J. Zool., Lond. (1998) 246, 215±231 # 1998 The Zoological Society of London Printed in the United Kingdom

Phylogenetic relationships of the Pomacanthidae (Pisces: Teleostei) inferred from allozyme variation

K. C. Chung and N. Y. S. Woo* Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong (Accepted 23 February 1998)

Abstract Phylogenetic relationships of 31 pomacanthid species representing all genera were investigated by horizontal starch gel electrophoresis. Nei's genetic distances (D) were calculated and dendrograms constructed from allozyme variation in 23 loci. The average D value was 0.03 between conspeci®c colour variants, 0.40 between congeneric subgenera, and 1.27 between confamilial genera. From the dendrograms the following were revealed: (1) the generic status of Apolemichthys arcuatus and Holacanthus venustus,as well as the generic and subgeneric status of Centropyge multifasciata were con®rmed; (2) the subgeneric status of Xiphypops was con®rmed; (3) Pygoplites diacanthus was found to be closely related to members of the subgenus Pomacanthus; (4) Pomacanthus zonipectus was found to be biochemically distinct from its con-subgeners; (5) the genera Pomacanthus and Holacanthus are more closely related than currently thought; (6) the Genicanthus species might warrant a subfamily of their own.

Key words: Pomacanthidae, biochemical systematics, phylogeny, heterozygosity, allozymes, electrophoresis, genetic distance

INTRODUCTION comprehensively revised the systematics of the group and proposed the probable af®nities between genera. He The Pomacanthidae consists of more than 80 brilliantly recognized seven genera: Chaetodontoplus, Pomacanthus coloured species that inhabit tropical coral reefs around (including the subgenera Pomacanthodes and Poma- the world. Family members vary in body size and canthus), Heteropyge (= Euxiphipops, including the pro®le, coloration, as well as in habitat and feeding, yet subgenera Heteropyge and Arusetta), Genicanthus, they are uni®ed in their strongly developed preopercular Holacanthus (including the subgenera Apolemichthys, spine, from which their family name is derived. Despite Holacanthus, Angelichthys, and Plitops), Centropyge its prominent status among the coral reef ®shes, studies (synonymized Xiphipops Jordan & Jordan) and Pygo- on the biology of the Pomacanthidae seem to receive plites. In a subsequent review, Smith (1955) gave full less attention than it deserves. There have been some generic rank to Apolemichthys and resurrected Xiphipops reports on the reproductive behaviour and biology to distinguish it from Centropyge, elevated Arusetta to (Bauer & Bauer, 1981; Thresher, 1982; Moyer, Thresher generic status, and erected the genus Pomacanthops. & Colin, 1983; Hourigan & Kelley, 1985), protogynous Smith also proposed separate family status, which was development (Shen & Liu, 1976; Hourigan & Kelley, supported by Munro (1955, 1967). This view was 1985; Moyer, 1987; Lutnesky, 1996), euryhalinity (Woo further justi®ed by Freihofer (1963) and Burgess (1974), & Chung, 1995), feeding habits and behaviour (Randall who contrasted structural differences between poma- & Hartman, 1968), and social organization (Moyer canthids and chaetodontids. Shen & Liu (1978) revised et al., 1983) of pomacanthids. Pomacanthidae, considering Euxiphipops as a junior The Pomacanthinae was previously included as a synonym of Pomacanthus, resurrecting Xiphipops as in subfamily in the family Chaetodontidae due to morpho- Smith (1955). They further divided the group into the logical similarities between the two groups (Fraser- subfamilies Pomacanthinae (containing the genera Brunner, 1933). Systematic work on the Pomacanthidae Chaetodontoplus and Pomacanthus) and Holacanthinae. was scattered and confusing until Fraser-Brunner (1933) Although Allen (1979) partly followed the systematics of Shen & Liu (1978) in his comprehensive book on pomacanthids, his resurrection of Xiphipops as proposed *All correspondence to: Dr N. Y. S. Woo, Biology Department, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. by Smith (1955) and Shen & Liu (1978) has not been E-mail: [email protected] widely accepted. 216 K. C. Chung and N. Y. S. Woo

