Supplementary File 1 – Extended Methods and Results of phylogenetic inference.
E XTENDED M ETHODS
To address conflicting phylogenetic results from the combination of morphological and molecular data, two separate analyses were conducted: (1) using morphological and molecular data alone, and (2) using a concatenated (total evidence) dataset with molecular and morphological data. These datasets were analyzed under
Parsimony using TNT v1.1 (Goloboff, P.A., Farris, J.S., et al. 2008) and under maximum
likelihood (ML) in RAxML v7.3 (Stamatakis, A. 2006). In addition, Bayesian analyses of
the molecular and the total evidence dataset were conducted in BEAST v1.8 (Drummond,
A.J., Suchard, M.A., et al. 2012) and MrBayes v3.2.2 (Ronquist, F., Klopfstein, S., et al.
2012), described in detailed in the main document.
TNT.– For the parsimony analyses, morphological characters were treated with equal weights and the multistate characters as non-additive. Heuristic searches were conducted using 1000 random addition sequence replicates with tree-bisection reconnection (TBR) branch swapping, and retaining all shortest trees. For the molecular and combined dataset, the search strategy employed in TNT analyzed the data under the new technology search option, selecting the sectorial search, ratchet, and tree fusing search methodologies, all with default parameters. Under this setting, iterations were run until the minimum length tree was found in 500 separate replicates, in order to hit as many tree islands as possible. To assess nodal support, bootstrap support was based on
1000 pseudoreplicates for each analysis using TBR branch swapping.

RAxML.– For the morphological analyses in a ML framework, all characters were
analyzed under the Mk model using a gamma distribution (Lewis, P.O. 2001) with 1000
replicates; commands employed were -m MULTIGAMMA -K MK –N 100. Because
RAxML cannot recognize polymorphic characters, those taxa in which character states were polymorphic were recoded as missing (“?”) for the RAxML runs. This recoding procedure, however, affected only 10 out of 14858 cells (0.06%) in the matrix. The analysis of the molecular alignments used a four-partition scheme: a separate partition for each codon position of exon markers plus one for 16S (commands employed were -m
GTRGAMMA). The GTR + Γ model was used for all data partitions. Branch support was assessed using the nonparametric rapid-bootstrapping algorithm, with automatic estimation of the sufficient number of replicates. The total evidence dataset including both DNA and morphology were analyzed under the GTRGAMMA plus Mk models, respectively (commands employed were -m GTRGAMMA -K MK), using five partitions: four for the molecular data (as explained above) and one for the morphological data. The final alignments were analyzed using ten independent runs.
E
XTENDED
R
ESULTS
The ML and ND analyses of the molecular dataset resulted in strongly supported trees (average Bootstrap support = 86.4% for RAxML and average Posterior probability
= 0.94–0.95 for MrBayes and BEAST, respectively; Fig. S1; Table S4), all of which resolve the monophyly of all extant tetraodontiform families as well as the subordinal
clades (sensu Betancur-R., R., Broughton, R.E., et al. 2013). A comparison of two
alternative classifications for tetraodontiform fishes is presented in Table S1. The inferred

relationships among the extant tetraodontiform families and suborders are largely
congruent to those estimated by other previous molecular studies (Holcroft, N.I. 2005,
Alfaro, M.E., Santini, F., et al. 2007, Yamanoue, Y., Miya, M., et al. 2008, Santini, F.,
Sorenson, L., et al. 2013). However, early-branching, intraordinal tetraodontiform
relationships are often weakly supported and incongruent among different analyses (Figs.
S1–S3). The parsimony and ML trees of the molecular dataset (Figs. S1a, b) differ primarily in that Moloidei is placed in the ML tree as sister group of Tetraodontoidei rather than the Balistoidei + Tetraodontoidei clade as in the parsimony tree, and in both cases the bootstrap support is relatively high (ML = 75–89%, parsimony = 90–99%).
Triacanthoidei and Triacanthodoidei are resolved as sister groups in the parsimony and
Bayesian trees whereas in the ML tree Triacanthoidei is closer to Balistoidei (parsimony
= 54%, ND MrBayes = 1–0.99, ND BEAST= 0.94–0.92, ML = 10%).
Trees (ML and TD) based on molecular and morphological data consistently obtain a clade containing Tetraodontoidei and Moloidei, similar to results obtained in
previous phylogenetic analyses of tetraodontiforms (e.g., Breder, C.M.J. and Clark, E.
1947, Winterbottom, R. 1974, Tyler, J.C. and Sorbini, L. 1996, Santini, F. and Tyler, J.C.
2003, Santini, F., Sorenson, L., et al. 2013). Monophyly of suborders is supported by
these analyses (Figs. S1–S3; Table S4), yet supraordinal interrelationships based on molecular data are incongruent with the results obtained from the morphological data
(based on both ML and parsimony analyses of the dataset compiled by Santini, F. and
Tyler, J.C. 2003; Fig. S2), but their support values are weak. At the family level, some
discrepancies also exist; for example, Triacanthodidae and Triacanthidae are resolved as

