Molecular Phylogeny of Osteoglossoids: A New Model for Gondwanian Origin and Plate Tectonic Transportation of the Asian Arowana

Yoshinori Kumazawa* and Mutsumi Nishida{dagger}

*Department of Earth and Planetary Sciences, Nagoya University, Nagoya, Japan; and
{dagger}Ocean Research Institute, University of Tokyo, Tokyo, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
One of the traditional enigmas in freshwater zoogeography has been the evolutionary origin of Scleropages formosus inhabiting Southeast Asia (the Asian arowana), which is a species threatened with extinction among the highly freshwater-adapted fishes from the order Osteoglossiformes. Dispersalists have hypothesized that it originated from the recent (the Miocene or later) transmarine dispersal of morphologically quite similar Australasian arowanas across Wallace's Line, but this hypothesis has been questioned due to their remarkable adaptation to freshwater. We determined the complete nucleotide sequences of two mitochondrial protein genes from 12 osteoglossiform species, including all members of the suborder Osteoglossoidei, with which robust molecular phylogeny was constructed and divergence times were estimated. In agreement with previous morphology-based phylogenetic studies, our molecular phylogeny suggested that the osteoglossiforms diverged from a basal position of the teleostean lineage, that heterotidines (the Nile arowana and the pirarucu) form a sister group of osteoglossines (arowanas in South America, Australasia, and Southeast Asia), and that the Asian arowana is more closely related to Australasian arowanas than to South American ones. However, molecular distances between the Asian and Australasian arowanas were much larger than expected from the fact that they are classified within the same genus. By using the molecular clock of bony fishes, tested for its good performance for rather deep divergences and calibrated using some reasonable assumptions, the divergence between the Asian and Australasian arowanas was estimated to date back to the early Cretaceous. Based on the molecular and geological evidence, we propose a new model whereby the Asian arowana vicariantly diverged from the Australasian arowanas in the eastern margin of Gondwanaland and migrated into Eurasia on the Indian subcontinent or smaller continental blocks. This study also implicates the relatively long absence of osteoglossiform fossil records from the Mesozoic.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Freshwater fishes form an important aspect of biogeographical studies, because they do not disperse easily through saltwater areas, and thus their evolution may be tightly linked to the geological histories of landmasses on which the evolution took place (Banarescu 1990Citation , pp. 11–55; Lundberg 1993Citation ). In such studies, it is very useful to have molecular clocks that can reliably date the corresponding evolutionary events. Previous studies (Thomas and Beckenbach 1989Citation ; Martin, Naylor, and Palumbi 1992Citation ; Adachi, Cao, and Hasegawa 1993Citation ; Martin and Palumbi 1993Citation ) have consistently suggested that molecular clocks run much more slowly in fishes than in mammals, presumably due to the lower metabolic rates and/or increased selectional constraints on the protein sequence evolution in poikilothermic fishes. However, a clock suitable for dating old divergences among bony fishes well over 100 MYA has not been fully developed, partly due to the supposed methodological difficulty in estimating large molecular distances by correcting multiple substitutions and the relative paucity of reliable fossil-based estimates of divergence times for bony fishes.

We have recently shown that gamma-corrected distances based on amino acid sequences of two mitochondrial protein genes, i.e., NADH dehydrogenase subunit 2 (ND2) and cytochrome b (cytb) genes, provide a good estimate of pairwise distances between rather distantly related animals (Kumazawa, Yamaguchi, and Nishida 1999Citation ; Kumazawa and Nishida 2000Citation ). These distances were then used to calibrate molecular clocks of bony fishes under some reasonable assumptions and to suggest that the familial radiation of perciform fishes considerably predated the Cretaceous/Tertiary boundary, after which their fossil records concertedly appear (Kumazawa, Yamaguchi, and Nishida 1999Citation ).

The present study focuses on fishes of the order Osteoglossiformes, one of the primary freshwater fish groups that are strictly intolerant of saltwater (Banarescu 1990Citation , pp. 48–55, 62–66). The osteoglossiforms are considered basal teleosts that preserve primitive anatomical features (e.g., the toothed tongue bones), but their individual members show peculiar specializations in morphology (e.g., elongate anal and dorsal fins), physiology (e.g., the air-breathing function of the swim bladder), and behavior (e.g., mouth brooding) (Nelson 1994Citation , pp. 90–97; Greenwood and Wilson 1998Citation ). These specializations or adaptations in morphology have contributed to obscure phylogenetic relationships of the osteoglossiforms (Bonde 1996Citation ; Li and Wilson 1996Citation and references therein). In a standard classification by Nelson (1994Citation , pp. 90–97), they were divided into two suborders, i.e., Osteoglossoidei and Notopteroidei (see table 1 ). The former comprises arowanas (the family Osteoglossidae) and the butterflyfish (the only species in the Pantodontidae), whereas the latter includes mooneyes (Hiodontidae), Old World knifefishes (Notopteridae), elephantfishes (Mormyridae), and the aba (the only species in the Gymnarchidae). Extant osteoglossiforms are adapted to various (mostly tropical or subtropical) freshwater habitats in continents of Gondwanian origin with some exceptions, i.e., hiodontids in North America, two notopterid genera from South to Southeast Asia, and the Asian arowana in Southeast Asia (Nelson 1994Citation , pp. 90–97).


