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Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Appendix S1 – Angelfish phylogenetics and divergence time estimation.

Methods

We downloaded sequences from GenBank for the six genes that have been sequenced for the greatest number of pomacanthid species, the mitochondrial 12s, 16s, cox1 and Cytb, as well as the nuclear markers Rag2 and S7 (table S1). We used MUSCLE (Edgar 2004) to align the sequences for each individual gene, inspected the alignments by eye for accuracy, and trimmed the sequences at the 3’ and 5’ ends to minimize missing characters. The final data matrix consisted of 610 bp for cox1, 593 bp for Cytb, 345 bp for 12s, 546 bp for 16s, 407 bp for Rag2 and 576 bp for S7, for a total of 3077 nucleotides. We used PartitionFinder (Version

1.1; Lanfear et al. 2012) to select the best partition scheme for the concatenated data and the best fitting models of sequence evolution using the Bayesian information criterion (BIC). We investigated only the models that can be utilized in BEAST 1.8 (Drummond and Rambaut,

2007) and did not include the proportion of invariant sites (I) parameter together with the gamma parameter (G) in the model tested, as I is already incorporated within the gamma parameter (Yang 2006). We partitioned the protein-coding loci (cox1, Cytb and Rag2) by codon position, but treated the other three loci as a single partition each, as they are non- protein coding and we deemed the fragment too short to be split as not enough sequence data would likely have been available to properly estimate the gamma. PartitionFinder selected 9 data partition out of the 12 potential ones.

We ran maximum likelihood analyses using RAxML (Stamatakis 2006), with each individual partition assigned a GTR+G model, the RAxML model closest to the

Partitionfinder results. We ran 1000 fast bootstrap replicates. We used MrBayes 3.2.3

(Ronquist et al. 2012) to perform Bayesian analyses using the partition scheme and models selected by Partition, with the exception of the TrN, which is not implemented in MrBayes,

1 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. and that we replaced with GTR. We ran multiple replicates with two analyses of 10 million generations each, with four chains (one cold, three heated) sampling every 1000 generations.

We used Tracer 1.6 (Drummond and Rambaut 2007) to inspect the trace files and verify that the chains had reached convergence, and discarded the first 25% of trees as burnin. We combined the post-burnin trees to obtain a 50% majority rule consensus tree and compared the topologies of the different replicates to each other to assess support for the results of the analyses.

To generate the timetree, we analyzed the concatenated alignment as nine unlinked gene partitions, after having assigned to each of these the model selected by Partitionfinder.

We used uncorrelated lognormal priors in BEAST 1.8 (Drummond and Rambaut 2007) and assigned a birth-death prior to the rates of cladogenesis. We ran four analyses of 50 million generations each, with sampling every 10000 generations. We used Tracer 1.6 (Drummond and Rambaut 2007) to inspect the trace files, ensuring that the chains had reached stationarity and the ESS values for all parameters were greater than 200. We removed the first 5-10% of the trees from each analysis as burnin, used LogCombiner to merge the files with the remaining trees, and TreeAnnotator (Drummond and Rambaut 2007) to obtain a timetree. No fossil older than the Pliocene can presently be assigned to the pomacanthids (Patterson 1993), so we used two acanthuroid fossils. Following Sorenson et al. (2013), we used the Middle

Eocene Sorbinithurus sorbinii from Monte Bolca (Italy) to assign a minimum age of 50 My

(Papazzoni and Trevisani 2006) to the split between Naso and Zanclus, and the basal Eocene

Kushlukia permira (Bannikov and Tyler 1995) from the lowermost layers of the middle part of the Danatinsk Suite, Uylya-Kushlyuk, southwest Turkmenistan, (Lower Eocene, Ypresian,

55.8 My; see Gavrilov et al. 2003) to date the split between Luvarus and the Naso+Zanclus clade. We used the Late Cretaceous age of the Calcari di Melissano area in the Lecce

2 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. province, southern Italy, which contains the oldest record of many percomorphs, to establish a soft upper boundary of 83.5 Ma (Schlüter et al 2008).

All phylogenetic analyses (RAxML, MrBayes and BEAST) were run on the Cipres portal v. 3.3 (Miller et al. 2010).

