Molecular phylogeny, systematics and morphological evolution of the acorn barnacles (Thoracica: Sessilia: Balanomorpha)

Molecular Phylogenetics and Evolution 81 (2014) 147–158

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Molecular phylogeny, systematics and morphological evolution of the acorn barnacles (Thoracica: Sessilia: Balanomorpha) Marcos Pérez-Losada a,b,c,⇑, Jens T. Høeg d, Noa Simon-Blecher e, Yair Achituv e, Diana Jones f, Keith A. Crandall b,c a

CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Rua Padre Armando Quintas 7, Universidade do Porto, Campus Agrário de Vairão, Vairão 4485-661, Portugal Computational Biology Institute, George Washington University, Ashburn, VA 20147, USA Department of Invertebrate Zoology, US National Museum of Natural History, Smithsonian Institution, Washington, DC 20013, USA d Marine Biology Section, Department of Biology, University of Copenhagen, Universitetsparken 4, DK-2100 Copenhagen, Denmark e The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, 52900 Ramat Gan, Israel f Perth Museums & Collections Western Australian Museum, 49 Kew Street, Welshpool, Western Australia 6106, Australia b c

a r t i c l e

i n f o

Article history: Received 10 March 2014 Revised 5 August 2014 Accepted 12 September 2014 Available online 27 September 2014 Keywords: Balanomorpha Barnacle DNA Morphology Phylogeny Systematics

a b s t r a c t The Balanomorpha are the largest group of barnacles and rank among the most diverse, commonly encountered and ecologically important marine crustaceans in the world. Paradoxically, despite their relevance and extensive study for over 150 years, their evolutionary relationships are still unresolved. Classical morphological systematics was often based on non-cladistic approaches, while modern phylogenetic studies suffer from severe undersampling of taxa and characters (both molecular and morphological). Here we present a phylogenetic analysis of the familial relationships within the Balanomorpha. We estimate divergence times and examine morphological diversity based on five genes, 156 specimens, 10 fossil calibrations, and six key morphological characters. Two balanomorphan superfamilies, eight families and twelve genera were identified as polyphyletic. Chthamaloids, chionelasmatoid and pachylasmatoids split first from the pedunculated ancestors followed by a clade of tetraclitoids and coronuloids, and most of the balanoids. The Balanomorpha split from the Verrucidae (outgroup) in the Lower Cretaceous (139.6 Mya) with all the main lineages, except Pachylasmatoidea, having emerged by the Paleocene (60.9 Mya). Various degrees of convergence were observed in all the assessed morphological characters except the maxillipeds, which suggests that classical interpretations of balanomorphan morphological evolution need to be revised and reinterpreted. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Within cirripedes the suborder Balanomorpha, or acorn barnacles, is by far the most species rich and important group in many marine communities. They get their vernacular name from lacking the fleshy stalk of their pedunculated relatives and from being encased in a more or less cone shaped receptacle of shell plates with its base firmly cemented to the substratum (Anderson, 1994). They are best known from their dominating presence in rocky intertidal habitats, although some species also inhabit the deep sea, while others (epibionts) attach to a variety of plants (tropical mangroves), animals and artificial structures. Epibiont barnacles are found on vertebrates (e.g., sea turtles, sea snakes

⇑ Corresponding author at: Computational Biology Institute, George Washington University, Ashburn, VA 20147, USA. E-mail address: [email protected] (M. Pérez-Losada). http://dx.doi.org/10.1016/j.ympev.2014.09.013 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

and whales) and invertebrates (e.g., mollusks, gorgonians, crustaceans, sponges and corals). None of these specialized barnacles are true parasites, but it is suspected that they have various deleterious effects on their host, including the survival and growth of threatened mangrove habitats (Satumanatpan and Keough, 1999). Balanomorphans are also known as primary foulers of man-made structures in the sea with enormous economic repercussions to human society, particularly to shipping and cooling systems of power and desalination plants (Schultz et al., 2011). Acorn barnacles are very diverse in their morphology (Anderson, 1994; Newman, 1987; Newman and Ross, 1976). Structurally, most species are volcano shaped, but the number of wall plates encircling the body can vary (8, 6, 4 plates), and in extreme cases the wall is concrescent with no evidence of separation into individual plates. There is also extensive variation in other hard characters associated with the wall plates, like the basis of the shell, which can be calcareous or membranous, or the interlocking

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between the wall plates and their connection to the opercular plates. Soft structures like the feeding appendages are also diverse in their morphology. Only their reproductive biology, which varies extensively in pedunculated cirripedes, seems rather conservative in the Balanomorpha inasmuch as the overwhelming majority of the species are hermaphrodites (Høeg and Møller, 2006). The large biological and structural diversity exhibited by the acorn barnacles makes them ideal models in studies of ecology and evolution. For example, the zonation of different acorn barnacle species and the recruitment of attached juveniles from larvae in the plankton are standard research areas in marine ecology (Høeg and Møller, 2006). Their specializations in adult structures, growth and feeding biology, on the other hand, have been the focus of intense research on the evolution of adaptation, including the seminal studies of Charles Darwin (Anderson, 1994; Crisp, 1983; Darwin, 1854, 1855; Newman, 1987; Schram and Høeg, 1995). Tremendous effort and scientific literature have also been devoted to the prevention of barnacle fouling. For example, the cement used in barnacle attachment has been intensively studied both for trying to emulate it technologically (underwater superglue) and for finding a means of preventing fouling (Kamino, 2013). Furthermore, the importance of the balanomorphans in all these areas has made the cyprid (the last larval stage before adulthood) the preferred model in studying settlement factors in marine larvae (Aldred and Clare, 2008; Chen et al., 2011; Kamino, 2013). Considering the biological and economic importance of acorn barnacles, a robust phylogenetic framework to study barnacle ecology and assess antifouling models is crucial. Similarly, a comprehensive phylogeny of the Balanomorpha would aid our understanding of how and when acorn barnacles evolved to exhibit their current diversity and would guide the development of a robust taxonomy and classification based on evolutionary relatedness. Unfortunately, the relationships among the main balanomorphan groups are still not well understood and their taxonomy has not been revised in nearly 40 years (Newman and Ross, 1976). Historically, morphology-based systematic studies have relied on noncladistic approaches where taxa were not defined in terms of apomorphies and character evolution was inferred from general ontogenetic patterns and fossil series (Buckeridge, 1995; Newman, 1987, 1996; Newman and Ross, 1976; Newman et al., 1969). This has resulted in para- and polyphyletic assemblages within the Balanomorpha, as identified in previous phylogenetic analysis (Pérez-Losada et al., 2008, 2012). The characters used in classical studies (mostly hard parts) are undoubtedly important, but to adequately resolve thoracican and in particular balanomorphan relationships, they must be re-evaluated and formally coded (e.g., Glenner et al., 1995), while new ones must be developed (e.g., Pitombo, 1999, 2004). Molecular characters, on the other hand, are straightforward and have greatly advanced thoracican systematics, providing consistent results across multiple studies (Linse et al., 2013; Pérez-Losada et al., 2008, 2004; Rees et al., 2014). But for the Balanomorpha, molecular phylogenies have until now either been limited on their taxonomic coverage of the suborder (Linse et al., 2013; Pérez-Losada et al., 2008, 2004; Rees et al., 2014), or confined to balanomorphan subgroups such as the coral barnacles (Malay and Michonneau, 2014; Simon-Blecher et al., 2007; Tsang et al., 2014), coronuloids (Hayashi et al., 2013) or chathamaloids (Fisher et al., 2004; Pérez-Losada et al., 2012; Wares et al., 2009). Consequently, a comprehensive and robust hypothesis of the evolutionary relationships of the Balanomorpha and estimates of divergence times for major clades within the suborder is still missing. Here we present an extensive phylogenetic analysis of the Balanomorpha based on five genetic loci and 156 specimens representing all of the twelve extant families and nine outgroups (Lithotrya and Verrucidae). We then combined our phylogeny with