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Table 1 Classification and Geographical Distribution of Extant Species in the Order Osteoglossiformes Based on Nelson (1994, pp. 90–97)

 
The Asian arowana (Scleropages formosus) has acquired a special status in Japan and some East Asian countries as a very popular but extremely expensive aquarium fish, which has led to its inclusion among species threatened with extinction (Goh and Chua 1999Citation , pp. 17–24). Several types of S. formosus with different color patterns inhabit separate regions of Southeast Asia (Borneo, Sumatra, and Indochina) that were probably connected through freshwater habitats during the Pleistocene glacial ages (Goh and Chua 1999Citation , pp. 17–24). An interesting question about this species from a biogeographical standpoint arises from the fact that two other species of the same genus inhabit Australasia (Australia and New Guinea), which is generally considered to belong to a zoogeographical region different from that of Southeast Asia (Banarescu 1995Citation , pp. 1349–1354). Was it possible for the arowanas of the primary freshwater fish category to disperse across Wallace's Line? This has been one of the greatest enigmas in freshwater zoogeography (Banarescu 1990Citation , p. 159; 1995, pp. 1349–1354, 1397).

Molecular phylogenetic approaches may provide new insights into this question. To our knowledge, however, molecular phylogeny of the osteoglossiforms has not been fully investigated, except for mormyrid electric fishes (Alves-Gomes 1999Citation ; Lavoué et al. 2000Citation ). In this study, we thus analyzed the osteoglossoid phylogeny and divergence times using the two mitochondrial protein sequences expected to function as useful molecular markers for these issues.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Samples and Sequence Determination
We determined the complete nucleotide sequences of the ND2 and cytb genes for 12 osteoglossiform species consisting of all extant species of the suborder Osteoglossoidei and two genera from each of the families Notopteridae and Mormyridae (see table 1 for classification and geographic distribution of these species). Although hiodontids and the aba were not sampled in this study, these 12 species cover most major groups of the order Osteoglossiformes. Fish specimens were obtained from either other investigators or local shops. The Asian arowana is a species threatened with extinction and thus protected by CITES (Goh and Chua 1999Citation , pp. 9–16). For this species, we used two individuals, here designated Asian arowana 1 and Asian arowana 2, that had died after being legally imported from Indonesia to Japan. They were cultivated individuals of a type of the so-called Bornean "Red arowana".

Amplification and sequence determination of the ND2 and cytb genes were carried out as previously described (Kumazawa, Yamaguchi, and Nishida 1999Citation ). Primers designed to amplify and sequence the same genes of perciforms (Kumazawa, Yamaguchi, and Nishida 1999Citation ) were useful in the present study. Amplified fragments purified with the QIAquick PCR purification kit (QIAGEN) were subjected to dye-terminator sequencing with the Applied Biosystems 373A DNA sequencer. Complete nucleotide sequences of the genes were unambiguously determined by reading both strands. The nucleotide sequence data reported in this study will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB035221AB035246.

Phylogenetic Analyses
Sequence alignment was conducted by eye with translated amino acid sequences of the osteoglossiforms as well as other taxa: the carp (Chang, Huang, and Lo 1994Citation ), the loach (Tzeng et al. 1992Citation ), the trout (Zardoya, Garrido-Pertierra, and Bautista 1995Citation ), African and neotropical cichlids (Kumazawa, Yamaguchi, and Nishida 1999Citation ), Amia calva (Kumazawa, Yamaguchi, and Nishida 1999Citation ), the coelacanth (Zardoya and Meyer 1997Citation ), sharks (Cao et al. 1998Citation ; Delarbre et al. 1998Citation ; Rasmussen and Arnason 1999aCitation ), and a ray (Rasmussen and Arnason 1999bCitation ). The alignment will appear in the EMBL database with the accession numbers ds43644 (ND2) and ds43645 (cytb). Amino acid sequences of the two genes were concatenated for phylogenetic analyses after gap sites were removed (723 alignable sites in total). Sharks and a ray were used as an outgroup.