sister groups in the ND, ML and Parsimony trees but not in the morphological tree (Figs.
S1, S2).
The relationships of the extant families of tetraodontiforms based on the ML and
TD are differ to those obtained with the molecular data alone (Figs. S2, S3; Table S4).
The sister relationship between Triacanthidae and Triacanthodidae is strongly supported for the ND analyses (1.0–0.92) and weaker for the TD analyses (MrBayes with lognormal root prior, 0.35–0.36), while in the other TD analyses they are in a polytomy with
†Moclaybalistidae. The monophyly of extant and fossil tetraodontiform families and suborders is also supported by the TD analyses; except for the apparent misplacement of
†
Eomola in the TD trees (Fig. S3c; Table 4), which renders Molidae paraphyletic.
Triodontidae is also paraphyletic in these trees (Fig. S3c; Table 4), consisting of two groups, one including only fossil genera († Eotetraodon and † Zignodon ), and one including extant and extinct species in the genus Triodon (Triodontidae is monophyletic in the ML tree; Fig. S3b). Not surprisingly, some fossil taxa show very different placement when molecular data are added, relative to the morphological trees. Most notably, the analysis derived from morphological data alone places triodontid and tetraodontid taxa in a polytomy, whereas addition of molecular data results in the monophyly of Tetraodontoidei. Similarly, the placement of the fossil families
†Eoplectidae, †Bolcabalistidae, †Moclaybalistidae, †Eospinidae and †Spinacanthidae was ambiguous depending on the reconstruction method (Figs. S1, S3), but the bootstrap support values are low (<70%).
Finally, bootstrap support values are significantly higher in the analyses of the molecular datasets alone (68.2–86.4%), relative to those based on morphological data

(38.6–53.1%) or the combined dataset (35.6–57.9%). Reconstruction methods also reveal substantial differences in this respect, with higher support values obtained with ML
(53.1–86.4%) than parsimony (35.6–68.2%; see details in Table S4).

Supplementary File 2 – Calibration points used for the node-dating calibrationdensity (ND-CD) analyses
A total of 9 or 12 calibration points were selected for node dating, based on the fossil placement obtained with analyses of combined data. Twelve calibration points were defined for the BEAST analyses, which allows the implementation of both stem and crown calibrations. Because MrBayes can only take crown calibrations, only nine points were used for this analysis. For direct comparison, placement of fossils is based on the topology obtained with tip dating in MrBayes, unless otherwise indicated (e.g.,
†
Eomola ). One calibration is based on a fossil not examined herein (calibration 12).
Following the recommendations of Parham, J.F., Donoghue, P.C., et al. (2012), the youngest fossil date from a biostratigraphic interval is used as the absolute age for minimum age constraint. The 95% soft maxima are based on lowly restrictive priors (i.e., relatively old dates).
(1) Tetraodontiformes + Antigonia (= root). MRCA: Antigonia, Takifugu . Hard minimum age: 95 Ma († Plectocretacicus clarae ). 95% soft maximum age: 122 Ma
(†
Acanthomorphorum forcallensis ). Prior settings: (a) Exponential distribution, mean=9.00 (crown calibration); (b) Lognormal distribution, mean=4.6789, st. dev.=0.0638.
(2) Tetraodontiformes.
MRCA: Triacanthodes, Tetraodon . Hard minimum age: 59 Ma
(†
Moclaybalistes danekrus ). 95% soft maximum age: 96.5 Ma († Plectocretacicus

clarae ). Prior setting: Exponential distribution, mean=12.18 Ma (crown calibration).
Comments: The position of † Moclaybalistes is unstable among different analyses and among previous studies. For instance, in ML (concatenated dataset) † Moclaybalistes branches off the stem Tricanthidae, whereas in parsimony it is sister to Triodontidae. In
Santini, F. and Tyler, J.C. (2003) † Moclaybalistes is placed within Balistoidei. Given this topological uncertainty, we use † Moclaybalistes as the oldest crown tetraodontiform.
(3) Triacanthodes (total group).
MRCA: Triacanthodes ethiops , T. anomalus . Hard minimum age: 23 Ma (†
Carpathospinosus propheticus ). 95% soft maximum age: 59 Ma
(†
Moclaybalistes danekrus ). Prior setting: Exponential distribution, mean=12.01 Ma.
Comments: Set as stem calibration in BEAST and as crown calibration in MrBayes, but placed one node below (i.e., MRCA: Tydemania, Triacanthodes ).
(4) Triacanthidae (total group).
MRCA: Triacanthus, Pseudotriacanthus . Hard minimum age: 49 Ma (†
Protacanthodes nimesensis
, †
Protacanthodes ombonii ). 95% soft maximum age: 96.5 Ma († Plectocretacicus clarae ). Prior setting: Exponential distribution, mean=15.52 Ma. Comments: Set as stem calibration in BEAST and as crown calibration in MrBayes, but placed one node below (i.e., MRCA: Triacanthus ,
Triacanthodes ).
(5) Balistidae.
MRCA: Xanthichthys, Balistapus . Hard minimum age: 30 Ma
(† Balistomorphus ovalis, † Balistomorphus orbiculatus, † Balistomorphus spinosus ). 95% soft maximum age: 59 Ma (†
Moclaybalistes danekrus ). Prior setting: Exponential

