Evolutionary Aspects of Gobioid Fishes Based Upon a Phylogenetic Analysis of Mitochondrial Cytochrome B Genes

Gene 259 (2000) 5–15 www.elsevier.com/locate/gene

Evolutionary aspects of gobioid ﬁshes based upon a phylogenetic analysis of mitochondrial cytochrome b genes

Akihito a, Akihisa Iwata b, Takanori Kobayashi c, Kazuho Ikeo d, Tadashi Imanishi d, Hiroaki Ono d, Yumi Umehara d, Chika Hamamatsu d, Kayo Sugiyama e, Yuji Ikeda e, Katsuichi Sakamoto e, Akishinonomiya Fumihito f, Susumu Ohno g,1, Takashi Gojobori d,* a The Imperial Residence, 1-1 Chiyoda Chiyoda-ku, Tokyo 100-0001, Japan b Graduate School of Asian and African Area Studies, Kyoto University, 46 Shimoadachi-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan c Fish Genetics Division, National Research Institute of Aquaculture, 422-1 Nakatsuhamaura, Nansei-cho, Watarai-gun, Mie 516-0108, Japan d Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan e Imperial Household Agency, 1-1 Chiyoda Chiyoda-ku, Tokyo 100-8111, Japan f Yamashina Institute for Ornithology, 115 Tsutsumine-aza, Konoyama, Abiko-shi, Chiba 270-1145, Japan g Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3000, USA Accepted 13 October 2000 Received by G. Bernardi

Abstract

The Gobioidei is a large suborder in the order Perciformes and consists of more than 2000 species belonging to about 270 genera. The vast number of species and their morphological specialization adapted to diverse habits and habitats makes the classification of the gobioid fishes very difficult. A comprehensive estimation of the evolutionary scenario of all gobioid fishes using only morphological information is difficult for two major reasons: first, in addition to wide ecological diversification, there is a trend towards specialization and degeneration of morphological characters among these species; second, an appropriate outgroup of gobioid fishes has not been recognized. Based upon nucleotide sequence comparisons of gobioid mitochondrial cytochrome b genes, we established the phylogenetic relationships of their differentiation into many groups of morphological and ecological diversity. The phylogenetic trees obtained show that most species examined have diverged from each other almost simultaneously or during an extremely short period of time. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Evolution; Gobioid ﬁshes; Mitochondrial cytochrome b; Phylogeny

1. Introduction of Akihito et al. (2000) (Table 2), which is close to that of Nelson (1994) except for the Gobiidae. Nelson (1994) The Gobioidei is a large suborder in the order recognized five subfamilies in the Gobiidae, which Perciformes comprising about 268 genera and approxi- included about 212 genera and roughly 1875 species, mately 2121 species (Nelson, 1994), and they have been but he remarked that his classification of subfamilies of variously classified into families and subfamilies in recent the Gobiidae was provisional. As the classification needs times as detailed data on them accumulated. These further study, no subfamilies are recognized in the classifications are shown in Table 1 (Miller, 1973; Hoese, Gobiidae in this paper. 1984; Akihito et al., 1984; Hoese and Gill, 1993; Pezold, The gobioid fishes are generally small fishes and 1993; Nelson, 1994; Akihito et al., 2000). The classifica- rarely exceed 50 cm. The smallest of all the fishes is tion of the gobioid fishes used in this paper follows that found among them, i.e a species identified as Trimmatom of the Gobiidae whose matured females are 8–10 mm * Corresponding author. in standard length (Winterbottom and Emery, 1981). E-mail address: [email protected] (T. Gojobori) The geographical distribution of the gobioid fishes is 1 Deceased. worldwide, and they are absent only from the Arctic

Table 1 Seven patterns of the classiﬁcation of families and subfamilies of the Gobioidei in recent time

Miller (1973) 2 families (7 subfamilies) Rhyacichthyidae, Gobiidae (Eleotrinae, Pirskeninae, Xenisthminae, Gobionellinae, Tridentigerinae, Gobiinae, Kraemeriinae) Hoese (1984) 6 families (6 subfamilies) Rhyacichthyidae, Eleotrididae, Xenisthmidae, Kraemeriidae, Microdesmidae (Microdesminae, Ptereleotrinae), Gobiidae (Oxudercinae, Amblyopinae, Sicydiinae, Gobiinae) Akihito et al. (1984) 3 families (2 subfamilies) Rhyacichthyidae, Gobiidae (Eleotridinae, Gobiinae), Gunnellichthyidae Hoese and Gill (1993) 6 families (5 subfamilies) Rhyacichthyidae, Odontobutidae, Gobiidae (Butinae, Eleotrinae, Gobiinae), Xenisthmidae, Kraemeriidae, Microdesmidae (Microdesminae, Ptereleotrinae) Pezold (1993) 2 superfamilies {6 families (5 subfamilies)} Rhyacichthyoidea {Rhyacichthyidae}, Gobioidea {Eleotrididae, Kraemeriidae, Microdesmidae, Xenisthmidae, Gobiidae (Amblyopinae, Gobiinae, Gobionellinae, Oxudercinae, Sicydiinae)} Nelson (1994) 8 families (9 subfamilies) Rhyacichthyidae, Odontobutidae, Eleotridae (Butinae, Eleotrinae), Gobiidae (Oxudercinae, Amblyopinae, Sicydiinae, Gobionellinae, Gobiinae), Kraemeriidae, Xenisthmidae, Microdesmidae (Microdesminae, Ptereleotrinae), Schindleriidae Akihito et al. (2000) 8 families (4 subfamilies) Rhyacichthyidae, Odontobutidae, Eleotridae (Butinae, Eleotrinae), Xenisthmidae, Gobiidae, Kraemeriidae, Microdesmidae (Microdesminae, Ptereleotrinae), Schindleriidae

