World-wide species distributions in the family Kyphosidae (Teleostei: Perciformes)

Accepted Manuscript World-wide species distributions in the family Kyphosidae (Teleostei: Perciformes) Steen Wilhelm Knudsen, Kendall D. Clements PII: DOI: Reference:

S1055-7903(16)30080-X http://dx.doi.org/10.1016/j.ympev.2016.04.037 YMPEV 5509

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

26 January 2016 10 April 2016 29 April 2016

Please cite this article as: Knudsen, S.W., Clements, K.D., World-wide species distributions in the family Kyphosidae (Teleostei: Perciformes), Molecular Phylogenetics and Evolution (2016), doi: http://dx.doi.org/ 10.1016/j.ympev.2016.04.037

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World-wide species distributions in the family Kyphosidae (Teleostei: Perciformes)

Steen Wilhelm Knudsen1 and Kendall D. Clements1

1) School of Biological Sciences, University of Auckland, Auckland, New Zealand

Corresponding author: Steen Wilhelm Knudsen Phone: (+64 9) 373 7599 ext. 88483, Fax.: (+64 9) 373 7417 (e-mail: [email protected])

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Running head: Evolution and distribution of sea chubs Type of paper: Full paper Number of text pages: 48 + x page with figure legends Number of figures: 3 Number of tables: 3 Number of supplementary figures: 9 Number of supplementary tables: 10 Number of supplementary data files: 3

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Abstract Sea chubs of the family Kyphosidae are major consumers of macroalgae on both temperate and tropical reefs, where they can comprise a significant proportion of fish biomass. However, the relationships and taxonomic status of sea chubs (including the junior synonyms Hermosilla, Kyphosus, Neoscorpis and Sectator) worldwide have long been problematical due to perceived lack of character differentiation, complicating ecological assessment. More recently, the situation has been further complicated by publication of conflicting taxonomic treatments. Here, we resolve the relationships, taxonomy and distribution of all known species of sea chubs through a combined analysis of partial fragments from mitochondrial markers (12s, 16s, cytb, tRNA -Pro, -Phe, -Thr and -Val) and three nuclear markers (rag1, rag2, tmo4c4). These new results provide independent evidence for the presence of several junior synonyms among Atlantic and Indo-Pacific taxa, demonstrating that several sea chub species are more widespread than previously thought.

In particular, our results can reject the

hypothesis of endemic species in the Atlantic Ocean. At a higher taxonomic level, our results shed light on the relationships between Girellidae, Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae and Scorpididae, with Scorpididae resolved as the sister group to Kyphosidae.

Keywords: sea chub; drummer; Kyphosus; Scorpis; Girella

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1. Introduction The increasing availability of molecular sequences for perciform clades has led to a greatly improved phylogenetic understanding of reef fishes (e.g., Miya and Nishida, 2014; Near et al., 2012a,b; Sanciangco et al., 2016), but the lack of a complete understanding of species level diversity remains an obstacle for elaborating the pattern of evolution in several clades of herbivorous reef fishes, e.g., scarines (Choat et al., 2012), acanthurids (Clements et al., 2004; Sorenson et al., 2013), and especially kyphosids. Kyphosid sea chubs are important macroalgal feeders on reefs in tropical and sub-tropical seas worldwide (Choat and Clements, 1998; Cvitanovic and Bellwood, 2009; Floeter et al., 2005; Howard, 1989; Vergés et al., 2012). Kyphosus species can comprise a large proportion of the fish biomass in some areas, especially Western Australia (Howard, 1989), where they are thought to have a considerable impact on algal biomass (Bennet et al., 2015; Vergès et al., 2012). Kyphosus is recorded as being strictly herbivorous (Choat et al., 2002, 2004; Clements and Choat, 1997; Horn, 1989; Moran and Clements, 2002), feeding on macroalgae, and most species display a specialised alimentary anatomy associated with microbial fermentation (Clements and Choat, 1997; Clements and Zemke-White, 2008; Crossman et al., 2005; Fishelson et al., 2014; Mountfort et al., 2002; Rimmer and Wiebe, 1987). Taxonomic confusion has plagued Kyphosus (Knudsen and Clements, 2013a, Sakai and Nakabo, 2014; Gilbert, 2015) and correct species identification has been problematical in numerous studies (e.g., Azzurro et al., 2013; Francour and Mouine 2008; Kiparissis et al., 2012; Ligas et al. 2011; Vergès et al. 2012; Mannino et al., 2015) to the extent that species have been considered indistinguishable in the Atlantic (Humann and DeLoach, 2002). Recently, two independent taxonomic treatments (Knudsen and Clements, 2013a; Sakai and Nakabo, 2014) presented conflicting views on the taxonomy of Kyphosus. Knudsen and Clements (2013a) reduced the number of valid species of Kyphosus to 12, whereas Sakai and Nakabo (2014) described two new species (Kyphosus atlanticus and K. bosquii) endemic to 4

the Atlantic Ocean, and retained two species, Kyphosus incisor and K. analogus, which were both considered junior synonyms of K. vaigiensis by Knudsen and Clements (2013a). Further, Sakai and Nakabo (2014) considered Kyphosus sectatrix to be a nomina dubia, and Gilbert (2015) listed a neotype for Kyphosus sectatrix. The discrepancies between these two recent taxonomic revisions of Kyphosus (Knudsen and Clements, 2013a; Sakai and Nakabo, 2014) means there is currently no consensus on the number or distribution of Kyphosus species. This situation is an impediment to understanding their ecology and evolution. Here, we re-examine morphological variation, global distribution and molecular variation in kyphosids to test between the two competing taxonomic treatments, and as such provide a framework for future study on the ecology and evolution of the group. Knudsen and Clements (2013a) considered the kyphosids (Kyphosinae sensu Nelson, 2006 or Kyphosidae sensu Randall, 2007) to consist of 12 species in the genus Kyphosus, including a new species of Kyphosus recently described from Western Australia (Knudsen and Clements, 2013b), and the three monotypic genera Hermosilla, Neoscorpis and Sectator, which were all considered junior synonyms of Kyphosus. Conversely, Sakai and Nakabo (1995, 2004, 2006, 2008, 2014) consider the genus Kyphosus to comprise 13 species, retaining Sectator and Hermosilla as valid monotypic genera. Kyphosids are found in the Indo-Pacific and the Atlantic Ocean, but are absent from the North East Pacific and the North West Atlantic Ocean (Allen and Robertson 1994; Knudsen and Clements, 2013a,b; Kuiter, 1997; Myers, 1999; Randall, 1983, 1999, 2007; Sakai and Nakabo, 1995, 2004, 2006; Tortonese, 1986). Juvenile kyphosids associate with floating objects (Luiz et al., 2015; Moore, 1962; Walker et al., 2004), and as a result can be found well offshore (Jokiel, 1990; Luiz et al., 2012). Kyphosids thus have the potential to disperse over great distances, and this could make their inter-ocean population connectivity greater than previously recognised. Because kyphosids occur worldwide, geminate species have been proposed across barriers such as the Panamanian Isthmus (Jordan, 1908; Thomson et al.,

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2001), but so far these ideas have not been tested. In this study we provide the first worldwide analysis of samples from all species of Kyphosus, including Kyphosus azureus and Kyphosus ocyurus (previously recognised as Hermosilla azurea and Sectator ocyurus, respectively), together with representatives of Atypichthys, Girella, Kuhlia, Microcanthus, Oplegnathus, Rhyncopelates and Scorpis. The work here provides a foundation for determining the extent of various species distributions, and allows us to test the validity of a monophyletic Kyphosidae sensu Nelson (2006). Nelson (2006) considered Kyphosidae to contain five subfamilies: Girellinae, Kyphosinae, Microcanthinae, Parascorpidinae, Scorpidinae, but acknowledged that these subfamilies might require recognition at family level, as their affinities remain obscure. A more common approach is to consider these as separate families (i.e. Girellidae, Kyphosidae, Microcanthidae, Parascorpididae and Scorpididae) (e.g., Randall, 2007). Molecular phylogenetic studies on perciform taxa that have included Kyphosus species indicate that the latter approach is more realistic (Near et al., 2012a; Yagishita et al., 2002, 2009; Yagishita and Nakabo, 2003; Sanciangco et al., 2016) (Fig. 1). The aims of this study are to: (1) resolve the evolutionary relationships among Kyphosus species on a global scale, (2) determine the number of valid species of Kyphosus world-wide based on both morphology and molecular data, (3) determine the level of molecular support for the recent taxonomic revision of Sakai and Nakabo (2014) compared to the previous taxonomy proposed by Knudsen and Clements (2013a), and (4) test whether the subfamilies in Kyphosidae sensu Nelson (2006) (i.e. Girellinae, Kyphosinae, Microcanthinae and Scorpidinae) form a clade or should be considered separate families sensu Randall (2007), and in doing so resolve the sister group to the sea chubs.