Phylogenetic analyses using either nucleotide or amino acid sequences were conducted by three different methods: the maximum-parsimony (MP) method with PAUP, version 4.0b2 (Swofford 1999Citation ), the neighbor-joining (NJ) method (Saitou and Nei 1987Citation ) with njboot in Takezaki's Lintre package (Takezaki, Rzhetsky, and Nei 1995Citation ), and the maximum-likelihood (ML) method with MOLPHY, version 2.3 (Adachi and Hasegawa 1996aCitation ). Detailed conditions for each analytical method are described in table 2 . In this study, all pairwise distances used in the NJ analyses were corrected using a gamma parameter representing the extent of rate heterogeneity over sites. The importance of incorporating this parameter into deep-branch phylogenetic analyses has been widely recognized in previous literature that dealt with broad taxonomic groups (see, e.g., Kumazawa and Nishida 1999, 2000Citation ; Mindell et al. 1999Citation ; Takezaki and Gojobori 1999Citation ).


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Table 2 Bootstrap Support for Each Nodal Relationship of the Osteoglossiform Phylogeny by Different Methods

 
Divergence Time Estimation
Estimation of divergence times was based on gamma-corrected ML or Poisson distances of the ND2/cytb amino acid sequences. These gamma-corrected distances among mammals (birds in part) were shown to correct multiple substitutions at the same sites most effectively even for divergences of a few hundred million years ago or 0.5–1.0 substitutions per site in the pairwise distance (Kumazawa, Yamaguchi, and Nishida 1999Citation ; Kumazawa and Nishida 2000Citation ). Gamma-corrected ML distances were obtained with PAML, version 2.0 (Yang 1999Citation ), using a gamma parameter ({alpha} = 0.44) estimated from the data set, whereas gamma-corrected Poisson distances averaged among possible species pairs at a node were obtained with tpcv in Takezaki's Lintre package (Takezaki, Rzhetsky, and Nei 1995Citation ).

The clock of bony fishes was calibrated as in our previous study (Kumazawa, Yamaguchi, and Nishida 1999Citation ), with minor modifications. First, we previously used eight sharks as chondrichthyan species, but here we used three sharks and a ray. This change had negligible effect on the calibration but saved computational time for the phylogenetic analyses. Second, we previously used PUZZLE, version 4.0 (Strimmer and von Haeseler 1996Citation ), for estimating gamma-corrected ML distances (Kumazawa, Yamaguchi, and Nishida 1999Citation ). However, these distances later turned out to be slightly overestimated due to errors in the mtREV24 matrix of PUZZLE 4.0, as noted in PUZZLE's online manual (http://www.tree-puzzle.de/manual.html). In this study, we thus used PAML 2.0 (Yang 1999Citation ) for estimating gamma-corrected ML distances and confirmed that they were consistent with the values obtained using PUZZLE, version 4.0.2, in which the mtREV error was fixed.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Molecular Phylogeny Using Two Mitochondrial Proteins
Among 723 amino acid sites of the ND2/cytb sequence data, 334 sites were constant among the 28 taxa used in figure 1 , but the remaining 389 variable sites included 309 parsimony-informative ones. Two individuals of the Asian arowana differed from each other at five amino acid sites, which represents the sequence polymorphism within species.



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Fig. 1.—A neighbor-joining tree constructed from concatenated amino acid sequences of ND2 and cytb genes using sharks and a ray as an outgroup. The gamma-corrected Poisson distance was used with njboot in Takezaki's Lintre package (Takezaki, Rzhetsky, and Nei 1995Citation ). Bootstrap probabilities from 300 replications are shown on the corresponding branches. Nodes a–l are specified for reference to tables 2 and 3

 
We built MP, NJ, and ML trees from both nucleotide and amino acid sequences of the ND2/cytb sequence data. Figure 1 shows an NJ tree using the amino acid sequences, and results from the other methods are summarized in table 2 . All of these molecular trees indicated the same osteoglossiform phylogeny, except for the topological relationship at node g. The robustness of the remaining topological relationships against methodological changes, as well as high bootstrap probabilities on several internal nodes, lend support to the reliability of the obtained molecular phylogeny in general.