fossil and morphological information to estimate divergence times across the Balanomorpha and reconstruct the evolutionary history of some structurally and ecologically important characters. Additionally, we discuss the implications of our results for interpreting barnacle morphological evolution. 2. Methods 2.1. Molecular analysis Newman and Ross (1976) recognized three superfamilies within the Balanomorpha, viz., the Chthamaloidea, the Balanomorphoidea (Bathylasmatidae + Tetraclitidae + Coronulidae), and the Balanoidea. But Newman (1996) split the two former resulting in six superfamilies, viz., the Chionelasmatoidea, Pachylasmatoidea, Chthamaloidea, Coronuloidea, Tetraclitoidea, and Balanoidea. These are the ones adopted here and also in Martin and Davis (2001) and, with minor differences, in the Worms Register of Marine Species (2014). Our sampling included 147 taxa representing at least 124 species from all of the six balanomorphan superfamilies and their twelve described families. Additionally, we used two pedunculates of the genus Lithotrya and seven verrucids as the outgroup (Supplementary Table 1 and Fig. 1). Our outgroup choice is supported by both molecular and morphological evidence (Pérez-Losada et al., 2008, 2004). Specimens were preserved in 70% EtOH and are housed at the Smithsonian National Museum of Natural History, the Zoological Museum of the Hebrew University of Jerusalem, and the Mina and Everard Goodman Faculty of Life Sciences (Bar Ilan University). Barnacle DNA extraction, amplification, and sequencing were performed as described in Pérez-Losada et al. (2004). The 18S rRNA (1822 bp), 28S rRNA (1742 bp), 12S (345 bp) and 16S (527 bp) genes, and the COI (670 bp) gene were sequenced using primers in Pérez-Losada et al. (2004) and Folmer et al. (1994), respectively. We generated 155 new sequences (GenBank Accession numbers: KM217412 to KM217565). 2.2. Phylogenetic analyses Nucleotide sequences from each gene region were aligned using MAFFT v6 (Katoh, 2008) under the global (G-INS-i) algorithm and default settings. Phylogenetic congruence among gene regions was assessed using Wiens’ (1998) protocol. No areas of strongly supported incongruence were observed among gene trees. All gene regions were analyzed as separate partitions (COI was subdivided into 1st + 2nd and 3rd codon positions) under the best-fit model of evolution selected by JModelTest v1.0.1 (Posada, 2009). The general time reversible model of evolution with proportion of invariable sites and gamma distribution was selected for each data partition (GTR + G + I). Maximum likelihood analysis of the concatenated partitions was performed in RAxML v7.2.0 (Stamatakis et al., 2008) using 1000 searches and 100 runs. Clade support was assessed using the non-parametric bootstrap procedure with 5000 bootstrap replicates run on the CIPRES Science Gateway portal (Miller et al., 2010). A likelihood topological test was conducted using the Shimodaira and Hasegawa (1999) test as implemented in RAxML. We also performed a Bayesian–Markov chain Monte Carlo (BMCMC) analysis of the concatenated partitions in MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003). Three independent BMCMC analyses were run in CIPRES with each consisting of four chains. Each Markov chain was started from a random tree and run for 107 cycles, sampling every 1000th generation. Model parameters were unlinked and treated as unknown variables with uniform default priors and they were estimated as part of the analysis. Convergence and mixing were monitored using Tracer v1.5

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(http://tree.bio.ed.ac.uk/software/). All sample points prior to reaching stationarity were discarded as burn-in. The posterior probabilities for individual clades obtained from separate analyses were compared for congruence and then combined and summarized on a 50% majority-rule consensus tree. 2.3. Divergence time estimation Balanomorpha time radiation was estimated in BEAST v1.7.4 (Drummond and Rambaut, 2007) under a lognormal relaxed clock (Drummond et al., 2006) and using the HKY + G model for each of the five gene partitions. A Yule speciation prior was used for the tree prior as recommended by the authors. We used nine of the calibrations in Pérez-Losada et al. (2008) (C6-root – C13; Table 1; Fig. 2) plus Austrobalanus antarticus (C14) from the Middle Eocene [38.0–47.8 Million years ago (Mya)]. The impact on barnacle time estimation of including and excluding these and other fossil calibrations has been tested in previous studies (Linse et al., 2013; Pérez-Losada et al., 2008, 2004). Here we selected the calibrations which reciprocal time estimates were congruent among each other. Internal calibrations were introduced as minimum ages using lognormal priors with the lower bound (offset) implemented as the lower end of the epoch containing the first fossil occurrence and a size (mean) adjusted to the duration of the epoch for all calibrations. The root of the tree (C6) was introduced as an interval based on two fossils. Two runs 2  107 generations long were completed and combined using LogCombiner v1.7.4 (part of the BEAST package). All the output generated by BEAST was analyzed in Tracer v1.5. We used the 2012 International Chronostratigraphic Chart at www.stratigraphy.org. 2.4. Character evolution We assessed the evolutionary history of six key morphological characters commonly used in balanomorphan taxonomy: wall plate number (1, 4, 6, 8 plates), maxilliped number (1, 2, 3 maxillipeds), basis (calcareous, membranous), and then presence/ absence of rostrum, latus and imbricating plates (Figs. 3–5). These characters were selected to specifically illustrate both the numerous difficulties, but also the potentially valuable phylogenetic information entailed in balanomorphan morphology. Ancestral state reconstruction was performed using the Bayesian approach implemented in BEAST v1.7.4. Error associated to the characters under study (mapping uncertainty) was taken into account by estimating posterior probabilities for the ancestral states. We used the same calibrations, models and priors implemented in the divergence time estimation analysis. Additionally, we used a symmetric model of trait substitution with an approximate continuous time Markov chain rate reference prior for the trait.clock.rate (Ferreira and Suchard, 2008). Such a prior is recommended when explicit

prior information is unavailable. An exponential prior (mean = 1 change/My) was also tested, but no significant differences in character state posterior probabilities were observed. Two independent runs of 2  107 generations were carried out and, as before, then combined in LogCombiner v1.7.4. Phenotypic characters were annotated in TreeAnnotator v1.7.4 (part of the BEAST package) and visualized in FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/).