2. Materials and methods 2.1. Taxonomic sampling and morphological comparison 6

All 12 valid species of Kyphosus (Knudsen and Clements, 2013a) were sampled for this study. In addition, we included several samples of xanthic specimens of Pacific K. sectatrix from the Kermadec and Hawaiian Islands (Suppl. Matr. Table 1), and examined the morphology of the supposed endemic xanthic K. lutescens from the Revillagigedo Islands (considered a synonym of K. sectatrix by Knudsen and Clements (2013a). A sample from Hawaiian K. sectatrix – identified as a supposed K. sandwicensis (Suppl. Matr. Table 1) – was also included, to shed more light on which kyphosid species occur in Hawaii now that comparison of morphological variation in museum material has revealed that the holotype of K. sandwicensis is indistinguishable from specimens of K. elegans (Knudsen and Clements 2013a). Samples obtained also include Hermosilla (Kyphosus azureus), Neoscorpis and Sectator (Kyphosus ocyurus). A broad range of samples from different parts of the Atlantic Ocean together with samples covering the morphological variation in Kyphosus (see Knudsen and Clements, 2013a) ensures that sampling also covers the variation found in the Atlantic species proposed by Sakai and Nakabo (2014). Furthermore, to examine the consistency of morphometric and meristic data between Knudsen and Clements, (2013a) and Sakai and Nakabo (1995, 2004, 2006, 2008, 2014), we also compared morphometric and meristic data of type material (Suppl. Matr Table 3, 7-10 and a Suppl. Matr. Fig. 8). Samples from Atypichthys, Girella, Graus, Kuhlia, Microcanthus, Oplegnathus, Rhyncopelates and Scorpis were included as representatives of families considered close to Kyphosidae (Near et al., 2012a, 2013; Yagishita et al., 2002, 2009), comprising a total of 115 tissue samples for the molecular study where 48 were vouchered specimens (Table 1, Suppl. Matr). For the phylogeny inferred from morphology we examined 584 museum specimens (see Knudsen and Clements 2013a for a list of museum specimens) representing Atypichthys, Bathystethus, Doydixodon, Dichistius, Girella, Graus, Kyphosus, Labracoglossa, Medialuna, Microcanthus, Neatypus, Scorpis and Tilodon. The ingroup for this study is here defined as Kyphosidae sensu Randall (2007) (i.e.

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Kyphosinae sensu Nelson 2006), and comprises Hermosilla, Kyphosus, Neoscorpis and Sectator. Unless otherwise specified in the following, all subsequent mention of Kyphosidae follows this definition, and Hermosilla and Sectator are considered junior synonyms of Kyphosus (see Knudsen and Clements, 2013a). Specimens were collected by colleagues or us by spear while diving, obtained from fish markets, or through loans of museum specimens (Suppl. Matr. Table 1). Tissue samples were cut from fin, muscle or gill filaments, and stored in 96% ethanol at 5°C. Where possible, voucher specimens for tissue samples were retained, however, due to remote collection localities and difficulties associated with storage and transport of large specimens, not all samples have corresponding voucher specimens. The voucher specimens are stored at AIM, AMS, KU, NMV, NMNZ, WAM and ZMUC; institutional abbreviations follow Fricke and Eschmeyer (2013). Species identification followed Tortonese (1986), Allen and Swainston (1988), Sakai and Nakabo (1995, 2004, 2006, 2008), Allen (1997), Knudsen and Clements (2013a), Kuiter, (2000), Randall (2007) and Randall et al. (1997), and by comparison with type material in museum collections stored at AMS, BMNH, BPBM, MCZ, MNHN, SMNS, QM, USNM, WAM and ZMB, and with additional material from AIM, AMS, BMNH, LACM, MNHN, NMV, NMNZ, QM, USNM, WAM, ZMB and ZMUC. The taxonomic identity of the groups obtained in the molecular analysis was determined by first matching vouchered specimens with museum type material, and then testing for correspondence between clusters of molecular and morphological variation following the approach sketched out by Feulner et al. (2007). This procedure allowed identification of diagnostic morphological characters for all species of kyphosids, and allowed us to test the species-hypotheses presented by Knudsen and Clements (2013a). Testing species-hypotheses using this integrative approach is in accord with the recommendations put forward by Pante et al. (2015).

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2.2. Selection of outgroup taxa and molecular markers Sequences from other perciform fish (Suppl. Matr. Table 1 and 5) obtained from the National Centre for Biotechnology Information (NCBI) GenBank were included to resolve the relationships between the various genera thought to be closely related to Kyphosus. The outgroup genera used were Atypichthys, Girella, Graus, Kuhlia, Medialuna, Microcanthus, Oplegnathus, Rhyncopelates and Scorpis. We also

included species representing

Acanthuridae, Centrolophidae, Chaetodontidae, Haemulidae, Kuhliidae, Monodactylidae, Oplegnathidae,

Percichthyidae,

Scatophagidae,

Scombridae,

Terapontidae

and

Tetraodontiformes to resolve relationships with related families (Near et al., 2012a, 2013; Nelson, 2006; Yagishita et al., 2009; Sanciangco et al., 2016). Extraction of DNA was performed either with the DNeasy blood and tissue extraction kit (QIAGEN, Bio-Strategy Ltd, New Zealand), following the supplied protocol, or by a phenol-

from mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were selected on the basis that (i) they allowed the incorporation of comparative sequences from the NCBI GenBank database, and (ii) these markers had proved useful in previous studies on perciform relationships (e.g., Clements et al., 2004, Davis et al., 2012; Quenouille et al., 2004; Smith et al., 2008). Partial and overlapping fragments of mtDNA genes were amplified for all samples and the sequences subsequently assembled to comprise one long fragment of 4180 bp, covering the 5’ end of the cytochrome b gene (cytb) to near the 3’ end of the 16s gene, and covering the four tRNA-genes in between. The mtDNA markers covered in this long fragment comprises: 5’ partial end of cytb, the control region (cr), the small ribosomal subunit (12s) and the partial 3’ end of the large ribosomal subunit (16s), and tRNA-Thr, tRNA-Pro, tRNAPhe and tRNA-Val (Meyer, 1993). Partial fragments of three nDNA genes, the putative uncharacterized protein Tmo-4c4 (tmo4c4) (Streelman and Karl, 1997) and the recombinase activating genes 1 and 2 (rag1 and rag2), were amplified for all sampled taxa (see Suppl.

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Matr. Table 5). Amplifications were performed with the primers listed in Suppl. Matr. Table 6.

2.3. PCR amplification and sequencing Small aliquots of extracted DNA (1-2 µL) were used as template for polymerase chain reaction (PCR). A touch down approach (Palumbi, 1996) was applied to optimize amplification of targeted gene-sequences. The reactions were standardised to a volume of 25 µL, containing 1 µL template (diluted to 100 ng µL-1, or less – depending on the final concentration from the initial extraction), 2.5 µL [1 x] reaction buffer, 1.25 µL [1.25 mM] MgCl2, 0.1 µL [80 µM] deoxynucleotide triphosphates, 0.05 µL [0.01 U µL -1] Taq polymerase and 18.15 µL double distilled RNase free water. All PCRs were performed on a Biometra T1 Thermocycler with an annealing temperature (T m) according to the primers listed in Suppl. Table 6. The amplifications were set with an initial denaturation m

-

m

1.6 % agarose gel tainted with either Gel Red or Ethidium bromide to check whether the PCR had yielded the expected product. Double stranded PCR products were purified for sequencing by using Exo-Shrimp Alkaline Phosphatase (SAP) -IT (USB). Sequencing reactions were completed with BigDye TM terminator chemistry v. 3.1. Sequencing kit. The sequencing was performed in both directions using the same primers as used for amplification. The single stranded products were then purified with CleanSeq, and finally run on a 16 capillary ABI3100 DNA Sequencer (Applied Biosystems Inc.).

2.4. Sequence analysis and reconstruction of phylogeny

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Sequences were visually inspected using Geneious 5.6 (Drummond et al., 2010). Sequence reads from both directions were assembled and ambiguities in nDNA sequences were coded as undetermined using IUPAC codes. Sequences were aligned in Geneious (Drummond et al., 2010) using MAFFT 6.822 (Katoh and Toh, 2010). Primer regions were identified and removed from the alignment. Alignments used for the phylogenetic analysis are supplied in the xml- and nexus-files in the supplementary material. The level of saturation on nucleotide sites in all partial sequences was estimated by comparing transition-transversion ratios across codon positions by obtaining an index of substitution saturation (Iss) with the software DAMBE v. 5.2.57 (Xia and Xie, 2001). Issvalues significantly larger than Iss-critical values indicate that nucleotide sites are saturated with substitutions. Tests of saturation were performed on all partial fragments of mtDNA and nDNA markers on all codon positions independently on the different datasets with different outgroup representatives. Codon positions that exhibited saturation were recoded as purinespyrimidines (RY-coding) in order to increase the phylogenetic signal on internal branches relative to external branches (Philips, 2001; Philips and Penny, 2003; Praz and Packer, 2014). The mtDNA control region was not included as discrepancy in alignments made it difficult to identify homologous sites in this region. PartitionFinder (Lanfear et al. 2012) was used to identify the best combination of gene partitions and optimal substitution models among the 56 models that are tested in PartitionFinder. A greedy search under the AICc (Akaike 1974) model selection was applied for all three nDNA genes and the mtDNA markers, and all codon partitions for each coding marker. PartitionFinder found the GTR+G+I model as best fit to some of the partitions, but only the GTR+G models were applied, as the gamma proportion of the model also models rate heterogeneity (Stamatakis, 2006). The same best-substitution model was inferred for 12s and 16s, and these two partitions were hereafter treated as a single partition. This partitioning scheme gave a total of seven partitions (mtDNA-12s-16s, mtDNA-tRNAs, mtDNA-cytb-

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codon-pos.1, mtDNA-cytb-codon-pos.2, nDNA-rag1, nDNA-rag2 and nDNA-tmo4c4) for the input files analysed in the software MrBayes v.3.2.2 x64 (Ronquist et al. 2012) and later on in BEAST v. 1.8.0 (Drummond et al., 2012). Even though a subdivision per codon position is preferred, as distinguishing between different codon positions when inferring phylogenies can affect final topology, especially when using Bayesian inference (Bofkin and Goldman, 2007; Li et al., 2008). Because the nDNA-rag2 partitions had an insufficient level of nucleotide variation to stabilize on all parameters with a HKY model in BEAST, we tried to combine all the nDNA 3-rd codon positions (i.e. the 3-rd codon positions from nDNA-rag1, nDNA-rag2 and nDNA-tmo4c4) into one partition, and join the 1st and 2nd codon positions from all three nDNA-markers in a second partition. However, the partition comprising the 1st and 2nd codon positions failed to stabilize in BEAST with an HKY model. Since rag1 and rag2 both encode enzymes that work together in the adaptive immune response in vertebrates, and likely share the same evolutionary origin (Kapitonov and Jurka, 2005), we concluded that rag2 was best placed in a partition with rag1, rather than placing rag2 in a partition with tmo4c4 or the mtDNA markers. We therefore analysed the nDNA-rag1 and nDNA-rag2 as a combined partition. We also analysed the four mtDNA-tRNA’s as one partition due to the limited number of informative nucleotide positions in these partitions, which otherwise would result in low values in the Effective Sample Size (ESS) of the parameters sampled in the ensuing Bayesian analysis. Initial attempts in BEAST with the GTR+G models failed to stabilize on all parameters, even though we performed the BEAST analysis with more than 2500 mio. generations when multiple log-files were combined. To ensure that sufficient high ESS values could be obtained for all priors, we therefore used HKY+G models in the analysis performed in both MrBayes and BEAST, rather than the GTR+G models suggested by PartitionFinder for the three nDNA markers, the mtDNA cytb and the four tRNAs, (see Suppl. Matr. Table 4). Although the analysis in MrBayes with the more complicated GTR+G models did stabilize on all parameters, we chose to apply the same substitution models (see Suppl.