distribution, mean=9.68 Ma (crown calibration). Comments: these fossils have been
considered stem balistid members by previous studies (Alfaro, M.E., Santini, F., et al.
2007, Dornburg, A., Santini, F., et al. 2008, Dornburg, A., Sidlauskas, B., et al. 2011) and
used for calibration one node below (i.e., split of Monacanthidae and Balistidae). While placement of † B. ovalis ,
†
B. orbiculatus is unstable among our different trees (stem members in the RAxML tree and crown members in the parsimony and Bayesian trees),
† B. spinosus is consistently placed as a crown balistid in all analyses derived from the combined dataset (Figs. 1,3).
(6) Balistoidei (Monacanthidae + Balistidae; total group).
MRCA: Aluterus, Balistes .
Hard minimum age: 49 Ma (†
Eospinus daniltshenkoi
, †
Bolcabalistes varii ). 95% soft maximum age: 96.5 Ma († Plectocretacicus clarae ). Prior setting: Exponential distribution, mean=15.52 Ma. Comments: Set as stem calibration in BEAST. This calibration was not used in MrBayes as crown calibration one node below (i.e., crown
Tetraodontiformes) because it was superseded by calibration 2 (based on the tip dating tree).
(7) Aracanidae (total group).
MRCA: Aracana, Capropygia . Hard minimum age: 49
Ma (†
Proaracana dubia ). 95% soft maximum age: 96.5 Ma († Plectocretacicus clarae ).
Prior setting: Exponential distribution, mean=15.52 Ma. Comments: Set as stem calibration in BEAST. This calibration was not used in MrBayes as crown calibration one node below (i.e., Aracanidae + Ostraciidae) because it was superseded by calibration 8
(based on the tip dating tree)

(8) Ostraciidae. MRCA: Lactoria, Ostracion . Hard minimum age: 49 Ma († Eolactoria sorbinii ). 95% soft maximum age: 96.5 Ma († Plectocretacicus clarae ). Prior setting:
Exponential distribution, mean=15.52 Ma (crown calibration).
(9) Diodontidae (total group).
MRCA: Diodon, Allomycterus . Hard minimum age: 49
Ma († Prodiodon erinaceus , † Prodiodon tenuispinus , † Heptadiodon echinus , † Zignodon ornasieroae ). 95% soft maximum age: 96.5 Ma († Plectocretacicus clarae ). Prior setting:
Exponential distribution, mean=15.52 Ma. Comments: Set as stem calibration in BEAST and as crown calibration in MrBayes, but placed one node below (i.e., MRCA: Diodon,
Takifugu ).
(10) Tetraodontidae (in part).
MRCA: Lagocephalus, Tetraodon.
Hard minimum age:
23 Ma († Archaeotetraodon winterbottomi ). 95% soft maximum age: 59 Ma
(†
Moclaybalistes danekrus ). Prior setting: Exponential distribution, mean=12.01 Ma.
(crown calibration). Comments: While †
Eomola bimaxillaria (41 Ma) is older than
† Archaeotetraodon winterbottomi , placement in this clade by the tip-dating analysis is incongruent with that obtained with RAxML and TNT as well as Santini, F. and Tyler,
J.C. (2003). We thus use † Eomola for calibration 11 (below).
(11) Moloidei (Molidae; total group).
MRCA: Mola, Ranzania . Hard minimum age: 41
Ma († Eomola bimaxillaria ). 95% soft maximum age: 96.5 Ma († Plectocretacicus clarae ). Prior setting: Exponential distribution, mean=18.03 Ma. Comments: set as stem

calibration in BEAST based on the results obtained with parsimony and ML on morphological data alone as well as combined data (see also comments under calibration
10). Given the variable placement of Moloidei among tetraodontiform suborders among different analyses, it is difficult to select a subtending node to assign † Eomola as crown constraint for MrBayes (not used).
(12) Mola and Masturus (total group). MRCA: Mola , Masturus . Hard minimum age: 22
Ma (
†Austromola ). 95% soft maximum age: 41 Ma (†
Eomola bimaxillaria ). Prior setting:
Exponential distribution, mean= 6.34 Ma. Comments: Although † Austromola was not examined here, its placement sister to the Mola + Masturus clade was obtained by the
parsimony analysis of 57 anatomical characters (Gregorova, R., Schultz, O., et al. 2009).
Set as stem calibration in BEAST and as crown calibration in MrBayes, but placed one node below (i.e., MRCA: Ranzania, Mola ).