Despite differing viewpoints, the current theory of Table 1. A list of the pomacanthid species studied in the pomacanthid phylogeny has not been seriously debated. present investigation. Allozymes from white muscle; liver; and However, as a result of several new species descriptions, heart were studied. The species are listed in alphabetical order. The total number of specimens sampled for a species (n)is including Centropyge debelius Pyle (Pyle, 1990a), C. indicated narcosis Pyle & Randall (Pyle & Randall, 1993), and C. boylei Pyle (Pyle, 1992; Pyle & Randall, 1993), and the Species investigated n erection of two genera Paracentropyge Burgess and Sumireyakko Burgess (Burgess, 1991), the classi®cation Apolemichthys arcuatus (Gray, 1831) 3 Apolemichthys trimaculatus (LaceÂpeÁde, 1831) 4 of the family has become more confused (Pyle, 1992; Apolemichthys xanthurus (Bennett, 1832) 3 Pyle & Randall, 1993, 1994). A taxonomic revision of Centropyge bicolor (Bloch, 1787) 5 the family is urgently required. Apparently certain Centropyge bispinosa (GuÈnther, 1860) (Paci®c Ocean) 6 problems related to pomacanthid phylogeny cannot be Centropyge bispinosa (GuÈnther, 1860) (Indian Ocean) 3 resolved by traditional morphological comparisons. The Centropyge ferrugata Randall & Burgess, 1972 4 purpose of this study is to estimate genetic divergence Centropyge heraldi Woods & Schultz, 1953 4 and relationships among pomacanthid species by using Centropyge loricula (GuÈnther, 1874) 9 Centropyge multifasciata (Smith & Radcliffe, 1911) 5 biochemical techniques. Isozyme electrophoresis was Centropyge nox (Bleeker, 1853) 3 employed because it is an established method of gener- Centropyge potteri (Jordan & Metz, 1912) 3 ating systematic data, as well as its power in delineating Centropyge tibicen (Cuvier, 1831) 4 species at various phylogenetic levels (Avise, 1974; Buth, Centropyge vrolikii (Bleeker, 1853) 3 1984). No previous studies using this technique for Chaetodontoplus duboulayi (GuÈnther, 1867) 5 pomacanthids have been published. Chaetodontoplus mesoleucus (Bloch, 1787) 4 Genicanthus semifasciatus (Kamohara, 1934) 7 Holacanthus ciliaris (Linnaeus, 1758) 8 Holacanthus passer Valenciennes, 1846 4 MATERIALS AND METHODS Holacanthus tricolor (Bloch, 1795) 8 Holacanthus tricolor (Bloch, 1795) Fish specimens (Brazilian colour variant) 3 Holacanthus venustus Yasuda & Tominaga, 1969 5 A total of 31 pomacanthid species including different Pomacanthus annularis (Bloch, 1787) 3 Pomacanthus arcuatus (Linnaeus, 1758) 5 colour variants from 2 species (Holacanthus tricolor Pomacanthus asfur (ForsskaÊl, 1775) 3 (Bloch): normal colour variant and the `Brazilian' colour Pomacanthus imperator (Bloch, 1787) 5 variant, and Centropyge bispinosa (GuÈnther): Paci®c Pomacanthus navarchus (Cuvier, 1831) 2 Ocean colour variant (Philippines) and Indian Ocean Pomacanthus paru (Bloch, 1787) 4 colour variant (Bali, Indonesia)) were investigated (Table Pomacanthus semicirculatus Cuvier, 1831 6 1). Species identi®cation was aided by Fraser-Brunner Pomacanthus sexstriatus (Kuhl & Van Hasselt, 1831) 3 (1933), Steene (1977), and Allen (1979). Among the Pomacanthus xanthometopon (Bleeker, 1853) 5 Pomacanthus zonipectus (Gill, 1862) 3 conspeci®c colour variants, the `Brazilian' colour variant Pygoplites diacanthus (Boddaert, 1772) 4 of H. tricolor differs from the normal colour variant by having a much thicker red margin in both dorsal and anal Total no. of specimens 146 ®ns, a much wider yellow stripe in the anal ®n, as well as having a yellow stripe of even thickness in the dorsal ®n which extends to the posterior margin. The Indian Ocean Individuals from which the tissues were removed were variant of C. bispinosa differs from the Paci®c one by ®xed in 10% formaldehyde. lacking the characteristic dark blue vertical stripes on the sides of the body. Specimens used in this study, repre- senting all pomacanthid genera and covering the major Allozymes investigated geographic area of their distribution, were obtained through the aquarium trade. As only the country of The following enzymes were studied: adenylate origin of the specimens rather than the exact site of kinase, alkaline phosphatase, aldolase, glucose-6-phos- collection was known, only specimens from the same phate isomerase, glucose-6-phosphate dehydrogenase, shipment were sampled as far as possible to avoid compli- glyceralde-hyde-3-phosphate dehydrogenase, isocitrate cations due to genetic differences among populations. dehydrogenase, l-lactate dehydrogenase, malate Fish were freshly dissected or temporarily stored at dehydrogenase, malic enzyme, phosphoglucomutase, 6- 770 8C before dissection. Liver, heart, and white phosphogluconate dehydrogenase, pyruvate kinase, and skeletal muscle were dissected from each specimen and superoxide dismutase (Table 2). The names of enzymes homogenized with 3±5 vols of extraction medium (0.1 M and Enzyme Commission numbers follow the recom- Tris-HCl, pH 7.0 containing 1 mM EDTA(Na2), 1 mM mendations of the Commission on Biochemical (b-mercaptoethanol and 0.05 mM NADP) as described Nomenclature (1973). Nomenclature for the loci was by Luczynski (1990). The homogenate was centrifuged adopted after Shaklee, Allendorf et al. (1990). Alleles at 20 000 g for 15 min at 4 8C; the supernatant was then were designated according to their relative electro- aliquoted and stored at 770 8C until electrophoresis. phoretic mobility. Pomacanthid phylogeny 217