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Matr. Table 4) in both MrBayes and BEAST to allow for a direct comparison of the final topologies. Phylogenetic trees were constructed with the software MrBayes v. 3.2.2 x64 (Ronquist et al., 2012) using a concatenated dataset from the 12s, 16s, cytb, the four tRNAs, and the three individual nDNA markers (this is referred to as dataset A1). Two additional datasets comprising only the mtDNA data (dataset A2) and only the three nDNA markers (dataset A3) were prepared separately. Finally, three datasets were prepared from each of the three nDNA markers individually (datasets A4-A6). Dataset A1-A6 comprised outgroup representatives from Girellidae, Kuhliidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae. A second version of dataset A1 (i.e. dataset A7) was prepared for an analysis using BEAST v. 1.8.0 (Drummond et al., 2012) and for an analysis using MrBayes v. 3.2.1 x64. The A7 dataset comprised taxa from Acanthuroidei, Balistidae, Centrolophidae, Chaetodontidae, Cichlidae, Girellidae, Haemulidae, Kuhliidae, Microcanthidae, Oplegnathidae, Percichthyidae, Scatophagidae, Scombridae, Scorpididae and Terapontidae as outgroup representatives. The A7 dataset only had single representatives for each taxon, whereas dataset A1-A6 had multiple representatives for both Kyphosidae and outgroup species from multiple oceanic regions. The MrBayes topologies from the separate datasets (dataset A1-A6) were compared by estimating the Robinson-Fould metric for the individual topologies (Robinson and Fould, 1981) using the software HashRF (Sul and Williams, 2008) (Suppl. Matr. Table 2). A decrease in the Robinson-Fould metric indicates higher similarity in the topologies compared. The Robinson-Fould metric measures the distance between the phylogenetic trees, and is a measure for how many operations it would require to convert one topology in to the other. As the topologies became less similar when the molecular markers were analysed separately (A2-A6) (Suppl. Matr. Table 2) compared to when all markers were analysed in a single combination (A1), a combined analysis of all molecular markers with an expanded outgroup

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representation (dataset A7) was preferred in the subsequent MrBayes and BEAST analysis. The taxon representation for these outgroup perciform families was limited to two or three extant representatives per family or order (dataset A7), as homologous NCBI sequences only were available for very few taxa in the perciform families most closely related to Kyphosidae.

2.5. Construction of phylogenies from sequence data The best substitution models inferred from PartitionFinder (Lanfear et al., 2012) was not applied for all partitions, due to low ESS values on some priors. Instead, more simple substitution models were applied in the MrBayes and the BEAST analysis for some of the partitions (Suppl. Matr. Table 4). The MrBayes analyses were run for four million generations, sampling every 400 generations, with four runs for each of the eight chains. The log files from the MrBayes analyses were combined using LogCombiner v. 1.8.0 (Drummond et al., 2012) and analysed in Tracer v. 1.6.0 (Rambaut and Drummond, 2007) to ensure that proper mixing and stability was obtained. Stability in the analysis was reached after 100,000 generations. For each run of the four runs the first 1000 trees of the 10,000 trees sampled were discarded (i.e. burn-in set to 10%) to produce the final consensus tree from 36,004 trees. These settings were applied to all MrBayes analysis of all datasets (dataset A1-A7). LogCombiner v. 1.8.0 (Drummond et al., 2012) was used to combine 36,004 trees, which then were analysed using TreeLogAnalyzer 1.8.0 (Drummond et al., 2012). The MrBayes inferred tree from the analysis of the combined mtDNA and nDNA dataset (dataset A7) with the expanded outgroup representation was used as a starting Newick tree for the subsequent BEAST-analyses by making the starting tree completely bifurcating by resolving eventual polytomies arbitrarily in Mesquite v. 2.75 (Maddison and Maddison, 2011), and importing the topology in BEAUti v. 1.8.0 (Drummond et al., 2012) together with the nucleotide matrix. Speciation rates were modelled with a birth-death model with incomplete sampling (Stadler, 2009), with a sample probability set as a beta prior (shape 2 and shapeB of

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100) to reflect that less than 10% of all extant outgroup species were included in this study. Priors were kept as gamma between 0 and infinity for all nucleotide substitution parameters with a GTR model. All priors for the mean of the uncorrelated lognormal relaxed clock were set as exponential with a mean of 1 and offset 0, to make it less likely that the clock rate inferred by BEAST would end up being higher than 1% substitutions per site per million years. An upper limit of 1% substitutions per site per million years is preferred, as chondrichthyans and teleosts have not been recorded with a divergence rate any higher than 1% substitutions/site/Myr (Martin and Palumbi, 1993). The xml-file was altered to allow nucleotide ambiguities to be interpreted. The BEAST analysis on dataset A7 was set to run for 160 million generations, sampling every 64,000 generations and returning 2501 trees. The resulting xml-file was run 8 times in parallel using multiple cpus on the Slurm cluster provided by the Auckland University e-research group. All 8 independent runs were finally combined using LogCombiner v. 1.8.0. The results from the 8 combined log-files were examined using the software Tracer v. 1.6.0. LogCombiner v. 1.8.0. was used to combine the 2501 trees from each of the 8 independent runs with a burn-in of 10% (i.e. 16 million generations) to generate a tree-file with 18,008 trees, this tree-file was used in TreeAnnotator 1.8.0 (Drummond et al., 2012) without a burn-in to create the consensus-tree based on 18,008 trees. The 18,008 trees was then analysed using TreeLogAnalyzer 1.8.0 (Drummond et al., 2012). To check the priors in our analysis for cross-prior influence we ran the analysis on empty alignments under the same settings as the dataset with nucleotide information. If any of the priors were defined improperly they will return different distributions of ESS values on the individual priors compared to when the analysis contains sequences (Sanders and Lee, 2007).

2.6. Phylogenetic analysis of morphological data The morphological characters examined on museum specimens included both meristic and

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morphometric characters (Suppl Matr. table 3, and suppl. matr nexus-files). All morphometric characters were first standardised to %SL to avoid discrepancy rising from allometric differences. Each character was divided in to a maximum of 10 bins. For each range of character-states a median character value was used in the subsequent parsimonious analysis in PAUP*4.10b (Swofford, 2002). As we did not have sequence data from all mtDNA and nDNA molecular markers from dichistiid species, this analysis of morphological characters allowed us to estimate the relationship with the other families included in this study (i.e. Girellidae, Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae). The final matrix comprised 43 taxa, prepared from examining 584 specimens for 152 characters (the matrix and character list is supplied in the suppl. matr. nexus-file). The analysis in PAUP*4.10b used a heuristic search approach with tree bisection reconnection (TBR) branch-swapping, and a bootstrap analysis performed with 1000 replications (Felsenstein, 1985) with an automatic increase to 100 replicates. The resulting topology was visualized using FigTree 1.4.0 (Rambaut, 2009).

2.7. Comparison of topologies using the stepping-stone approach To test which of the families could be considered as the sister-group to Kyphosidae we constrained various taxon sets as monophyletic in the BEAST analysis. We reran the BEAST analysis with a subsequent multiple stepping-stone (SS) analysis to obtain a marginal likelihood estimation (MLE), allowing us to compare various constrained topologies under a relaxed clock model (Drummond et al., 2006) and thus introduce extra parameter space for inferring the relationships between the families. The SS analysis was performed with seven different constraints – set in BEAUti, with Kyphosidae constrained to be monophyletic with Girellidae (1), Kuhliidae (2), Microcanthidae (3), Oplegnathidae (4), Terapontidae (5), or Scorpididae (6), or to be unconstrained (7). All seven constrained SS analyses were each run for 50 path steps, 5 mio. chains, and logging the likelihood for every 500th iteration. All seven

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SS analysis were each set to run four times in parallel to provide four independent MLE values per constrained topology. The MLE values obtained from the SS analysis indicate how the model and the constrained topology are matching the data. The most positive MLE value will indicate which topological constraint matches the data best. An average for all for MLE values was calculated, and then used to calculate ln Bayes Factors (Kass and Raftery, 1995) between the different constrained topologies, to see how different the seven constrained models were from each other (Table 1).