3. Results 3.1. Balanomorpha systematics Our ML and Bayesian analyses generated very similar topologies, hence only the ML tree with bootstrap proportions (BP) and posterior probabilities (PP) is presented in Fig. 1. Our phylogenetic analyses revealed that many traditionally recognized taxa at all levels from genus to superfamily did not form monophyletic assemblages. The Chthamaloidea fell into two main clusters, one well supported (BP P 70% or PP P 0.95) basal clade (node C) comprising the majority of the members of the Chthamalidae; and a second cluster (node E) weakly supported (BP < 70% and PP < 0.95) including the chthamalid Pseudoctomeris, Catophragmidae, the only member of the Chionelasmatoidea and the well supported Pachylasmatoidea monophylum. Three Chthamalidae genera (Octomeris, Chthamalus and Microeuraphia) were polyphyletic in our analyses. The remaining Balanomorpha constituted a well supported clade (node F), with the archaeobalanid (Balanoidea) genus Elminius (node G) at the base, followed by two large well supported sister clades including the monophyletic Tetraclitoidea + Coronuloidea (node I) and the rest of the Balanoidea (node L), making the Balanoidea polyphyletic. Within the Tetraclitoidea, the Tetraclitidae (node J) did not form a clade since the Bathylasmatidae (represented here by Hexelasma velutinum) was nested within them. The tetraclitid genera Tetraclitella was monophyletic, while Epopella and Tetraclita were not. Within the Coronuloidea (node K), the Platylepadidae and Coronulidae mingled together, while the third coronuloid family (Chelonibiidae) was depicted as monophyletic. Stomatolepas and Chelonibia were monophyletic, while Platylepas and Cylindrolepas were polyphyletic. None of the three extant Balanoidea families, Pyrgomatidae, Archaeobalanidae and Balanidae, formed monophyletic assemblages, with the latter two showing the greater intermixture of lineages. The pyrgomatids Cantellius and Nobia formed a clade, but Galkinia and Trevathana did not. All the archaeobalanid genera but Conopea were monophyletic. Finally, the balanid Austromegabalanus and Megabalanus were monophyletic, while Balanus and Amphibalanus were not. Hence, of the 10 Balanomorpha families represented by more than one species in our analyses, only Pachylasmatidae (two genera) and Chelonibiidae (one genus) were

Table 1 Species and ages of fossils used as calibrations for divergence time estimations. L = Lower, M = Middle, U = Upper. Calibrated nodes are indicated in Fig. 2. All calibrations were introduced as minimum ages, except C6, which was introduced as an interval (minimum and maximum ages). Nodes are numbered after Pérez-Losada et al. (2008) except C14. Species

Reference

Geologic age (Mya)

Node

Proverruca vinculum Pynolepas rigida Verruca tasmanica Metaverruca sculpta Pachydiadema (Catophragmus) cretacea Chamaesipho brunnea Tetraclitella sp. cf. purpurascens Palaeobalanus lindsayi Austromegabalanus victoriensis Austrobalanus antarticus

Newman et al. (1969) Newman et al. (1969) Buckeridge (1983) Buckeridge (1983) Buckeridge (1983) Buckeridge (1983) Buckeridge (1983) Buckeridge (1983) Buckeridge (1983) Buckeridge and Newman (2010) and Buckeridge (1983)

U. Cretaceous (Senonian) (72.1–89.8) U. Jurassic (145.0–163.5) U. Cretaceous (Santonian-Campanian) (72.1–86.3) Neogene–L. Miocene (Aquitanian) (20.4–23.0) U. Cretaceous (Senonian) (72.1–89.8) Neogene–L. Miocene (16.0–23.0) Neogene–L. Miocene (Aquitanian) (20.4–23.0) Paleogene–M. Eocene (38.0–47.8) Neogene–M.–L. Miocee (11.6–23.0) Neogene–M. Eocene (38.0–47.8)

C6-root C6-root C7 C8 C9 C10 C11 C12 C13 C14

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VERRU Verruca spengleri

75

0.99

100 1.0

C

A

<70 0.95

B

<70 <0.95

<70 <0.95

E

D

77 G 1.0 73 J 0.98

98 1.0

96 1.0

I

F

98 1.0

<70 0.98

K

H 80 1.0

<70 1.0

<70 <0.95

M

L

N

Lithotrya sp.