Table 2. Summary of the enzymes examined in this research

Enzyme (abbreviation) Sa Locib E.C. no. Typec Tissued

Adenylate kinase (AK) 1 AK-1*,2*,3* 2.7.4.3 t M Alkaline phosphatase (ALP) 2 ALP* 3.1.3.1 h L Aldolase (ALD) 4 ALD* 4.1.2.13 l L Glucose-6-phosphate isomerase (GPI) 2 GPI-1*,2* 5.3.1.9 i M Glucose-6-phosphate dehydrogenase (GDH) 2 GDH* 1.1.1.49 o H Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 4 GAPDH* 1.2.1.12 o L Isocitrate dehydrogenase (IDH) 2 IDH-1*,2* 1.1.1.42 o M l-Lactate dehydrogenase (LDH) 4 LDH-1*,2* 1.1.1.27 o M, H Malate dehydrogenase (MDH) 2 MDH-1*,2* 1.1.1.37 o M Malic Enzyme (NADP+)(ME) 4 ME* 1.1.1.40 o M Phosphoglucomutase (PGM) 1 PGM-1*,2* 5.4.2.2e t L 6-Phosphogluconate dehydrogenase (6-PGDH) 2 6-PGDH* 1.1.1.44 o M Pyruvate kinase (PK) 1 PK* 2.7.1.40 t M Superoxide dismutase (SOD) 2 SOD-1*,2* 1.15.1.1 o L a Number of subunits. b Nomenclature for the loci was adopted after Shaklee et al. (1990). c Enzyme types: H, hydrolases; I, isomerases; L, lyases; O, oxidoreductases; T, transferases. d Tissue types: H, heart; L, liver; M, white muscle. e Former EC number, 2.7.5.1.

Table 3. Summary of genetic variability over 23 loci scored in each species

a Species P0.99 Ho He AIL AAL (Mean ‹ SE) (Mean ‹ .se) (Mean ‹ se)