2.7. Test of a monophyletic Kyphosidae sensu Nelson (2006) We performed partitioned maximum-likelihood (ML) analysis on the different mtDNA and nDNA markers using RAxML v. 7.0.4 (Stamatakis, 2006) on both a topology constrained according to a monophyletic Kyphosidae (i.e. Kyphosidae sensu Nelson, 2006) (H0) (i.e. Girellidae, Kyphosidae, Microcanthidae and Scorpididae) and an unconstrained topology that allowed Girellidae, Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae to group together (H1), reflecting the topologies obtained from the unconstrained MrBayes analysis (Fig. 2) and the BEAST analysis (Fig. 3). The constrained topology (H0) would reflect the close relationship among these taxa indicated by larval morphology (Freihofer, 1963; Johnson and Fritzsche, 1989; Neira et al., 1997) which led to the monophyletic Kyphosidae proposed by Nelson (2006). The ML analysis were performed with a GTRGAMMA model for the six datasets (A1-A6), using first the –E option and –q option to specify partitions and the –g option for constraining the topology, and finally comparing the best topologies using the –f g and the –z options. We used the likelihood-based approximately unbiased (AU) test implemented in the software package CONSEL v. 0.1i (Shimodaira and Hasegawa, 2001) to compare the best ML- trees obtained (Table 3). A low likelihood from an ML analysis signifies the best tree, the difference in ln-likelihood indicates the level of differences between the constrained topologies, and the AU test is used to check

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whether the difference in likelihood values is significant.

3. Results 3.1. Molecular markers used in the phylogeny The assembled mtDNA dataset consisted of 3167 bp from 126 individuals. The mtDNA sequence data covered 310 bp of 3’ end of cytb, 985 bp of 12s and 1560 bp of 16s, and 97 bp of tRNA-Thr, 73 bp of tRNA-Pro, 70 bp of tRNA-Phe and 72 bp of tRNA-Val. The nDNA sequence data consisted of 2093 bp comprising 524 bp of tmo4c4, 823 bp of rag1 and 746 bp of rag2. Inspection of sequences did not reveal any unexpected stop codons, frame shifts or abrupt indels in any of the coding mtDNA- and nDNA-sequences. A partial fragment of cytb (Ksp_Be_060, insert accession number) was obtained from a specimen of K. cinerascens collected in the tropical Western Atlantic. This sequence is not included in the topologies presented in this study, as the phylogenetic analyses were based on multiple mtDNA and nDNA markers, but the cytb fragment (Ksp_Be_060, insert accession number) from the Atlantic individual matches the cytb fragment in the Indo-Pacific K. cinerascens (Suppl. Matr. Tabel 1). Individual preliminary phylogenetic analysis of each marker placed every species within the expected genus based on identification at point of capture. The nucleotide sequences were registered at NCBI GenBank with accession numbers (Suppl. Matr. Table 1). The mtDNA fragments were registered as one long continuous fragment per individual (Suppl. Matr. Table 1). Substitution saturation was indicated only by significant Iss.c-values obtained for cytb third codon positions in the datasets with broad outgroup representation (dataset A7, with Perciformes and Tetraodontiformes), and the third codon cytb positions was therefore RYrecoded in the analysis with the comprehensive outgroup representation (dataset A7). No saturation was detected in any of the nDNA markers regardless of which representative outgroups were included in the dataset (i.e. Perciformes and Tetraodontiformes), and no 18

saturation was detected in cytb third codon positions in the dataset with a limited outgroup representation (dataset A1-A6). The third codon cytb positions were therefore left unchanged in these datasets. The ESS values obtained from the BEAST analysis and the BEAST analysis with the SS analysis were each >200 for all priors. The combined log file from all of the parallel runs performed on the empty alignment returned posteriors distributed within the intervals set for original prior distributions in BEAUti, indicating that no priors were set improperly.

3.2. Outgroup relationships The topologies retrieved from the Bayesian analysis of all datasets (A1-A7) resolved Kyphosidae as monophyletic (Fig. 2, 3, Suppl. Matr. Fig. 1-6). The tmo4c4 nDNA (dataset A6) was unable to resolve relationships within Kyphosidae, but placed the girellids as sister group to the kuhliids, kyphosids, microcanthids, oplegnathids, scorpidinids and the terapontids (Suppl. Matr. Fig. 6). Kyphosidae sensu Nelson (2006) is comprised of girellids, kyphosids, microcanthids and scorpidinids, but none of the topologies retrieved in the present study were consistent with such a clade. Rather, four different topologies were resolved: (a) a lineage containing Kuhliidae, Microcanthidae, Oplegnathidae and Terapontidae resolved as the sister lineage to Kyphosidae + Scorpididae, with Girellidae as the sister group to this combined clade (Fig. 3), (b) Kuhliidae, Microcanthidae, Oplegnathidae, Terapontidae and Scorpididae combined in a clade that was sister to a clade containing Girellidae and Kyphosidae (Suppl. Matr. Fig. 1-3 and 5), (c) Girellidae was sister to a polytomic clade containing Microcanthidae, Kuhliidae, Oplegnathidae, Scorpididae and Terapontidae, and this was itself sister to Kyphosidae (Suppl. Matr. Fig. 6), or (d) Girellidae, Kuhliidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae fell in a clade that was sister to Kyphosidae (Fig. 2 and Suppl. Matr. Fig. 4). Although girellids, kyphosids, microcanthids and scorpidids formed a clade in all topologies, kuhliids, oplegnathids and terapontids always

19

also belonged to this clade (Fig. 2 and 3, and Suppl. Matr. Fig. 1-6). The topology with respect to Girellidae, Kuhliidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae varied depending on the molecular marker examined and the combination of the molecular markers used (Suppl. Matr Fig. 1-6). Bayesian analysis of the combined nDNA markers (dataset A3) (i.e. rag1, rag2 and tmo4c4) resolved the GirellaGraus-clade as the sister to Kyphosidae (Suppl. Matr. Fig. 3). A MrBayes analysis of these three nDNA markers independently (dataset A4, A5 and A6) of each other could not resolve the relationship between the families Girellidae, Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae. Support for all MrBayes topologies and for BEAST-inferred topologies was generally high across all branches, with the lowest support values situated around the split between Girellidae and Scorpididae-Microcanthidae (Fig. 2 and 3, and Suppl. Matr. Fig. 1 and 2). Comparisons were made between the topologies from the six different datasets (A1-A6), both constrained according to a monophyletic Kyphosidae sensu Nelson (2006) (H0) and unconstrained with respect to the relationships among a monophyletic Girellidae, Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae (H1). For five out of six datasets (A1, A2, A3, A5 and A6) the best likelihood values (-ln L) from the ML analysis were obtained for the unconstrained topology with all seven families forming a clade (H1) (Table 3). Only the nDNA rag1 dataset (dataset A4) found the -ln L value from the ML analysis for the constrained topology (i.e. Kyphosidae sensu Nelson, 2006) more likely than the -ln L value for unconstrained topology (Table 3), but the AU-test showed that ML -ln L for this partition (A4) did not differ significantly between the constrained and the unconstrained topology. The ML -ln L value for an unconstrained topology (H1) was 40003.9 for the full dataset (A1) and 27171.9 for the mtDNA partition (A2) (Table 3), and both likelihood values differed significantly from ML -ln L values for the constrained topologies (H0). A monophyletic Kyphosidae sensu Nelson (2006) (H0) was thus not favoured by any

20

significant likelihood values from the data partitions analysed (Table 3), although only the full dataset (A1) and the mtDNA partition (A2) were able to reject a monophyletic Kyphosidae sensu Nelson (2006) (H0). The nDNA based datasets (A3-A6) did not yield significantly different likelihood values for the constrained (H0) and unconstrained (H1) topologies. A comparison of the constrained topologies analysed in an SS approach in BEAST showed that the best MLE (-48894.2) was obtained with Scorpididae constrained as the sister group to Kyphosidae. The next best topology (-48897.0) had Girellidae constrained as sister to Kyphosidae (Table 1), but this topology was an lnBF=16 worse than Scorpididae as sister to Kyphosidae.

3.3. Ingroup relationships The monotypic Neoscorpis was consistently found to be the sister group to Kyphosus (Fig. 2 and 3, and Suppl. Matr. Fig. 1-6). There was a strongly supported relationship between Kyphosus azureus and K. cornelii, which together formed the sister lineage to the clade containing the remaining Kyphosus species, including Kyphosus ocyurus (Fig. 2 and 3). Within this latter clade the temperate species K. gladius and K. sydneyanus together formed the sister group to the tropical/subtropical Kyphosus species (i.e. other than K. cornelii). The sub-tropical and temperate species K. azureus, K. cornelii, K. sydneyanus, K. gladius and Neoscorpis can thus be considered sister to the sub-tropical/tropical species of Kyphosidae (i.e. K. bigibbus, K. cinerascens, K. elegans, K. hawaiiensis, K. ocyurus, K. sectatrix and K. vaigiensis). Specimens of K. sectatrix from the Atlantic Ocean fell out in a clade with K. pacificus from the Pacific and Indian Oceans, as sister-species to K. hawaiiensis. The presence of Atlantic specimens of K. bigibbus (previously regarded as restricted to the IndoPacific) indicates that these species are more widespread than currently perceived (Suppl. Matr. Fig. 1-6) (Knudsen and Clements, 2013a). No support was found in Kyphosus for