VERRU Verruca stroemia VERRU Verruca laevigata VERRU Altiverruca sp. VERRUCIDAE VERRU Metaverruca recta VERRU Rostratoverruca krugeri VERRU Rostratoverruca sp CHTHA Chamaesipho columna CHTHA Chamaesipho brunnea CHTHA Chamaesipho sp. CHTHA Chamaesipho tasmanica CHTHA Octomeris angulosa CHTHA Notochthamalus scabrosus CHTHA Octomeris brunnea CHTHA Caudoeuraphia caudata CHTHA Hexochamaesipho pilsbryi CHTHA Nesochthamalus intertextus CHTHA Chthamalus anisopoma CHTHA Chthamalus proteus CHTHA Chthamalus fragilis CHTHAMALOIDEA CHTHA Tetrachthamalus oblitteratus CHTHA Chthamalus montagui CHTHA Chthamalus stellatus CHTHA Chthamalus dentatus CHTHA Chthamalus malayensis CHTHA Chthamalus antennatus CHTHA Microeuraphia sp1 CHTHA Microeuraphia sp2 CHTHA Euraphia sp1 CHTHA Euraphia sp2 CHTHA Chthamalus challengeri CHTHA Microeuraphia rhizophorae CHTHA Microeuraphia withersi CHTHA Jehlius cirratus CHTHA Chthamalus bisinuatus CHTHA Microeuraphia depressa CATOP Catomerus polymerus CHION Eochionelasmus ohtai CHIONELASMATOIDEA CATOP Catophragmus imbricatus CHTHA Pseudoctomeris sulcata PACHY Pachylasma giganteum PACHY Pachylasma japonica PACHYLASMATOIDEA PACHY Eutomolasma sp2 PACHY Eutomolasma sp1 ARCHA Elminius kingii BALANOIDEA ARCHA Elminus covertus ARCHA Elminius modestus TETRA Austrobalanus imperator 2 TETRA Austrobalanus imperator 1 BATHY Hexelasma velutinum TETRA Epopella plicata TETRA Epopella simplex TETRA Tetraclitella divisa TETRA Tetraclitella purpurascens TETRA Tetraclitella pilsbryi TETRACLITOIDEA TETRA Tetraclitella multicostata TETRA Tetraclita stalactifera TETRA Newmanella vitiata TETRA Tetraclita rufotincta TETRA Tetraclita achituvi TETRA Tetraclita japonica TETRA Tesseropora rosea TETRA Tetraclita squamosa TETRA Tetraclita sp. TETRA Tetraclita serrata PLATY Stephanolepas muricata CHELO Chelonibia caretta 2 CHELO Chelonibia caretta 1 CHELO Chelonibia testudinaria 2 CHELO Chelonibia testudinaria 1 CHELO Chelonibia patula CHELO Chelonibia manati PLATY Stomatolepas elegans 1 PLATY Stomatolepas elegans 2 PLATY Stomatolepas praegustatur PLATY Stomatolepas transversa PLATY Stomatolepas gracilis CORONULOIDEA PLATY Stomatolepas sp. PLATY Cylindrolepas sinica CORON Xenobalanus globicipitis CORON Cryptolepas rhachianecti CORON Coronula diadema CORON Chelolepas cheloniae PLATY Platylepas decorata PLATY Cylindrolepas darwiniana PLATY Platylepas sp. PLATY Platylepas ophiophilus PLATY Platylepas hexastylos PYRGO Ceratoconcha domingensis PYRGO Megatrema anglicum ARCHA Armatobalanus allium 1 ARCHA Armatobalanus allium 2 PYRGO Cantellius sp1 PYRGO Cantellius sp2 PYRGO Cantellius sp3 PYRGO Cantellius pallidus PYRGO Nobia grandis 2 PYRGO Nobia sp. PYRGO Nobia grandis 1 PYRGO Pyrgoma cancellata 1 PYRGO Pyrgoma cancellata 2 PYRGO Galkinia indica PYRGO Hiroa stubbingsi PYRGO Darwiniella conjugatum 1 PYRGO Galkinia sp. PYRGO Darwiniella conjugatum 2 PYRGO Trevathana sp2 PYRGO Savignium crenatum PYRGO Trevathana sp1 PYRGO Pyrgopsella youngi PYRGO Hoekia sp. PYRGO Neotrevathana elongatum PYRGO Wanella milleporae BALAN Balanus perforatus ARCHA Acasta sp. ARCHA Acasta pertusa BALAN Balanus trigonus BALAN Menesiniella aquila BALAN Balanus nubilus BALAN Balanus balanus BALANOIDEA ARCHA Striatobalanus amaryllis BALAN Balanus crenatus BALAN Balanus glandula ARCHA Semibalanus balanoides ARCHA Semibalanus cariosus ARCHA Conopea sp1 ARCHA Conopea sp3 BALAN Balanus sp3 BALAN Balanus sp4 BALAN Balanus sp1 BALAN Balanus sp2 BALAN Balanus poecilotheca BALAN Balanus cirratus BALAN Amphibalanus eburneus BALAN Amphibalanus amphitrite BALAN Balanus sp6 BALAN Balanus sp5 BALAN Balanus reticulatus ARCHA Neocasta laevigata BALAN Austromegabalanus nigrescens BALAN Austromegabalanus psittacus ARCHA Conopea cymbiformis ARCHA Conopea sp2 ARCHA Conopea calceola BALAN Megabalanus ajax BALAN Megabalanus sp2 BALAN Megabalanus sp1 BALAN Megabalanus spinosus BALAN Megabalanus stultus BALAN Megabalanus californicus BALAN Megabalanus occator BALAN Megabalanus tintinnabulum BALAN Megabalanus volcano BALAN Megabalanus coccopoma

Fig. 1. Maximum likelihood phylogeny of Balanomorpha. Branch lengths are shown proportional to the amount of change along the branches. Bootstrap proportions (if P70%) or Bayesian posterior probabilities (if P0.95) are indicated by thicker lines on the tree and shown for the backbone nodes. Similar color tones are used for families belonging to the same superfamily. Family codes for each taxon are as follows: VERRU = Verrucidae, CHTHA = Chthamalidae, CATOP = Catophragmidae, CHION = Chionelasmatidae, PACHY = Pachylasmatidae, TETRA = Tetraclitidae, BATHY = Bathylasmatidae, PLATY = Platylepadidae, CORON = Coronulidae, CHENO = Chelonibiidae, PYRGO = Pyrgomatidae, ARCHA = Archaeobalanidae, and BALAN = Balanidae. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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7 8 10

