DOI: 10.5061/dryad.2jm63xsn6
title: Electronic supplementary information: Independent and adaptive evolution of phenotypic novelties is driven by coral symbiosis in barnacle larvae

description: The invasion of novel habitats is recognized as a major promotor of
 adaptive trait evolution in animals. We tested whether similar ecological
 niches entail independent and adaptive evolution of key phenotypic
 structures related to larval host invasion in distantly related taxa. We
 use disparately related clades of coral barnacles as our model system
 (Acrothoracica: Berndtia and Thoracica: Pyrgomatidae). We analyze the
 larval antennular phenotypes and functional morphologies facilitating host
 invasion. Extensive video recordings show that coral host invasion is
 carried out exclusively by cypris larvae with spear-shaped antennules.
 These first exercise a series of complex probing behaviors followed by
 repeated antennular penetration of the soft host tissues, which
 subsequently facilitates permanent invasion. Phylogenetic mapping of
 larval form and function related to niche invasion in 99 species of
 barnacles (Thecostraca) compellingly shows that the spear-phenotype is
 uniquely associated with corals and penetrative behaviors. These features
 evolved independently in the two coral barnacle clades and from ancestors
 with fundamentally different antennular phenotypes. The larval host
 invasion system in coral barnacles likely evolved adaptively across
 millions of years for overcoming challenges associated with invading and
 entering demanding coral hosts.   Key words: adaptive host
 invasion, larval phenotypes, coral barnacle, barnacle phylogeny
description: CORAL BARNACLE COLLECTIONS Live corals of
 <i>Leptastrea purpurea </i>and <i>Psammocora
 profundacella </i>bearing burrowing and adult females of
 <i>Berndtia</i> barnacles were collected by SCUBA diving on
 coral reefs in the vicinity of the Northeast (NE) coast of Taiwan. Adult
 specimens on approximately 3 cm<sup>2</sup> 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 <i>Darwiniella angularis</i> (Thoracica:
 Pyrgomatidae; inhabiting <i>Cyphastrea chalcidicum</i>)
 originate from Liu et al. (37). <i>Larval culture
 and imaging</i> Hatched nauplius larvae of
 <i>Berndtia </i>from the field-collected females were cultured
 in autoclaved seawater in petri dishes at ~26°C. As the larvae of
 <i>Berndtia</i> 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, &gt;50
 nauplii (all instars) and &gt;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.<br> <br> <i>Larval
 experiments</i> To track how
 the <i>Berndtia </i>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
 (<i>Baccalaureus</i> 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
 <i>Trypetesa lampas</i> (Acrothoracica: Trypetesidae; Video
 1), <i>Peltogaster paguri </i>(Rhizocephala: Peltogastridae;
 Video 1; Fig 1B) and <i>Scalpellum scalpellum </i>(Thoracica:
 Scalpellidae; Video 1; Fig 1C) were collected in the vicinity of the
 Kristineberg Marine Biological Station in Southern Sweden. Hermit crabs
 (<i>Pagurus bernhardus</i>) were removed, and their shells
 cracked open using a hammer. The shell pieces were examined under a
 dissection microscope for female <i>T. lampas</i> specimens
 (often identified by exhibiting a pink tint <i>in vivo</i>).
 The free hermit crabs were screened for infestation with <i>P.
 paguri. </i>The specimens of <i>S. scalpellum</i> 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,
 <i>Galkinius altiapiculus</i> and <i>Trevathana
 savignium</i> 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 <i>Galkinia</i>
 barnacles were collected using a hammer and chisel at 0-20m. The females
 and their broods were processed as described above for
 <i>Darwiniella angularis.</i> The cyprids of
 <i>Trevathana magaretae </i>were reared from females attached
 to the coral <i>Platygyra lamellina</i> in the Gulf of Aqaba
 as described for <i>Galkinia.</i> The cyprids were reared as
 described for <i>Darwiniellaangularis</i> 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 <i>sp. </i>9 to maintain
 consistency with the naming by (37)) were photographed alive and fixed in
 95% ethanol. Additionally, we collected 163 specimens of
 <i>Hansenocaris itoi</i> 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 <i>sp. </i>9and 14 of
 <i>Hansenocaris itoi</i> 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 ddH<sub>2</sub>O,
 4.0uL Fast-Run<sup>TM</sup> 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.