21

geminate species with a separation across the Panamanian Isthmus as proposed by Jordan (1908) and Thomson et al. (2001), as K. elegans was sister to K. cinerascens rather than K. sectatrix (Fig. 2 and 3, and Suppl. Matr. Fig. 1, 2, 3 and 5). Kyphosus cinerascens was recently recorded from the western Atlantic Ocean (Knudsen and Clements, 2013a), and could perhaps reflect an alternative geminate species to K. elegans. Kyphosus analogus from the east Pacific Ocean, K. vaigiensis from the west Pacific and Indian Ocean, and K. incisor from the Atlantic Ocean all fell out as a single clade (Fig. 2 and 3, and Suppl. Matr. Fig. 1 and 2). Specimens identified upon collection as xanthic K. pacificus from the Kermadec Islands and misidentified as K. sandwicensis from Hawaii (e.g. Randall 2005, 2007; Randall and Cea, 2010, see Knudsen and Clements 2013a: 41) fell out together with both K. sectatrix from the Atlantic (Suppl. Matr. Fig. 1-3) and K. pacificus from Japan (identified by K. Sakai), a result that is also supported by morphology (Knudsen and Clements, 2013a). Kyphosus ocyurus was the sister species to K. vaigiensis (Fig. 2 and 3), and comprised a tropical clade together with the two other exclusively tropical species K. cinerascens and K. elegans and the antitropical K. bigibbus. Synonyms and valid names in Knudsen and Clements (2013a) are consistent with the present phylogenetic results, since (a) several taxa (i.e. K. bigibbus, K. sectatrix, K. vaigiensis) are here shown to include what were previously considered to be regional endemics (Suppl. Matr. Fig. 1, 2 and 3), and (b) Hermosilla and Sectator sit within Kyphosus. Our comparison of meristic data from our taxonomic revision (Knudsen and Clements, 2013a) and the meristic data provided by Sakai and Nakabo (1995, 2004, 2006, 2008, 2014) (Suppl. Matr. Table 3) revealed differences between studies in counts for Atlantic specimens, but not for Indo-Pacific specimens. Our total gill raker counts for Atlantic K. bigibbus (19-23) and K. sectatrix (22-25) (Knudsen and Clements, 2013a) were lower than those reported by Sakai and Nakabo (2014) for Atlantic K. atlanticus (23-26) and K. bosquii (24-27) (Suppl. Matr. table 8). Counts of longitudinal scale rows from Atlantic kyphosid specimens also differed between the two studies. Sakai and Nakabo (2014) reported a lower number of

22

longitudinal scale rows in the species they described as K. atlanticus (50-56) than we found in Atlantic K. bigibbus (54-67) and K. sectatrix (60-69) (Suppl. Matr. table 9).

4. Discussion We examined the evolutionary relationships of sea chubs to other taxa using a combination of mitochondrial and nuclear markers. A close relationship between Kyphosidae and Girellidae, Microcanthidae and Scorpididae is apparent, but Kuhliidae, Oplegnathidae and Terapontidae are also closely related to Kyphosidae (Fig. 2 and 3, and Suppl. Matr. Fig. 1-6). The complete taxon sampling in the present study allowed us to generate a more complete picture of kyphosid relationships than previous studies have been able to provide (e.g. Yagishita et al., 2002, 2009; Sanciango et al., 2016). From the topologies inferred in this study it is clear there are only 12 valid species of Kyphosus, a finding inconsistent with the taxonomy of Sakai and Nakabo (2014). Although we did not have Atlantic material of K. cinerascens for the phylogenetic analysis, we do have mtDNA support for the Atlantic specimen being conspecific with the Pacific populations (Ksp_Be_060, insert accession number), corroborating our previous finding that K. cinerascens is present in the Atlantic Ocean (Knudsen and Clements, 2013a).

4.1. Relationships of Kyphosidae to other taxa A combined analysis of the seven gene-dataset (dataset A7) (Fig. 2 and 3) showed that Kyphosidae forms part of a clade containing Girellidae, Kuhliidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae. All inferred topologies (Fig. 2 and 3, and Suppl. Matr. Fig. 1-6) thus support previous studies demonstrating that Kyphosidae sensu Nelson (2006) is paraphyletic (Near et al., 2012a; Yagishita et al., 2002, 2009). Furthermore, our results do not support the hypothesis that the RLA-10 nerve pattern establishes arripidids, girellids, kyphosids, microcanthids and scorpidinids as a clade (Freihofer, 1963; Neira et al., 23

1997). The close relationship between Girellidae and Scorpididae suggested by Johnson (1984) based on a distinctive shared larval form was not corroborated in our BEAST topology (Fig. 3). The nDNA alone (dataset A3-A6) did not have adequate phylogenetic signal to reject or support a monophyletic Kyphosidae sensu Nelson (2006) (H0), but the AU-test (Table 3) showed that inclusion of the mtDNA (dataset A1 and A2) favoured a Kyphosidae sensu Randall (2005), and only by inclusion of the mtDNA data was it possible to reject a monophyletic Kyphosidae sensu Nelson (2006) (H0). The AU-test can perhaps be argued as limited for discriminating between the most reliable topologies, but it is important to note that both the BEAST and MrBayes topologies independently supported Kyphosidae sensu Randall (2005) (Fig. 2 and 3). Even though we used the same taxa, gene markers, partitions and substitution models (Suppl. Matr. Table 4) in the MrBayes and BEAST analyses, these two approaches yielded distinct topologies (Fig. 2 and 3, Table 1 and 2). However, obtaining different topologies from MrBayes and BEAST is not uncommon (Ludt et al., 2015). This is most likely due to the extra parameter space introduced by the relaxed clock in BEAST (Drummond et al. 2006), which allows for a more comprehensive search among different topologies. Further support for the topology obtained with BEAST (Fig. 3) was obtained through the SS analysis, which favoured Scorpididae as the sister to Kyphosidae (Table 1). This is consistent with the finding that simulation studies show the relaxed clock model to be the most effective at recovering the correct tree (Drummond et al., 2006; 2012). In contrast, the topology obtained from MrBayes (Fig. 2) yielded a clade containing Girellidae Kuhliidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae as sister to Kyphosidae. However, neither the BEAST analysis nor the parsimony analysis of the morphological data (Suppl. Matr. Fig. 7) favoured a close relationship between Girellidae and Kyphosidae, supporting Scorpididae as the most likely sister family to Kyphosidae. The South African taxon Dichistius displays strong affinities with both kyphosids and

24

scorpidids in osteology, larval characters, and in sharing pattern 10 of the ramus lateralis accessorius facial nerve (Johnson and Fritzsche, 1989; Leis and van der Lingen, 1997; Neira et al., 1997; Smith, 1935) and in molecular variation (Sanciangco et al., 2016) (Fig. 1H). Our phylogenetic analysis of morphological characters (Suppl. Matr. Fig. 7) suggested that Dichistiidae is more closely related to Girellidae than the other families examined (i.e. Kuhliidae, Kyphosidae, Microcanthidae, Oplegnathidae, Scorpididae and Terapontidae), although its precise phylogenetic relationships are unclear from our study. Previous molecular studies (e.g. Betancur-R et al., 2013, 2014; Sanciangco et al., 2016) also found that Kyphosidae was close to Kuhliidae, Oplegnathidae and Terapontidae, but these did not include Scorpididae, resolved in the present study as the sister clade to Kyphosidae (Fig. 3, table 1). The recent analysis of Sanciango et al. (2016) resolved Dichistiidae as sister to a clade containing Kyphosidae, Oplegnathidae, Kuhliidae and Terapontidae (Fig. 1H), consistent with the more distant relationship between Kyphosidae and Dichistiidae retrieved in the analysis of morphological data in the present study.

4.2. Relationships within Kyphosidae Neoscorpis lithophilus is sister to the rest of the kyphosids, and bears a strong resemblance in body shape to Scorpis as reflected in its original description (Gilchrist and Thompson, 1908). The reduced number of dorsal fin spines, absence of a serrated edge on the preoperculum (serrated in Scorpis), and difference in tooth shape in N. lithophilus led Smith (1931) to erect Neoscorpis. The zebra-perch sea chub K. azureus, which occurs from Monterey Bay to the Gulf of California in the eastern Pacific Ocean, is sister species to K. cornelii, which occurs from Cape Leeuwin to Coral Bay in Western Australia (Fig. 2 and 3, and Suppl. Matr. Fig. 7). The morphology of K. azureus and K. cornelii reflects the close relationship inferred by the molecular data, as both share having a low number of vertebrae (25 in K. cornelii and K. azureus vs. 26 in other species of Kyphosus), a low number of gill

25

rakers (17-21 in K. cornelii and K. azureus vs. 19-32 in other species of Kyphosus), and the lack of scales on the interorbital and preorbital region (other Kyphosus have scales both dorsally and on the interorbital region). This relationship prompted the decision to treat Hermosilla as a junior synonym of Kyphosus (Knudsen and Clements, 2013a). All molecular topologies derived from both combined and individual molecular markers where multiple representatives per species were included cluster K. sectatrix from the Atlantic together with the Indo-Pacific K. sectatrix (Suppl. Matr. Fig. 1-6), and this clade of K. sectatrix was always sister clade to K. bigibbus (Fig. 2. and 3). This corroborates the clustering of specimens identified as K. atlanticus, K. pacificus, K. sectatrix and Pimeleptrus gallveii based on morphological variation in gill rakers, interorbital width and pectoral fin length (Knudsen and Clements, 2013a, Fig. 6, and Suppl. matr. Fig. 8). Overall, this lends further support for the circumglobal distribution of K. sectatrix, as acknowledged by Gilbert (2015), Knudsen and Clements (2013a) and Mannino et al. (2015). Examination of museum material showed that the xanthic Revillagigedo Islands endemic K. lutescens is also a synonym of K. sectatrix, which also has xanthic variants at the Hawaiian and Kermadec Islands (Knudsen and Clements, 2013a). Our molecular analysis supported this view (Fig. 2 and 3, and Suppl. Matr. Fig. 1-6). The recent designation of a neotype for Kyphosus sectatrix (Gilbert et al., 2015) does not challenge the decision to treat Indo-Pacific and Atlantic populations of K. sectatrix as a single species (Knudsen and Clements, 2013a), consistent with molecular and morphological data (Suppl. Matr. Fig. 1-6, 8). Kyphosus incisor (Cuvier, 1831), K. analogus (Gill, 1862) and K. vaigiensis (Quoy and Gaimard, 1825) grouped together in all topologies, and without any distinct phylogenetic separation (Fig. 2 and 3, and Suppl. Matr. Fig. 1, 2 and 3). This and the lack of any morphological characters separating these three taxa indicate that these should be regarded as a single and widely distributed species, recognised by the senior synonym K. vaigiensis (Quoy and Gaimard, 1825) as proposed in the recent taxonomic revision of Kyphosidae