6

9

14 11

12

13

175

150

125

100

75

50

25

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Lithotrya valentiana Lithotrya sp. VERRU Verruca spengleri VERRU Verruca stroemia VERRU Verruca laevigata VERRU Altiverruca sp. VERRU Metaverruca recta VERRU Rostratoverruca krugeri VERRU Rostratoverruca sp CHTHA Chamaesipho columna CHTHA Chamaesipho brunnea CHTHA Chamaesipho sp. CHTHA Chamaesipho tasmanica CHTHA Octomeris angulosa CHTHA Notochthamalus scabrosus CHTHA Octomeris brunnea CHTHA Caudoeraphia caudata CHTHA Hexochamaesipho pilsbryi CHTHA Nesochthamalus intertextus CHTHA Chthamalus anisopoma CHTHA Chthamalus proteus CHTHA Chthamalus fragilis CHTHA Tetrachthamalus oblitteratus CHTHA Chthamalus montagui CHTHA Chthamalus stellatus CHTHA Chthamalus dentatus CHTHA Chthamalus malayensis CHTHA Chthamalus antennatus CHTHA Microeuraphia sp1 CHTHA Microeuraphia sp2 CHTHA Euraphia sp1 CHTHA Euraphia sp2 CHTHA Chthamalus challengeri CHTHA Microeuraphia rhizophorae CHTHA Microeuraphia withersi CHTHA Jehlius cirratus CHTHA Chthamalus bisinuatus CHTHA Microeuraphia depressa CATOP Catomerus polymerus CHION Eochionelasmus ohtai CATOP Catophragmus imbricatus CHTHA Pseudoctomeris sulcata PACHY Pachylasma giganteum PACHY Pachylasma japonica PACHY Eutomolasma sp2 PACHY Eutomolasma sp1 ARCHA Elminius kingii ARCHA Elminus covertus ARCHA Elminius modestus TETRA Austrobalanus imperator 2 TETRA Austrobalanus imperator 1 BATHY Hexelasma velutinum TETRA Epopella plicata TETRA Epopella simplex TETRA Tetraclitella divisa TETRA Tetraclitella purpurascens TETRA Tetraclitella pilsbryi TETRA Tetraclitella multicostata TETRA Tetraclita stalactifera TETRA Newmanella vitiata TETRA Tetraclita rufotincta TETRA Tetraclita achituvi TETRA Tetraclita japonica TETRA Tesseropora rosea TETRA Tetraclita squamosa TETRA Tetraclita sp. TETRA Tetraclita serrata PLATY Stephanolepas muricata CHELO Chelonibia caretta 2 CHELO Chelonibia caretta 1 CHELO Chelonibia testudinaria 2 CHELO Chelonibia testudinaria 1 CHELO Chelonibia patula CHELO Chelonibia manati PLATY Stomatolepas elegans 1 PLATY Stomatolepas elegans 2 PLATY Stomatolepas praegustatur PLATY Stomatolepas transversa PLATY Stomatolepas gracilis PLATY Stomatolepas sp. PLATY Cylindrolepas sinica CORON Xenobalanus globicipitis CORON Cryptolepas rhachianecti CORON Coronula diadema CORON Tubicinella cheloniae PLATY Platylepas decorata PLATY Cylindrolepas darwiniana PLATY Platylepas sp. PLATY Platylepas ophiophilus PLATY Platylepas hexastylos PYRGO Ceratoconcha domingensis PYRGO Megatrema anglicum ARCHA Armatobalanus allium 1 ARCHA Armatobalanus allium 2 PYRGO Cantellius sp1 PYRGO Cantellius sp2 PYRGO Cantellius sp3 PYRGO Cantellius pallidus PYRGO Nobia grandis 2 PYRGO Nobia sp. PYRGO Nobia grandis 1 PYRGO Pyrgoma cancellata 1 PYRGO Pyrgoma cancellata 2 PYRGO Galkinia indica PYRGO Hiroa stubbingsi PYRGO Darwiniella conjugatum 1 PYRGO Galkinia sp. PYRGO Darwiniella conjugatum 2 PYRGO Trevathana sp2 PYRGO Savignium crenatum PYRGO Trevathana sp1 PYRGO Pyrgopsella youngi PYRGO Hoekia sp. PYRGO Neotrevathana elongatum PYRGO Wanella milleporae BALAN Balanus perforatus ARCHA Acasta sp. ARCHA Acasta pertusa BALAN Balanus trigonus BALAN Menesiniella aquila BALAN Balanus nubilus BALAN Balanus balanus ARCHA Striatobalanus amaryllis BALAN Balanus crenatus BALAN Balanus glandula ARCHA Semibalanus balanoides ARCHA Semibalanus cariosus ARCHA Conopea sp1 ARCHA Conopea sp3 BALAN Balanus sp3 BALAN Balanus sp4 BALAN Balanus sp1 BALAN Balanus sp2 BALAN Balanus poecilotheca BALAN Balanus cirratus BALAN Amphibalanus eburneus BALAN Amphibalanus amphitrite BALAN Balanus sp6 BALAN Balanus sp5 BALAN Balanus reticulatus ARCHA Neocasta laevigata BALAN Austromegabalanus nigrescens BALAN Austromegabalanus psittacus ARCHA Conopea cymbiformis ARCHA Conopea sp2 ARCHA Conopea calceola BALAN Megabalanus ajax BALAN Megabalanus sp2 BALAN Megabalanus sp1 BALAN Megabalanus spinosus BALAN Megabalanus stultus BALAN Megabalanus californicus BALAN Megabalanus occator BALAN Megabalanus tintinnabulum BALAN Megabalanus volcano BALAN Megabalanus coccopoma

0 My

Fig. 2. Bayesian chronogram of the Balanomorpha radiation. Time 95% high posterior density intervals are shown for each node. Calibrations C6–C14 are detailed in Table 1. Similar color tones are used for families belonging to the same superfamily. See Fig. 1 for superfamily names and family codes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Evolution of wall plate number (left) and rostrum (right). All nodes showed character state posterior probabilities P0.95 unless marked by an asterisk. Different colors are used for each superfamily. See Fig. 1 for superfamily names and family codes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Evolution of latus (left) and imbricating plates (right). All nodes showed character state posterior probabilities P0.95 unless marked by an asterisk. Different colors are used for each superfamily. See Fig. 1 for superfamily names and family codes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Evolution of maxilliped number (left) and basis (right). All nodes showed character state posterior probabilities P0.95 unless marked by an asterisk. Different colors are used for each superfamily. See Fig. 1 for superfamily names and family codes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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monophyletic (Fig. 1). The less diverse families (Catophragmidae; two genera) and all of the more species-rich families (Chthamalidae, Tetraclitidae, Platylepadidae, Coronulidae, Pyrgomatidae, Archaeobalanidae and Balanidae) were polyphyletic. Our Bayesian analysis of the Balanomorpha radiation based on five genes and 10 calibrations showed relatively narrow confidence intervals for most nodes in the chronogram (Fig. 2). The origin of the Sessilia (root) was placed in the Upper Jurassic, 161.8 (145.0– 190.3) Mya, while its split into asymmetric barnacles (Verrucidae) and Balanomorpha occurred in the Lower Cretaceous, 139.6 (113.4–164.5) Mya. Radiation of the Balanomorpha commenced in the Lower Cretaceous, 105 (91.3–125.7) Mya; and by the Paleocene, 60.9 (49.4–71.9) Mya, all the major balanomorphan lineages (Chthamaloidea, Tetraclitoidea, Coronuloidea, Balanoidea) had already appeared. Only the smaller lineage Pachylasmatoidea would have diversified in the Oligocene, 31.2 (17.5–48.8) Mya; although additional pachylasmatoid genera need to be analyzed to confirm our time estimate. 3.2. Balanomorpha morphological evolution To examine key morphological characters integral to balanomorphan taxonomy and evolution, we applied a Bayesian approach to assess the evolution of the following three pairs of morphological characters: wall plate number and rostrum (Fig. 3), latus and imbricating plates (Fig. 4), and basis and maxilliped number (Fig. 5). Ancestral states with PP P 0.95 were estimated for most nodes in the ML trees. Our analyses suggested convergent evolution in all characters except maxilliped number; convergence was mainly explained by homoplasy, but to a certain extent, also by the complexity of the chosen characters and the missing information for some of the coded species. 4. Discussion 4.1. Balanomorpha systematics This study represents the most comprehensive analysis of familial balanomorphan relationships (as listed in Martin and Davis, 2001). Previous molecular phylogenetic analyses of the Balanomorpha have had either limited taxonomic representation (Linse et al., 2013; Pérez-Losada et al., 2008, 2004; Rees et al., 2014) or focused on particular groups within the suborder (Hayashi et al., 2013; Malay and Michonneau, 2014; PérezLosada et al., 2012; Simon-Blecher et al., 2007; Tsang et al., 2014; Wares et al., 2009). A previous morphological phylogenetic study by Newman and Ross (2001) used also a large suite of larval characters and species to infer balanomorphan relationships, but their results lacked resolution due to both extensive homoplasy in the data and their outgroup choice (Pérez-Losada et al., 2004). 4.1.1. Balanomorpha taxonomy Multiple Balanomorpha genera, families and superfamilies in Newman and Ross’ comprehensive classification (1976) and subsequent modifications (Martin and Davis, 2001; Newman, 1987, 1996) were demonstrated to be polyphyletic (Fig. 1). But several of those taxa could be reestablished as monophyletic by relatively ‘‘benign’’ taxonomic rearrangements. The largest superfamily, the Balanoidea, is polyphyletic because Elminius (node G) splits earlier in the Balanomorpha tree. Similarly, the Chthamalidae is polyphyletic because Pseudoctomeris falls off the main clade. The Coronulidae is polyphyletic because Chelolepas clustered with the platylepadids. Our Pyrgomatidae clade (node M) encompasses all coral barnacles except Wanella, but it also includes the balanid Armatobalanus. A redefined Tetraclitidae subsuming the small