Table 2 Species of gobioid groups examined in the present study

Accession Species Family and subfamily Collecting locality Collection Habitata no. date

AB021230 Rhyacichthys aspro (Ra) Rhyacichthyidae Iriomote Island, Okinawa, Japan 13 July 1996 F AB021231 Bostrychus sinensis (Bs) Eleotridae Butinae Ishigaki Island, Okinawa, Japan 14 July 1996 B AB021232 Butis amboinensis (Ba) Eleotridae Butinae Iriomote Island, Okinawa, Japan 12 July 1996 B AB021233 Calumia godeﬀroyi (Cg) Eleotridae Eleotrinae Amami Island, Kagoshima, Japan 1998 S AB021234 Dormitator maculatus (Dm) Eleotridae Eleotrinae (purchased from aquarium shop) May 1998 F AB021235 Eleotris acanthopoma (Ea) Eleotridae Eleotrinae Ishigaki Island, Okinawa, Japan 7 April 1996 F AB021236 Eleotris fusca (Ef ) Eleotridae Eleotrinae Iriomote Island, Okinawa, Japan 30 May 1996 F AB021237 Eleotris melanosoma (Em) Eleotridae Eleotrinae Ishigaki Island, Okinawa, Japan 3 April 1996 B AB021238 Eleotris oxycephala (Eo) Eleotridae Eleotrinae Yoshida-cho, Shizuoka, Japan 1993 F AB021239 Gobiomorphus hubbsi (Gh) Eleotridae Eleotrinae Ashley River, New Zealand 28 July 1998 F AB021240 Hypseleotris compressa (Hc) Eleotridae Eleotrinae (purchased from aquarium shop) October 1997 F AB021241 Micropercops swinhonis (Ms) Odontobutidae Samrye-up, Chollabuk-do, Korea 8 May 1996 F AB021242 Mogurnda mogurnda (Mm) Eleotridae Eleotrinae (purchased from aquarium shop) December 1997 F AB021243 Odontobutis obscura (Oo) Odontobutidae Kochi, Japan 15 August 1995 F AB021244 Ophieleotris sp. Eleotridae Eleotrinae Iriomote Island, Okinawa, Japan 2 Nov. 1995 F (Akihito et al., 1984) (Osp) AB021245 Ophiocara porocephala (Op) Eleotridae Butinae Ishigaki Island, Okinawa, Japan 3 April 1996 B AB021246 Oxyeleotris marmorata (Om) Eleotridae Butinae Bangkok, Thailand June 1997 F AB021247 Tateurndina ocellicauda (Toc) Eleotridae Eleotrinae (purchased from aquarium shop) December 1997 F AB021248 Xenisthmus sp. ( Xsp) Xenisthmidae Zamami Island, Okinawa, Japan April 1997 S AB021249 Acanthogobius ﬂavimanus (Af ) Gobiidae Chiba, Japan 3 November 1996 S AB021250 Kraemeria cunicularia ( Kc) Kraemeriidae Ishigaki Island, Okinawa, Japan 14 July 1996 B AB021251 Periophthalmus argentilineatus (Par) Gobiidae Ishigaki Island, Okinawa, Japan 14 July 1996 B AB021252 Ptereleotris heteroptera (Ph) Microdesmidae Iriomote Island, Okinawa, Japan 20 July 1997 S Ptereleotrinae AB021253 Taenioides limicola (Tl ) Gobiidae Iriomote Island, Okinawa, Japan 22 July 1997 B AB021254 Tridentiger bifasciatus (Tb) Gobiidae Lake Shinji, Shimane, Japan 2 December 1996 B AB021255 Tridentiger obscurus (Tob) Gobiidae Mie, Japan May 1997 B AB021256 Gunnellichthys monostigma (Gm) Microdesmidae Iriomote Island, Okinawa, Japan 20 July 1997 S Microdesminae AB021257 Protogobius attiti (Pat) Unassigned Province SUD, New Caledonia 10 November 1997 F