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(Knudsen and Clements, 2013a). The recent record of a K. vaigiensis in the Mediterranean (Mannino et al., 2015) further supports this view. Kyphosus bigibbus material from the Atlantic was genetically and morphologically indistinguishable from K. bigibbus from the Indo-Pacific, suggesting a wide distribution encompassing the Western Pacific, Indian and Atlantic Oceans. Thus the historical problem of confounding K. pacificus and K. bigibbus, resolved by Sakai and Nakabo (2004) in the Pacific, appears to have been repeated in the Atlantic and Indian Oceans. In the Atlantic Ocean K. bigibbus has likely been confused with K. sectatrix (e.g. Canas et al. 2005), while in Australia K. bigibbus has been confused with K. sydneyanus (e.g. Downie et al., 2013; Gomon, 2008; Michael et al., 2013). Kyphosus cinerascens and K. elegans share a relatively high number of gill rakers (25-30 and 24-27, respectively), and until recently K. cinerascens was thought to be restricted to the Indo-Pacific Ocean (Knudsen and Clements, 2013a). However, multiple photographic records from the tropical Western Atlantic (Knudsen and Clements, 2013a; D.R. Robertson pers. com.) show that K. cinerascens is present in the Atlantic Ocean. A partial cytb fragment from a specimen from Belize (Ksp_Be_060, insert accession number) and a museum specimen (MOCH0001519 at the Oceanographic Museum of the Federal University of Pernambuco) from Saint Pauls Rocks (see Suppl. Matr. Fig. 9) confirm the presence of K. cinerascens in the Atlantic Ocean (Suppl. Matr. Tabel 1). Sanciangco et al. (2016) also found a close relationship between K. cinerascens and K. elegans. Jordan and Fessler (1893) were in doubt whether to regard Sectator as a genus or a subgenus. Later, Jordan and Evermann (1903) decided to regard Sectator as a genus, and Sectator has since been considered valid. The shallow divergence between Kyphosus ocyurus and K. vaigiensis (Fig. 2 and 3, and Suppl. Matr. Fig. 1-6) together with the blue and golden colours found along the body of K. vaigiensis, the soft dorsal fin ray count (15 in K. ocyurus and 13-15 in K. vaigiensis), and the total number of pterygiophores (39 in K. ocyurus and 36-

27

38 in K. vaigiensis) supports Sectator being a junior synonym of Kyphosus (Knudsen and Clements, 2013a). Finding the elongate K. ocyurus nested inside Kyphosidae resembles the autapomorphic pattern of Inemia vittatum (now Haemulon vittatum) nested in Haemulidae (Rocha et al., 2008), elongate fusiliers (Caesioninae) nested inside Lutjanidae (Miller and Cribb, 2007), and Gomphosus nesting within Thalassoma (Bernardi et al., 2004).

4.3. Taxonomy of valid kyphosid species The results of the present study are inconsistent with the recognition of two newly described species of Kyphosus from the Atlantic Ocean (i.e. Kyphosus bosquii and K. atlanticus) (Sakai and Nakabo, 2014). We had previously shown that K. bosquii was a junior synonym of K. bigibbus based on morphological characters (Knudsen and Clements, 2013a), i.e. the number of gill rakers, the interorbital width and pectoral fin length (see Knudsen and Clements, 2013a, Fig. 6, Suppl. Matr. Fig. 8, Suppl. Matr. Table 3, 7-9). It could be argued that our sampling from the Atlantic Ocean was limited (we were unable to obtain tissue samples from Atlantic K. bigibbus, although we examined many museum specimens of K. bigibbus from the Atlantic Ocean) and thus possibly failed to include these newly described species in both the present study and our taxonomic revision (Knudsen and Clements, 2013a). However, out of 61 specimens of Kyphosus bigibbus and 65 specimens of K. sectatrix collected worldwide, 14 K. bigibbus and 8 K. sectatrix are from the Atlantic Ocean (Knudsen and Clements, 2013a). We examined the longitudinal scale rows – the character Sakai and Nakabo (2014) used for distinguishing between K. bosquii (61-66 scales) and K. atlanticus (50-66 scales) – and found that in these specimens from the Atlantic, K. bigibbus had 54-66 scales and K. sectatrix had 60-67 scales along the longitudinal row. These Atlantic specimens of K. bigibbus and K. sectatrix still group together with either the type for K. bigibbus or K. sectatrix, respectively (Suppl. Matr. Fig. 8 and Knudsen and Clements, 2013a), and these specimens also group with the vouchered and sequenced specimens included in the present

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study (Suppl. Matr. Fig 1-6). A principal component analysis of the most variable morphological characters in Pacific and Atlantic material of K. bigibbus and K. sectatrix (see Knudsen and Clements, 2013a) yielded two separate groups (Suppl. Matr. Fig. 8), each of which corresponded to vouchered and sequenced specimens of K. bigibbus from the Pacific Ocean and K. sectatrix from the Pacific and Atlantic Oceans, respectively. We therefore argue that our failure to resolve K. atlanticus and K. bosquii in our topologies is a result of these taxa being junior synonyms of K. sectatrix and K. bigibbus, respectively, and not a result of limited sampling. It is noteworthy that Sakai and Nakabo (2014) failed to find K. cinerascens in the Atlantic. They found a relatively high gill raker count (23-26) and low longitudinal scale counts (5056) in K. atlanticus, compared to our results from Atlantic specimens of K. bigibbus and K. sectatrix (Suppl. Matr. Table 8-9). A possible explanation for this discrepancy is that Sakai and Nakabo included specimens of K. cinerascens in the material on which they based their description of K. atlanticus. Gilbert (2015) erected a neotype (USNM 116963) for K. sectatrix – which also happens to be the holotype for K. atlanticus described by Sakai and Nakabo (2014) – to stabilize the name Kyphosus sectatrix. Gilbert (2015) made a sound decision establishing a neotype for K. sectatrix, as it is unclear whether the remaining syntype for K. sectatrix (MNHN 0000-2977) (see Knudsen and Clements, 2013a for a discussion on the two syntypes) matches the species in the drawing made by Catesby (1743) on which Linneaus (1758) based his description of Perca sectratrix (i.e. Kyphosus sectatrix). But regardless of which type specimen is associated with this taxon (see Knudsen and Clements, 2013a) - i.e. whether it is MNHN 0000-2977, USNM 116963, AMS.IB 5332 or BMNH 1910.9.9:8, type specimens for Kyphosus sectatrix, K. sectatrix, K. pacificus and Pimelepterus gallveii, respectively – all share a similar gill raker count, interorbital width and pectoral fin length, and all cluster together in a principal component analysis (Suppl. Matr. Fig. 8) with vouchered and sequenced specimens from both

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the Atlantic and the Indo-Pacific. We agree with Gilbert (2015) that the oldest valid name available (i.e. Kyphosus sectatrix) should be applied. Neither Gilbert (2015) nor Mannino et al. (2015) supported the hypothesis that kyphosid species in the Atlantic Ocean should be regarded as endemic and thus distinct from the IndoPacific K. sectatrix and K. bigibbus, as suggested by Sakai and Nakabo (2014). Gilbert (2015) implies that Knudsen and Clements (2013a) identified two holotypes for K. sectatrix, but this is not the case. Knudsen and Clements (2013a) examined the two syntypes of K. sectatrix (MNHN 0000-2977 and MNHN 0000-9601), and failed to discriminate between the type status of MNHN 0000-2977 based on its specimen label and its type status following Bauchot (1963). Bauchot (1963) lists MNHN 0000-2977 as a syntype. Desoutter (1990) regarded Kyphosus sectarix as a valid name. Neither Bauchot (1963) nor Desoutter (1990) claimed that MNHN 0000-2977 was a holotype. The label on the jar containing MNHN 0000-2977 specifies that MNHN 0000-2977 is a syntype, as also indicated by Bauchot (1963) who regarded K. sectatrix as a valid name.

4.4. Distribution of Kyphosidae Neoscorpis is a monotypic genus endemic to the subtropical Southeast African coast between southern Mozambique and Cape Agulhas (Heemstra and Heemstra, 2004; van der Elst and King, 2007). The two temperate species K. sydneyanus and K. gladius are restricted to Australasia. Kyphosus cinerascens reach Hawaii, but have not been recorded along the west coast of Middle America. The distributions of several Kyphosus species are clearly much more widespread than previously assumed. Kyphosus cinerascens are present in the Indo-Pacific Ocean, the Red Sea and the tropical Atlantic Ocean. Kyphosus sectatrix and K. vaigiensis are both found circumtropically from the eastern Pacific across the Pacific Ocean to the Indian Ocean, the Red Sea and the eastern and western Atlantic and the Mediterranean. Kyphosus

