NATIONAL SCIENCE FOUNDATION
TOKYO REGIONAL OFFICE

The National Science Foundation's (NSF) Tokyo Regional Office periodically receives and disseminates reports on research developments in Japan that are related to the Foundation's mission. It also provide occasional reports on developments in other East Asian Countries (http://www.twics.com/~nsfasia/as-reports.htm).

These reports present information for the use of NSF program officers and policy makers; they are not statements of NSF policy..

Special Scientific Report #01-04 (October 12, 2001)

Historically, the taxonomy of Daphnia galeata (Crustacea, Branchiopoda) has been confused. This was due to unclear original descriptions, the occurrence of numerous different morphological races, and the presence of many intermediates. In the classical works of Ueno (i.e.1933, 1938), Japanese D. galeata were often confused with D. longispina and D. hyalina. Finally, after the taxonomy of this species group had been clarified in Europe, Tanaka (1992) concluded that the Daphnia found in Lake Biwa and Lake Kisaki were indeed D. galeata. Although D. galeata from North America and Europe have now been described with both mitochondrial and nuclear markers, Japanese D. galeata have never been genetically characterized.

The main goal of this project was to examine the genetic differences between Daphnia galeata of North America, Europe, and Japan. We wanted to find out if D. galeata of Japan were more similar to European or North American D. galeata. This research has important evolutionary implications on the divergence of North American D. galeata mendotae from European D. galeata galeata. This divergence is said to have occurred mostly as a result of the introgression of genes from D. dentifera (Taylor et al. 1996), a species thus far only found in North America. An arctic glacial refugium seems like a likely place for this introgression, and the introgressed populations probably then dispersed after the last glacial recession. If the refugium in question was the Bering land bridge, then introgressed animals may have dispersed down both the east and west coasts of the Pacific, with some appearing in Japan.

Genetic variation was examined using PCR-based RFLPs (restriction fragment length polymorphisms). Both 12S-16S mitochondrial DNA and ITS2 (second internal transcribed spacer, nuclear ribosomal DNA) were amplified, and the PCR products were digested with restriction enzymes. The resulting bands were visualized using agarose gel electrophoresis and ethidium bromide staining. Sequence differences at the enzyme cut sites caused different banding patterns on the gel. The protocol for ITS2 digestion with Rsa1 has been developed by H. Sprenger and D. Taylor (unpublished) and methods for the digestion of the 12S-16S region were borrowed from Taylor and Hebert (1993). Allozymes were also used to look at genetic variation using the methods of Hebert & Beaton (1993). This involved electrophoresis of animal homogenate on a cellulose acetate gel, and visualization through staining with the appropriate reagents. This method can distinguish proteins that have different amino acids, and thus different charges. Much of my time was spent teaching the above methods to Seiji Ishida, my collaborator.

Casual morphologic observations indicate that Japanese D. galeata share some characteristics with North American D. galeata. They seem to have broadly pointed or sometimes high and rounded helmets, and large compound eyes.

Digestion of the 12S-16S region (mtDNA) with the restriction enzyme Rsa1 showed no differences among individuals from Europe, North America, and Japan. Digestion of the same region with Taq1 was difficult to interpret because many more bands appeared than were expected based on the literature from which the method was borrowed. Although we found differences in banding patterns between individuals, there did not seem to be any fixed differences between continents. Digestion of ITS2 with Rsa1 gives characteristically different banding patterns for North American and European D. galeata (Fig 1., based on unpublished previous analysis of several populations from each continent). Digestion of Lake Biwa (Japan) ITS2 with Rsa1 showed the North American banding pattern. This part of the project is ongoing, and recent results show the presence of the D. galeata X D. galeata mendotae and D. dentifera banding patterns in Lake Biwa.

Figure 1. Species specific banding patterns for Rsa1 
digestion of ITS2. A) D. dentifera B) D. dent. X D. gal. 
mend. C) D. galeata mendotae D) D. gal. mend. X D. gal.
gal. E) D. galeata galeata

Note: this method may not always be reliable in discriminating
between D. dentifera and D. galeata mendotae in the Alaska 
area, where recently some D. dentifera have been shown to 
have the D. galeata mendotae pattern.

Initially, allozyme analysis indicated that Japanese D. galeata may be similar to European D. galeata galeata (Fig 2). This is especially evident at the locus GPI, where there are strong differences between the two subspecies. The most common allele in D. galeata galeata is b (allele frequency = 0.906), while c is most common in D. galeata mendotae (allele frequency = 0.913) (Taylor & Hebert 1993). You can see that the Lake Biwa (Japan) laboratory clone had a genotype of bb. When wild samples were analyzed, however, they appeared to have the bc genotype. This agrees with the ITS2 RFLP data that there might be D. galeata galeata X D. galeata mendotae hybrids in Lake Biwa.

* Upper case from Taylor et al. 1996
**Lower case from Taylor & Hebert 1993

A definitive conclusion based on this evidence is premature. Since the change of a single base pair can change the banding pattern of RFLPs, we expect them to be noisier than allozyme data, as seen with the Alaskan D. dentifera. Analysis of additional allozyme loci, increased sample sizes, and collection of sequence data are in progress in order to complete this project.

The possibility that D. galeata mendotae may be present in Asia allows us to reexamine our hypotheses about the evolution of the species. If the group diverged from D. galeata galeata mainly as a result of introgression from D. dentifera, where or when did this happen? If the initial divergence began in the Beringian refugium during the Pleistocene, it’s possible that animals dispersed down into both Asia and North America. European populations, then, must have been populated from a different source.

This research has sparked the beginning of a very interesting project looking at the evolution of the species D. galeata mendotae. Future directions of this collaboration include increasing sample sizes and number of loci of the allozyme data, examining the egg bank to follow the historical progression of Daphnia in Lake Biwa, and collecting sequence data to further examine the genetic relationships between D. galeata galeata and D. galeata mendotae. We are also looking into the possibility of collecting more Asian samples in order to further explore the range of D. galeata mendotae and examine questions about its origin. Also, the collections we made this summer of Bosmina will allow Prof. Derek J. Taylor, my advisor at SUNY/Buffalo, to include those Japanese specimens in his studies.

Tanaka S (1992) Morphology and variation of Daphnia galeata SARS from Lake Biwa and Lake Kisaki in Japan. Jpn. J. Limnol. 53(1): 47-54.

Taylor DJ & Hebert PDN (1993) Habitat-dependent hybrid parentage and differential introgression between neighboringly sympatric Daphnia species. Proc. Natl. Acad. Sci. 90: 7079-7083.

Taylor DJ, Hebert PDN, & Colbourne JK (1996) Phylogenetics and evolution of the Daphnia longispina group (crustacea) based on 12S rDNA sequence and allozyme variation. Molecular Phylogenetics and Evolution 5(3): 495-510.

Ueno M (1933) The freshwater branchiopoda of Japan. IV Genus Daphnia of Japan: 2. Local Races of Japanese Daphnia. Memoirs of the College of Science, Kyoto Imperial University, Series B, Vol. IX, No. 4, Art 9, p 322-346.

Ueno M (1938) Japanese freshwater cladocera: a zoogeographical sketch. Annto. Zool. Japon. 17(3,4): 283-294.

 

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