a S: sea; F: fresh water; B: brackish water. Akihito et al. / Gene 259 (2000) 5–15 7 and Antarctic areas. Most gobioid fishes have bottom- 2.2. DNA amplification and sequencing dwelling habits in the sea and rivers, and their habitats show a wide spectrum, from the sea-floor at a depth of We extracted DNA from each of these 28 species of over 300 m to mountain streams. gobioid fishes. First, the tissue fragments were incubated The adaptations to these habitats have developed overnight at 60°C in TNES-6M buffer and proteinase various morphological specializations. A notable adap- K. Then, samples were deproteinized and total DNA tation to the benthic way of life is the development of was extracted from an individual sample, using the a sucker formed by uniting the pelvic fins to cling to a method of Asahida et al. (1996). We designed primers substratum. The sucker is found in a large number of specific to the cytochrome b gene that was used for PCR species of the Gobiidae. Sicyopterus japonicus has a (polymerase chain reaction) analysis (see Fig. 2). short round sucker whose adhesive power is extremely PCR reactions were conducted under the following strong, and it clings to the rocks in torrential streams conditions: 1 min at 94°C, followed by 30 cycles of 30 s by utilizing the sucker. It even climbs the walls of at 94°C, 30 s at 50°C, and 1 min at 72°C, and finally, a waterfalls, utilizing the mouth and sucker like an inch- single extra extension step of 5 min at 72°C. The PCR worm. On the other hand, Barbuligobius boehlkei lives products were purified using an Ultra Clean PCR on sandy and unstable bottoms in the sea, utilizing its Clean-up Kit (MO BIO). large sucker as an anchor. The purified PCR products were sequenced by the In addition to the morphological adaptations to these cycle sequencing dye-primer method, using an ABI 377 habitats, the gobioid fishes have undergone degeneration DNA sequencer. For the cytochrome b gene of each of the body lateral line, cephalic sensory canals, and species examined, sequencing was completed six times bones from the state found in the suborder Percoidei, for both strands to make sure of the sequence quality. which may be the basal evolutionary group from which Every effort was made during the entire process of the the other perciform groups, including the Gobioidei, experiments to avoid mislabelling and contamination. have been derived (Nelson, 1994), and these degenera- All sequence data obtained have been deposited in the tions are found within the gobioid fishes (Fig. 1). DDBJ/EMBL/GenBank International DNA sequence Though there is a trend towards degeneration of such data bank under accession numbers AB021230– characters as mentioned above, as well as morphological AB0212257 (Table 2). diversifications adaptive to habitats, it is difficult to estimate comprehensively the evolutionary scenario of 2.3. Sequence analysis all gobioid fishes using only morphological information. One reason for this problem is the controversy over the The nucleotide sequences and the deduced ancestral form of this group, though the monophyletic 380-residue-long amino acid sequences of the mito- origin of the gobioid fishes has been unanimously recog- chondrial cytochrome b genes from the 28 representative nized. An appropriate outgroup has not been identified gobioid species were obtained in the present study. (Winterbottom, 1993). These sequences were aligned with each other using In an attempt to resolve the various problems associ- CLUSTAL-W program packages (Thompson et al., ated with the gobioid fishes, we conducted a molecular 1994). The multiple alignments of these sequences were phylogenetic analysis using cytochrome b gene sequences subsequently mutually refined. Our attempts to obtain of mitochondrial DNA derived from 28 species of the a phylogenetic tree of gobioid fish utilized two indepen- gobioid fishes. Much to our surprise, we found that, for dent methods (Nei, 1987): (A) the maximum likelihood most species in the phylogenetic network obtained, the (ML) method (Felsenstein, 1981, 1989, 1995; Phylip branch lengths leading to each of the species examined 3.5c), and (B) the neighbour-joining (NJ) method from its centre were almost equal. This implies that (Saitou and Nei, 1987). We produced 100 bootstrap these species diverged simultaneously during a short replicates for ML trees and 1000 bootstrap replicates period at a given past time. Along with these observa- for NJ trees (Felsenstein, 1985). Because of the lack of tions, the taxonomic problems will also be addressed an appropriate outgroup, we constructed unrooted trees from the viewpoint of morphological and molecular instead of rooted trees. Various reasonably similar trees evolution. involving the 28 gobioid fish species were obtained by these two methods. The trees were constructed from all codon positions 2. Materials and methods of nucleotide sequences by these two methods. The trees were also constructed from the first and second codon 2.1. Materials positions (data not shown). Whether these trees were constructed from nucleotide substitutions involving all Materials used for the molecular analysis are shown three codon positions or from substitutions occurring in Table 2. only at the first and second positions made surprisingly 8 Akihito et al. / Gene 259 (2000) 5–15 ically from Akihito and coworkers (Akihito, ., 1984), and our original data. Some characters without scientific names are drawn schematically. et al Fig. 1. Some examples of morphological characters mentioned in the present study, which verify those of Table 3 except for vertebrae; modified schemat 1969, 1986; Akihito Akihito et al. / Gene 259 (2000) 5–15 9

