri (Rhizocephala: Peltogastridae; Video 1; Fig 1B) and Scalpellum scalpellum (Thoracica: Scalpellidae; Video 1; Fig 1C) were collected in the vicinity of the Kristineberg Marine Biological Station in Southern Sweden. Hermit crabs (Pagurus bernhardus) were removed, and their shells cracked open using a hammer. The shell pieces were examined under a dissection microscope for female T. lampas specimens (often identified by exhibiting a pink tint in vivo). The free hermit crabs were screened for infestation with P. paguri. The specimens of S. scalpellum were collected from thecate hydroids in the same area by dredging with an Agassiz-trawl. Adult specimens of the tree species were subsequently placed in small, aerated culture vessels. Upon release, the non-feeding nauplius larvae of several female/hermaphrodite broods of the three species were cultured in separate cultures with their conditions being assessed regularly. The cyprids were upon emergence photographed and live recorded under a LEICA DMRXA microscope fitted with an EVOLUTION© camera. Lastly, we live recorded live cyprids of two additional acorn coral barnacles, Galkinius altiapiculus and Trevathana savignium to put further emphasis on the larval phenotypes of acorn coral barnacles. The former (Video 12) was collected around Taiwan (Suao, Kending, Turtle Island, Green Island and Siao Liou Chiou Island). Small pieces of corals with adult female Galkinia barnacles were collected using a hammer and chisel at 0-20m. The females and their broods were processed as described above for Darwiniella angularis. The cyprids of Trevathana magaretae were reared from females attached to the coral Platygyra lamellina in the Gulf of Aqaba as described for Galkinia. The cyprids were reared as described for Darwiniellaangularis with the exception of using 20-24°C and at 13L:11D light cycle using LED-lamps. The nauplii are non-feeding and were not fed. Cypris larvae were live recorded using a Sony Power HAD and as for the Kristineberg-material. COLLECTION, DNA EXTRACTION AND PCR OF Y-LARVAE The Facetotecta is an enigmatic taxon for which no single-specimen voucher exists. To adequately root our trees outside the focal group of interest it was necessary to expand the marker coverage of the Facetotecta. We deployed plankton nets along the pier of Gong Guan Harbor, Green Island, Taiwan between 15:30-18:30 in September and October. Y-larvae were picked under an Olympus SZX7 stereo microscope with Pasteur pipettes and placed in small Petri dishes. +100 specimens of new, unnamed y-larva species (here referred to as Facetotecta sp. 9 to maintain consistency with the naming by (37)) were photographed alive and fixed in 95% ethanol. Additionally, we collected 163 specimens of