Results

We present a new and robust phylogenetic hypothesis, and produce a time-calibrated phylogeny that includes 67 species, i.e. 76% of the extant pomacanthid diversity. The maximum likelihood analysis performed in RAxML and the Bayesian analyses from MrBayes produce very similar topologies, with the only disagreements limited to relationships among some of the most recently evolved lineages. We only present the MrBayes consensus tree here and include in the figure (figure S3) both posterior probabilities (PP) and bootstrap proportions (BSP) from the RAxML tree. However, before describing the relationships inferred in our study, we wish to clarify that while it is known that some closely-related angelfish species can hybridize (Pyle and Randall 1994), there is currently no indication that the degree of such events across the entire group might have obscured broad patterns of phylogenetic relationships within this clade. Furthermore, sequences obtained from known hybrid individuals in many groups of reef fishes are usually indicated as such in GenBank and none of these were included in this study, thus minimizing the risk that some of the relationships recovered in our trees might be due to hybridization.

Monophyly of the Pomacanthidae is strongly supported, with a PP of 1 and BSP of

100. Marine angelfishes appear to be comprised by two main subclades: the first includes a monophyletic genus Chaetodontoplus sister to an equally monophyletic Pomacanthus.

Support for both of these genera, as well as for the subclade they comprise, is high in both

Bayesian (PP=1) and likelihood (BSP=100) trees. The remaining subclade includes the bulk

3 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. of the angelfish diversity: the monotypic genus Pygoplites is the first lineage to branch off, followed by a group composed by the Banded angelfish Apolemichthys arcuatus and one clade of Centropyge (C. Centropyge). Support for the monophyly of this clade is not high, with a PP lower than 0.9 and a BSP of 56. The next lineage to branch off the main pomacanthid subclade is composed by the genus Holacanthus, followed by a group formed by the remaining species of Apolemichthys included in our sampling (A. kingi, A. xathurus and A. trimaculatus). These groups are highly supported with PP above 0.99 and BSP over 93. The remaining angelfishes in our study belong to two additional clades of Centropyge and to the monophyletic Genicanthus that appears to be nested within the former. The first and the second Centropyge clades include species from the subgenus C. Paracentropyge and C.

Xiphypops, respectively.

The topology of the Bayesian analysis performed in BEAST is highly congruent with those of the MrBayes and RAxML analyses (Figures 1 & S4); the only significant difference is in the position of the Royal angelfish Pygoplites diacanthus, which appears to be nested within the second pomacanthid subclade, instead of sister to it and appears as the sister group to Holacanthus, albeit with very low support (PP=0.73).

Pomacanthids split from their outgroups ~ 99 million years ago (Ma). As the sister group of angelfishes remains uncertain, this age might not be a reliable stem age of pomacanthids and simply represent the age of the split between pomacanthids and the other lineages included in our study. The crown age of angelfishes dates to the earliest part of the

Paleocene, ~ 66 Ma (45-94 Ma 95% highest posterior density or HPD). The crown age of the first pomacanthid subclade is ~ 60 Ma (40-86 Ma 95% HPD), with both Chaetodontoplus and

Pomacanthus originating in the Early Oligocene (respectively 33 Ma, 21-51 Ma HPD, and 31

Ma, 20-45 Ma 95% HPD).

4 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Much of the diversification within the second pomacanthid subclade appears to have occurred during the Middle to Late Eocene, with the entire subclade dating to ~ 43 Ma (30-60

Ma 95% HPD), Apolemichthys arcuatus + first Centropyge clade dating to 41 Ma (27-57 Ma

95% HPD), Pygoplites + Holacanthus dating to 34 Ma (22-49 Ma 95% HPD) and the split between Apolemichthys and the remaining Centropyge + Genicanthus clade dating to 34 Ma

(23-48 Ma 95% HPD). In spite of the relatively old ages of many clades, several Miocene or younger radiations can also be identified, including that of Holacanthus (21 Ma, 13-32 Ma

95% HPD); Apolemichthys kingi + A. xathurus and A. trimaculatus, dated at 19 Ma (10-30

Ma 95% HPD); Genicanthus (17 Ma, 10-25 Ma 95% HPD). Several clusters of very recently evolved species are also evident: the Holacanthus bermudensis and H. ciliaris split is only ~

0.4 Ma while the group formed by H. limbaughi, H. passer and H. clarionensis is only ~ 0.67

Ma. The Centropyge “acanthops” group, which in our sampling is represented by C. acanthops, C. resplendens, C. aurantonota, C. argi, C. fisheri and C. flavicauda, is only ~ 1.8

Ma, while the split between C. ferrugata and C. shepardi dates to 0.5 Ma and the group of C. joculator, C. multicolor and C. nahackyi is 0.9 Ma.