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sectatrix is, furthermore, found at the Revillagigedo Islands in the East Pacific (where it was described as K. lutescens), at the Hawaiian Islands (incorrectly referred to as K. sandwicensis by Randall, 2005; Clements and Knudsen 2013a: 41), at Easter Island in the central Pacific Ocean, and south to northern New Zealand. Both K. sectatrix and K. vaigiensis appear to be able to spread across oceanic barriers (Briggs, 1961; Ekman, 1953; Lessios and Robertson, 2006; Luiz et al., 2012). Rafting by juvenile stages (Jokiel, 1990) is likely the explanation behind the ability of Kyphosus populations to maintain connectivity across the Pacific Ocean and across the Atlantic Ocean (Luiz et al., 2012, 2015). Kyphosus ocyurus have been found on both sides of the Pacific Ocean (Araga, 1984; Gotshall, 1998; Lessios and Robertson, 2006), and are probably able to raft from the east Pacific to the west Pacific across the East Pacific oceanic barrier (EPB) – as this barrier does not appear to be a major hindrance for distribution of K. ocyurus (Lessios and Robertson, 2006). Kyphosus analogus in the East Pacific has turned out to be a junior synonym of K. vaigiensis (Knudsen and Clements, 2013a), and K. vaigiensis is known for long distance dispersal (Sakihara et al., 2015; Welsh and Bellwood, 2014). This species is thus also likely to disperse across the Pacific Ocean from the Indo-Pacific Ocean to the eastern Pacific Ocean (Suppl. Matr. Fig. 1-3). Juveniles of K. bigibbus and K. sectatrix also disperse widely from the Indo-Pacific Ocean across the Benguela upwelling into the Atlantic Ocean. Kyphosus cinerascens has a wide distribution from the central Pacific Ocean to the Indian Ocean, the Red Sea and the Atlantic Ocean, but its sister species, K. elegans, is largely restricted to the eastern Pacific Ocean and could thus be limited by the EPB, although the holotype (MNHN 0000-9818) of K. sandwicensis – which was shown to be a specimen of K. elegans by Knudsen and Clements (2013a) – indicates that K. elegans does occasionally make it west to Hawaii (Knudsen and Clements, 2013a). The finding that four species of a herbivorous group of fishes (i.e. Kyphosus bigibbus, K. cinerascens, K. sectatrix, K. vaigiensis) are widely distributed in all oceans across the world

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is surprising, as such wide distributions are usually found among pelagic carnivorous species such as scombrids (Durand et al., 2005) and Coryphaena (Díaz-Jaimes et al., 2010). The dispersal of Kyphosus across the Pacific Ocean westward or eastward does not appear to be obstructed by an Eastern Pacific oceanic barrier (EPB) (Briggs, 1961; Lessios and Robertson, 2006; Robertson and Cramer, 2009) as dispersal can be in other species (e.g., Doryrhamphus excisus and Cirrhitichthys oxycephalus) (Lessios and Robertson, 2006) where the EPB is a significant barrier. Kyphosid dispersal more closely resembles that seen in trumpetfish (Aulostomus), which are also able to disperse across oceanic barriers with reoccurring reconnections between populations by long distance dispersal (Bowen et al., 2001). Finding widespread distribution of K. vaigiensis (i.e. K. analogus, K. incisor and K. vaigiensis), K. sectatrix (i.e. K. pacificus, K. sandwicensis and K. sectatrix), K. bigibbus and K. cinerascens through comparison of molecular and morphological variation (Knudsen and Clements, 2013a) shows that several species of kyphosids are far more widespread than previously believed, and that the Western Indian Ocean and the East Atlantic share the same kyphosid species. Wide dispersal and broad connectivity are also observed in other reef fish families (e.g., Balistidae, Carangidae and Sparidae) (Floeter et al., 2008; Gaither et al., in press) that share species between the tropical east Atlantic Ocean and the South-Western Indian Ocean across the Benguela Barrier. The common route of dispersal appears to be from the Indian Ocean to the Atlantic Ocean (Gaither et al. 2015). Floeter et al. (2008) recorded 47 species of reef fish shared across this barrier. Robertson et al. (2004) also pointed out that there is connectivity between reef fish faunas in the western Pacific Ocean and the East Pacific, mainly by eastward migration. Craig et al. (2004) found Serranidae in the eastern Pacific Ocean with sister species in the eastern Indian Ocean, rather than the proposed geminate ‘sister’ on the Atlantic side of the Panamanian Isthmus. Broad dispersal of juveniles can be so efficient that oceanic islands might not even be required as stepping stones (Joyeux et al., 2001). A higher number of kyphosid species found at oceanic islands can also be correlated

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with the age of the island (Hachich et al., 2015). Briggs and Bowen (2013) speculated that the number of trans-Atlantic species shared between the Central East and Central West Atlantic Ocean would diminish, as future genetic studies reveal cryptic species across the East-West Atlantic Ocean. However, Kyphosus bigibbus, K. cinerascens, K. sectatrix and K. vaigiensis in the Atlantic Ocean (Knudsen and Clements, 2013a) add to the number of shared species across the Atlantic and to the number of shared species between the Indian Ocean and the Atlantic Ocean.

5. Conclusions The inclusion of multiple outgroups in this study has shown that the family designation and subfamily groupings of Kyphosidae presented by Nelson (2006) should be abandoned. Instead, strong support was found for the previous view (Yagishita et al., 2002, 2009) that considers Girellidae, Kyphosidae, Microcanthidae and Scorpididae as separate families. Testing of various topologies in the present study supports Scorpididae as the sister group to Kyphosidae. The taxonomic relationships among the kyphosid genera Hermosilla, Kyphosus, Neoscorpis and Sectator have also been resolved consistent with the taxonomic revision of Kyphosidae presented in Knudsen and Clements (2013a). The present study found no support for the endemic Atlantic species described by Sakai and Nakabo (2014).

Acknowledgments We thank the following for contributing and helping with samples and materials, museum visits and support. Gerry Allen (WAM), Morten Allentoft, Peter Bartsch (ZMB), Phillippe Bearez (MNHN), Andrew Bentley (KU), Giacomo Bernardi (University of California), Dianne Bray (NMV), Paulo Buckup (MNRJ), Patrick Campbell (BMNH), Kent E. Carpenter (Old Dominion University), Romain Causse (MNHN), Oliver Crimmen (BMNH), Brady Doak (University of Auckland), Alexei Drummond (University of Auckland), Rick Feeney 33

(LACM), Claude Fereraz (MNHN), Carlos E. L. Ferreira and Beatrice Ferreira (Biologia Marinha, Universidade Federal Fluminense, Brazil), Malcolm Francis (NIWA, New Zealand), Ronald Fricke (SMNS), Michael Gillings (Macquarie University), Martin Gomon (NMV), Daniel Gotshall, Euan Harvey (University of Western Australia), Amanda Hay (AMS), Jeffrey Johnson (QM), Nicolai Konow (Brown University), Rudie Kuiter (NMV), Denise Kühnert (University of Auckland), Shane Lavery (University of Auckland), Alessandro Ligas (Consorzio per il Centro Interuniversitario di Biologia Marina ed Ecologia applicata), Jeffrey Leis (AMS), Daniel Lippi (Biologia Marinha, Universidade Federal Fluminense, Brazil), James McClaine (BMNH), Frazer McGregor (Coral Bay Research Station, Murdoch University), Mark McGrouther (AMS), Lynn McIlwain (Applecross Marine Laboratory, Perth), Jennifer McIlwain (University of Guam), Tammes Menne (ZMUC), Malene Møhl, Peter Rask Møller (ZMUC), Sue Morrison (WAM), Hiroyuki Motomura (KAUM-I), Jørgen Nielsen (ZMUC), Stephan Nylinder (NRM), Loren O’Hara (BPBM), John Paxton (AMS), Jan Yde Poulsen (Bergen University), Patrice Provust (MNHN), Jack Randall (BPBM), Sandra Raredon (USNM), Sally Reader (AMS), Clive Roberts (NZNM), Erik Schlögl (university of technology, Sydney), Andrew Stewart (NZNM), Carl Struthers (NZNM), Arnold Suzumoto (BPBM), Tom Trnski (AIM), Edward O. Wiley (KU), Jeffrey Williams (USNM), Gabsi Zora (MNHN). The crew on the Danish Galathea 3 expedition. The crew on S/Y ‘Nordkaperen’. The New Zealand eScience Infrastructure high-performance computing facilities and the staff at the Centre for eResearch at the University of Auckland, URL http://www.nesi.org.nz. Financial support for this PhD project was provided by the New Zealand Government funded NZIDRS administered by Education New Zealand, the Faculty Research development Fund of the University of Auckland, the Julie von Müllens Fond (Kongelige Danske Videnskabernes Selskab [Royal Danish Society of Sciences]) and the Australian Museum Geddes Postgraduate Award, and the NZ Ministry of Research and Innovation through Te Papa’s subcontract within NIWA’s Biodiversity and Biosecurity Science Programme

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(previously NIWA’s FORST contract: C01X0502).

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Figure legends Fig. 1. Reproduced topologies from phylogenetic studies that have included Kyphosidae. (A) Reproduced topology based on neighbour-joining, and on (B) maximum likelihood, both from Yagashita et al. (2002) and both inferred from mtDNA nd2. (C) Reproduced topology based on neighbour-joining, and on (D) maximum likelihood, both from Yagashita and Nakabo (2003) and both inferred from mtDNA nd2. (E) Reproduced excerpt from topology presented by Yagahshita et al. (2009) from a maximum likelihood analysis of a dataset with mtDNA genomes. (F) Reproduced excerpt from topologies presented by Near et al. (2012a) from a maximum likelihood analysis of a dataset with 10 nDNA markers. (G) Reproduced excerpt from topology presented by Near et al. (2013) from a maximum likelihood analysis of a dataset with 10 nDNA markers. (H) Reproduced excerpt from topology presented by Sanciangco et al. (2016) from a maximum likelihood analysis of a dataset with 23 gene markers.

Fig. 2. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on mtDNA and nDNA (i.e. 16s, 12s, cytb, tRNA-Val, tRNA-Pro, tRNA-Thr and tRNA-Phe, rag1, rag2 and tmo4c4) (dataset A7). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted. Photos of D. pictum, M. strigatus and A. latus were kindly provided by Erik Schlögl. Photo of K. azureus were kindly provided by Daniel Gotshall. Photo of N. lithophilus were kindly provided by Giacomo Bernardi. Photo of O. woodwardi were kindly provided by Tom Trnski.

Fig. 3. BEAST inferred phylogeny of Kyphosidae and related sister families based on mtDNA and nDNA (i.e. 16s, 12s, cytb, tRNA-Val, tRNA-Pro, tRNA-Thr and tRNA-Phe, rag1, 51

rag2 and tmo4c4) (dataset A7). Consensus tree post-burnin created from all trees sampled per analysis. Nodes are supported by 100% posterior probability unless otherwise denoted. Photos of D. pictum, M. strigatus and A. latus were kindly provided by Erik Schlögl. Photo of K. azureus were kindly provided by Daniel Gotshall. Photo of N. lithophilus were kindly provided by Giacomo Bernardi. Photo of O. woodwardi were kindly provided by Tom Trnski.