Supplementary File 3 – Calibration file used for the node-dating fossilized birthdeath process (ND-FBD) analyses in DPPDiv v1.1
The ND-FBD analyses in DPPDiv used 36 fossils in addition to root calibration.
Placement of fossils is based on the topology obtained with tip dating in MrBayes, with some exceptions noted in Supplementary File 1. The FBD approach requires the specification of absolute ages for fossils, and the current recommendation is to sample their ages from a uniform distribution given by their stratigraphic range (Table 3), in
order to approximate random recovery (future implementations of the program will treat the ages of fossils as random variables by placing prior densities on occurrence times;
Heath, T.A., Huelsenbeck, J., et al. 2013). Instead, we selected the absolute ages of
fossils obtained with tip dating in MrBayes (Table S2). One calibration is based on a
-t
-t
38
-t
-t
-t
-t fossil not examined herein (see calibration 12 in Supplementary File 2). The calibration file follows the specification of the program’s format. The number of calibrations used is given in the first row of the file; the following rows specify the “-t” flag, two MRCA taxa subtending the placement of the fossil, and the fossil’s absolute age. root 95.44621
Caproidae_Antigonia_capros_T0001 Ostraciidae_Lactoria_cornuta_T0025
95.22325
Caproidae_Antigonia_capros_T0001 Ostraciidae_Lactoria_cornuta_T0025
74.7302
Caproidae_Antigonia_capros_T0001 Ostraciidae_Lactoria_cornuta_T0025
95.48307
Triacanthodidae_Parahollardia_lineata_T0095
Triacanthodidae_Tydemania_navigatoris_T0096
Triacanthodidae_Triacanthodes_anomalus_T0059
Triacanthodidae_Tydemania_navigatoris_T0096
26.49457
28.9661

-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
-t
Triacanthidae_Trixiphichthys_weberi_T0063
Triacanthodidae_Tydemania_navigatoris_T0096 49.93452
Triacanthidae_Trixiphichthys_weberi_T0063
Triacanthodidae_Tydemania_navigatoris_T0096 49.12948
Triacanthidae_Trixiphichthys_weberi_T0063
Triacanthodidae_Tydemania_navigatoris_T0096 33.83157
Triacanthidae_Trixiphichthys_weberi_T0063
Triacanthodidae_Tydemania_navigatoris_T0096 29.95205
Triacanthidae_Trixiphichthys_weberi_T0063
Triacanthodidae_Tydemania_navigatoris_T0096 23.20983
Triacanthidae_Trixiphichthys_weberi_T0063
Triacanthodidae_Tydemania_navigatoris_T0096 32.57545
Tetraodontidae_Sphoeroides_maculatus_T0047
Triacanthodidae_Tydemania_navigatoris_T0096 54.7633
Tetraodontidae_Sphoeroides_maculatus_T0047
Triacanthodidae_Tydemania_navigatoris_T0096 51.03538
Tetraodontidae_Sphoeroides_maculatus_T0047
Triacanthodidae_Tydemania_navigatoris_T0096 52.98431
Tetraodontidae_Sphoeroides_maculatus_T0047
Triacanthodidae_Tydemania_navigatoris_T0096 61.03195
Tetraodontidae_Sphoeroides_maculatus_T0047
Triacanthodidae_Tydemania_navigatoris_T0096 52.68475
Balistidae_Balistapus_undulatus_T0009
Balistidae_Abalistes_stellatus_T0002
Balistidae_Balistapus_undulatus_T0009
29.24497
Balistidae_Abalistes_stellatus_T0002
Balistidae_Balistapus_undulatus_T0009
Balistidae_Abalistes_stellatus_T0002
33.43563
31.42342
Balistidae_Balistapus_undulatus_T0009
Balistidae_Abalistes_stellatus_T0002 32.50394
Aracanidae_Aracana_aurita_T0007 Ostraciidae_Lactoria_cornuta_T0025
51.09196
Ostraciidae_Ostracion_rhinorhynchos_T0036
Ostraciidae_Lactoria_cornuta_T0025 54.35557
Ostraciidae_Ostracion_rhinorhynchos_T0036
Ostraciidae_Lactoria_cornuta_T0025 30.30148
Tetraodontidae_Takifugu_ocellatus_T0088
Triodontidae_Triodon_macropterus_T0062 41.25104
Tetraodontidae_Sphoeroides_maculatus_T0047
Triacanthodidae_Tydemania_navigatoris_T0096 49.88452
Tetraodontidae_Takifugu_ocellatus_T0088
Triodontidae_Triodon_macropterus_T0062 50.09464
Tetraodontidae_Takifugu_ocellatus_T0088
Triodontidae_Triodon_macropterus_T0062 53.99818
Tetraodontidae_Lagocephalus_laevigatus_T0027
Tetraodontidae_Sphoeroides_maculatus_T0047 16.31247