Hansenocaris itoi Kolbasov and Høeg 2003 in the waters off the White Sea Marine Biological Station, Russia (66°34’N, 33°08′E). All larvae were captured by vertically dragging 72μm mesh net with a 40 cm mouth opening from 40-0 m. 17 naupliar specimens were fixed alive in 95% ethanol. Eight specimens of Facetotecta sp. 9and 14 of Hansenocaris itoi were transferred individually to sterile Eppendorf tubes in 1.0uL ethanol drops. The DNA was then extracted by adding 40uL AE-Buffer and 4uL Protease K (QIAGEN™) and incubation at 56ºC for 1h and 75ºC for 12min. The exuviae of the specimens left at the tube bottom were subsequently mounted on glass slides in glycerine jelly and photographed in a Zeiss AX10 light microscope fitted with differential interference contrast (DIC) optics. The slides are stored at the Natural History Museum Denmark. We used the polymerase chain reaction (PCR) to amplify partial sequences of 12S ribosomal DNA (rDNA, 400bp), 16S rDNA (330bp), 18S rDNA (1100bp), 28S rDNA (770bp), cytochrome oxidase subunit I (COI, 650bp) and Histone-3 (H3, 330bp). Primers and PCR conditions are listed in Supplementary Table 2. We pipetted 0.4uL of each 10uM primer, 12.2uL ddH2O, 4.0uL Fast-RunTM Taq 5x Master Mix with Dye (Protech Technology Enterprise Co., Ltd., Taiwan) and 3.0uL DNA template into each Eppendorf tube. PCR was conducted in a DNA Engine Thermal Cycler (Bio-Rad, Richmond, California, USA), and the products were visualized by electrophoresis on 1.5% agarose gel in 1 × TAE buffer. DNA purification and Sanger sequencing were performed by Genomics BioSci & Tech Ltd. (New Taipei City, Taiwan). The sequences were assembled and edited in Geneious Prime 2020.1.1. We uploaded curated sequences to NCBI GenBank (Accession: XXXX-YYYY). These sequences represent the first single-specimen-vouchered nucleotide sequences of the Facetotecta.珊瑚藤壶样本采集：采自中国台湾东北（NE）沿海附近珊瑚礁的紫薄孔珊瑚（Leptastrea purpurea）与深生沙珊瑚（Psammocora profundacella）活体样本，其体表带有钻孔型贝氏藤壶（Berndtia barnacle）的雌性成体，采集方式为水肺潜水（SCUBA diving）。将约3 cm²大小、携带成体藤壶的宿主珊瑚活体转移至实验室，置于盛有曝气过滤海水的1 L烧杯中培养，光照周期设置为10小时光照/14小时黑暗，搭配LED灯（LED lamps）照明。每日更换养殖海水，定期维护养殖容器。每日检查所有养殖容器，观察是否有无节幼体（naupliar larvae）释放——无节幼体是入侵型腺介幼体（cyprid larva）的前期发育阶段。幼体释放后，利用聚光光源浓缩无节幼体，再通过玻璃移液管转移至灭菌后的曝气培养皿中。 查尔西达尔文藤壶（Darwiniella angularis，Thoracica: Pyrgomatidae；寄居在查尔西脊孔珊瑚Cyphastrea chalcidicum）的幼体材料引自Liu等（文献37）。 幼体培养与成像：野外采集的贝氏藤壶雌性成体孵化出的无节幼体，置于高压灭菌海水中的培养皿内，于约26℃环境下培养。贝氏藤壶幼体为卵黄营养型（lecithotrophic，非摄食型；视频2），培养期间无需投喂饵料。每日更换海水，直至无节幼体变态为腺介幼体——此时可观察到幼体两侧出现复眼，取代原单眼（图3A）。为评估培养中幼体的游泳行为与整体结构，分别使用搭载微分干涉差（DIC）光学系统的奥林巴斯SZX7体视显微镜（SM）与蔡司AX10光学显微镜（LM），对无节幼体与腺介幼体进行活体录像与拍照。所有观测幼体均来自多只雌性成体的混合幼群。为获得更高分辨率的幼体形态结构，按照文献(8, 29)的方法，固定超过50只各龄期无节幼体与超过100只腺介幼体，用于光学显微镜与扫描电子显微镜（SEM）观测。图像后期编辑使用"Corel PHOTO-PAINT X8"完成，照片拼版通过"Corel DRAW X8"制作，视频编辑使用ACDSee Photo Studio。 