Phylogenetic Relationship Within Osteoglossiformes
The molecular trees (fig. 1 and table 2 ) support monophyly of the osteoglossiform species with high bootstrap values in relation to the other teleostean groups used in this study (see node i). Consistent with current ichthyological classification based on morphology (Nelson 1994Citation , pp. 1–5), the osteoglossiforms branched off basally from teleosts before the divergence of ostariophysans (carp and loach) from the lineage leading to protacanthopterygians (trout) and acanthopterygians (cichlids). Within the order Osteoglossiformes, monophyly of the osteoglossid species was supported with high bootstrap probabilities at node e. However, the relationship among the osteoglossids, notopterids, and mormyrids remained unresolved in our molecular analyses, because different tree-building methods supported different clustering patterns among them (table 2 ).

The molecular trees consistently showed an unexpected phylogenetic affinity of the butterflyfish with the notopterids, although bootstrap support at node j varied considerably depending on the method used (table 2 ). Morphological studies suggested that this species could be the sister group of either one (osteoglossines) or both (osteoglossines and heterotidines) osteoglossid subfamilies (Taverne 1979Citation ; Lauder and Liem 1983Citation ; Nelson 1994Citation , pp. 90–97; Bonde 1996Citation ; Li and Wilson 1996Citation ). If this untraditional relationship really is the case, a number of synapomorphies defining the suborders Osteoglossoidei and Notopteroidei (see, e.g., Lauder and Liem 1983Citation ; Li and Wilson 1996Citation ) need to be reconsidered. Thus, although the phylogenetic affinity of the butterflyfish to the notopterids is currently the most straightforward interpretation of the molecular data, it will need to be further evaluated in the future. Within the family Osteoglossidae, two distinct lineages representing the osteoglossines and heterotidines were recognized, albeit with weaker bootstrap support for the osteoglossine monophyly. This is in agreement with previous morphological studies (Lauder and Liem 1983Citation ; Bonde 1996Citation ; Li and Wilson 1996Citation ). The Osteoglossinae and the Heterotidinae have distinct morphological characteristics with regard to, e.g., the presence or absence of mandibular barbels and the number of branchiostegal rays (Nelson 1994Citation , pp. 90–97).

As for the topological relationships among the five osteoglossine species, the molecular trees were in good agreement with the traditional classification (Nelson 1994Citation , pp. 90–97). Two species of Osteoglossum make a sister clade to the Scleropages species, among which two morphologically more similar species in Australasia form a sister clade to the Asian species. Moderately high (nodes a and b) to very strong (node d) bootstrap support was obtained. In spite of these topological consistencies with the traditional classification, the molecular phylogeny had an unexpected feature. The divergences among three Scleropages species (nodes a and b) were much deeper than expected from the fact that they were classified within the same genus (see fig. 1 ). This became conspicuous by comparison with the depth of divergences between mormyrid genera and among cichlid genera. The three Scleropages species may thus be more appropriately classified into different genera, at least from the molecular standpoint. In contrast, the divergence between two Osteoglossum species is reasonably shallow in light of their classificatory status. These observations, in turn, highlight the exceptionally high morphological conservation among the Scleropages species. Further support for this argument was obtained by quantitative evaluation of the depth of each divergence point (see below). Of course, we are aware that taxonomic ranks should not be determined from molecular divergence values only. Further study should be expected to scrutinize our proposition to revise the classificatory status of the genus Scleropages.

Rate Constancy Test, Clock Calibration, and Divergence Time Estimation
In order to use molecular sequence data as the molecular clock, the homogeneity of modes and rates of sequence evolution among lineages must be carefully examined. PUZZLE 4.0.2 (Strimmer and von Haeseler 1996Citation ) uses a chi-square analysis to test at the 5% level whether the amino acid composition of each taxon is identical to the average composition among all taxa. We performed this test using the ND2/cytb amino acid sequences and confirmed that no taxon had a significantly deviated amino acid composition (data not shown).

Relative-rate tests (the two-cluster test; Takezaki, Rzhetsky, and Nei 1995Citation ) were then conducted using the gamma-corrected amino-Poisson distances among the 28 taxa. Since sharks and a ray were used as an outgroup, evolutionary rates were compared between clusters created by all nonchondrichthyan nodes of the tree shown in figure 1 . The test showed, with a 99% significance level, that the lineage leading to the northern barramundi may have experienced a significantly accelerated molecular evolution compared with that leading to the spotted barramundi after their divergence at node b (data not shown). This rate inequality was not found between clusters at any other internal node. We thus excluded the northern barramundi from subsequent analyses. After that, the clock hypothesis held firm for all of the internal nodes.