-t
-t
-t
-t
-t
-t
-t
-t
-t
Tetraodontidae_Arothron_hispidus_T0008
Tetraodontidae_Sphoeroides_maculatus_T0047 33.16794
Tetraodontidae_Lagocephalus_laevigatus_T0027
Tetraodontidae_Sphoeroides_maculatus_T0047 4.51564
Diodontidae_Allomycterus_pilatus_T0071
Tetraodontidae_Sphoeroides_maculatus_T0047 41.9064
Diodontidae_Allomycterus_pilatus_T0071
Triodontidae_Triodon_macropterus_T0062 51.17608
Diodontidae_Allomycterus_pilatus_T0071
Triodontidae_Triodon_macropterus_T0062 50.5678
Diodontidae_Allomycterus_pilatus_T0071
Triodontidae_Triodon_macropterus_T0062 54.88407
Diodontidae_Allomycterus_pilatus_T0071
Triodontidae_Triodon_macropterus_T0062 54.23441
Tetraodontidae_Takifugu_ocellatus_T0088 Molidae_Ranzania_laevis_T0042
41.53452
Molidae_Ranzania_laevis_T0042 Molidae_Masturus_lanceolatus_T0028
22

Table S1. Comparison of tetraodontiform classifications based on most recent studies. Each cell in the table indicates the familial composition for each suborder.
Suborder
†Plectocretacicoidei
Triacanthodoidei
Santini, F. and Tyler, J.C. (2003)
†Cretatriacanthidae, †Plectocretacicidae, †Protriacanthidae
Triacanthodidae, Triacanthidae
Betancur-R., R., Broughton, R.E., et
_ al. (2013)
Triacanthodidae
Triacanthoidei _ Triacanthidae
Balistoidei
Ostracioidei
Balistidae, Monacanthidae, Aracanidae, Ostraciidae,
†Bolcabalistidae, †Eospinidae, † Spinacanthidae,
†Moclaybalistidae, †Protobalistidae
_
Tetraodontidae, Diodontidae, Molidae, †Eoplectidae
Balistidae, Monacanthidae
Aracanidae, Ostraciidae
Tetraodontoidei Tetraodontidae, Diodontidae
Moloidei _ Molidae
Triodontoidei * _ Triodontidae
*Triodontoidei was not formally classified by Betancur-R., R., Broughton, R.E., et al. (2013) but a recent update of the
classification scheme added this taxon (Betancur-R., R., Wiley, E.O., et al. 2013).

Table S2.
Tetraodontiform fossils included in the present study and associated metadata (Santini, F. and Tyler, J.C. 2003).
Voucher* Species
MCSNV 1377
MCSV S.L.1 and 2
IGPUB 1FDC29
Cretatriacanthus guidottii
Plectocretacicus clarae
Protriacanthus gortanii
ZPALWr A/2096-2099 Prohollardia avita
ZPALWr A/3000-3004 Carpathospinosus propheticus
MHNN 002
IGUP 10.901/2
Protacanthodes nimesensis
Protacanthodes ombonii
BMNH P 524
BMNH P 454/3974
MSNPN 157
IPB P1686
MNHN LP 10918
MCSNV T 21/2
MCSNV 4 434
GMUC 16-5631
Acanthopleurus collettei
Acanthopleurus serratus
Acanthopleurus trispinosus
Cryptobalistes brevis
Spinacanthus cuneiformis
Protobalistum imperialis
Bolcabalistes varii
Moclaybalistes danekrus
PIN 2179-101
NSKG 2688
IGUN 288
BMNH P 3973/P 500
PIN 1413-17
MCSNV T 8/T 63
MCSNV T 6/T 7
MM Ge 26828
BMNH P.62702
Eospinus daniltshenkoi
Balistomorphus orbiculatus
Balistomorphus ovalis
Balistomorphus spinosus
Oligobalistes robustus
Proaracana dubia
Eolactoria sorbinii
Oligolactoria bubiki
Triodon antiquus
Age (Ma) Locality of origin
76.5-70.0
Nardò, Italy
96.9-95 Hakel, Lebanon
95.5-93.0 Comen, Slovenia
27-24
29-28
Rzeszow, Poland
Przysietnica, Poland
55.0-49.0 Monte Bolca, Italy
55.0-49.0 Monte Bolca, Italy
33.9-28.4 Kanton Glarus, Switzerland
33.9-28.4 Kanton Glarus, Switzerland
33.9-23.03 Muntele Cozla, Romania
33.9-28.4 Kanton Glarus, Switzerland
55.0-49.0 Monte Bolca, Italy
55.0-49.0 Monte Bolca, Italy
55.0-49.0 Monte Bolca, Italy
65.5-59 Mo-clay, Denmark
55.8-48.6 Turkmenistan
33.9-28.4 Kanton Glarus, Switzerland
33.9-28.4 Kanton Glarus, Switzerland
33.9-28.4 Kanton Glarus, Switzerland
33.90-32.25 Caucasus, Ukraine
55.0-49.0 Monte Bolca, Italy
55.0-49.0 Monte Bolca, Italy
33.9-28.4 Moravia, Czech Republic
55.8-33.9 Great Britain
Completeness**(%)
38.5
41.5
55.5
45
22
15.5
53.5
36.5
96.5
55.7
50.5
69
66
58.5
68.5
12
49
54.5
39.5
54.5
20.5
8.5
17
21