3. Results and discussion

3.1. Explosive divergence of gobioid ﬁshes

The phylogenetic trees for 28 gobioid fishes were constructed by the ML and NJ methods (Fig. 3a and b respectively), and were constructed from all codon positions. Unrooted trees are presented because of the lack of an appropriate outgroup, since the root for each of the trees could not be determined definitely. As shown in Fig. 3a and b, the phylogenetic relationships among the species in the ML tree were virtually identical to those in the NJ tree except for two species: Od. obscura (Oo), and Xenisthmus sp. ( Xsp). They are separated into six clusters as follows, and the two species are not included in these clusters. Cluster 1: R. aspro (Ra), Pr. attiti (Pat) Cluster 2: D. maculatus (Dm), H. compressa (Hc), E. melanosoma (Em), E. fusca (Ef), E. acanthopoma (Ea), E. oxycephala (Eo), C. godeffroyi (Cg), Go. hubbsi (Gh) Cluster 3: Ox. marmorata (Om), Op. porocephala (Op), Bo. sinensis (Bs), Bu. amboinensis (Ba) Cluster 4: Tr. obscurus (Tob), Tr. bifasciatus (Tb), Ac. flavimanus (Af ), Pe. argentilineatus (Par), Ta. limicola (Tl), Mi. swinhonis (Ms) Cluster 5: Pt. heteroptera (Ph), Gu. monostigma (Gm), Kr. cunicularia (Kc). Cluster 6: Mo. mogurnda (Mm), Ophieleotris sp. (Osp), Fig. 2. Primer sequences used for PCR analysis of cytochrome b gene. Ta. ocellicauda (Toc) H, Y, D, and R represent ‘A, T, or C’, ‘C or T’, ‘A, T, or G’, and ‘A The distances between any pair of the six clusters are or G’ respectively. much shorter than those between each species within the clusters, and each species has almost the same little difference. While it is true that the substitution rate distance from the neighbouring cluster. Furthermore, at the third codon positions is usually ten times higher all bootstrap values between any pair of these six clusters than at the first or second positions, this extraordinarily are very small. This result implies that the six clusters high rate of substitutions at third codon positions cre- must have diverged almost at the same time or during ated its own problem: the saturation effect of nucleotide an extremely short period of time. substitutions. However, this effect was not serious when Gobioid fishes might have acquired various niches, the numbers of nucleotide substitutions at the first and mostly on the bottom of rivers and seas, including the second positions were included. The substitution method water stratum near the bottom. To accomplish this, they used for the construction of the ML tree shown in must have effectively used their small body size and Fig. 3a was that of Hasegawa et al. (1985). The ML benthic way of life, mostly with the attaching ability of trees were constructed under two different assumptions their suckers. These features would have made important regarding the substitution rate at each nucleotide site. contributions to the explosive divergence of gobioid Namely, a substitution rate was assumed (1) to be equal fishes. for all sites (Nei, 1987) and (2) to vary across sites Adult gobioid fishes spawn adhesive eggs and are following the gamma distribution (Ota and Nei, 1994; generally benthic dwellers, but they have a planktonic Takezaki and Gojobori, 1999). However, the trees life stage in their larval period. Therefore, passive obtained did not differ much from each other. Thus, migrations of larvae via currents might also have con- Fig. 3a shows the ML tree in the case of (1). tributed to the worldwide distribution of gobies. They Fig. 3b shows the NJ tree that was constructed from have planktonic life stages of variable duration during all codon positions. For the same reasons, the NJ tree their larval period (Akihito et al., 1984). Thus, this may was also constructed using the numbers of transversion explain why E. fusca, which takes up life at the bottom substitutions only. However, the tree obtained was not at a larger size than E. acantohopoma, has a wider much different from that constructed using all codon geographical distribution than E. acanthopoma. In addi- positions (data not shown). tion, some freshwater genera, for example Odontobutis 10 Akihito et al. / Gene 259 (2000) 5–15 ). Each species is indicated by two- or three-letter representations (see Table 1). Species b ) and the NJ method ( a Fig. 3. The phylogenetic trees for 28 gobioid fishes constructed by the ML method ( names with black and white circles show those six and five branchiostegal rays respectively. The bootstrap values were computed by 100 replications. Akihito et al. / Gene 259 (2000) 5–15 11 and some species of Rhinogobius, could have positively pelvic fins. Its appearance resembles some genera of the advanced into continental inland areas, changing their Balitoridae (Homalopteridae) of the Cypriniformes. Pr. life style from amphidromous (presence of planktonic attiti lives in slower areas of swift, clear streams and larval period in the sea) to fluvial (direct development has a slightly depressed head without thick pectoral and without planktonic larval period) type (Akihito et al., pelvic fins. Its appearance resembles Gobio and its 1984). related genera of the Cyprinidae of the