Table legends Table 1. Different marginal likelihood values and ln BF obtained for different topological constraints in stepping stone analysis in BEAST. The most positive marginal likelihood favors the best topological constraint - i.e. with Kyphosidae and Scorpididae as a monophyletic group. The ln BF values are obtained by taking the exponential to the absolute value of the different marginal likelihood values subtracted from each other. A ln BF >1 indicates siginificant difference between the two models compared - i.e. the greatest difference between topologies is found between (Kyphosidae, Scorpididae) as monophyletic versus (Kyphosidae, Terapontidae) as monophyletic. Forcing (Kyphosidae, Girelidae) to be monophyletic is an ln BF of 16 worse than having (Kyphosidae, Scorpididae as monophyletic

Table 2. Percentage of different topologies from the 95% most credible trees obtained from MrBayes and BEAST by analysing the same dataset (A7) under the same substitutions models and same partitions (see Suppl. Matr. Table 4).

Table 3. Comparisons of ML log likelihood values among alternative tree topologies obtained from different partitions. ML -ln L is the obtained best likelihood value for the topology. Diff -ln L

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is the difference between the best of the two topologies (H0 and H1) compared in the partition. p is the significance level for the likelihood values for the best tree is indicated by the letter ‘p’.

Supplementary material Figures in supplementary material Fig. suppl. matr. 1. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on the mtDNA markers (16s, 12s, cytb, tRNA-Val, tRNA-Pro, tRNA-Thr and tRNA-Phe) (dataset A2). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted.

Fig. suppl. matr. 2. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on both the mtDNA and nDNA markers (16s, 12s, cytb, tRNA-Val, tRNA-Pro, tRNA-Thr, tRNA-Phe, rag1, rag2 and tmo4c4) (dataset A1). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted.

Fig. suppl. matr. 3. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on three nDNA markers (rag1, rag2 and tmo4c4) (dataset A3). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological

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characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted.

Fig. suppl. matr. 4. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on the rag1 nDNA marker (dataset A4). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted.

Fig. suppl. matr. 5. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on the rag2 nDNA marker (dataset A5). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted.

Fig. suppl. matr. 6. Phylogeny of Kyphosidae and related sister families inferred with MrBayes based on the tmo4c4 nDNA marker (dataset A6). Consensus tree post-burnin created from all trees sampled per analysis. Groups indicate clusters of species where morphological characters match the type material for the species. Voucher specimen numbers are listed next the species names at the tips. Nodes are supported by 100% posterior probability unless otherwise denoted.

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Fig. suppl. matr. 7. Phylogeny of Kyphosidae and related sister families inferred with PAUP from 152 morphological characters. Analysed in a parsimonious analysis in a heuristic search with 1000 bootstrap replicates. Nodes are supported by 100% bootstrap support unless otherwise denoted.

Fig. suppl. matr. 8. Principal component (PC) plot of K. bigibbus and K. sectatrix. PC plot prepared from the following morphological characters: Total gill raker count, upper gill raker count, lower gill raker count, interorbital width (%SL), pectoral fin length (%SL). Squares indicate museum specimens without corresponding tissue sample available (i.e. no DNA information available), triangles indicate specimens with matching tissue sample available (i.e. DNA sequence obtained), crosses indicate type specimens. All points, apart from those marked with red catalog numbers, are based on morphometric and meristic data collected and presented by Knudsen and Clements (2013a). Red catalog numbers mark points based on data as presented by S&K (Sakai and Nakabo 2004, 2014), their corresponding points based on data collected by K&C (Knudsen and Clements, 2013a) are marked with blue catalog numbers.

Fig. suppl. matr. 9. Photograph of K. cinerascens MOCH0001519 from Saint Pauls Rocks. Deposited in the Marine Vertebrate Collection (CHORDATAMAR - Coleção de Cordados Marinhos) of the Museu de Oceanografia. Photo by Daniel Lippi (Biologia Marinha, Universidade Federal Fluminense, Brazil).

Legends for supplementary material, tables Supplementary material – Table 1.

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List of samples and species used in this study, collecting locations, tissue catalog numbers and GenBank Accession numbers.

Supplementary material – Table 2. Robinson-Fould metric values for topologies obtained from various partitions of markers analyzed in MrBayes with multiple representatives per species, compares the distance between the different topologies. A lower RF metric between two topologies thus reflects more similar topologies.

Supplementary material – Table 3. Comparison of counts and measurements of Kyphosus spp. made by Sakai and Nakabo (2014) and Knudsen and Clements (2013a)

Supplementary material – Table 4. Best fit models for substitution and best partitions as suggested by PartitionFinder, and models applied in the subsequent analysis. The p-number following the markers refer to the partition as divided by codons.

Supplementary material – Table 5. List of NCBI GenBank sequences for species representing outgroups.

Supplementary material – Table 6. Primer sequences used for amplification and sequencing in this study

Supplementary material – Table 7. Frequency distributions of meristics of Kyphosus.

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Supplementary material – Table 8. Frequency distributions of meristics of Kyphosus.

Supplementary material – Table 9. Frequency distributions of meristics of Kyphosus.

Supplementary material – Table 10. Differences in assignments of Atlantic Kyphosus type specimens to different species, according to Knudsen and Clements (2013a) and Sakai and Nakabo (2014).

Additional supplementary material: 1) Input xml-file for the BEAST analysis prepared in BEAUti. 2) Input nexus-file for the PAUP analysis of the morphological data. 3) Input nexus-file for the MrBayes analysis of the datasets A1 to A7.

57

58

59

60

(Kyphosidae, Girellidae)

(Kyphosidae, Scorpididae)

(Kyphosidae, Microcanthidae)

(Kyphosidae, Kuhliidae)

(Kyphosidae, Terapontidae)

(Kyphosidae, Oplegnathidae)

no constraints

-48897.0

-48894.2

-48904.3

-48924.4

-48909.6

-48903.9

marginal likelihood

-48919.6

no constraints

-48919.6

1

(Kyphosidae, Girellidae)

-48897.0

6.2E+09

1

(Kyphosidae, Scorpididae)

-48894.2

1.0E+11

1.6E+01

1

(Kyphosidae, Microcanthidae)

-48904.3

4.2E+06

1.5E+03

2.4E+04

1

(Kyphosidae, Kuhliidae)

-48924.4

1.3E+02

7.8E+11

1.3E+13

5.3E+08

1

(Kyphosidae, Terapontidae)

-48909.6

2.2E+04

2.8E+05

4.6E+06

1.9E+02

2.7E+06

1

(Kyphosidae, Oplegnathidae)

-48903.9

6.3E+06

9.8E+02

1.6E+04

1.5E+00

7.9E+08

2.9E+02

1

61

Clade

BEAST

MrBayes

(%)

(%)

(Out of 36004 trees in MrBayes) (Out of 18008 trees in BEAST) Kuhliidae+Terapontidae

100.0

99.7

Oplegnathidae+Kuhliidae+Terapontidae

100.

99.7

Oplegnathidae+Microcanthidae+Kuhliidae+Terapontidae

99.7

96.1

*<5.0

80.9

25.6

35.6

*<5.0

*<5.0

56.9

*<5.0

*<5.0

5.3

97.7

8.9

100.0

100.0

Scorpididae+Girellidae Kyphosidae+Oplegnathidae+Microcanthidae+Kuhliidae+Terapontidae Kyphosidae+Girellidae Kyphosidae+Scorpididae Kyphosidae+Girellidae+Oplegnathidae+Microcanthidae+Kuhliidae+Terapontidae Kyphosidae+Scorpididae+Oplegnathidae+Microcanthidae+Kuhliidae+Terapontidae Kyphosidae+Scorpididae+Girellidae+Oplegnathidae+Microcanthidae+Kuhliidae+Terapontidae *not found in less than 5% of the trees

62

Constrained topology

Data partition**

ML -ln L

Diff -ln L

p (AU tes)t

Kyphosidae sensu Nelson (2006) (H0) A1 (mtDNA, rag1, rag2, tmo4c4)

40017.6

13.6

0.183

Unconstrained (H1)

40003.9

best

0.000*

Kyphosidae sensu Nelson (2006) (H0) A2 (mtDNA)

27185.2

13.3

0.183

Unconstrained (H1)

27171.9

best

0.000*

Kyphosidae sensu Nelson (2006) (H0) A3 (rag1, rag2, tmo4c4)

12106.7

0.9

0.469

Unconstrained (H1)

12105.8

best

0.533

Kyphosidae sensu Nelson (2006) (H0) A4 (rag1)

4755.3

best

0.606

Unconstrained (H1)

A4 (rag1)

4759.9

4.6

0.290

Kyphosidae sensu Nelson (2006) (H0) A5 (rag2)

4516.1

1.1

0.371

Unconstrained (H1)

4515.0

best

0.104

Kyphosidae sensu Nelson (2006) (H0) A6 (tmo4c4)

2591.9

2.2

0.094

Unconstrained (H1)

2589.7

best

0.451

A1 (mtDNA, rag1, rag2, tmo4c4)

A2 (mtDNA)

A3 (rag1, rag2, tmo4c4)

A5 (rag2)

A6 (tmo4c4)

*) significantly different p < 0.05 - i.e. constrained topology H0 rejected at 5% confidence level. **) data partitions are explained in the text.

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Research highlights Relationships, taxonomy and distribution of all known species of Kyphosus is resolved. Testing of various topologies supports Scorpididae as the sister group to Kyphosidae Subfamily groupings of Kyphosidae should be abandoned. No support was found for Atlantic endemic species of Kyphosus.

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Graphical Abstract

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