Ichthyol Res (2010) 57:71–77 DOI 10.1007/s10228-009-0125-y
FULL PAPER
Cryptic diversification of the swamp eel Monopterus albus in East and Southeast Asia, with special reference to the Ryukyuan populations Seiji Matsumoto Æ Takeshi Kon Æ Motoomi Yamaguchi Æ Hirohiko Takeshima Æ Yuji Yamazaki Æ Takahiko Mukai Æ Kaoru Kuriiwa Æ Masanori Kohda Æ Mutsumi Nishida
Received: 3 April 2009 / Revised: 25 June 2009 / Accepted: 28 June 2009 / Published online: 19 August 2009 Ó The Ichthyological Society of Japan 2009
Abstract The swamp eel Monopterus albus is widely distributed in tropical and subtropical freshwaters ranging from Southeast Asia to East Asia, and is unique in its ability to breathe air through the buccal mucosa. To examine the genetic structure of this widespread species, molecular phylogenetic analyses of mitochondrial 16S rRNA sequence (514 bp) were conducted for 84 specimens from 13 localities in Southeast and East Asia. The analyses showed clearly that this species can be genetically
S. Matsumoto Kashihara City Museum of Insect, 624 Minamiyama, Kashihara, Nara, 634-0024, Japan T. Kon (&) M. Yamaguchi H. Takeshima Y. Yamazaki T. Mukai K. Kuriiwa M. Nishida Department of Marine Bioscience, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan e-mail:
[email protected] M. Kohda Department of Biology and Geosciences, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Present Address: Y. Yamazaki Department of Biology, Faculty of Science, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan Present Address: T. Mukai Department of Regional Policies, Faculty of Regional Studies, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Present Address: K. Kuriiwa National Museum of Nature and Science, 3-23-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan
delineated into three clades based on geographical populations [China–Japan (Honshu ? Kyushu), Ryukyu Islands, and Southeast Asia clades], with each clade exhibiting its own reproductive behavior. Therefore, ‘‘M. albus’’ is believed to be composed of at least three species. The Southeast Asia clade with the highest genetic diversity may include more species. The Ryukyu clade was estimated to have diverged more than 5.7 million years ago, suggesting that the Ryukyuan ‘‘M. albus’’ is native. In contrast, in the China–Japan clade, all haplotypes from Japan were closely related to those from China, suggesting artificial introduction(s). Keywords Monopterus albus Ryukyu Islands Mitochondrial DNA Endemic species Artificial introduction(s)
Introduction Our understanding of the large genetic divergence that is present within freshwater fish species has increased following the development of molecular phylogenetic analytical techniques. For example, in one of the most widespread primary freshwater fish species, common carp Cyprinus carpio, distributed across Eurasia, a large genetic divergence [2.4–3.3% in the mitochondrial cytochrome b gene (cytb) and control region (CR) sequences] has been found between the wild form of carp from Lake Biwa, Japan, and the ‘‘Eurasian’’ wild and domesticated forms from other geographical areas (Mabuchi et al. 2005, 2007). Large genetic divergences within freshwater fish species were also found in various species, even in those with more restricted distributions (Avise 2000; Watanabe et al. 2006). The determination of phylogenetically independent groups
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is very important when defining conservation units such as the evolutionarily significant unit (ESU) (Moritz 1994). The swamp eel Monopterus albus (Synbranchiforms: Synbranchidae) is a primary freshwater fish distributed widely across East India (Greater Sunda Islands, the Malay Peninsula, and the Indochinese Peninsula), the Philippines, and the southern part of East Asia (southeastern China, the Korean Peninsula, the western Japanese Archipelago) (Rosen and Greenwood 1976; Baˆnaˆrescu 1990). It inhabits muddy substrates in marshes, ponds, rice paddies, brooks, and slow-flowing streams. This fish is morphologically and ecologically unique in having an elongated body, similar to that of a snake, covered with mucus and devoid of scales and fins, an ability to breathe air through the buccal mucosa, and in exhibiting protogynous hermaphroditism (Liu 1944; Liem 1963; Chan and Phillips 1967). It has been suggested that its ability to breathe air may have