Cypriniformes. These observations can explain the interesting enigma In spite of these differences, they share such common that, although gobioid groups appear unsuitable for characters as the body lateral line, bones, and the ocean navigation because of their specialized morphologi- transforming cteni on the scales (Fig. 1, Table 3). The cal features, their geographical distribution is global and body lateral line is only found in these two species in their ecological niches are immensely diverse. This finding the gobioid fishes. The loss of bones in these species is suggests that they became widely distributed around the the least in the gobioid fishes, except for the lack of the world, undergoing evolutionary specialization with many lower postcleithrum, and they have an infraorbital bone, morphological changes. This must have been accom- a dorsal extension of the scapula, three epurals, and plished by their advancement to diverse ecological niches transforming cteni, which are rarely found in other during an extremely short period of time. gobioid fishes (Fig. 1, Table 3). There is a difference Additional explanations are needed for an overview between them in the cephalic sensory canal system. That of the evolutionary trends of gobioid fishes. In the case of R. aspro is the least degenerate in the gobioid fishes, of Rhinogobius, they have only minor distinguishable with the presence of the infraorbital canal, a connection morphological differences except for colour patterns, but between the oculoscapular canal and the preopercular they have a distinct segregation of ecological niches, such canal, and an extension of the preopercular canal to the as currents and still waters, large and small rivers, lower jaw, whereas that of Pr. attiti has none of these upstream and downstream, fast and slow currents, and parts (Watson and Po¨llabauer, 1998), but that of Pr. deep and shallow depths, and show egg size diversity attiti is ranked less degenerate than other gobioid fishes (Akihito et al., 1984). The number of nucleotide substitu- except for the species included in Cluster 3. Contrary to tions of cytochrome b genes among ten species of the respective different appearances of these species, the Rhinogobuis except for R. giurinus which is thought to be above-mentioned morphological characters show that different genus, is 3.0±1.4% (Y. Aonuma, personal they are closely related. Our molecular research also communication). Aonuma et al. (1998) suggested that confirms that the relation between these species is very Rhinogobius had undergone the speciation process very close, as the ratios of nucleotide substitutions for R. recently. Taking into account the fact that the number aspro and Pr. attiti do not appear to be much different of nucleotide substitutions among 28 gobioid fishes was from the ratios within the species of Eleotris and about 10% in Fig. 3b, and that the gobioid group com- Tridentiger. prises more than 10% of presently existing fishes, we Cluster 2 comprises five genera of the Eleotrinae reasonably speculate that gobioid fishes repeatedly experi- of the Eleotridae; Dormitator, Hypseleotris, Eleotris, enced multiple adaptive radiations during their evolution. Calumia, and Gobiomorphus. As shown in Table 3, the As stated before, the phylogenetic trees obtained in genera of Cluster 2 share common characters of bones the present study strongly imply that an extremely high and 15 segmented caudal rays, but these characters are number of different species of gobioid fishes could have also found in two of three genera of Cluster 6. Thus, diverged during a very short period of time. Although a the genera of Cluster 2 are not morphologically sepa- similar phenomenon has been observed for cichlids in rated from those of Cluster 6. Our molecular research Lakes Malawi and Victoria (Meyer et al., 1990; Kocher shows that, apart from the fact that the relation between et al., 1993, 1995), global radiation of such a huge variety the two species of Eleotris, E. melanosoma and E. fuscus, of species as gobioid fishes has never been reported is closer, the genetic distances between the species of before. Furthermore, the divergence of Rhinogobuis and Eleotris are larger than those between Dormitator and cichlids is a much more recent phenomenon. Hypseleotris and between Calumia and Gobiomorphus. As species of Eleotris strongly resemble each other, the 3.2. Comparison between morphological characters and result of our molecular research shows that the morpho- molecular phylogenetic trees logical similarities do not always correlate with the genetic distances. Cluster 1 comprises R. aspro of the Rhyacichthyidae Cluster 3 comprises four genera of the Butinae of the and Pr. attiti, which has not yet been assigned to a Eleotridae: Oxyeleotris; Ophiocara, Bostrychus, and family (Watson and Po¨llabauer, 1998). They are Butis. They have no body lateral line, but the extension different in appearance and habitats. R. aspro lives in of the cephalic sensory canals is the same as those of torrential streams and holds the body close to the rocks Pr. attiti, and less degenerate than in other gobioid