A. arcuatus 0.088 ‹ 0.010 0.087 0.119 2.96 ‹ 0.01 1.22 ‹ 0.42 A trimaculatus 0.067 ‹ 0.008 0.087 0.118 3.87 ‹ 0.01 1.26 ‹ 0.45 A. xanthurus 0.130 ‹ 0.014 0.130 0.125 3.00 ‹ 0.00 1.22 ‹ 0.42 C. bicolor 0.196 ‹ 0.012 0.304 0.257 4.65 ‹ 0.03 1.57 ‹ 0.51 C. heraldi 0.121 ‹ 0.011 0.159 0.166 3.96 ‹ 0.01 1.35 ‹ 0.49 C. multifasciata 0.211 ‹ 0.012 0.333 0.190 4.74 ‹ 0.02 1.43 ‹ 0.51 C. nox 0.228 ‹ 0.015 0.304 0.199 4.00 ‹ 0.00 1.43 ‹ 0.51 C. tibicen 0.152 ‹ 0.013 0.203 0.134 4.00 ‹ 0.00 1.39 ‹ 0.50 C. vrolikii 0.101 ‹ 0.011 0.101 0.078 3.00 ‹ 0.00 1.22 ‹ 0.42 C. bispinosa (I) 0.153 ‹ 0.014 0.217 0.154 4.26 ‹ 0.06 1.35 ‹ 0.49 C. bispinosa (P) 0.165 ‹ 0.014 0.232 0.171 4.22 ‹ 0.06 1.43 ‹ 0.51 C. ferrugata 0.143 ‹ 0.011 0.188 0.132 3.96 ‹ 0.01 1.30 ‹ 0.47 C. loricula 0.140 ‹ 0.008 0.348 0.185 7.48 ‹ 0.06 1.48 ‹ 0.59 C. potteri 0.101 ‹ 0.010 0.101 0.130 3.00 ‹ 0.00 1.26 ‹ 0.45 Ch. duboulayi 0.057 ‹ 0.005 0.087 0.131 4.61 ‹ 0.03 1.30 ‹ 0.47 Ch. mesoleucus 0.098 ‹ 0.010 0.130 0.180 4.00 ‹ 0.00 1.39 ‹ 0.58 G. semifasciatus 0.062 ‹ 0.006 0.116 0.091 5.48 ‹ 0.05 1.22 ‹ 0.42 H. ciliariis 0.113 ‹ 0.009 0.261 0.183 6.83 ‹ 0.05 1.48 ‹ 0.59 H. tricolor (Bra) 0.097 ‹ 0.010 0.101 0.107 3.13 ‹ 0.03 1.22 ‹ 0.42 H. tricolor 0.101 ‹ 0.008 0.232 0.139 6.70 ‹ 0.06 1.35 ‹ 0.57 H. passer 0.066 ‹ 0.007 0.087 0.126 3.96 ‹ 0.01 1.26 ‹ 0.45 H. venustus 0.066 ‹ 0.008 0.101 0.081 4.61 ‹ 0.02 1.17 ‹ 0.39 P. asfur 0.078 ‹ 0.006 0.101 0.154 3.91 ‹ 0.01 1.39 ‹ 0.50 P. navarchus 0.152 ‹ 0.015 0.101 0.109 2.00 ‹ 0.00 1.17 ‹ 0.39 P. sexstriatus 0.124 ‹ 0.009 0.159 0.109 3.87 ‹ 0.02 1.35 ‹ 0.57 P. xanthometapon 0.099 ‹ 0.007 0.159 0.128 4.83 ‹ 0.02 1.35 ‹ 0.49 P. annularis 0.087 ‹ 0.013 0.087 0.099 3.00 ‹ 0.00 1.17 ‹ 0.39 P. imperator 0.097 ‹ 0.008 0.159 0.130 4.91 ‹ 0.01 1.30 ‹ 0.47 P. semicirculatus 0.127 ‹ 0.007 0.232 0.145 5.39 ‹ 0.03 1.48 ‹ 0.59 P. zonipectus 0.087 ‹ 0.007 0.087 0.122 3.00 ‹ 0.00 1.74 ‹ 2.07 P. arcuatus 0.097 ‹ 0.007 0.145 0.107 4.30 ‹ 0.04 1.30 ‹ 0.47 P. paru 0.067 ‹ 0.010 0.087 0.069 3.91 ‹ 0.01 1.17 ‹ 0.39 Py. diacanthus 0.135 ‹ 0.013 0.174 0.130 3.78 ‹ 0.02 1.30 ‹ 0.47 a P0.99, proportion of polymorphic loci at the 0.99 level; Ho, observed heterozygosity; He, expected heterozygosity; AIL, average number of individuals scored per locus; AAL, average number of alleles scored per locus. 218 K. C. Chung and N. Y. S. Woo ; o H ; HC, Centropyge Pomacanthus ; CM, Pomacanthus asfur ; PS, (Paci®c Ocean); CF, ; PA, Genicanthus semifasciatus ; GS, Centropyge heraldi Centropyge bispinosa Pomacanthus imperator ; CH, Holacanthus venustus represents the number of specimens sampled; ; PI, N ; HV, . Centropyge bicolor representing muscle, liver, and heart origin of the enzyme. Loci Chaetodontoplus mesoleucus (Indian Ocean); CBP, h ; CB, , and Holacanthus passer l , ; CHM, Pomacanthus annularis Pygoplites diacanthus m ; HP, ; PA, ; PYD, Centropyge bispinosa ; CBI, Apolemichthys xanthurus Holacanthus tricolor Chaetodontoplus duboulayi Pomacanthus paru ; AX, ; PP, ; CHD, Pomacanthus xanthometopon Centropyge vrolikii ; PX, ; CV, Centropyge potteri Pomacanthus arcuatus Apolemichthys trimaculatus (`Brazilian colour variant'); HT, ; CP, ., 1990. Abbreviations of enzymes as in Table 2 ; AT, ; PAR, Centropyge tibicen et al Pomacanthus sexstriatus ; CT, ; PS, Holacanthus tricolor Centropyge loricula ; CL, Apolemichthys arcuatus Centropyge nox ; HTB, Pomacanthus zonipectus ; PZ, ; CN, . Relative allele frequencies of the loci scored * * * Pomacanthus navarchus * semicirculatus nomenclature follows Shaklee, Allendorf multifasciatus Holacanthus ciliaris PN, Table 4 Abbreviations: AA, represents the observed heterozygosity for each locus. The name of each locus is preceded by the pre®x Centropyge ferrugata Locus m-AK-1 m-AK-2 m-AK-3 l-ALD Pomacanthid phylogeny 219 * * * * l-GAPDH h-GDH m-GPl-l Locus l-ALP 220 K. C. Chung and N. Y. S. Woo * * * * * * Locus m-GPI-2 m-IDH-1 m-IDH-2 h-LDH m-LDH-1 m-LDH-2 Pomacanthid phylogeny 221 * * * * * Locus m-MDH-1 m-MDH-2 m-ME l-PGM-1 l-PGM-2 222 K. C. Chung and N. Y. S. Woo * * * * m-PK l-SOD-1 l-SOD-2 Locus l-6PGDH Pomacanthid phylogeny 223 ith Nei's correction for . Genetic distance (D) and genetic identity (I) of the pomacanthid species investigated. Genetic distance values (above diagonal) were calculated w small sample size. Genetic identity values are placed below diagonal. I, P, B: colour morphs from Indonesia, Philippines, and Brazil, respectively. Table 5 224 K. C. Chung and N. Y. S. Woo