facilitated its wide dispersion in natural and/or artificial ways (Matsumoto 1997; Matsumoto and Iwata 1997; Matsumoto et al. 1998). Monopterus albus, however, has remained little studied in terms of its evolutionary biology, including its phylogeny and population genetics, although this fish is often a target species for ecological and physiological studies. It is uncertain whether the genetic structure of this widespread fish is homogeneous, as a result of its high dispersal (or artificial introductions), or heterogeneous, similar to that of the common carp. To elucidate the dispersal processes of M. albus (from East Asia to Southeast Asia), the present study aims to examine the phylogenetic relationships and genetic diversity among the geographical populations of M. albus using
mitochondrial DNA (mtDNA) sequencing. In Japan, this species is distributed in southwestern Honshu and Kyushu, two of the main islands, as well as the Ryukyu Islands located southward from Kyushu. It is generally accepted that M. albus is non-native in the main islands; it was introduced artificially, as known from its discontinuous distribution (Imatani 1980; Matsumoto et al. 1998; Nakabo 2002). However, M. albus may be native in the Ryukyu Islands considering its continuous distribution from the islands of Amami to Iriomote (Matsumoto et al. 2007). Therefore, in the present study, specimens were collected from various localities in Japan, including the Ryukyu Islands, as well as several East Asian sites (i.e., Taiwan, Shanghai, Fuzhou and Hainan in China, and Java in Indonesia).
Materials and methods Sampling. A total of 84 specimens of Monopterus albus were collected from 13 localities in East Asia and Southeast Asia from August 1999 to August 2005 (Table 1, Fig. 1). All specimens were collected from a stream, rice field, marsh, or pond using a hand net, excluding four specimens that were bought from the commercial market of Puli in Taiwan. In dry rice fields, a shovel was used to dig specimens from the mud. The tail tip (ca. 1 cm) of each specimen was cut and preserved in 99.5% ethanol. Most specimens were immediately released at the same site after collecting tail samples. Twenty-three specimens were fixed in 10% formalin under anesthesia immediately after
Table 1 Location and date of sampling, and the number of specimens examined Sampling locations
Sampling date
Number of individuals examined
Preanal length (mm)
Haplotypes observed (no. of individuals)
a. Sakurai City, Nara, Honshu, Japan
1–10 August 1999
10
263–433
Ma06 (6), 11 (1), 12 (3)
b. Oki Tawn, Fukuoka, Kyushu, Japan
20 November 2001
5
223–280
Ma02 (4), 05 (1)
c. Izena I., Ryukyu Is., Japan d. Okinawa I., Ryukyu Is., Japan
24 July 2005 23 September 2001
2 9
142–183 162–204
Ma15 (2) Ma13 (3), 15 (5), 16 (1)
e. Kume I., Ryukyu Is., Japan
21 September 2001
3
80–115
Ma14 (3)
f. Ishigaki I., Rykyu Is., Japan
3 August 2001
5
117–160
Ma14 (1), Ma15 (1), Ma17 (3)
g. Taipei, Taiwan
29 August 2005
2
180–245
Ma07 (2)
h. Puli, Taiwan
30 August 2005
4
270–311
Ma20 (3), 25 (1)
i. Hengchuen, Taiwan
31 August 2005
1
80
Ma28 (1)
j. Shanghai, China
8 December 2001
15
229–290
Ma01 (1), 03 (5), 04 (3), 08 (1), 09 (4), 10 (1)
k. Fuzhou, China
21 July 2002
10
230–256
Ma26 (1), 27 (4), 29 (5),
l. Haikou, Hainan Is., China
23 July 2002
8
210–245
Ma18 (1), 19 (7)
m. Yogyakarta, Java Is., Indonesia
8 August 2002
10
142–211
Ma21 (4), 22 (3), 23 (2), 24 (1)
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Cryptic diversification of a swamp eel
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Fig. 1 Left: maximum-likelihood tree of 29 haplotypes (84 specimens) of the swamp eel Monopterus albus based on the mitochondrial DNA 16S ribosomal RNA gene (514 bp), obtained using the TrN ? C model. Numbers beside major internal branches indicate
bootstrap probabilities based on NJ (1,000 replicates), MP (100 replicates), and ML (100 replicates). Right: distribution ranges of the three clades of ‘‘M. albus,’’ and sampling localities (a–m, see Table 1)