MCSNV IG 91170
IGUP 6789
IGUP 6890/6891
PIN 287-9
PIN 3363 111/111a
USNM 290643
PIN 4424-31
BMNH P 10426
BMNH P 10735
NMW 1853.XXVII.6a-
6b
IGUP 6894
Eoplectus bloti
Zignoichthys oblongus
Eotetraodon pygmaeus
Archaeotetraodon jamestyleri
Archaeotetraodon winterbottomi
Sphoeroides hyperostosus
Pshekhadiodon parini
Prodiodon erinaceus
Prodiodon tenuispinus
Heptadiodon echinus
Zignodon fornasieroae
55.0-49.0
55.0-49.0
55.0-49.0
16.5-15.0
33.90-32.25
5.3-3.6
42-41
55.0-49.0
55.0-49.0
55.0-49.0
55.0-49.0
PIN 4425/1a Eomola bimaxillaria 42-41
*Additional vouchers may be available (see Santini, F. and Tyler, J.C. 2003)
Monte Bolca, Italy
Monte Bolca, Italy
Monte Bolca, Italy
Crimea, Ukranie
Caucasus, Ukraine
North Carolina, USA
Caucasus, Ukraine
Monte Bolca, Italy
Monte Bolca, Italy
Monte Bolca, Italy
Monte Bolca, Italy
Caucasus, Ukraine
46
34
41
40.5
31
33
33
25
20
29.5
15
5

Table S3. Markers examined and abbreviations used throughout the text.
16S rRNA
Ectoderm-neural cortex protein 1
Cardiac muscle myosin heavy chain 6
Early growth response 1
G protein-coupled receptor 85
Glycosyltransferase
Interphotoreceptor retinoid-binding protein
Mitochondrial ribosomal
Nuclear exon
Nuclear exon
Nuclear exon
Nuclear exon
Nuclear exon
Nuclear exon
Myeloid/lymphoid or mixed-lineage leukemia Nuclear exon
Patched domain containing 4
Pleiomorphic adenoma gene-like 2
Nuclear exon
Nuclear exon
Recombination activation gene 1
Rhodopsin si:dkey-174m14.3
Similar to SH3 and PX domain containig 3 gene
T-box brain 1
Nuclear exon
Nuclear exon
Nuclear exon
Nuclear exon
Nuclear exon
ZIC family member 1 Nuclear exon
16S
ENC1
MYH6
EGR1
SREB2
GLYT
IRBP
MLL
PTR
PLAGL2
RAG1
RH
SIDKEY
SH3PX3
TBR1
ZIC1

Table S4. Summary of topological congruence and bootstrap support values derived from the phylogenetic analyses of different datasets and optimality criteria. n/a, insufficient taxonomic sampling to test hypothesis; gray cells, relationships not recovered from analysis.
Morphological Molecular Combined
Taxon/Clade
Tetraodontiformes 100
Plectocretacicoidei
(Cretatriacanthidae +
Protriacanthidae + Plectocretacidae) 44
Tetraodontiformes without
Plectocretacicoidei 100
Triacanthodoidei (Triacanthodidae)
Triacanthoidei (Triacanthidae)
45
83
Triacanthodidae + Triacanthidae
Bolcabalistoidea (Eospinidae +
Bolcabalistidae)
Monacanthidae
Balistoidei (Monacanthidae +
Balistidae)
Balistidae
Aracanidae
22
100
93
76
100 100 100 1
74
100 n/a n/a n/a n/a n/a n/a 77 57
42 100 100 1
72 100 100 1
56 n/a n/a n/a
54 59 1
100 100 100 1
91 100 100 1
57 100 100 1
41 100 100 1
1 n/a
1
1
1
1 1 100 100 1 n/a n/a n/a n/a n/a n/a 4 66 0.74
1
1
1
1 n/a
1
1
0.92 0.93
1
1
1
1 n/a
1
1
1
1
17
73
52
94
97
93
72
85
99
87
0.61
1
0.99
0.99
0.96
0.84
1 92 82 0.99
1 21 65 0.71
1
0.99
0.39
0.99
0.99
0.36
0.74
0.97
0.88
0.99
0.69
1 1
1
0.46
0.99
0.99
0.77
0.95
0.76
0.99
0.99
0.99
0.84
1
0.99
0.73
0.99
0.81
1
1