Electrophoresis nera, n = 39), with an average value of 0.52. The average values of genetic distances between genera ranged from The enzymes were analysed by horizontal starch gel 1.14 ‹ 0.30 (between Pomacanthus to other genera, electrophoresis at 4 8C using 12% starch (Sigma n = 39) to 1.39 ‹ 0.25 (between Chaetodontoplus to other Chemical Co., St. Louis, U.S.A., Lot no. 114H0286), at genera, n = 9), with an average value of 1.27. 70 V (20 mA per gel) for the initial 20 min, then for 17 h Based upon the genetic distance values phenetic at 110 V (38 mA per gel, or 2.3 mA per cm gel width). dendrograms were constructed by using the PHYLIP All the chemicals for allozyme electrophoresis were package (Figs 1 & 2). purchased form Sigma. The sliced gels were stained for allozymes according to Shaw & Prasad (1970) and Harris & Hopkinson (1976) with slight modi®cations. DISCUSSION In all electrophoretic runs the buffer employed was 188 mM Tris / 50 mM citrate pH 8.6 (Harris & Due to limited sample availability relatively few speci- Hopkinson, 1976), with 15 mg NADP added at the mens per species were examined in this study. The cathode compartment. The gel buffer was obtained by number of loci examined (23), however, was relatively 10-fold dilution of the tank buffer. high. The analyses of genetic distances and hence the dendrograms generated should therefore be robust since estimates of genetic divergence depend more on the Comparison between taxa number of loci examined than on sample size (Nei, 1978; Gorman & Renzi, 1979; Shaklee, Tamaru & Allele frequencies, proportion of polymorphic loci at Waples, 1982). the 0.99 level (P0.99), and average heterozygosity per The average value of genetic distances between con- locus (Ho) were calculated. Average expected heterozyg- speci®c colour variants, congeneric species, intergeneric osity (He) was calculated using Nei's (1978) correction species, and genera accorded well with reported values for small sample size. Genetic distances of Nei (1972) (Shaklee, Tamaru et al., 1982; Cross, Mills & Williams, between taxa were calculated from the allele frequencies 1992). by using GENDIST (Felsenstein, 1993), which was The average value of heterozygosities among the modi®ed to include the correction for small sample size specimens studied (16.7%) is higher than the typical (Nei, 1978). Phenetic dendrograms were constructed values for ®sh in general (Ward, Skibinski & Wood- with FITCH, KITSCH, and UPGMA in the PHYLIP mark, 1992) and marine ®sh in particular (Smith & package (Felsenstein, 1993) based on Nei's genetic Fujio, 1982, Ward, Woodmark & Skibinski, 1994), distance. which averages around 5.3%. This abnormally high value of heterozygosity might partly be due to the small sample size employed. As evidently this value agreed RESULTS well with the average value of expected heterozygosity (14.1%) calculated with Nei's (1978) correction for small Genetic variability of the scored loci sample size. Alternatively, such a high heterozygosity could be accounted for by the habitat specialist- A total of 23 loci was resolved from the enzymes generalist model (Valentine, 1976; Smith & Fujio, 1982) assayed. Several measures of genetic variability in- in which the level of heterozygosity tends to be higher in cluding the proportion of polymorphic loci at the 0.99 habitat specialists and lower in habitat generalists. level (P0.99), observed heterozygosity (Ho), expected Specialist species would represent an accumulation of heterozygosity (He), average number of individuals several narrow-range alleles and is characterized by high scored per locus (AIL), and average number of alleles heterozygosities so as to allow a greater degree of ®ne scored per locus (AAL) are provided in Table 3. The tuning to the environment. In generalist species, the allele frequencies of the 23 loci scored are tabulated in population or species would consist of a few wide-range Table 4. alleles and be characterized by low heterozygosities so that those individuals are capable of surviving over a wide range of conditions. In such highly specialized Genetic distance between taxa (feeding habits, morphology, colouration, and beha- viour, etc.) teleosts as the pomacanthids, it is predicted Genetic distance values between conspeci®c colour var- by the habitat specialist-generalist model that they are iants was minute (Table 5), with 0.01 between the characterized by a high heterozygosity value. Holacanthus tricolor colour variants (n = 11), and 0.05 Of the three dendrograms derived from the algo- between the Centropyge bispinosa colour variants rithms (Figs 1 & 2), the two derived from KITSCH and (n = 9). The average value of genetic distances between FITCH are identical, while several features are common congeneric subgenera (Tables 6 & 7) ranged from to the three dendrograms: 0.40 ‹ 0.12 (mean ‹ sd) (between Holacanthus a. The phenetic relationships in the genus Pomacanthus (Holacanthus) to other subgenera, n = 28) to 0.61 ‹ 0.06 LaceÂpeÁde basically conform with current pomacanthid (between Pomacanthus (Pomacanthus) to other subge- systematics, this supports the synonymizing of Euxiphi- Pomacanthid phylogeny 225 l . Genetic distance (D) and genetic identity (I) among pomacanthid taxa. Genetic distance is placed above diagonal and genetic identity below diagona Table 6 226 K. C. Chung and N. Y. S. Woo