幼体行为实验：为实验探究贝氏藤壶腺介幼体的寄主探索与入侵行为，我们将不同幼群的6~50只活体腺介幼体，投放至盛有寄主珊瑚的10 L透明方形水族箱中，所有水族箱均通过气泵与无菌玻璃移液管曝气。过滤海水每日更换。三种藤壶的腺介幼体均为非摄食型。实验期间，每日白天每隔10~15分钟，使用体视显微镜（SM）检查腺介幼体的存活状态，该显微镜搭载松下Lumix G8数码相机。活体录像的拍摄间隔为15分钟，单次拍摄时长4~30分钟，拍摄时段为每日06:30~23:00，部分实验连续观测24小时。该实验针对三种藤壶重复开展，共计32天。为记录幼体与稚体的变态过程，幼体入侵后每隔30分钟进行观测与录像。 其他藤壶幼体的采集、饲养与成像：我们在中国台湾绿岛东北海岸的公馆港（Gongguan Harbor）布设光陷阱，采集到了阿斯克托藤壶（ascothoracid，Baccalaureus sp.；视频1；图6B）、钻孔型藤壶（Acrothoracica；视频1）与藤壶型藤壶（acorn barnacle，Balanomorpha sp.；视频1）的腺介幼体。在奥林巴斯SZX7体视显微镜下，于培养皿中分拣浮游生物，选取10只阿斯克托藤壶、1只钻孔型藤壶与1只藤壶型藤壶的活体腺介幼体，使用奥林巴斯IX70光学显微镜进行录像记录。按照前文所述方法，选取5只阿斯克托藤壶腺介幼体制备扫描电子显微镜样本。 钻穴藤壶Trypetesa lampas（Thoracica: Trypetesidae；视频1）、寄居蟹根头藤壶Peltogaster paguri（Rhizocephala: Peltogastridae；视频1；图1B）与铠茗荷Scalpellum scalpellum（Thoracica: Scalpellidae；视频1；图1C）的样本采集自瑞典南部克里斯汀堡海洋生物站（Kristineberg Marine Biological Station）附近海域。将寄居蟹（Pagurus bernhardus）取下后，用锤子敲开其螺壳，在体视显微镜下检视雌性T. lampas成体（活体状态下通常呈粉色，可作为识别特征）；对自由生活的寄居蟹进行筛查，检测是否被P. paguri寄生。通过阿加西拖网（Agassiz-trawl）在同一区域的鞘状水螅体上采集S. scalpellum成体样本。随后将成体样本置于小型曝气培养容器中。幼体释放后，三种藤壶的多只雌性/雌雄同体成体的非摄食型无节幼体，分别置于单独的培养体系中培养，并定期评估其生长状态。腺介幼体孵化后，使用搭载"EVOLUTION©"相机的"LEICA DMRXA"显微镜进行拍照与活体录像。 此外，我们还对另外两种珊瑚藤壶：高顶加勒廷藤壶Galkinius altiapiculus（视频12）与萨维尼特雷瓦撒藤壶Trevathana savignium的腺介幼体进行活体录像，以进一步凸显珊瑚藤壶的幼体表型。高顶加勒廷藤壶的采集地涵盖中国台湾地区的苏澳、垦丁、龟山岛、绿岛与小琉球岛（Siao Liou Chiou Island）。使用锤子与凿子在0~20 m水深采集带有雌性加勒廷藤壶成体的珊瑚小块组织，雌性成体及其幼群的处理方法与前文所述的查尔西达尔文藤壶一致。特雷瓦撒藤壶Trevathana magaretae的腺介幼体，采自亚喀巴湾附着在扁脑珊瑚Platygyra lamellina上的雌性成体，其饲养方法与加勒廷藤壶一致，仅将培养温度调整为20~24℃，光照周期设置为13小时光照/11小时黑暗，搭配LED灯照明。其无节幼体为非摄食型，无需投喂饵料。腺介幼体的活体录像使用Sony Power HAD相机完成，拍摄方法与克里斯汀堡采集的样本一致。 Y形幼体的采集、DNA提取与PCR扩增：Facetotecta是一类神秘的甲壳动物类群，目前尚无单一标本凭证。为确保系统发育树的外类群定根合理，我们需要扩大Facetotecta类群的标记覆盖范围。于9月与10月的15:30~18:30期间，在中国台湾绿岛公馆港码头沿线布设浮游生物网采集样本。使用巴斯德移液管在奥林巴斯SZX7体视显微镜下挑取Y形幼体，置于小型培养皿中。超过100只新发现的未命名Y形幼体物种（本文参照文献(37)的命名方式，暂称为Facetotecta sp. 9）经活体拍照后，固定于95%乙醇中。此外，我们在俄罗斯白海海洋生物站（66°34′N，33°08′E）附近海域采集到163只Hansenocaris itoi Kolbasov & Høeg, 2003样本。所有幼体均通过垂直拖网采集：使用网口直径40 cm、网孔72 μm的浮游生物网，从40 m水深拖至水面。其中17只无节幼体样本经活体固定后置于95%乙醇中。选取8只Facetotecta sp.9样本与14只Hansenocaris itoi样本，分别转移至添加1.0 μL乙醇的无菌Eppendorf管中。随后加入40 μL AE缓冲液与4 μL蛋白酶K（"QIAGEN™"），于56℃孵育1小时，再于75℃孵育12分钟完成DNA提取。留在管底的标本蜕壳随后用甘油明胶封片于载玻片上，使用搭载微分干涉差（DIC）光学系统的蔡司AX10光学显微镜进行拍照。封片标本保存于丹麦自然历史博物馆。 我们使用聚合酶链式反应（PCR）扩增12S核糖体DNA（rDNA，400 bp）、16S rDNA（330 bp）、18S rDNA（1100 bp）、28S rDNA（770 bp）、细胞色素氧化酶亚基I（COI，650 bp）与组蛋白3（H3，330 bp）的部分序列。引物序列与PCR反应条件详见补充表2。向每个Eppendorf管中加入0.4 μL每条10 μM的引物、12.2 μL双蒸水、4.0 μL "Fast-Run™ Taq 5x"预混染料缓冲液（Protech科技企业股份有限公司，中国台湾）与3.0 μL DNA模板。PCR反应在DNA Engine热循环仪（"Bio-Rad"，美国加利福尼亚州里士满）中完成，扩增产物通过1.5%琼脂糖凝胶电泳、1×TAE缓冲液染色后进行可视化检测。DNA纯化与桑格测序由Genomics BioSci & Tech Ltd.（中国台湾新北市）完成。序列拼接与编辑使用"Geneious Prime 2020.1.1"软件完成。我们将整理后的序列上传至"NCBI GenBank"（登录号：XXXX-YYYY）。这些序列是首个带有单一标本凭证的Facetotecta类群核苷酸序列。