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monogeneric family Bathylasmatidae would be monophyletic, as would a taxon consisting of the Catophragmidae and the Chionelasmatidae. In contrast, species-rich families and genera such as the Platylepadidae, Balanidae, Achaeobalanidae, Tetraclita, Chthamalus and Balanus are very far from being monophyletic, thus of limited taxonomic value. 4.1.2. Balanomorpha phylogeny The backbone structure of our Balanomorpha tree confirms some previous morphological hypotheses and phylogenetic results in other molecular studies. Most ‘‘chthamaloid’’ taxa are aggregated at the base of the tree as indicated before (Linse et al., 2013; Newman, 1996; Pérez-Losada et al., 2008, 2004; Rees et al., 2014), and the same is true for the chionelasmatoid and pachylasmatoids that were also regarded as a part of the chthamaloids by Newman and Ross (1976). Slightly higher in the tree, the well supported clade comprising all Tetraclitoidea and Coronuloidea (node I) corresponds to the superfamily Balanomorphoidea recognized by Newman and Ross (1976), but later abandoned (Martin and Davis, 2001; Newman, 1996). A Tetraclitidae + Coronuloidea clade was also confirmed by Hayashi et al. (2013) in their molecular analyses. Finally, the Balanoidea, which has usually been considered the more ‘‘advanced’’ form of the Balanomorpha (Anderson, 1994), is similarly falling in a more derived position in our phylogenetic tree. Within the Chthamaloidea, our phylogenetic trees are largely comparable to the results in Pérez-Losada et al. (2012), but since the authors only focused on chthamaloid species, they could not test the monophyly of the superfamily. At the morphological level, we can also confirm that reductions and increases in shell plate number occurred multiple times (Fig. 3). Moreover, the 8 wall plates, traditionally regarded as the plesiomorphic condition (Newman, 1996; Newman and Ross, 1976), arose multiple times from the 6 wall plates for an updated Chthamaloidea as well as for the acorn barnacles as a whole (Fig. 3). Within the Coronuloidea, our phylogenetic analyses largely agree with the evolutionary patterns in Hayashi et al. (2013). As discussed below for coral barnacles, the monophyly of the coronuloids suggests that barnacle epibiosis on marine vertebrates (represented here by whales, turtles and dugongs) originated as a single evolutionary event. Morphologically the coronuloids are characterized by at least one structural apomorphy, viz., the non-occlusion of the opercular plates. This may relate to the fact that, unlike the many intertidal balanomorphan species, coronuloids are never exposed to prolonged desiccation and thus have less need for a tight closure of the mantle cavity. Our analyses of the Pyrgomatidae agree with those already published (Malay and Michonneau, 2014; Simon-Blecher et al., 2007; Tsang et al., 2014) in that all balanomorphans associated with stony corals form a monophyletic group (excluding Wanella but including Armatobalanus). Exclusion of Wanella from such a redefined Pyrgomatidae clade is well warranted on biological grounds. This genus is associated with hydrocorals rather than stony corals (Simon-Blecher et al., 2007; Tsang et al., 2014). It is then most parsimonious to assume that the association between balanomorph barnacles and stony corals evolved as a single evolutionary event that later entailed profound specializations in the armature of shell plates. The ‘‘archaeobalanid’’ Armatobalanus allium is characterized by a narrow carino-lateral, which is aborted in the four plated coral barnacles. In this feature Armatobalanus may therefore represent the plesiomorphic state of the stony coral associated clade. As suggested by Darwin (1854 p. 282) ‘‘this species [Armatobalanus allium] shows passage to the coral inhabiting genus Creusia’’. Interestingly, the cypris larvae of stony coral associated barnacles have an antennule that is uniquely spear-shaped, probably as an adaptation to settlement on the live epidermis of their coral host