When the significance level was lowered to 95%, external lineages leading to the Nile arowana and to the pirarucu were also found to possibly have different evolutionary rates. However, no other clusters created by any internal node, including those for cichlids, were found to have different rates (data not shown). We retained both of the heterotidines for subsequent analyses, allowing a somewhat unreliable estimation of the divergence time at node f. However, this did not affect the reliability of estimated times at the other internal nodes. Removal of either of the Nile arowana or the pirarucu had a negligible effect on them (data not shown). Taken together, these results indicate that there is no significant rate difference between the osteoglossiforms and other groups of fishes, which can justify the use of the clock calibrated using nonosteoglossiform fishes for estimating divergence times among the osteoglossiforms.

For the time estimation, we used gamma-corrected ML and Poisson distances of the ND2/cytb amino acid sequences that have been shown to correct multiple substitutions most effectively (see Materials and Methods). Figure 2 shows that the clock for bony fishes can be calibrated consistently using the reasonable assumption of continental vicariance of cichlids and two external calibration points based on reliable time estimates from independent molecular and/or paleontological evidence. A regression line through the origin (5.4 x 10-4 substitutions/site/Myr) was used as the clock to infer divergence times among the osteoglossiform taxa (table 3 ). Similar divergence times were obtained using two independent distance measurements, i.e., gamma-corrected ML and Poisson distances.



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Fig. 2.—Calibration of the molecular clock. ND2/cytb amino acid sequences of the 28 taxa (see fig. 1 ) were used. Gamma-corrected ML distances averaged among the corresponding species pairs were plotted against estimated divergence times. Plotted data represent the divergence of African and Neotropical cichlids at the time of the continental breakup of the African and South American landmasses (100 MYA), sarcopterygian versus actinopterygian divergence at 450 MYA and chondrichthyan versus osteichthyan divergence at 528 MYA. Recent molecular (e.g., Streelman et al. 1998Citation ; Farias et al. 1999Citation ; unpublished data) and morphological (Stiassny 1991Citation ) studies indicate that the African and Neotropical cichlids are monophyletic relative to each other and that the Indian-Malagasy species make an outgroup of the African + Neotropical clade, strongly supporting the vicariant divergence of the continental cichlid clades on the Gondwanaland breakup (see Kumazawa, Yamaguchi, and Nishida [1999Citation ] for more detailed discussion). The latter two divergence times were from independent molecular time estimates using multiple nuclear gene sequences (Kumar and Hedges 1998Citation ). The earliest fossil records for sarcopterygians and actinopterygians were, respectively, from the Lower Devonian and the Upper Silurian (Benton 1993Citation , pp. 611–613, 657–659), indicating that they diverged from each other in the Silurian (409–439 MYA) or earlier (see open arrows in the figure) and that the molecular time estimate for the divergence (450 MYA) is not considerably overestimated, if it is overestimated at all. Together with the earliest chondrichthyan fossils from the Middle Devonian (Benton 1993Citation , pp. 593–595), these paleontological records also suggest Silurian or earlier divergence between chondrichthyans and osteichthyans. A regression line through the origin (R2 = 0.997) was obtained for the molecular clock of this study (5.4 x 10-4 substitutions/site/Myr). Note that even if the calibrations were made as consistently as possible with the minimum divergence times from the fossils (see the broken line), the clock rate would increase by only 13%, and this would not change our basic arguments about the historical biogeography of osteoglossiforms

 

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Table 3 Divergence Times Among Osteoglossiforms Estimated in this Study

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Historical Biogeography
Geological evidence (Smith, Smith, and Funnell 1994Citation ; the Plates Project 1998Citation ) shows that all continents remained united as the supercontinent Pangea during Triassic times (208–245 MYA). Terrestrial faunas were similar worldwide, and there were probably few geographical barriers that hindered their dispersal on Pangea (see, e.g., Briggs 1995Citation , pp. 61–65). In the middle Jurassic (157–178 MYA), Pangea was split into Laurasia and Gondwanaland, which were further fragmented into smaller landmasses: Eurasia, North America, and Greenland from the former, and Africa, South America, Australia, Antarctica, Madagascar, and India from the latter (Smith, Smith, and Funnell 1994Citation ; the Plates Project 1998Citation ). Plate tectonics has continuously reshaped these landmasses to the present arrangement.

Although extant osteoglossiforms inhabit terrestrial regions mostly of Gondwanian origin, fossil evidence suggests their once worldwide distribution (Lundberg 1993Citation ; Bonde 1996Citation ; Li and Wilson 1996Citation ). This is consistent with our molecular evidence suggesting that not only did the order Osteoglossiformes originate before Pangea began to be fragmented, but also its diversification into individual (sub)families (see values at nodes e, g, i, and j in table 3 ). On the other hand, the estimated divergence time between two osteoglossine genera (172 ± 19 MYA at node c, table 3 ) overlaps the period when Pangea separated into Laurasia and Gondwanaland (Smith, Smith, and Funnell 1994Citation ; the Plates Project 1998Citation ). It thus seems possible that the two genera originated and evolved primarily on Gondwanaland. The divergence time between black and silver arowanas at node d (26 ± 6 MYA) was small enough to deduce that they evolved within the isolated South American landmass.