Table 7. Average genetic distances between pomacanthid genera and subgenera

Genera and subgenera Genetic distance (mean ‹ sd) Genetic distance (mean ‹ sd)

Apolemichthys 1.38 ‹ 0.25 (n = 146) Centropyge (Centropyge) 0.53a (n = 49) Centropyge (Xiphypops) 0.53a (n = 49) Centropyge 1.25 ‹ 0.25 (n = 146) 0.53b (n = 49) Chaetodontoplus 1.39 ‹ 0.25 (n = 146) Genicanthus 1.38 ‹ 0.22 (n = 146) Holacanthus (Angelichthys) 0.41 ‹ 0.13a (n = 28) Holacanthus (Holacanthus) 0.40 ‹ 0.12a (n = 28) Holacanthus (Plitops) 0.56 ‹ 0.10a (n = 28) Holacanthus (undetermined) 0.53 ‹ 0.13a (n = 28) Holacanthus 1.25 ‹ 0.42 (n = 146) 0.48 ‹ 0.13b (n = 28) Pomacanthus (Arusetta) 0.52 ‹ 0.02a (n = 39) Pomacanthus (Euxiphipops) 0.56 ‹ 0.08a (n = 39) Pomacanthus (Pomacanthodes) 0.53 ‹ 0.08a (n = 39) Pomacanthus (Pomacanthus) 0.61 ‹ 0.06a (n = 39) Pomacanthus 1.14 ‹ 0.30 (n = 146) 0.56 ‹ 0.07b (n = 39) Pygoplites 1.15 ‹ 0.25 (n = 146) All genera or subgenera 1.27 ‹ 0.28d (n = 146) 0.52 ‹ 0.07c (n = 116) a Genetic distance between congeneric subgenera. b Average genetic distance between all congeneric subgenera. c Average genetic distance between all subgenera among all genera. d Average genetic distance between all genera. pops Fraser-Brunner (and Arusetta Fraser-Brunner) assign P. imperator, P. annularis and P. semicirculatus to with Pomacanthus by Shen & Liu (1978). As indicated by the subgenus Arusetta Fraser-Brunner, which manifests genetic distance the subgenus Pomacanthus LaceÂpeÁde is higher biochemical af®nity, although P.(Arusetta) asfur presented as the outgroup of its congeners. This is is presented as the outgroup of the latter species. Alter- justi®ed from the differences of this subgenus in general natively, it can be seen from the dendrograms that P. morphology and in dorsal spine and ®n ray counts from zonipectus is biochemically closer to P. asfur and that of other subgenera (IX, 30 vs XIII, 20±24 in other members of the subgenus Pomacanthus than to other congeners). The grouping of subgenus Pomacanthus with members of the subgenus Pomacanthodes. The three Pygoplites diacanthus (Boddaert) (then with other Poma- groups of species are allopatric but uni®ed by their canthus species studied, and subsequently with the genus biochemical af®nity and juveniles that manifest yellow Holacanthus LaceÂpeÁde), however, is unexpected since the colouration and cleaning habits, it is tentatively believed two groups are usually placed in different subfamilies. that they should have evolved from a common ancestor. In other Pomacanthus subgenera, P.(Arusetta) asfur Regarding this, members of the subgenus Poma- (ForsskaÊl) groups with members of subgenus Poma- canthodes other than P. zonipectus might warrant a canthodes Gill, which in turn forms a cluster with subgenus of their own. members of subgenus Euxiphipops. P.(Pomacanthodes) b. The genera Holacanthus and Pomacanthus are zonipectus (Gill), is biochemically divergent from its con- clustered together in the clustering algorithms. This subgeners and is presented as the outgroup of all Poma- coincides with the proposal of Fraser-Brunner (1933) canthus subgenera except subgenus Pomacanthus that the genera Pomacanthus and Holacanthus might be LaceÂpeÁde. This discordance from current pomacanthid monophyletic. The af®nities between the two genera can systematics is supported by several features of P. zoni- be justi®ed from their resemblance in unique characters pectus which are atypical of the subgenus, such as dorsal such as large body size, spongivorous feeding habits spine count (XI vs XIII typical of the subgenus), pre- (Randall & Hartman, 1968), and ontogenic dichroma- sence of yellow coloured band in juveniles (which can tism (a phenomenon only observed also in the also be seen in juveniles of P.(Arusetta) asfur and those Chaetodontoplus species, which is closely related to the of subgenus Pomacanthus; Allen, 1979), ectoparasite- two genera). Moreover, some juveniles of both genera cleaning habit of the juveniles (a characteristic also were reported to set up cleaning stations that serve to shared by juveniles of the subgenus Pomacanthus and remove external parasites from other ®sh (Kerstitch, some Holacanthus species; Kerstitch, 1977), and atypical 1977). Further evidence is given by the clustering of body colouration and dimensions of the adults (the P. diacanthus in the algorithms with members of protrusion at the forehead region and the body height to Pomacanthus. The monotypic genus Pygoplites was standard length ratio is higher than for its con-subge- erected by Fraser-Brunner (1933) and was described as ners; Smith & Heemstra, 1986). Considering the related to Holacanthus, with differences mainly in the biochemical distinctiveness of P. zonipectus from all cheek-scales, form of preorbital and interoperculum, other con-subgeners, it might be more appropriate to and caudal ®n pro®le. P. diacanthus and most Pomacanthid phylogeny 227