Ostracioidei (Aracanidae +
Ostraciidae)
Ostraciidae
Triodontidae
Moloidei (Molidae)
Diodontidae
Tetraodontidae
Triodontidae + Molidae +
Diodontidae + Tetraodontidae
Molidae + Diodontidae +
Tetraodontidae
Tetraodontoidei + Balistoidei +
Moloidei
Tetraodontoidei (Diodontidae +
Tetraodontidae)
79
73
38
42
49
32
7
92 100 100 1
77 100 100 1
1
1
1
1
1 81 92 0.99
1 64 70 0.97
0.99
0.78
0.94
0.99
1
0.97 n/a n/a n/a n/a n/a n/a 9 23
51 100 100 1
64 100 100 1
45 100 100 1
1
1
1
1
1
1
1
1
1
24
56
45
40
63
46
0.53*,
0.99**
0.98
0.86
0.99*,
0.47**
0.98
0.93
0.99*,
0.70**
0.99*** 0.99*** 0.99***
0.99
0.92
0.99*,
0.65**
1***
0.99
0.92
59
44
0.82 0.85 0.65 0.65 20 54
42 1
98 75
1 1 1 9 21
21 39 100 100 1 1 1 1 38 45 0.80
Average support values (across all nodes) 36.5 53.1 76.8 86.4 0.94 0.95 0.94 0.94 46.1 57.9 0.85
* Triodontidae including extant and extinct species of the genus Triodon
** Triodontidae including only fossil genera †
Eotetraodon and †
Zignodon
*** Excluding †
Eomola
0.75
0.86
0.81
0.81
0.85
0.53
0.84
0.80

Table S5. Comparisons of divergence time for major tetraodontiform clades estimated with node dating methods (mean and 95% HPD, in
Ma). ND-CD: node-dating calibration-density approach; ND-FBD: node-dating, fossilized birth death process (tree model); UGR: uncorrelated gamma-distributed rates (clock model); DPP: Dirichlet process prior (clock model).
Taxon/Clade
ND-CD -
MrBayes
Exponential
ND-CD -
MrBayes -
Lognormal
ND-CD -
BEAST -
Exponential
ND-CD -
BEAST -
Lognormal
ND-FBD - UGR ND-FBD - DPP
Tetraodontiformes
Triacanthodoidei
(Triacanthodidae)
Triacanthoidei (Triacanthidae)
Diodontidae
96.4 (113.4-84.3) 94.1 (112.5-80.2) 89.0 (100.5-76.4) 93.9 (108.4-79.1) 86.5 (104.2-71.1) 93.7 (118.7-75.0)
35.8 (49.2-25.4) 34.5 (50.1-22.3) 30.1 (39.6-23.8) 30.2 (39.4-23.7) 38.0 (53.5-28.2) 42.2 (59.9-29.8)
32.5 (51.9-15.3) 30.7 (49.7-12.5) 25.8 (42.7-11.1) 27.2 (44.0-12.6) 16.1 (30.5-6.0) 15.8 (26.4-7.1)
Triacanthodidae + Triacanthidae 73.9 (93.1-55.1) 71.3 (95.6-53.2) 67.4 (82.8-49.0) 69.9 (88.4-49.3) 72.0 (88.3-54.4) 72.1 (92.5-52.3)
Balistoidei (Monacanthidae +
Balistidae) 78.4 (97.1-63.3) 75.5 (99.4-66.2) 63.8 (76.8-49.6) 71.2 (86.6-56.5) 58.8 (73.6-45.4) 63.6 (80.0-47.1)
Monacanthidae
Balistidae
Ostracioidei (Aracanidae +
Ostraciidae)
Aracanidae
Ostraciidae
Moloidei (Molidae)
Molidae + Tetraodontoidei
Tetraodontoidei (Diodon. +
Tetraodon.)
66.8 (82.9-50.3) 69.1 (84.3-51.5) 47.5 (60.6-35.4) 50.3 (62.3-37.4) 34.3 (45.9-22.7) 38.9 (61.0-24.5)
35.6 (45.1-30.0) 33.2 (43.3-30.0) 33.5 (39.5-30.0) 34.0 (41.3-30.0) 40.2 (50.4-32.3) 40.4 (50.2-32.2)
66.7 (80.8-54.8)
27.5 (43.0-14.4)
57.3 (68.0-49.9) 57.9 (66.3-50.1) 57.6 (64.9-51.1) 58.1 (66-51.6)
27.3 (22.0-38.4) 29.4 (22.4-39.9) 26.5 (22-34.8) 26.7 (35.1-22.0)
61.4 (69.9-54.2)
30.0 (45.2-22.0)
55.1 (68.9-36.3)
33.2 (45.6-22.0)
87.1 (108.3-72.9) 89.2 (108.3-72.1) 79.0 (90.9-65.5) 83.4 (98.8-68.4) 75.0 (92.3-59.3) 78.8 (98.9-59.9)
78.6 (97.0-61.6)
67.6 (81.4-53.2)
25.6 (43.2-13.5)
79.2 (96.0-63.4)
66.1 (77.4-56.5)
12.3 (21.2-5.3)
68.6 (81.2-55.7)
67.2 (78.6-56.6)
12.6 (20.9-5.4)
72.5 (86.6-57.7)
68.7 (79.8-58.1)
6.5 (11.5-2.6)
62.3 (77.8-46.8)
64.1 (76.1-52.0)
9.1 (19.6-3.1)
65.8 (84.4-50.3)
31.7 (45.3-19.3) 32.5 (46.6-20.2) 20.8 (31.6-11.5) 21.1 (31.7-11.5) 12.4 (20.0-5.9) 15.9 (28.2-7.1)
Tetraodontidae 64.2 (81.1-49.3) 62.7 (82.3-50.4) 52.3 (63.2-41.4) 55.4 (67.7-44.3) 40.5 (51.1-30.0) 44.2 (61.7-30.2)