collection and preserved in 70% ethanol. These specimens have been deposited in the Osaka Natural History Museum (OMNH-P32081–P32103). DNA preparation, amplification, and sequencing. Total genomic DNA was isolated from the muscle tissue of each specimen using DNeasy Tissue Kit (Qiagen). The partial mitochondrial 16S ribosomal RNA gene was amplified by the polymerase chain reaction (PCR) using the universal primers L1567 (50 -AAG GGG AGG CAA GTC GTA-30 ) (present study) and H2196 (50 -GTC TGA GCT TTA ACG CTT TCT-30 ) (Yamaguchi et al. 2000). PCR was carried out in a 15 ll reaction volume containing 8.3 ll sterile and distilled H2O, 1.5 ll buffer (TaKaRa), 1.2 ll dNTP (4 mM), 1.5 ll of each primer (5 lM), 0.07 ll of 5 unit/ll Taq DNA polymerase (Ex Taq; TaKaRa), and 1 ll template on a
thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) for 35 cycles, with the following thermal cycle profile: preheating at 96°C for 4 min, denaturing at 94°C for 10 s, annealing at 50°C for 10 s, and extension at 72°C for 30 s. Before sequencing, the double-stranded DNA obtained through PCR was purified using ExoSAP-IT (USB), consisting of exonuclease I and shrimp alkaline phosphatase. Direct sequencing of the purified DNA using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit v.3.0 and 3.1 (Applied Biosystems) was performed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). All sequences were deposited in DDBJ/EMBL/GenBank (accession nos. AB494967–494995). Phylogenetic analysis. The DNA sequences were initially aligned using CLUSTAL X, version 1.81 (Thompson
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et al. 1997); the aligned sequences were realigned using ProAlign ver. 0.5 (Lo¨ytynoja and Milinkovitch 2003) and the regions with posterior probabilities of C70% were used for the phylogenetic analyses. Gaps were excluded from all analyses as missing data. The hierarchical likelihood-ratio test approach (Huelsenbeck and Crandall 1997) was used to select the model of DNA evolution that best fitted the data, as implemented in the program Modeltest 3.6 (Posada and Crandall 1998). Modeltest was also used to estimate the parameters of the model of evolution for input into PAUP* 4.0b10 (Swofford 2002). The appropriate model of nucleotide substitution was TrN ? C (Tamura and Nei 1993). The base frequencies were estimated to be A = 0.3971, C = 0.2288, G = 0.1708, and T = 0.2033. Gamma shape parameter C = 0.4458. Specified alternative rates were [A–C] = 1.0000, [A–G] = 2.4842, [A–T] = 1.0000, [C– G] = 1.0000, [C–T] = 8.0576, and [G–T] = 1.0000. We used Mastacembelus favus and Synbranchus marmoratus (DDBJ/EMBL/GenBank accession numbers AP002946 and AP004439, respectively) to root the trees. Phylogenetic relationships among the haplotypes detected were inferred by the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods using PAUP*4.0b10. NJ analysis was performed with the distance matrix calculated using TrN ? C. To evaluate the robustness of the internal branches of the NJ tree, 1,000 bootstrap replications (Felsenstein 1985) were conducted for each, using the same algorithm as that of each tree search. For the ML analysis, the appropriate substitution model was calculated using TrN ? C. MP analysis was performed with the tree bisection reconnection (TBR) routine implemented in PAUP*. To evaluate the robustness of the internal branches of the MP and ML trees, 100 bootstrap replications were conducted for each, using the same algorithm as that of each tree search. Haplotype network analysis. A statistical parsimony network (SPN) for Clade A (consisting of Japanese, Chinese, Taiwanese, and the Ryukyuan specimens) of the above phylogenetic trees was constructed using TCS 1.18 (Clement et al. 2000), which employs the method of Templeton et al. (1992). Divergence time estimation. To gain a rough estimate of the divergence time between subclades A1 and A2, time estimation analysis was conducted using two methods, the molecular clock method and the nonparametric rate smoothing (NPRS) approach (Sanderson 1997). In the molecular clock method, a divergence rate of 0.15–0.23% per million years between the fishes’ mitochondrial rRNA genes (Bargelloni et al. 1994; Alves-Gomes 1999) was applied to the average p distance (uncorrected average sequence divergence) between sub-clades A1 and A2. The NPRS approach based on the ML tree was performed using the r8s ver. 1.71 program (Sanderson 2002) because a high
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rate of heterogeneity between the A and B clades of Monopterus was observed by the two-cluster test (LINTREE) (Takezaki et al. 1995). In this method, in order to roughly calibrate the NPRS tree, the above molecular clock was applied to the origin of Monopterus.