1.5 1.0 0.5 0.0 Genetic distance Fig. 1. Phenetic dendrogram of the 31 pomacanthid species based upon FITCH and KITSCH analysis of genetic distance data corrected for small sample size. * Centropyge bispinosa (colour variant from Indonesia), ** Holacanthus tricolor (colour variant from Brazil).

Holacanthus species share common characters such as that members of Pomacanthus LaceÂpeÁde, which are dorsal spine count and dentate preoperculum. Gas biochemically close to P. diacanthus, be clustered with bladders in both genera are bifurcated and distinguished Holacanthus. only in the number of pyloric caeca, which is three to Further investigations on the phylogenetic relation- four in Holacanthus and 22 in Pygoplites (Shen & Liu, ship between the genera Pomacanthus, Holacanthus, and 1978). Based on the morphological resemblance Pygoplites would be crucial in resolving pomacanthid between Pygoplites and Holacanthus, it is reasonable phylogeny and evolution. As P. diacanthus is allopatric 228 K. C. Chung and N. Y. S. Woo

1.5 1.0 0.5 0.0 Genetic distance Fig. 2. Phenetic dendrogram of the 31 pomacanthid species based upon UPGMA analysis of genetic distance data corrected for small sample size. * Centropyge bispinosa (colour variant from Indonesia), ** Holacanthus tricolor (colour variant from Brazil). with both members of the subgenus Pomacanthus, (1984) and tentatively considered to be a member of P. arcuatus and P. paru, it is tentatively believed that Holacanthus (Pyle, 1989), always clustered with its con- P. diacanthus may represent an intermediate form geners, A. trimaculatus and A. xanthurus, though it is an between Holacanthus and Pomacanthus that emerged as outgroup of the latter species. Evidence supporting the the two groups began to diverge. generic status of A. arcuatus includes body pro®le and c. Apolemichthys arcuatus, which was placed in the colour pattern (dark patch in anal ®n and scattered newly erected genus Desmoholacanthus by Fowler spots on body), which are typical of the genus. Fowler (1941), excluded from Apolemichthys by Heemstra (1941) distinguished the genus Desmoholacanthus Pomacanthid phylogeny 229