Discussion

The phylogenetic intra-relationships of pomacanthids have received relatively scarce attention when compared to many other groups of coral reef fishes, such as wrasses (Westneat and

Alfaro 2005; Alfaro et al. 2009; Kazancioglu et al. 2009; Cowman and Bellwood 2011); pufferfishes (Alfaro et al. 2007; Santini et al 2013a); triggerfishes (Dornburg et al. 2011;

Santini et al 2013b) and damselfishes (Cooper et al. 2009; Frédérich et al. 2013). While a number of studies have been published over the past few years dealing with relationships within some pomacanthid subclades (Alva-Campbell et al. 2010; Hodge et al. 2013; Gaither et al. 2014), very few studies have investigated the relationships of the major angelfish

5 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. lineages. Bellwood et al. (2004) sequenced portions of the mitochondrial 12s and 16s ribosomal loci for 24 angelfish species, plus selected outgroups (including chaetodontids, scatophagids and kyphosids), and recovered a pectinate maximum likelihood topology in which Pomacanthus appeared as the sister group to all remaining pomacanthids, followed by a branching pattern that saw Chaetodontoplus, and Pygoplites + Holacanthus as sequential sister groups to a large clade that included the three groups of Centropyge that appear in our phylogenies, as well as Genicanthus and Apolemychthys (Bellwood et al. 2004). Bellwood et al. (2004) also performed penalized likelihood analyses of divergence times among the pomacanthid species in their study, and inferred a crown age of 38 Ma in their preferred tree.

While their topology and the age of origin of the angelfish clade show some significant differences with our results, there are several areas of agreement between their study and our own. However, the ages for most pomacanthid clades are significantly greater in our study.

The age of the penalized likelihood study of Bellwood was based on having a fixed age for the root of only 50 Ma, an age that we now know is a major underestimation for the age of many coral reef groups (Near et al. 2013). For example while the age of crown Pomacanthus in our study (~ 30 Ma) is similar to the 28 Ma inferred by Bellwood and colleagues, they infer an age of 23 Ma for both the Pygoplites + Holacanthus clade, and for the Centropyge +

Paracentropyge group, which we both estimate to be ~ 34 Ma.

All other studies of pomacanthid relationships focused on taxonomically restricted subclades. The broadest sampling among other studies is that of the pygmy angelfishes

(Centropyge) by Gaither et al. (2014). Sequences for 2272 bp from five markers (the mithochondrial cox1 and Cytb, and the nuclear Tmo, Rag2, and S7) were used to infer the relationships among 102 pomacanthid individuals, revealing the existence of a topology rather similar to the one we recovered. Gaither et al. (2014) found Chaetodontoplus mesoleucus to be the sister group to all other specimens in their sampling, including a clade formed by three

6 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. species of Pomacanthus, Pygoplites diacanthus, and finally three clades of Centropyge.

Among these several lineages can be identified: a first Centropyge clade that includes

Apolemichthys arcuatus, a second Centropyge clade includes additional Apolemichthys species as well as Genicanthus, and within which three different groups of Centropyge species can be recognized including the C. Paracentropyge group and the “acanthops” species group, corresponding to the traditional Xiphypops subgenus. Finally a third clade of

Centropyge is recognized with at least eight species, including the flavissima complex, which likely includes many currently unrecognized species that are genetically highly distinct from one another. Gaither et al. (2014) performed a Bayesian time calibration analysis that relied on secondary calibration points inferred from the Bellwood et al. (2004) study. The ages are thus similar to the ones inferred by Bellwood and colleagues, and are much younger than the ages found in our study; for this reason we will not discuss them further. In a study of the biogeography and speciation in Pomacanthus, Hodge et al. (2013) sequenced partial fragments of the loci 12S, 16S and S7 and produced a topology that is identical to the one we found. Pomacanthus zonipectus + (P. arcuatus, P. paru) are sister to the remaining

Pomacanthus, followed by a branching pattern of P. navarchus + (P. sexstriatus+xanthometopon), P. imperator + P. annularis and finally a subclade formed by P. asfur, P. rhomboides, P. maculosus, P. chysurus and P. semicirculatus. They also used fossils of the stem acanthuroids from the Early Eocene, including Avitoluvarus, to time-calibrate their molecular phylogeny by providing ages for the split of the outgroup taxa. They inferred an age of ~ 25 Ma for the crown of Pomacanthus, which is slightly younger than the 30 Ma of our study. The last study that used DNA sequences to investigate pomacanthid intra- relationships is that of Alva-Campbel et al. (2010) which used 2283 bp for four loci (12S,

16S, Cytb and S7) to investigate the relationships of seven species of the genus Holacanthus.