Origin and Migrational Pathway of the Asian Arowana
As discussed above, the genus Scleropages possibly originated and evolved on Gondwanaland after the breakup of Pangea. However, one of its members, the Asian arowana, now inhabits a part of Eurasia. How can this be explained? Figure 3 illustrates three models for this zoogeographically interesting question, and we discuss the validity of each model in light of the molecular and geological evidence.



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Fig. 3.—Models of the origin and migrational pathway of the Asian arowana. Paleogeographical maps at approximately 170, 138, 45, and 20 MYA (Smith, Smith, and Funnell 1994Citation ) are shown on which three models (A–C) are indicated by arrows. The three models are (A) early-middle Jurassic (157–208 MYA) origin and migration via Laurasia, (B) recent (e.g., Miocene, 5–23 MYA) origin and migration across Wallace's Line, and (C) early Cretaceous (112–146 MYA) origin and migration on the Indian subcontinent

 
Model A of figure 3 shows that an ancestor of the Asian arowana originated when Laurasia and Gondwanaland were still connected and that it dispersed via freshwater habitats from Gondwanaland to Laurasia (Banarescu 1995Citation , pp. 1349–1354). However, the estimated divergence time between the Asian and Australasian arowanas (138 ± 18 MYA; table 3 ) makes this model unlikely. Geological evidence shows that Laurasia and Gondwanaland had been almost completely separated by 160 MYA (Smith, Smith, and Funnell 1994Citation ; the Plates Project 1998Citation ), implying that freshwater fishes could not have migrated between the two supercontinents after 160 MYA. In addition, if this scenario were the case, it would be reasonable to propose that ancestors of the genus Scleropages inhabited regions connecting the two supercontinents (i.e., Africa, North America, and South America). However, no fossil records for Scleropages have been found from these regions, and the habitats of extant Scleropages species (Southeast Asia and Australasia) correspond to the two eastern extremities of Laurasia and Gondwanaland.

Model B of figure 3 shows that the Asian arowana arose relatively recently via the transmarine dispersal of an Australasian arowana after plate tectonics moved Australia and New Guinea into the proximity of Southeast Asia (Banarescu 1995Citation , pp. 1349–1354, 1400). If this model were the case, the divergence time between the Asian and Australasian arowanas would be quite recent (<23 MYA). However, the molecular time estimate (138 ± 18 MYA) was much older (table 3 ), making this model unlikely. Another argument against the dispersal of a Scleropages species in this model comes from the fact that Southeast Asia and Australasia belong to two distinct zoogeographical regions bordered by Wallace's Line (Banarescu 1995Citation , pp. 1349–1354) and that the freshwater ichthyofauna was considered to be one of the most conspicuous indices discriminating the two zoogeographical regions (Briggs 1987Citation , pp. 45–55). Some authors (see, e.g., Lundberg 1993Citation ; Briggs 1995Citation , pp. 290–292) suggested the transmarine dispersal of euryhaline Scleropages populations by pointing out the existence of osteoglossid fossils found in saltwater beds (i.e., some of the Late Cretaceous to Eocene phareodontines, reviewed in Bonde [1996Citation ]). However, this is not a straightforward idea. Extant Scleropages species are highly adapted to freshwater, and their fossils are found only in freshwater beds (Sanders 1934Citation ).

Model C of figure 3 is a new model that is consistent with the molecular evidence. It assumes that ancestors of the Asian and Australasian arowanas diverged in the eastern margin of Gondwanaland during the Early Cretaceous (112–146 MYA) and that the former was transported northward across the Tethys Sea (the paleo-Indian Ocean) on the Indian subcontinent. During the Jurassic, India-Madagascar and Australia were connected through Antarctica in the eastern margin of Gondwanaland (Smith, Smith, and Funnell 1994Citation ; the Plates Project 1998Citation ). India-Madagascar was separated from Gondwanaland 120–130 MYA (Smith, Smith, and Funnell 1994Citation ; the Plates Project 1998Citation ) or somewhat more recently (Krause et al. 1997Citation ), whereas Australia remained close to Antarctica during the Cretaceous and even the Early Tertiary. The estimated divergence time between the Asian and Australasian arowanas (138 ± 18 MYA) is close to or slightly larger than the probable time of the India-Madagascan separation from Gondwanaland, which is consistent with the idea that the Asian arowana originated on a part of Gondwanaland and was carried by the Indian subcontinent. Moreover, model C can naturally explain the peculiar localization of extant Scleropages species, and there is no need to invoke a saltwater adaptation of this primary freshwater fish group.