with the strongly depressed head and thick pectoral and fishes. The loss of bones in Ox. marmorata and Bo. 12 Akihito et al. / Gene 259 (2000) 5–15 ) ) # ) # # 9( )in / ) ) ) 27 ( ) ) ) ) ) ) # # # # # # # # # # ) = # ( ) 17 ) # # + IIIIII0 / ++ sp. 34 ~ 15 10 30 + )2( = 17 + # ( 17 ~ d ~ +++ 33 ~ 16 15 or 30 + = 16 ++ 18 ~ : ~ + present present absent ( ) variable variable 3 # ) 9( / # ) present present absent ( ) absent absent present ( ) absent absent present ( ) present present present ( ) absent present absent ( ) 16 13 ) absent absent absent ( # # # # # # # # ( 28 ( + )171717( = 12 + )2 2 # # 17 ~ II II II I 0 / ~ ++ 93 / 28 11 = 17 + + II II I II 0 / ) absent absent ( 26 11 83 # / = 16 + II II I I / degenerate degenerate degenerate marmorata aspro attiti obscura swinhonis c ) present ( 26 10 83 # / = phala 16 + II II I I / : extending dorsally between upper proximalradial and cleithrum. + 27 10 83 / = 16 + II II I I / 27 ~ 14 11 83 / +++++++++ 26 + or a = 13 16 ~ II II I I / degenerate degenerate degenerate ~ ++ ) # )3 ) absent absent absent absent present present ( ) present present present ( ) present present present present present present ( ) present present present present present present( # # 17 10 ) present present present present present present ( ) absent present absent present present present ( ) less less less less least less absent degenerate degenerate ( # # # # # # ) 32 ( ~ # ( )171717171717( ~ 16 # 31 + . (1988), Hayashi and Hayashi (1994), Hoese and Gill (1993), and Miller (1973) with our original data ( = ++ 36 et al ) present ( ) present ( ) present ( ~ 16 15 # # # ) 29 + # ( = 16 20 ~ ~ mogurnda ocellicauda amboinensis sinensis poroce 25 13 = 15 + present present ( sp. : ossified and incompletely surrounds the foramen. . (1984), Birdsong + 26 ~ 10 10 8 variable variable variable ( / b et al 25 + = 15 12 ~ IIIIII / ~ 93 / 15 14 +++ ++ ++ 27 + or = 12 16 ~ IIIIIII0 / ~ maculatus compressa 25 11 83 / = 15 + IIIIII / ++ ++ (four species) ) present present present present present ( ) present present present present present ( ) present present present 25 10 83 # # # / ) = # royi ( 15 ff + IIIIII / ++ present ( 31 ) present ( ) present ( # ~ 16 10 ( # ) b b 27 + # ( = 12 : ossified and completely surrounds the foramen. 19 ~ ~ variable 3 ++ Go.hubbsi C. gode Eleotris D. H. Ophieleotris Mo. Tat. Bu. Bo. Op. Ox. R. Pr. Od. Mi. Xenisthmus ++ e Modified from Akihito (1967, 1969, 1971, 1986), Akihito Absent in some speciesWithout of pore the G. same genus. Some individuals have oneRelation or between three. dorsal spine pterygiophores and neural spines. c e a b d Vertebrae 11 line P–V pattern Body lateral absent absent absent absent absent absent absent absent absent absent absent absent present present absent absent absent ( cteni Epural number 2 2 2 2 2 2 2 2 2 2 2 2 3 3 ( bones Transforming absent absent absent absent absent absent absent absent absent absent absent absent present present ( Supratemporal absent absent absent absent absent absent absent absent ( Middle radial absent absent absent absent absent absent absent absent absent absent absent absent present present present present absent ( postcleithrum Ossified scapula caudal rays Interneural gap present absent absent absent absent absent present variable ( Segmented 15 15 15 15 15 15 15 17 ( postcleithrum Lower present Upper present ( bone Endopterygoid present Infraorbital absent absent absent absent absent absent absent absent ( Table 3 Morphological characters of gobioid fishes with six branchiostegal rays mentioned in the present study Sensory canal degenerate absent absent absent degenerate degenerate absent absent ( this study. Akihito et al. / Gene 259 (2000) 5–15 13 sinensis (most specimens of which have an infraorbital Cluster 5 comprises each genus of the Ptereleotrinae bone) is the least in the gobioid fishes, because they and Microdesminae of the Microdesmidae and one have the lower postcleithrum, which is lacking in R. genus of the Kraemeriidae; Ptereleotris, Gunnelichthys, aspro and Pr. attiti, though their epurals are one fewer and Kraemeria. There is no consensus among researchers than in R. aspro and Pr. attiti. Ophiocara and Butis on the taxonomical position of Ptereleotris. The have no infraorbital bone, but other bones are present. Ptereleotris group and Microdesmus group including In addition to the above-mentioned characters, they Gunnellichthys share a very long posterior pelvic process. share such common characters as 17 segmented caudal Hoese (1984) proposed two subfamilies, Ptereleotrinae rays and the same P–V pattern (relation between dorsal and Microdesminae, in the family Microdesmidae. On spine pterygiophores and neural spines). Our molecular the other hand, Akihito et al. (1984) treated the research confirms that the relation among them is close Ptereleotris group as a member of the subfamily enough to classify them in the subfamily Butinae of the Gobiinae because of large differences in body shape, Gobiidae (Hoese and Gill, 1993) or that of the vertebra and dorsal spine number, and P–V pattern Eleotridae (Nelson, 1994) as far as the four genera are from the Gunnellichthyidae