Their maximum likelihood topology also matches our inferred topologies, with a splitting

7 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. pattern of Holacanthus africanus, H. tricolor, H. bermudensis+H. ciliaris, and H. limbaughi+H. passer+H. clarionensis.

It is unsurprising that all these studies show similar topologies, as they were mostly based on similar sets of loci. Our study, however, is the most comprehensive to date, as it includes all loci used in these previous studies (with the exception of Tmo-4c4 from the

Gaither et al. (2014) study, as these sequences are exclusively available for the species of

Centropyge and are not available for the other genera). The sampling of our study (~ 75% of the extant diversity) is also more comprehensive than in previous studies and, for the first time, we provide a time-calibrated phylogeny based on the use of fossils for closely related groups. Furthermore, findings from phylogenetic (Gaither et al. 2014, present study) and comparative analyses (body size and shape evolution; see main text of the present study) highlight the need for additional work on morphological evolution in angelfishes, and suggest the need for future continuous systematic treatment of the family Pomacanthidae.

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Alva-Campbell A., Floeter S.R., Robertson D.R., Bellwood D.R., Bernardi G. 2010 Evolutionary history and modes of speciation of Holacanthus angelfishes (Pomacanthidae). Mol. Phylogenet. Evol. 56, 456–461.

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Bellwood D.R., Van Herwerden L., Konow N. 2004 Evolution and biogeography of marine angelfishes (Pisces: Pomacanthidae). Mol. Phylogenet. Evol. 33, 150–155.

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Cooper W.J., Smith L.L., Westneat M.W. 2009 Exploring the radiation of a diverse reef fish family: Phylogenetics of the damselfishes (Pomacentridae), with new classifications based on molecular analyses of all genera. Mol. Phylogenet. Evol. 52(1), 1-16.

Cowman P.F., Bellwood D.R. 2011 Coral reefs as drivers of cladogenesis: expanding coral reefs, cryptic extinction events, and the development of biodiversity hotspots. J. Evol. Biol. 24, 2543-2562.

DiBattista J.D., Waldrop E., Bowen B.W., Schultz J.K., Gaither M.R., Pyle R.L., Rocha L.A. 2012 Twisted sister species of pygmy angelfishes: discordance between taxonomy, coloration, and phylogenetics. Coral Reefs 31, 839–851.

Dornburg A., Sidlauskas B., Santini F., Sorenson L., Near T.J., Alfaro M.E. 2011 The influence of an innovative locomotor strategy on the phenotypic diversification of triggerfish (family: Balistidae). Evolution 65(7), 1912-1926.

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Frédérich B., Sorenson L., Santini F., Slater G.J., Alfaro M.E., 2013. Iterative ecological radiation and convergence during the evolutionary history of damselfishes (Pomacentridae). Am. Nat. 181, 94-113.

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Gaither M.R., Schultz J.K., Bellwood D.R., Pyle R.L., DiBattista J.D., Rocha L.A., Bowen B.W. 2014. Evolution of pygmy angelfishes: Recent divergences, introgression, and the usefulness of color in taxonomy. Mol. Phylogenet. Evol. 74, 38–47

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Appendix S2 – Ancestral state reconstruction of body size.

Methods

In order to assess the ancestral size of marine angelfishes, we used continuous trait data on body size. We performed an estimation of the Maximum Likelihood ancestral states for size

(log-transformed Total Length) using the function fastAnc in the R-package phytools (Revell

2012).

Results

Reconstruction of body size evolution showed that the ancestral marine angelfishes would be medium-sized in comparison with extant taxa. These results suggested that the evolutionary history of Pomacathidae is characterized by shifts to large-sized species (e.g. Pomacanthus and Holacanthus) and small-sized species, i.e. pygmy lineages from the genus Centropyge.

12 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Reference

Huelsenbeck J.P., Nielsen R., Bollback J.P. 2003 Stochastic mapping of morphological characters. Syst. Biol. 52(2), 131-158.