There are some fossil records of Scleropages which should be considered in evaluating the migrational history of the Asian arowana. Freshwater beds of Eocene times (35–57 MYA) in central Sumatra yielded fossils that could be attributed to the genus Scleropages (Sanders 1934Citation ). The Indian subcontinent became connected to Eurasia by the late Early Eocene (Jaeger, Courtillot, and Tapponnier 1989Citation ; Metcalfe 1999Citation ). Thus, the fossilized Scleropages in central Sumatra could have come from India through terrestrial freshwater habitats soon after their disembarkation from the Indian subcontinent.

Another intriguing possibility is that the Asian arowana was carried by smaller terranes (continental blocks) (e.g., the Sikuleh, Natal, and Bengkulu) that drifted from the Australian part of Gondwanaland in the Late Jurassic (146–157 MYA) and were annexed to Southeast Asia in the Late Cretaceous (65–112 MYA) (reviewed in Metcalfe 1999Citation ). These three terranes actually accreted to Sumatra, the place where the fossilized Scleropages was found (Sanders 1934Citation ). Given the Late Jurassic separation of these terranes, the estimated divergence time between the Scleropages species (138 ± 18 MYA) may appear somewhat late to support this explanation. However, because the history of Gondwanian landmasses in Southeast Asia has not fully been revealed (Metcalfe 1999Citation ), we do not exclude the possibility that model C holds with one such terrane rather than with the Indian subcontinent.

Molecular Clocks of Bony Fishes
Molecular clocks of bony fishes have been studied using a variety of taxa, genes, and assumptions (see, e.g., Martin and Palumbi 1993Citation ; Ortí et al. 1994Citation ; Murphy and Collier 1996Citation ; Bermingham, McCafferty, and Martin 1997Citation ; Penzo et al. 1998Citation ; Zardoya and Doadrio 1999Citation ). Since most of these clocks are based on nucleotide sequences of relatively closely related taxa, little is known as to whether these clocks can be reliably extrapolated for divergences well over 100 MYA in time. For these old divergences, it seems reasonable to use amino acid sequences or nucleotide sequences without third codon positions, which are prone to be saturated quickly (Kocher et al. 1995Citation ).

In this study, we thus used amino acid sequences of two mitochondrial protein genes. In spite of the general idea that quickly evolving mitochondrial sequences are not suited for dating deep divergences, gamma-corrected distances from the mitochondrial protein sequences were shown to correct multiple substitutions efficiently (Kumazawa and Nishida 2000Citation ). The rate of the molecular clock for bony fishes using these distances was found to be nearly the same as or slightly faster than that for sharks but about three times as slow as that for mammals (Kumazawa, Yamaguchi, and Nishida 1999Citation ). This profile for the rate difference between fishes and mammals is consistent with previous work using transversion substitutions at fourfold-degenerate sites (Martin, Naylor, and Palumbi 1992Citation ) and restriction fragment length polymorphisms (Martin and Palumbi 1993Citation ).

Long Lack of Fossil Records?
Our molecular evidence suggested a Paleozoic origin of the order Osteoglossiformes (table 3 ), which is considerably older than the first osteoglossiform fossil record in the Late Jurassic (Benton 1993Citation , p. 624). Although the molecular data did not resolve the relationship among the osteoglossids, notopterids, and mormyrids, these groups are likely to have diverged during Permian-Triassic times (table 3 ). Fossil records for the three families and some supposedly related extinct groups have been found from the Cretaceous or later (Benton 1993Citation , p. 624; Bonde 1996Citation ). Thus, there appears to be a time gap between molecular and fossil evidence. We interpret this apparent discrepancy to be indicative of the paucity of osteoglossiform fossil records rather than the inferiority of our molecular time estimates.

In this respect, it should be emphasized that our estimates were based on a reasonably calibrated molecular clock using well-corrected distances (Kumazawa, Yamaguchi, and Nishida 1999Citation ; Kumazawa and Nishida 2000Citation ; fig. 2 ) and that the rate constancy test was carefully carried out. Another argument is that the clock rate is consistent with the fossil evidence for the divergence point between sarcopterygians and actinopterygians (see the legend of fig. 2 ). Finally, in order to reconcile the molecular and paleontological time estimates on the osteoglossiforms, the clock rate would have to be roughly twice that of figure 2 . However, this would elevate the fish rate to close to the mammalian one and cause clear inconsistency with previous work (Thomas and Beckenbach 1989Citation ; Martin, Naylor, and Palumbi 1992Citation ; Adachi, Cao, and Hasegawa 1993Citation ; Martin and Palumbi 1993Citation ).