of Akihito et al. (1984). concerned. Miller (1973) also included them in the same subfamily, Cluster 4 comprises four genera of the Gobiidae and i.e. Gobiinae. In the present study, we have shown that one genus of the Odontobutidae; Tridentiger, Acantho- Pt. heteroptera is closer to Gunnellichthys monostigma gobius, Periophthalmus, Taenioides,andMicropercops. on the basis of molecular phylogenetic trees, and the Among the genera of Cluster 4, there is a distinct relative numbers of nucleotide substitutions for the two ff di erence between Micropercops and the other genera. species also do not appear to be much different from Micropercops is less degenerate in the loss of bones in the relative numbers between the two Tridentiger conge- the gobioid fishes, such as having an infraorbital bone, ners. Gunnellichthys and Kraemeria are morphologically the endopterygoid, upper postcleithrum, six branchioste- different. Although Kraemeria has a short body with a gal rays, scapula with the dorsal extension completely P–V pattern and the number of vertebrae common to surrounding the foramen, middle radial in the first ptery- the general Gobiidae, the tip of its lower jaw is elongated giophore of the second dorsal fin, and transforming cteni and cone-like and it lives a mid-sand life at the tidal on the scales (Fig. 1, Table 3). Thus the morphological line in brackish water. Gunnellichthys lives a free-swim- characters are similar to those of Cluster 1 or Od. obscura. ming life near the sea bottom. There is no consensus on The other genera have five branchiostegal rays and none the taxonomical position of the kraemerid gobies among of the above-mentioned characters of Micropercops.In researchers. Hoese (1984) proposed the family spite of these morphological differences, our molecular Kraemeriidae, and Miller (1973) proposed the subfamily research shows that both ML and NJ trees include Kraemeriinae, but the kraemerid gobies were included Micropercops in Cluster 4, though the genetic distance in the subfamily Gobiinae by Akihito et al.(1984). Our between Micropercops and the other genera of Cluster 4 molecular phylogenetic trees show that Kraemeria is is larger than that within the other genera and the bootstrap value between Micropercops and the other closer to Gunnellichys and Ptereleotris. genera of Cluster 4 is small. Further research on this Cluster 6 comprises three genera of the Eleotrinae of problem is needed. The other genera of Cluster 4 live in the Eleotridae; Mogurnda, Ophieleotris, and Tateurndina. ff diversified habitats and have diversified habits. Tridentiger But these three genera belong to di erent groups, as and Acanthogobius generally live on the bottom of brack- shown by Birdsong et al. (1988) and mentioned below. ish waters, whereas Periophthalmus leads a very active We also found that Gobiomorphus and Calumia, life out of the water, hopping about on land, using its Dormitator and Hypseleotris, and Ophieleotris and pectoral fins with their elongate muscular base, and with Mogurnda each formed smaller groups. However, ff bulging eyes, and Taenioides is eel-like in shape with the di erences in the cephalic sensory canals, the P–V dorsal and anal fins connected with the caudal fin and patterns, and the numbers of vertebrae were observed with degenerate eyes, burrowing into the soft mud in the within each group (Fig. 1, Table 3). Birdsong et al. brackish water. The morphological differences between (1988) grouped the gobioid fishes by the numbers of Periophthalmus and Taenioides adaptedtotheirrespective vertebrae and characteristics of the P–V patterns, creat- lifestylesledthemtobeclassifiedindifferent subfamilies ing, for example, the Dormitator group (Bostrychus, of the Gobiidae, Oxudercinae and Amblyopinae (Hoese, Dormitator, and Ophieleotris), the Eleotris group 1984; Nelson, 1994). However, Harrison (1989) placed (Calumia and Eleotris), the Gobiomorphus group them in the Gobionellinae, because they share similar (Gobiomorphus and Mogurnda), and the Hypseleotris features of the suspensorium. The molecular analysis group (Hypseleotris). Furthermore, Tateurndina was pre- does not appear to be much different from the ratios sented as an unassigned genus. Our results are not within the species of Eleotris and Tridentiger. This result consistent with their groupings. supports Harrison’s hypothesis that they comprise a Odontobutis of the Odontobutidae and Xenisthmus of monophyletic group. the Xenisthmidae do not belong to any cluster. Our 14 Akihito et al. / Gene 259 (2000) 5–15 molecular research supports the assignment of these mens and photographs, Dr Robert M. McDowall of the genera to their respective families by Nelson (1994). National Institute of Water and Atmospheric Research, Odontobutis and Micropercops were included in the New Zealand, Mr Tetsuo Yosino of the University of Eleotrididae (Hoese, 1984; Birdsong et al., 1988) or the the Ryukyus, Dr Ronald E. Watson of Florida, USA, Eleotrinae (Miller, 1973; Akihito et al., 1984). But Mr Hitoshi Sato of the Shimane Prefectural Hoese and Gill (1993) proposed the family Government, Mr Hiroshi Sakurai of the