Fowler from Apolemichthys Fraser-Brunner on the basis g. Genetic distance between the colour variants of H. of the greatly contrasted colouration of this species, tricolor and C. bispinosa are 0.01 and 0.05, respectively. together with characters such as longer body, denticu- The biochemical differences between the colour variants lated shoulder girdle, and small spines at the lower edge can be resolved by increasing the sample size as such of preopercle. These characters, however, are not con- assays involve extensive screening of the polymorphic sidered to be much different from members of loci among colour variants. As there are at least four Apolemichthys and should not warrant either generic or colour variants of C. bispinosa in the Indo-Paci®c region subgeneric recognition. (Steene, 1977), it is interesting to investigate the extent d. Holacanthus venustus Yasuda & Tominaga, a much of diversity between the various colour variant popula- debated species, was placed in Genicanthus by Shen & tions, which might possibly provide hints on the pattern Lim (1975), in Holacanthus by Allen (1979) with undeter- of pomacanthid diversity and evolution. mined subgeneric status, in Sumireyakko by Burgess h. Genicanthus Swainson is presented as the outgroup (1991) but placed in Centropyge Kaup and suggested to of all other pomacanthid genera. As judged from the represent a link between C. multifasciata and the rest of high average genetic distance from other pomacanthid Centropyge (Pyle & Randall, 1993). However, H. venustus genera, and the possession of some unique characters always clustered with members of Holacanthus. H. ve- among pomacanthids such as external morphology nustus and the group containing H.(Angelichthys) ciliaris (much longer body length, emarginate to lunate caudal (L.) and H.(Holacanthus) tricolor (Bloch) form a cluster, ®n), shorter and smaller teeth (Fraser-Brunner, 1933; which then group with H.(Plitops) passer Valenciennes. Randall, 1975), mid-water planktivorous mode of nutri- The genetic distance between H. venustus and other tion, conspicuous permanent sexual dimorphism Holacanthus subgenera, together with its meristic differ- (Randall, 1975), promiscuous social organization ences from its congeners (fewer ®n ray counts and smaller (Moyer, 1984; Pyle, 1990b), and unique oesophageal body size; Burgess, 1991) seem to warrant separate sub- papillae (Howe, 1993), this genus warrants separate generic, rather than generic status. On the contrary, the subfamilial designation. As only one Genicanthus genetic distance between H.(Angelichthys) ciliaris and H. species was investigated in this research, support for this (Holacanthus) tricolor might be too small to support the argument requires more extensive sampling of other separation of the two species into different subgenera. Genicanthus species. e. The two Centropyge subgenera, Centropyge Kaup Dendrograms derived from UPGMA differ from that and Xiphypops Jordan & Jordan, form a group with an generated by the other two algorithms only in the average genetic distance of 0.53. This con®rms the clustering of Apolemichthys Fraser-Brunner, which is current subgeneric status of the two groups and thus presented with Chaetodontoplus Bleeker in the former, resurrection of the subgenus Xiphypops to full generic and with Centropyge Kaup in the latter algorithms. In status as proposed by Smith (1955) and Shen & Liu spite of the differing results, the algorithms did con®rm (1978) is negated. a close relationship among the three groups. Although In the subgenus Xiphypops, C. potteri (Jordan & they belong to different subfamilies and vary in body Metz) and C. loricula (GuÈnther) form a cluster, C. size, they are morphologically similar, and including the ferrugata Randall & Burgess and C. bispinosa (GuÈnther) possession of rounded unpaired ®ns. The genus Chaeto- form another cluster. This grouping is supported by the dontoplus may represent a crucial group for determining phenotypic resemblance (in terms of colouration, body the phylogenetic relationship between the currently re- size, and morphology) among members of the groups. cognized subfamilies, and tentatively, with the third In the subgenus Centropyge the species form a hierarchy putative subfamily containing the genus Genicanthus of clusters, with C. heraldi Woods & Schultz and C. Swainson. bicolor (Bloch) genetically more diverse from other members of the subgenus. The phenetic relationships of the Centropyge species as revealed in the present den- Acknowledgements drograms, however, are far from complete since only 10 of the 32 currently recognized Centropyge species were The authors acknowledge the hospitality of Drs Dave sampled. Additional investigations are necessary in Aldeson and Philip Moore of CSIRO, Sydney, for order to clearly de®ne the phenetic and phylogenetic permission to carry out part of the electrophoresis work relationships within the genus. in their laboratories. Special thanks are also extended to f. Centropyge (Centropyge) multifasciata (Smith & many individuals from CSIRO at Sydney, in particular Radcliffe), which was placed in the newly erected genus Dr Noriko Nishimura, Yuk Lin Wong, Bronwyn Paracentropyge Burgess (1991) based on morphological Kelley, Kathy Isaacs, and Pat Pisansarkit, whose exten- differences with its congeners, and whose generic status sive assistance have been invaluable. We are also was considered as undetermined by Pyle & Randall grateful to Dr K. H. Chu for his constructive criticisms (1993), always clustered with members of subgenus on the manuscript. This study was supported in part by Centropyge Kaup. Thus, the generic as well as subge- a Direct Grant for Research awarded to NYSW by The neric status of C. multifasciata is con®rmed through its Chinese University of Hong Kong. biochemical af®nity with its con-subgeners. 230 K. C. Chung and N. Y. S. Woo

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