Fossils of bony fishes are not necessarily considered well preserved in general. Of 425 teleostean families, 181 (43%) are completely lacking in their fossil record, and 58 (24%) of the remaining 244 families having recognizable fossil records occur with only otoliths (Benton 1993Citation , pp. 621–622). We thus suspect that there is a long unrecorded history for the osteoglossiforms in the Mesozoic. A similar lack of fossil records has also been suggested for perciform families (Kumazawa, Yamaguchi, and Nishida 1999Citation ), some teleostean orders (Kumazawa, Yamaguchi, and Nishida 1999Citation ), and mammalian and avian orders (Janke et al. 1994Citation ; Hedges et al. 1996Citation ; Cooper and Penny 1997Citation ; Janke, Xu, and Arnason 1997Citation ; Kumar and Hedges 1998Citation ; Waddell et al. 1999Citation ). The history of vertebrates based on the paleontological evidence has not been substantially changed in its broadscale pattern since the 19th century (Benton 1998Citation ). However, these lines of molecular studies may cast a doubt on the accuracy of the well-accepted vertebrate histories or, at least, they may call for the histories to be questioned and reexamined by multidisciplinary approaches, including molecular evolutionary ones.


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Freshwater zoogeography in general has been investigated primarily on the basis of faunal comparison using both extant and extinct species (Banarescu 1990Citation , pp. 11–47). Although such comparisons suggested an evolutionary affinity of the Asian arowana to the Australasian ones, definite evolutionary models which are consistent with both the geological and physiological (freshwater-adapted) conditions could not be envisaged due to the lack of corroborative evidence from molecules or fossils (for relevant discussions, see, e.g., Banarescu 1990Citation , p. 159; 1995, pp. 1349–1354, 1397; Taki 1993Citation , pp. 117–130). The present study provided strong molecular evidence to propose a novel evolutionary model for the Asian arowana. More generally, it demonstrated that molecular data can effectively combine with paleogeographical (or paleontological) information to gain new zoogeographical insights. Previous paleontological studies suggested that the latest Cretaceous-Paleocene Indian fauna and flora which survived extensive volcanic activities was almost completely replaced by the diverse and relatively advanced biota of tropical Asia upon the India-Asia collision (see, e.g., Briggs 1987Citation , pp. 123–137; Prasad and Khajuria 1995Citation ). The present study may thus provide the rare corroborative evidence to support a hypothesis that India or smaller continental blocks could serve as a cradles to convey Gondwanian freshwater faunas.

At present, the time estimates in table 3 are based on only two protein genes and a few calibration points and thus may be considered approximate estimations. However, due to the difficulties in the clock calibration for deep-branch osteichthyan groups as outlined earlier, even a rough framework of their divergence times has not been established by molecular approaches. We consider that our time estimates should be scrutinized in the future with more sequences or with other calibrations and revised if necessary. Nevertheless, our conclusion drawn herein about the origin and migrational pathway of the Asian arowana seems robust, because the three possible models of figure 3 propose quite different divergence times between the Asian and Australasian arowanas.

Since the Asian arowana is highly valued as a noble aquarium fish in Asian countries, it has been threatened with extinction in its native localities due to overfishing (or illegal fishing) and trading (Goh and Chua 1999Citation , pp. 9–24). Given the premium that this species points to the dynamic plate tectonics across the paleo-Indian Ocean, more attention than ever should be paid to its conservation.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
We are grateful to KK Istana Arowana and Messrs. E. Tajima and C. P. Jacoby for providing fish tissues. We thank Drs. M. Yamaguchi, Y. Yabumoto, T. Ueno, T. Kon, R. Nomura, and K. Tamaki and members of the Geobiology laboratory of Nagoya University for providing useful information and discussion on the results reported herein. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan to Y.K. (grants 09214102 and 12640680) and M.N. (grants 10660189 and 12460083).


    Footnotes
 
Masami Hasegawa, Reviewing Editor

1 Keywords: Osteoglossiformes teleost fish mitochondrial DNA historical biogeography fossil-based divergence time molecular clock Back

2 Address for correspondence and reprints: Yoshinori Kumazawa, Department of Earth and Planetary Sciences, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. E-mail: h44858a{at}nucc.cc.nagoya-u.ac.jp Back


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Accepted for publication July 25, 2000.





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