Results Sequences. DNA segments of 514 base pairs (bp) of the 16S rRNA gene were successfully sequenced and aligned for 84 specimens. A total of 179 nucleotide positions (35% in 514 bp) varied in the ingroup; these variations defined 29 haplotypes. The mean percentage base composition of the ingroup was A, 36.9; C, 22.7; G, 17.9%; and T, 22.4%. The p distances (±SD) between the ingroup and outgroups, Mastacembelus favus and Synbranchus marmoratus, were 22.0 ± 1.2 and 17.6 ± 1.4%, respectively, and the average ML distances based on TrN ? C between them were 45.8 ± 4.7 and 33.1 ± 4.0%, respectively. Phylogenetic analysis. The ML tree based on the TrN ? C model demonstrated that those 29 haplotypes could be divided into two major clades (A and B) (Fig. 1). Both the NJ and MP analyses produced basically similar tree topologies. Clades A and B were supported by relatively high bootstrap values of 60–99 and 100%, respectively (Fig. 1). Clade A was composed of two subclades (A1 and A2) supported by relatively high bootstrap values of 76–88%, excluding subclade A1 on MP analysis. The average p distances were 8.8 ± 0.4, 8.9 ± 0.6, and 1.3 ± 0.5% between subclade A1 and clade B, subclade A2 and clade B, and subclades A1 and A2, respectively. The average ML distances (TrN ? C) were 11.5 ± 0.7, 11.7 ± 0.9, and 1.5 ± 0.4%, respectively, between the pairs mentioned above. The geographical distributions of the haplotypes in the two major clades, A and B, were restricted. Haplotypes of clades A and B were found in East Asia (Nara, Fukuoka, Izena Island, Okinawa Island, Kume Island, Ishigaki Island, Taipei, and Shanghai) and Southeast Asia (Puli, Hengchun, Shanghai, Fuzhou, Haikou, and Yogyakarta), respectively. It was noteworthy that the haplotypes of subclade A1 were found in two Japanese localities, Nara and Fukuoka, as well as in Taipei in Taiwan and in Shanghai in China, and that the haplotypes of subclade A2 were found only in the Ryukyu Islands (Izena Island, Kume Island, Okinawa Island, and Ishigaki Island). The average p distances within clades A and B were 1.4 ± 0.6 and 2.5 ± 1.0%, respectively. Haplotype network of clade A. To enhance the accuracy of the haplotype network analysis for clade A, the 16S rRNA sequences were realigned for the clade (584 bp). A total of 29 nucleotide positions (5%) varied in the ingroup; these variations defined the same 17 haplotypes as in the
Cryptic diversification of a swamp eel
Fig. 2 Haplotype network of clade A of Monopterus albus, based on 584 bp of the mitochondrial DNA 16S ribosomal RNA gene. Each ellipse represents one haplotype. The size of the ellipse is proportional to the haplotype frequency (1–6 specimens) in Fig. 1. A small black dot represents putative (missing) haplotypes that were not observed
phylogenetic analysis. The haplotype network of clade A (Fig. 2) showed a ‘‘dumbbell-like phylogeny’’ consisting of 51 specimens collected from the Japanese main islands, China, Taiwan, and the Ryukyu Islands. It consisted of two subclades (A1 and A2) that differed by a minimum of 11 mutations. Although no haplotype was shared by the specimens from the Japanese main islands (n = 15), China (n = 15), and Taiwan (n = 2) in subclade A1, the haplotypes from the Japanese main islands (Ma02, 05, 06, 11 and 12) were connected to one of the Chinese haplotypes by very short branches with 1–3 mutations. Subclade A2 consisted of the Ryukyuan haplotypes only. Divergence time. The divergence time based on the average p distance between subclades A1 and A2 was estimated as 5.7–8.7 million years ago (Mya) (molecular clock) and 20.7–32.0 Mya (NPRS).
