DOI: 10.5061/dryad.3r2280gn1
title: A new molecular phylogeny of salps (Tunicata: Thalicea: Salpida) and the evolutionary history of their colonial architecture

description: Salps are marine pelagic urochordates with a complex life cycle including
 a solitary and colonial stage composed of asexually-budded individuals.
 These colonies develop into species-specific architectures with distinct
 zooid orientations, including transversal, oblique, linear, helical, and
 bipinnate chains; as well as whorls, and clusters. The evolutionary
 history of salp colony architecture has remained obscured due to the lack
 of a homology-based ontology to characterize architectures, as well as a
 lack of phylogenetic taxon sampling and resolution of critical nodes. We
 (1) collected and first-time sequenced eight species of salps, (2)
 inferred the phylogenetic relationships among salps, and (3) reconstructed
 the evolutionary history of salp colony architecture. We collected salp
 specimens via offshore SCUBA diving, dissected tissue samples, extracted
 their DNA, amplified their 18S gene, and sequenced them using Sanger
 technology. We inferred a new molecular phylogeny using both Maximum
 Likelihood and Bayesian approaches. Using this phylogeny, we reconstructed
 the ancestral states of colony architecture using a Bayesian ordered
 Markov model informed by the presence and absence of specific
 developmental mechanisms that lead to each architecture. We find that the
 ancestral salp architecture is either oblique or linear, with every other
 state being derived. Moreover, linear chains have evolved independently at
 least three times. While transversal chains are developmentally basal and
 hypothesized to be ancestral, our phylogenetic topology and
 reconstructions strongly indicate that they are evolutionarily derived
 through the loss of zooid torsion. These traits are likely critical to
 multijet locomotory performance and evolving under natural selection. Our
 work showcases the need to study the broader diversity of salp species in
 order to gain a comprehensive understanding of their organismal biology,
 evolutionary history, and ecological roles in pelagic ecosystems.
description: To build a well-resolved molecular phylogeny, we primarily used
 the 18S gene accession list for salps and outgroups from Govindarajan et
 al. 2011 with a few modifications. We expanded taxon sampling by
 collecting tissue samples from understudied salp species
 (<em>Metcalfina hexagona, Ihlea punctata, Helicosalpa virgula,
 Helicosalpa younti, Cyclosalpa bakeri, Cyclosalpa pinnata,
 </em>and<em> Ritteriella amboinensis</em>) using tissue
 samples from specimens we collected while bluewater SCUBA diving (Haddock
 & Heine, 2005) from a small vessel off the coast of Kailua-Kona
 (Hawai’i Big Island, 19°42'38.7" N 156°06'15.8" W),
 over 2000m of offshore water. When possible, we sampled a variety of
 tissues from the zooid excluding the gut to avoid contamination from food
 particles, as well as the tunic to avoid clogging the DNA extraction
 columns. These samples were preserved in ethanol at room temperature until
 the point of DNA extraction in the lab. We aligned these sequences using
 MUSCLE 5.1 (Edgar 2004) with default settings. As a sensitivity analysis,
 we alternatively aligned them with MAFFT 7.419 (Katoh et al. 2009) with
 default settings. In addition, we experimented with post-processing these
 alignments with GBLOCKS 0.91b (Castresana 2000) with default settings
 except for allowing half-gap positions (as used in Govindarajan et al.