Tokyo Sea Life Odontobutidae, to which they assigned the genera Park, Dr Harumi Sakai of the National Fisheries Odontobutis, Micropercops, and Percottus, on the basis University, Shimonoseki, Mr Takashi Urano of the of the character state of the scapula, the anterior procur- University Museum, University of Tokyo, Dr Christine rent cartilage, the middle radial of the first pterygiophore Po¨llabauer of ERBIO, New Caledonia, Mr Kensaku of the second dorsal fin, and the transforming cteni on Azuma of the Nishinihon Institute of Technology, the scales, and that the family formed a second sister Kochi, Dr Koichi Shibukawa of the Matsudo Municipal group to the remaining gobioid fishes after the Museum, Dr Prachya Musikasinthorn of Kasetsart Rhyacichthyidae. Mi. swinhonis has an infraorbital bone, University, Thailand, Mr Kazunori Kumon of the Japan and some specimens also have three epurals (our unpub- Sea-Farming Association, Mr Hidenori Yoshigou of the lished data) like R. aspro and Pr. attiti. Furthermore, Naigai Technos Corporation, Hiroshima, and Mr only M. swinhonis has a small dorsal procurrent cartilage Masami Furuta of the Toba Aquarium. We are grateful in the Odontobutidae (our unpublished data). Our to Dr Ichiro Nakayama of the National Research results do not support the proposition of Hoese and Institute of Aquaculture, Mie for translating from Gill (1993), and the phylogenetic status of Odontobutis French, and Dr Frank Pezold of Northeast Louisiana and Micropercops is not clear in our phylogenetic trees. University, USA, for providing information about the These observations are interesting in understanding geographical distribution of gobioid fishes. We also the relationships between morphological and molecular thank Dr Richard C. Goris of Yokohama City phylogenies. However, our present molecular data seem University, Dr David Swinbanks of Nature Japan K.K., insufficient to conduct a firm discussion about the rela- Dr. Yoshimasa Aonuma of National Research Instutute tion between morphological and molecular phylogenies, of Fisheries Sciences Ueda and Mr Katsusuke Meguro because the number of nucleotides composed is still of the Imperial Household Agency for their cooperation. small. Moreover, we express our special thanks to Dr Toru Seven parties in recent time have propounded their Fuwa, the vice president of Wakunaga Pharmaceutical own views on the classification of families and sub- Company, for his encouragement to S.O. and T.G. families, as shown in Table 1. Thus, it seems extremely Finally, we note that the computation was done on a difficult to classify particular genera into an appropriate supercomputer system at the National Institute of family or subfamily. This difficulty is mainly because it Genetics, Mishima. is not always easy to identify morphological characters showing phylogenetic information, since gobioid fishes have undergone both morphological specialization and References degeneration. The molecular phylogenetic trees suggest that, because the six clusters of gobioid fishes examined Akihito, Prince, 1967. Additional research on the scapula of gobiid have diverged almost at the same time, it is no wonder fishes. Japan. J. Ichthyol. 14 (4/6), 167–182. in Japanese. that their classification is so difficult. Akihito, Prince, 1969. A systematic examination of the gobiid fishes Finally, we would like to emphasize that the role of based on the mesopterygoid, postcleithra, branchiostegals, pelvic morphological studies will become more important with fins, scapula and suborbital. Japan. J. Ichthyol. 16 (3), 93–114. the advancement of molecular phylogenetic studies. in Japanese. Akihito, Prince, 1971. On the supratemporals of gobiid fishes. Japan. More thorough and detailed studies of morphological J. Ichthyol. 18 (2), 57–64. in Japanese. and molecular evolution will give us significant insight Akihito, Prince, 1986. Some morphological characters considered to into the evolution of gobioid fishes. be important in gobiid phylogeny. In: Uyeno, T., Arai, R., Taniuchi, T., Matsuura, K. (Eds.), Indo-Pacific Fish Biology. Pro- ceeding of the Second International Conference on Indo-Pacific Fishes. The Ichthyological Society of Japan, Tokyo, pp. 629–639. Acknowledgements Akihito, Prince, Hayashi, M., Yoshino, T., Shimada, K., Yamamoto, T., Senou, H., 1984. Suborder Gobioidei. In: Masuda, H.Amaoka, We thank Dr Naoko Takezaki at the Graduate K., Araga, C., Uyeno, T., Yoshino, T. (Eds.), The Fishes of the University for Advanced Studies for her help. Special Japanese Archipelago. Tokai University Press, Tokyo, pp. 236–289. thanks are given to the following people for providing English text; plates 235–258 and 353–355. Akihito, Sakamoto, K., Ikeda, Y., Iwata, A., 2000. Suborder Gobi- invaluable specimens: Mr Atsushi Ono of the Dive oidei. In: Nakabo, T. (Ed.), Fishes of Japan with Pectorial Keys Service Ononini, Mr Kiyoshi Hagiwara of the Marine to the Species, second edition. Tokai University Press, Tokyo, Biotechnology Institute Corporation for providing speci- pp. 1139–1310. Akihito et al. / Gene 259 (2000) 5–15 15

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