Discussion Genetic diversity of the swamp eel. Our molecular phylogenetic analyses of nucleotide sequences of mtDNA
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clearly showed that populations of the swamp eel Monopterus albus collected from various areas are genetically clustered into two major clades: clade A from East Asia and clade B from Southeast Asia (Fig. 1). Clade A was divided into two subclades: A1 from China–Japan and A2 from the Ryukyu Islands. Conventionally, this species has been considered a single species with a wide distribution (Fig. 1). From the present results, however, it is conceivable that the swamp eel ‘‘M. albus’’ is a species complex consisting of three monophyletic groups with high geographical specificity. Previous field observations, as well as our own, show that the reproductive behavior of ‘‘M. albus’’ differs significantly among the three geographical populations. First, in the China–Japan population, which probably belongs to subclade A1, a breeding male digs a nest hole and then makes a foam mass. The eggs are spawned into this foam mass, where they are fertilized. After hatching, the male keeps the larvae in his buccal cavity until they begin to breathe air (Matsumoto and Iwata 1997). Second, in the Ryukyu population, which probably belongs to subclade A2, a breeding male does not keep his larvae in his buccal cavity, which is rather narrow. This lack of mouth brooding is a significant difference between the reproductive behavior of the China–Japan population (subclade A1) and that of the Ryukyu population (A2), although they show similar reproductive behavior otherwise (Matsumoto et al. 2007). Finally, the reproductive behavior of the Southeast Asia population, which probably belongs to clade B, is very different from that of the East Asia population (clade A; subclades A1 ? A2). Liu and Hu (1980) observed the reproductive behavior of ‘‘M. albus’’ in Taiwan, in the distribution range of clade B, and reported that the eggs were spawned not in the foam mass but at the roots of the common water hyacinth, Eichhornia crassipes, floating on the water surface. The embryos developed normally into larvae without parental care (Liu and Hu 1980). In fact, during our field survey in Java, Indonesia, several local farmers said that they frequently observed that the eggs of ‘‘M. albus,’’ which probably belongs to clade B, were spawned on plant leaves underwater, and that the embryo developed into the larvae without parental care. In addition, the larvae of ‘‘M. albus’’ were observed swimming freely in a rice field there (S. Matsumoto, unpublished data). This fact may be associated with the lack of mouth brooding by males of the Southeast Asia population, which probably belongs to clade B. Such differences observed in their mitochondrial genomes and their reproductive behavior imply that ‘‘M. albus’’ is composed of at least three distinct species. One of the above three species, clade B, has an average within-species sequence divergence (p distance = 2.5%) that is much higher than the between-species value
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between subclades A1 and A2 (1.3%). This result strongly suggests that clade B includes more taxonomic entities, consistent with the suggestion that the ‘‘M. albus’’ introduced into the southeastern United States includes at least three species, two of which were found to be the same as the species from Southeast Asia based on molecular phylogenetic analysis (Collins et al. 2002). There are seven junior synonyms in M. albus (sensu lato) distributed widely in Asia (Rosen and Greenwood 1976). Detailed taxonomic studies are needed for the ‘‘M. albus’’ species complex. Taiwanese ‘‘M. albus’’. Two alternative explanations have been proposed for the origins of Taiwanese swamp eels. First, ‘‘M. albus’’ is non-native in Taiwan, where it was introduced artificially after 1940 (P. K. Liu, personal communication). Second, ‘‘M. albus’’ is native (C.S. Tzeng, personal communication). Our results indicate that two clades (species) of the ‘‘M. albus’’ complex are distributed in Taiwan. Two specimens collected from Taipei belong to subclade A1 (China–Japan clade), and four specimens