 2011). The alignment contained every sequence except for
 <em>Cyclosalpa</em> <em>bakeri</em> specimen
 D27-Cbak-B-1, which appeared truncated and non-comparable after
 post-processing. GBLOCKS retained 43% of sites when aligning with MUSCLE
 and 45% of sites when aligning with MAFFT. To make an ML inference from
 these alignments, we used IQTree 1.6.12 (Nguyen et al. 2015) with 1000
 bootstrap replicates (SM Fig. 1). Node support was reported using
 bootstrap support (BS). The consensus trees obtained using the model
 selected by the best Bayesian Information Criterion (TIM3e+R5: transition
 model with equal base frequency, with 5 rate categories) and using
 GTR+I+Gamma are congruent, regardless of whether MAFFT or MUSCLE was used
 for alignment. However, the consensus trees with GBLOCKS were not
 congruent with these trees by several nodes which had low support, due to
 many trimmed sequences appearing identical. We suspect that GBLOCKS is
 removing critical phylogenetic signal from the data, and therefore decided
 not to use it for downstream analyses. We collected between one and five
 specimens of adult blastozooid colonies from each target species via
 bluewater SCUBA diving. We took photographs of these colonies using a
 Nikon DSLR camera with a 75mm lens facing downwards on a tripod with the
 colonies fully submerged in glass dishes and a ruler for scale. We
 anesthetized the salp specimens using 0.2% MS-222 prior to photographing
 them in order to avoid swimming motion in the dishes. We coded the colony
 architecture for each species we encountered in the field based on our
 photographs and observations. In addition, we complemented these
 observations with published records such as Madin (1990) for species we
 did not encounter, such as <em>Pegea confoederata, Pegea bicaudata,
 Thalia democratica, Thalia orientalis, Thetys vagina, Cyclosalpa
 floridiana, Salpa younti, </em>and<em> Salpa
 </em>thompsoni. In addition to categorizing each architecture as
 transversal, helical, whorl, cluster, oblique, linear, or bipinnate; we
 also measured the dorsoventral zooid-stolon (zooid oral-aboral axis to
 stolon axis) angle. To do so, we photographed salp colonies from three
 homologous orthogonal planes of observation defined in Damian-Serrano
 & Sutherland (2023) as the oral-aboral-normal (normal sensu
 perpendicular) plane, the dorsoventral-normal plane, and the stolon-normal
 plane. Using the photographs taken from the dorsoventral-normal plane, we
 measured the zooid-stolon angle in ImageJ using the aligned endostyle and
 gill bar as a proxy for the zooid oral-aboral angle, and the line
 connecting the opaque guts of serially neighboring zooids as a proxy for
 the stolon angle. We measured at least three zooids per colony and between
 one and three individual colonies per species.  We used the Bayesian time tree to reconstruct the
 ancestral states of colonial architectures as a categorical character. We
 ran the ancestral reconstruction using a Bayesian ordered Markov model
 (OMM) that constrains the rate matrix to allow only transitions between
 states that are adjacent in the developmental ontology. To do this, we
 hard-coded the transition rates between non-adjacent states (e.g. between
 helical and linear architectures) to be zero, thus requiring states
 changes across developmental pathways to transition back to a transversal
 architecture (representing the loss of specific developmental mechanisms)
 and then shift towards a different pathway following the required order of
 underlying mechanism gains and losses. This model estimated twelve rate
 parameters allowing for asymmetrical rates of gain and loss for each
 transition between architecture states. Alternatively, we repeated this
 analysis estimating a single rate for all transitions, while still
 constraining non-adjacent transitions (SM Figure 4). We used RevBayes for
 this analysis, adapting the categorical Markov model ancestral state
 reconstruction protocol described in the “morph_ase” tutorial on the
 RevBayes website (scripts available in the Dryad repository).
 Alternatively, we reconstructed the ancestral states using stochastic
 mapping with simpler “equal rates” (single rate parameter for all state
 transitions with 100 simulations, SM Figure 5) and “all rates different”
 (42 independent rate parameters with 25 simulations, one for each rate
 transition in each direction, SM Figure 6) models in the R package
 phytools (Revell 2012). While a myriad of alternative models could be
 estimated and compared, this is beyond the scope of this study.
 While colonial architecture as a whole can be
 conceptualized as a categorical trait, some architectures differ from each
 other across a gradient in a continuous trait that drives their structural
 differences. The dorsoventral zooid-stolon angle is one such trait, which
 drives the streamlining of salp chains. This continuous trait ranges from
 one extreme with zooids arranged perpendicularly (90°) to the stem of the
 colony (transversal, helical, whorl, and cluster architectures), all the
 way to linear chains with zooids arranged in parallel to the stem (as in
 the linear chains of <em>Soestia</em>
 <em>zonaria</em>), with a gradation of more or less oblique
 intermediate forms. We reconstructed the evolutionary history of the
 dorsoventral zooid-stolon angle of salp colonies on the Bayesian time tree
 using tip values measured from our photographs, and a Brownian Motion
 model in the R package phytools (Revell 2012). We used an ML ancestral
 state reconstruction with 95% confidence intervals in the R package ape
 5.6 (Paradis & Schliep 2019). We matched the
 sequences that form the tips of the molecular phylogeny and the species of
 the specimens from which we took the morphological and kinematic data. In
 the case of <em>Pegea</em>, the species we observe off Hawaii
 has intermediate traits between <em>P. confoederata</em>
 (blastozooid morphology) and <em>P. socia</em> (oozoid
 morphology), possibly representing either a new species, a hybrid, or
 phenotypic variation within either species. Since we are confident that
 this is a <em>Pegea</em> species and that the genus
 <em>Pegea</em> is likely monophyletic, the branch length for
 this specimen should be congruent to that of any other
 <em>Pegea</em> species as long as it is the only
 <em>Pegea</em> species in the tree. Thus, we mapped the
 morphological and speed data to the species tip of
 <em>Pegea</em> <em>confoederata</em>.
description: R, RStudio, RevBayes, IQTree, GBLOCKS, FigTree