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(Brickner and Høeg, 2010). This larval feature may well be synapomorphic for Pyrgomatidae redefined as above; testing this hypothesis would require to study the cypris larvae of the hydrocoral associated species Wanella. The strongest disagreement between our analyses and the established taxonomy concerns the Balanoidea, where a fundamental revision is needed at both family and generic levels. Both the Archaeobalanidae and the large family Balanidae are polyphyletic and with their constituent species intermingled with each other in two of the four subclades at node N. A solid phylogeny of the present members of the Balanidae is important, since species such as Semibalanus balanoides and Amphibalanus amphitrite and A. improvisus are models in both intertidal ecology and in research on larval settlement and antifouling prevention. If we were to generalize inferences from these model species to barnacles at large, accurately determining their phylogenetic position in the Balanidae subtree becomes critical. Pitombo (2004) made a serious attempt at this in his morphological cladistic analysis, but his conclusions were based on the assumption that the entire Balanidae was monophyletic, a hypothesis rejected by both our topology test and Bayesian analysis (P and PP < 0.001). 4.1.3. Balanomorpha time radiation Our Bayesian dating analysis provides divergence time estimates for balanomorphan groups like the Pachylasmatoidea and Pyrgomatidae; it also provides more robust estimates (as expected from higher number of taxa, genes and calibrations) than previous studies for other major groups like the Chthamaloidea, Tetraclitidae and Balanoidea and all the taxa within. The mean time estimates generated here for the Sessilia (139.6 Mya) and Balanomorpha (105.0 Mya) radiations are approximately 10 Million years (My) older than those in Linse et al. (2013) and 10 and 20 My younger, respectively, than those in Pérez-Losada et al. (2008). Within the Balanomorpha, few relevant comparisons can be made with other studies; Linse et al. (2013) and Pérez-Losada et al. (2008) reported a mean age for the Chthamalidae crown node younger (<25 My) and older (>30 My), respectively, than our current estimate (92.8 My; excluding Pseudoctomeris). Similarly, Hayashi et al. (2013) reported almost the same estimate as us for the Coronuloidea (59.6 Mya), but a younger estimate (<8 My) than ours (73.4 Mya) for the Coronuloidea + Tetraclitoidea clade. Differences among these four studies are likely due to a wider taxon sampling of the Balanomorpha in our study, but also to the higher number of calibrations used here, differences in prior constraints, number and type of genes (3 vs. 5 genes; mtDNA vs. nDNA) and alignments analyzed, and the Bayesian (BEAST vs. Thorne and Kishino, 2002) approach and molecular models (substitution and clock rate) chosen to build the chronograms. Integration of existing and new fossil evidence (Bracken-Grissom et al., 2014; Oakley et al., 2013; Porter et al., 2005; Sauquet et al., 2012), paleontological data (Wilkinson et al., 2011) and new gene regions (Mulcahy et al., 2012) into the dating analysis could well modify the time estimates that we have provided here; but until then, we recommend using this chronogram (Fig. 2) for further studies on Balanomorpha radiation. 4.2. Balanomorpha morphology 4.2.1. Evolution of morphological characters The morphological information used in balanomorphan phylogenetics derives from a rather limited character set concerning the disposition and structure of the shell plates and the structure of the mouthparts and cirri. As currently defined and used, the characters from hard parts are problematic, since they are often subject to a priori assumptions about character evolution and very prone to homoplasy. But there is potential for greatly expanding the list

of characters by using the numerous details involved in shell plates (e.g., Pitombo, 1999, 2004). Moreover, there is still much unused information for large scale phylogeny in soft body characters from mouthparts and cirri, especially if studied with SEM as in Høeg et al. (1994), Chan et al. (2008) and Walley (2012). These body parts are also interesting because they relate intimately to the evolution of feeding ecology in the taxon. In the next section, we discuss the phylogenetic evolution of six key morphological characters commonly used in balanomorphan systematics. Wall plate number (Fig. 3). Balanomorphans can have four, six or eight wall plates in their shell, or a single concrescent shell. The maximum number of 8 wall plates comprises carina (C), rostrum (R) and paired rostro-lateral (RL), lateral (L), and carino-lateral (CL) plates, and this condition has been assumed as plesiomorphic for the Balanomorpha. The hypothesis of an eight plated ancestor derives primarily from studies of species (including fossils) that were assumed to have a very plesiomorphic morphology (Buckeridge, 1995, 1996; Buckeridge and Newman, 1992; Newman, 1996; Newman and Yamaguchi, 1995; Yamaguchi and Newman, 1990, 1997a, 1997b). Contrary to this view, our analysis suggests that 6 wall plates is the ancestral condition, with 8 wall plates appearing as a derived state in at least four independent events. A future analysis including DNA sequences from Neobrachylepas relicta and Waikalasma boucheti, both previously argued to be morphologically very plesiomorphic (Buckeridge, 1996; Newman and Yamaguchi, 1995), may change this view. But even if 8 wall plates is reconstructed as the ancestral condition, our analysis shows that plate numbers must have evolved convergently in the acorn barnacles, as also shown in the Chthamaloidea (Pérez-Losada et al., 2012). Plate numbers were never proposed by themselves as homologous character states, since different pathways can lead to six or four plates. Each individual plate needs to be coded as a separate character as we have done for the rostrum. But such coding suffers from many uncertainties concerning plate identity because individual wall plates in balanomorphans have either been assumed to evolve de novo, fuse with adjacent plates or become lost altogether. Thus, plates in similar positions and termed by similar names may not necessarily be homologous, either when compared to pedunculated outgroups or within balanomorphans. This is especially true for the lateral plates (see also Glenner et al., 1995). Moreover, in some coral barnacles individual wall plates cannot be distinguished at all, making it virtually impossible to establish the ancestral condition by direct comparison. Our analysis shows that independent reductions in plate number have occurred both within the basal clades of chthamaloids and in other large groups (Tetraclitoidea and Pyrgomatidae). Such reductions have long been suggested in the evolution of the balanomorphan wall, but our analysis shows these plate transitions in a phylogenetic context. Rostrum (Fig. 3). Our analysis illustrates that presence of the rostral plate is a plesiomorphic feature for all the Balanomorpha, but also that it is subject to homoplasy higher in the tree. This is probably due to the complex nature of the balanomorphan rostrum, which may represent an independent plate (R) homologous to that in the outgroups – a compound fusion of the original rostrum and the two adjacent rostro-lateral plates (RL–R–RL) as in Coronula, or even a fusion of the two rostro-laterals under complete loss of the original rostrum itself (RL–RL). Early shell plate development immediately after cypris settlement may offer important clues to the nature of the ‘‘rostrum’’, but such information is only available for a mere handful of species (Glenner and Høeg, 1993; Runnström, 1925; Shalaeva, 1996). A more reliable coding of the ‘‘rostum’’ is therefore much needed. Latus (Fig. 4). Our analyses indicate that the evolution of the lateral plates involves some convergence. But the true situation may be even more complex. According to Newman and