bought in Puli and one specimen collected from Hengchun belong to clade B (Southeast Asia clade) (Table 1, Fig. 1). Although it is possible that both groups are native species, we cannot reject the possibility of one group being native and the other being non-native, or both clades being non-native. Detailed phylogeographic analysis may provide information regarding the origin of Taiwanese swamp eels. Japanese ‘‘M. albus’’. All specimens from the Japanese main islands (Honshu and Kyushu) belong to subclade A1, along with the Chinese specimens (Fig. 1). Although no haplotype was shared by the Japanese and Chinese populations, the Japanese haplotypes (Ma02, 05, 06, 11, and 12) were connected to one of the Chinese haplotypes (Ma01, 03, 04, 08–10) by short branches with 1–4 mutations (Fig. 2). The existence of these closely related haplotypes suggests that gene flow between Japan and China must have occurred until recently on an evolutionary timescale, or that swamp eels were introduced artificially from China to Japan and vice versa. In freshwater fish, the divergence times of sister species in Japan and China have usually been estimated as being older than the late Miocene (less than about 5 Mya) (Watanabe et al. 2006). Thus, there appears to be a few possibilities for recent gene flow between Japan and China. Primarily, the population of swamp eels in Nara Basin is thought to have been introduced artificially from the Korean Peninsula in the early twentieth century. This is based on a testimony describing the introduction of many swamp eels ([10 individuals) into Nara, Japan (Imatani 1980). Considering the genetic evidence and this testimony, we conclude that ‘‘M. albus’’ in the main islands of Japan is non-native and was introduced from continental northeast Eurasia (Matsumoto et al. 1998).
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Native ‘‘M. albus’’ in the Ryukyu Islands. All Ryukyuan specimens belong to subclade A2 (Fig. 1). This finding of an endemic clade of ‘‘M. albus’’ distributed in the Ryukyu Islands is completely new and is particularly interesting. There was a suggestion that ‘‘M. albus’’ was introduced artificially from China or Southeast Asia into the Ryukyu Islands because this fish is a popular food in East and Southeast Asia. A belontid paradise fish Macropodus opercularis (Kuroiwa 1927) may have been frequently traded to the Ryukyu Islands in the age of the Ryukyu Kingdom (1429–1879). However, the present study estimated that the divergence time between Subclades A2 (the Ryukyu Is.) and A1 (China–Japan) was more than 5.7 Mya. This divergence time is too great to accept that the Ryukyuan ‘‘M. albus’’ was introduced artificially. Therefore, we conclude that the Ryukyuan ‘‘M. albus’’ is a native species endemic to the region. The present finding of swamp eels that are endemic to the Ryukyu Islands further highlights the uniqueness of the freshwater fish fauna of this area. In 1991, the Ryukyuan ‘‘M. albus’’ was listed as a ‘‘Threatened Local Population (LP)’’ (a species that exists in an isolated local population and is in danger of extinction) in the Red List by the Ministry of the Environment (Environmental Agency of Japan 1991). The evolutionary independence and uniqueness of the Ryukyuan ‘‘M. albus’’ warrant its conservation. Acknowledgments We thank H. Fujimoto (Yaeyama Agriculture and Forestry High School), K. Takehara (Okinawa Prefectural Museum), F. Sato (Kumejima Firefly Pavilion), and M. Taira (Ishigaki City Office) for their cooperation with the collection of specimens in the Ryukyu Islands. We are also grateful to B. Ridersius (Murakabi Biana K. K.) for helping with the sampling in Yogyakarta, as well as to C.S. Tzeng (National Tsing Hus University) and T.L. Tao (Taiwanese Freshwater Fish Museum) for their help with sampling in Taiwan. We also thank two anonymous reviewers for their constructive comments aimed at improving the manuscript. This study was supported in part by grants-in-aid for the Encouragement of Scientists awarded to S.M. (17916021) and for Scientific Research to M.N. (15380131, 19207007) by the Japan Society for the Promotion of Science.
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