Revell L.J. 2012 phytools: An R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3(2), 217-223.

13 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Appendix S3 – Details on models of shape evolution and stochastic mapping.

We used the R-package mvMORPH v.1.0.7 (Clavel et al. 2015) to compare the fit of four models of shape evolution accounting for sampling variance. The models included: (a) a single rate (σ2) Brownian Motion model (i.e. a pure drift process – BM1), (b) a OU model with a single optimum for all species (OU1), (c) BM models with different rate parameters for body size optima inferred from SURFACE (BMM_size) and (d) OU models with separate optima (θ) for body size optima inferred from SURFACE (OUM_size) (table 1). If body size influences the tempo and/or the mode of shape diversification, then we would expect

BMM_size or OUM_size to best fit our data. Conversely, if size has no influence on body shape the other models should fit better.

In OUM_size model, we considered that groups of lineages evolved independently

(i.e. both the drift parameter σ and the strength of selection α were restricted to be diagonal).

We fitted models using mean species scores along the first three PC axes on shape variables to reduce the number of parameters. To feed these models, we used stochastic character mapping (Huelsenbeck et al. 2003) to infer possible histories of body size. The stochastic mapping was produced using the function make.simmap in the PHYTOOLS package v.0.5.38

(Revell 2012). For the parameterization of make.simmap, we used the estimated ancestral state and the best model for the transition matrix from our empirical data. To assess the best model for the transition matrix, we fitted a model with equal rate of transition between states and a model with all rates different using the function ace in the R-package ape. The likelihood of these two models was then compared using likelihood ratio tests, asking for the use of unequal rates for size evolution (p = 0.043).

Through stochastic mapping, we sampled 50 character histories for each of the 100 trees generating 5,000 character maps. These character maps allowed us to incorporate

14 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al. uncertainty associated with tree topology, branch length, and timing of the transitions among states.

References

Clavel, J., Escarguel, G., and Merceron, G. 2015. mvMORPH: an R package for ﬁtting multivariate evolutionary models to morphometric data. Methods Ecol. Evol. 6, 1311-1319.

Huelsenbeck, J.P., Nielsen, R., and Bollback, J.P. 2003. Stochastic mapping of morphological characters. Syst. Biol. 52, 131-158.

Revell, L.J. 2012. phytools: An R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217-223.

15 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Table S1. List of studied species and their maximum total length (TL). The number of used

X-rays (n) and the Genbank accession numbers are also provided.

This supplementary material is provided as a separate excel file.

16 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Figure S1. The homologous landmarks (LMs) used in the analysis of body shape variation, here illustrated for Centropyge flavissima: (1) mouth tip; (2) ventral extremity of the lacrymal; (3) dorsal extremity of the lacrymal; (4) top of the supraoccipital crest; (5) base of the first spiny dorsal fin ray; (6) base of the last spiny dorsal fin ray; (7) base of the last soft dorsal fin ray; (8) dorsal base of the caudal fin; (9) ventral base of the caudal fin; (10) posterior insertion of the anal fin; (11) last posterior vertebrae; (12) anterior insertion of the anal fin; (13) insertion of the pelvic fin; (14) ventral extremity of the cleithrum; (15) point of maximum curvature of the inner edge of the preoperculum; (16) quadrato-mandibular articulation; (17) hyomandibular-neurocranium articulation; (18) neurocranium-vertebrate articulation.

17 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Figure S2. Number of body size optima (i.e. number of distinct regimes after collapsing convergent regimes = k_prime; See Ingram & Mahler 2013 for more information) estimated by SURFACE when using the consensus time-tree including 65 angelfish species (red line) and the 100 sampled trees from posterior distribution generated by BEAST (white histograms).

18 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Figure S3. Bayesian phylogenetic hypothesis based on the analysis of the concatenated dataset using MrBayes 3.2. Posterior probability and bootstrap percentage are shown above branches.

19 Body shape convergence driven by small size optimum in marine angelfishes - Frédérich et al.

Figure S4. Pomacanthid timetree based on a Bayesian relaxed clock approach implemented in BEAST 1.8. Horizontal bars on each node indicate 95% HPD confidence intervals. Posterior probabilities (PP) greater than 0.5 indicated near corresponding nodes, nodes without any indication of nodal support have PP below 0.5. Timescale at bottom of figure is in million years. Fish images modified under Creative Commons license from original photographs by J.E. Randall (retrieved from http://www.fishbase.org).