Table S6. Comparisons of divergence time for major tetraodontiform clades estimated with tip dating methods (mean and 95% HPD, in Ma).
TD: tip dating.
Taxon/Clade
Tetraodontiformes
Triacanthodoidei (Triacanthodidae)
Triacanthoidei (Triacanthidae)
Triacanthodidae + Triacanthidae
Balistoidei (Monacanthidae + Balistidae)
Monacanthidae
Balistidae
Ostracioidei (Aracanidae + Ostraciidae)
Aracanidae
Ostraciidae
Moloidei (Molidae)
Molidae + Tetraodontoidei
Tetraodontoidei (Diodon. + Tetraodon.)
Diodontidae
Tetraodontidae
TD - MrBayes -
Exponential
TD - MrBayes -
Lognormal
TD - BEAST -
Exponential
TD - BEAST -
Lognormal
135.3 (164.2-109.6) 119.4 (132.8-106.6) 185.2 (383.6-167.9) 136.7 (150.7-123.5)
74.5 (106.8-48.9) 65.3 (89.3-45)
46.3 (70.8-26.3) 76.3 (101.6-59.1)
52.7 (188.5-31.1)
97.4 (232.7-56.4)
45.0 (59.9-32.4)
68.1 (88.9-51.7)
_ 101.6 (120.2-82.8) _ _
119.0 (149.1-95.1) 104.1 (120.6-87.1) 118.1 (297.4-100.1) 97.5 (126.4-89.0)
107.0 (137.4-78.4) 91.9 (113.4-72.6)
90.2 (118.6-63.0) 77.6 (99.9-56.8)
95.4 (250.1-70.0)
58.4 (203.9-39.3)
68.0 (84.5-50.7)
50.2 (65.5-36.3)
102.4 (134.6-76.2) 90.1 (112.6-69.5)
47.1 (24..6-73.8) 71.8 (95.4-49.8)
84.2 (112.6-60.6) 74.2 (95.0-56.0)
53.9 (86.2-29.4) 46.8 (72.3-19.5)
68.3 (245.1-64.7)
16.0 (158.8-10.1)
62.3 (205.8-51.8)
62.7 (190.7-21.5)
76.5 (96.3-58.6)
70.3 (81.8-49.0)
60.5 (74.0-49.0)
37.4 (60.0-17.7)
_ _ 135.4 (335.1-132.7) _
122.7 (151.1-98.1) 108.4 (124.4-93.7) 129.5 (320.6-119.0) 104.7 (119.8-87.1)
51.1 (75.1-29.5) 91.2 (111.4-70.4) 91.5 (254.8-64.7) 78.4 (99.6-59.8)
114.8 (144.4-90.0) 102.4 (119.6-86.80) 102.1 (269.7-86.0) 84.9 (95.5-65.5)

Figure S1.
Summary of molecular phylogenetic hypotheses of extant tetraodontiform taxa obtained from alternative analyses: (a) Parsimony analysis (strict consensus of 36 trees with 23994 steps; IC = 0.41, IR = 0.76). (b) ML analysis. (c) Bayesian analysis
(under MrBayes).

Figure S2.
Summary of morphological phylogenetic hypotheses of extant and fossil tetraodontiform taxa obtained from alternative analyses: (a) Parsimony analysis (strict consensus of 699 trees with 570 steps; IC = 0.56; IR = 0.81). (b) ML analysis. The dagger symbol ('†') denotes fossil families.

Figure S3.
Summary of phylogenetic hypotheses combining morphological and molecular data from alternative analyses: (a) Parsimony analysis (strict consensus of 1828 trees with 23981 steps; IC=
0.31, IR= 0.65). (b) ML analysis. (c) Bayesian analysis (under MrBayes). The dagger symbol ('†') denotes fossil families.

Figure S4.
Marginal distributions for several clade ages obtained from the analyses of 12 calibrations
(black; priors and data), root calibration only (blue; data and no priors), and empty data (red; priors and no data) under node dating in BEAST. Except for the root constraint (a), which is common to all analyses, the remaining 11 comparisons show the effects of priors on age estimates. In five cases, marginal distributions of analyses with root calibration only are moderately to substantially different with those obtained with all calibrations (with and without data, which are in turn similar to each other), suggesting that priors have a moderate effect on this analysis.

Figure S5.
Chronograms for Tetraodontiformes based on the node dating calibration density analyses (ND-CD; red, MrBayes; blue,
BEAST; exponential root priors), including calibration points and 95% HPD intervals (see details on Supplementary File 2).

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