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Yamaguchi (1995) the latus (upper median latus) of pedunculated barnacles is not homologous to the latus found in the Balanomorpha. We have accordingly coded the latus as absent in the outgroup Lithotrya. By an elaborated suite of arguments, Newman and Yamaguchi (1995) instead argued that the balanomorphan latus evolved by duplication of the rostro-lateral plate and therefore called it RL2 (see character discussion in Glenner et al., 1995). But evidence emerging from fossil species near the base of the Balanomorpha may lead to a total reinterpretation of all the lateral wall plates (Andrew Gale, Univ. Portsmouth, pers. comm.). If so, the carina may be the only wall plate that can be coded legitimately in all balanomorphans. Imbricating plates (Fig. 4). The presence of one or several whorls of small plates between the base and the true wall plates has long been considered an ancestral feature within the Balanomorpha (Glenner et al., 1995). Such imbricating plates are not only present in the closest pedunculated relatives (Pollicipes, Lithotrya), but also in the Brachylepadomorpha, which are assumed to represent part of the Sessilia ‘‘stem group’’. Nevertheless, our analyses show imbricating plates as an apomorphy for a small clade of catophragmids and chionelasmatoids nested into the balanomorphan tree; hence, if the position of such clade is confirmed, imbricating plates were either convergently lost several times or they have reappeared in the aforementioned clade. Maxilliped number (Fig. 5). The number of maxillipeds (or mouth cirri) is a phylogenetically highly informative character with no homoplasy. Cirripede maxillipeds are both shorter and morphologically distinct from the more posterior appendages that form the suspension feeding cirri, and they serve to pass food from this feeding basket to the mandibles, first and second maxillae. Lepadomorph barnacles have only a single pair of maxillipeds. All Balanomorpha are characterized by having two pairs (Chthamaloidea, Chionelasmatoidea and Pachylasmatoidea) or three pairs, a synapomorphy for the clade comprising all Coronuloidea, Tetraclitoidea and Balanoidea. Basis (Fig. 5). A membranous, as opposed to a calcareous, basis is habitually used in diagnosis of balanomorphan taxa. Our analysis indicates that the membranous state has evolved first in the Balanomorpha, while a solid basis evolved later several times, or was secondarily lost. Pitombo (2004) also concluded that this character was homoplasic. The adaptive significance of the character is not clear since upper-intertidal barnacle species living in a high-energy environment have both calcareous (e.g., Balanus improvisus and Tetraclita) and membranous basis (Chthamalus, Semibalanus and Elminius). 4.2.2. Balanomorphan morphology and homoplasy Our analysis reveals various degrees of convergent evolution in almost all the morphological characters studied. Even characters normally considered primitive and unproblematic, such as eight wall plates and imbricating plates do not show a simple pattern of evolution in our tree. This indicates that morphological characters often used in balanomorphan systematics are more prone to homoplasy than previously believed. Several large clades are indeed characterized by distinct morphological changes, but these are convergently repeated elsewhere in the tree. Such characters can still be used for diagnostic purposes as long as this takes place in a phylogenetic framework that is otherwise considered robust based on other evidence. But to adequately use morphology in balanomorphan phylogenetics, we will need a much larger and more refined character matrix than is presently available. Pitombo (1999) took important steps in this direction by implementing a very detailed list of characters applicable to this task and later executing a cladistic analysis of the Balanidae (Pitombo, 2004). Unfortunately, he coded higher-level taxa (e.g., Balaninae) rather than species exemplars; which impedes comparison with our results

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because his higher-level input taxa may not be monophyletic. We recommend that, wherever possible, characters always be coded for single species to avoid any a priori assumptions about their evolution. 5. Conclusions We have demonstrated that some aspects of balanomorphan phylogeny conform to existing ideas. Yet, we have also found new and surprising phylogenetic patterns and that numerous balanomorphan taxa at all levels are polyphyletic. The present analysis offers a comprehensive and fairly robust phylogeny to discuss the evolution of both structural and ecological traits within the Balanomorpha and provides the basis for tracing how balanomorphans evolved from their sessilian ancestors. Our phylogeny can be used to evaluate morphological characters in terms of homologies, homoplastic events and the extent to which morphology is correlated to the diverse habitats presently occupied by balanomorphans. Finally, we have developed a phylogenetic framework for revising balanomorphan systematics based on evolutionary relatedness. This again will prove highly valuable when extrapolating results from model species used in ecology and larval studies. Acknowledgments Specimens collected by us were identified by Y.A., A.J. Southward and J. Buckeridge. Samples were also provided and identified by B.K.K. Chan, T. Yamaguchi, R. Hayashi, and A. Biccard. This research was supported by the following grants: PTDC/BIA-BEC/ 098553/2008 to M.P.-L, Y.A. and J.T.H.; NSF DEB-0236135 to K.A.C. and M.P.-L.; NSF DEB-1301820 to K.A.C.; the U.S.-Israel Binational Science Foundation (BSF) 2004-239 to Y.A. and K.A.C.; Israel Science Foundation (ISF) 574/10 to Y.A. J.T.H. received support from Danish Natural Science Foundation, the Carlsberg Foundation, and the SYNTHESYS 1, 2 and 3 Projects http://www.synthesys.info/ financed by the European Community Research Infrastructure Action under the FP-6 & 7 ‘‘Capacities’’ Program.’’ We are very thankful to Philippe Lemey for his help with the beta version of BEAST used in this study. S. Zilinsky (BIU) helped with the lab work. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2014.09. 013. References Aldred, N., Clare, A.S., 2008. The adhesive strategies of cyprids and development of barnacle-resistant marine coatings. Biofouling 24, 351–363. Anderson, D.T., 1994. Barnacles – Structure, Function, Development and Evolution. Chapman and Hall, London. Bracken-Grissom, H.D., Ahyong, S.T., Wilkinson, R.D., Feldmann, R.M., Schweitzer, C.E., Breinholt, J.W., Bendall, M., Palero, F., Chan, T.-Y., Felder, D.L., Robles, R., Chu, K.-H., Tsang, L.-M., Kim, D., Martin, J.W., Crandall, K.A., 2014. The emergence of the lobsters: phylogenetic relationships, morphological evolution and divergence time comparisons of an ancient group (Decapoda: Achelata, Astacidea, Glypheidea, Polychelida). Syst. Biol. 63, 457–479. Brickner, I., Høeg, J.T., 2010. Antennular specialization in cyprids of coral-associated barnacles. J. Exp. Mar. Biol. Ecol. 392, 115–124. Buckeridge, J.S., 1983. Fossil barnacles (Cirripedia: Thoracica) of New Zealand and Australia. NZ Geol. Surv. Paleontol. 50, 1–151+pls. Buckeridge, J.S., 1995. Phylogeny and biogeography of the primitive Sessilia and a consideration of a Tethyan origin for the group. In: Schram, F.R. (Ed.), New frontiers in barnacle evolution. A.A. Balkema, Rotterdam, The Netherlands, pp. 225–267. Buckeridge, J.S., 1996. A living fossil Waikalasma boucheti n.sp. (Cirripedia, balanomorpha) fromVanuatu (new Hebrides), Southwest Pacific. Bull. Mus. Natl. Hist. Nat. 4. Ser. A 18, 447–457.

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