Bulletin of Japanese Society of Microbial Ecology Vol. 8, No. 1, 35-42, 1993

Intraspecific Crossing, Zygote Germination and F1 Progeny of pectorale

KESHAB SHRESTHA, YOSHIHIKO SAKO and YUZABURO ISHIDA*

Laboratory of Microbiology, Department of Fisheries, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

(Received November 25, 1991-Accepted April 10, 1993)

Intraspecific crossing, zygote germination and production of Fr progenies of heterothallic strains of Gonium pectorale isolated from Tibet, Nepal and the Ryukyu Islands (Japan) were carried out. The results were compared with similarity coefficients in isozyme pattern for each cross. It was revealed that the strains from Nepal and the Ryukyu Islands which had high sexual affinity for each other had high similarity coefficients (0.80-1.00 and 0.60-0.80), while Tibetan strains which had low sexual affinity with the strains from the other areas had low similarity coefficients below 0.60 in most crosses with them. It is possible that Tibetan strains are isolated by mountain barrier and have developed a different feature in their gene from the strains of the other strains. There was a decreasing tendency in gametic compatibility and in the ability to produce Fr progenies during routine culture maintenance. Ecological or evolutional meanings of the alteration of sex competence were discussed.

Key words: Gonium pectorale, , green , sexual affinity, geographical feature

Introduction lated from Nepal and Tibet. According to him, Tibetan strains have low sexual affinities with Gonium pectorale Muller is a heterothallic fresh Nepalese strains. The same was suggested by water green alga with some exceptional case of preliminary crossing experiment between Tibetan homothallism9). This primitive member of strains and a few strains of Ryukyu Islands Volvocales (Chlorophyceae) has a pair of motile (Japan). In addition, it was shown that sexual flagella6). In vegetative phase, this alga is char- ability in G. pectorale was decreased during vege- acterized by 16 colonial biflagellated cells. All tative growth or stock cultures. After collecting a the cells in a colony are active and have potential considerable number of strains from the Ryukyu to develope into reproductive cells. The sexual Islands, Sako et al.8) reported that strains isolated and asexual reproduction is prominent in this from Tibet (China) had a different isozyme pat- species. Stein9) reported the phenomena of sexual terns comparing with those from Nepal and the isolation among the populations from U.S.A., Ryukyu Islands. The present work deals with the Canada and U.K. Clonal isolation and a mating sexual affinity and similarity value in isozyme type survey in a number of clones obtained from pattern between the strains from Tibet, Nepal and such a different localities were also investigated by the Ryukyu Island in an effort to define the her. phylogenetic relationship between these three local Saito7) conducted a study of sexual affinity in groups. The significance of the alteration of sex- mating clones of colonial green alga Gonium iso- ual ability are discussed.

* Corresponding author 36 SHRESTHA et al.

Materials and Methods Zygote germinated after 4-7 days of innoculation into the fresh medium. From the germinated Experiment on fusion and zygote formation zygote clonal F1 cells were isolated by using Thirty six clonal strains of G. pectorale used in micropipette. The mating type of F1 progenies the previous paper8) were employed in this study. were determined by the back cross with their The designation of each strain followed the abbre- parental strains. Similarity coefficients of isozyme viation used in that paper. Strains for stock cul- band patterns between pairs of strains calculated in ture were grown axenically in M6 medium under a the previous paper8) was used to examined the 14: 10h LD regime and 2000 lux from cool white correlation with sexual affinities. fluorescent bulbs. For crossing experiments, cells were grown in a series of cultures: pre-preculture, Results preculture and main culture. Pre-preculture con- sisted of 0.3ml of stock cells grown in a tube The crossing experiments between representative containing 10ml of M6 medium at a temperature clones from Tibet (3 clones), Nepal (8 clones) and of 25C and light intensity of 5000 lux on a 14: 10 the Ryukyu Islands (7 clones) were carried out to LD cycle. When growth reached the late check whether or not they produce zygotes and the exponential phase, pre-preculture was innoculated zygotes germinate (Table 1 and Fig. 1). In our into a 100ml of flask containing 50ml of the same experiments the zygote germination was observed medium and incubated under the same conditions. in only four crosses; N-8(+) x T-3(-) of Preculture cells were again innoculated into a flask Nepal-Tibet; N-8(+) x T-1(-) also of Nepal- containing 11 of M6 medium for main culture. Tibet; N-8(+) x N-8(-) of Nepal-Nepal and Cells from the flask containing plus and minus Ir-7(+) x O-11 (-) of Ryukyu Islands-Ryukyu cells were separately harvested at the late Islands. The zygotes from other crosses failed to exponential phase by centrifugation of 4000Xg for germinate even if a sufficient number of zygotes 5min at 20C. Then harvested cells were suspend- were formed. In the intragroup crosses of Tibet ed into nitrogen free M6 medium to induce (Tibet x Tibet), no zygote between T-1 (+) and gametogenesis. After 24h of gamete induction, T-3(-), and between T-4(+) and T-3(-) was 0.1ml of cells (1-2 x 106 cells/ml) from each formed. In other crosses in which zygotes wer mating clone was poured into a well of microplate formed, germination did not occur. In the inter- (Nuns, microwell 96F). Clones without mixing group crosses of Nepal x Tibet, only pair of N- were poured in a well side by side as a control to 8(+) x T-1(-) and N-8(+) x T-3(-) formed compare with the fused one. Fusion and zygote zygotes which germinated successfully, but in the formations were observed by an inverted micro- other crosses, zygotes were not formed or even if scope (Nikon, Diaphot-TMD). zygotes were formed, no germination was obser- ved. In the intragroup crosses of Nepal (Nepal x Germination and sexual affinity Nepal) only N-8(+) x N-8(-), produced matur- Zygotes were allowed to mature for a few weeks ed zygotes which could germinate, whereas in the to three months until color changed into brown other crosses zygotes did not germinated at all. In red. Then matured zygotes obtained were dried the Nepal x Japan crosses except O-11(+) x K- up in the clean bench for several hours. A dried 3(-) parental pair all produced zygotes but none zygote was transferred into fresh medium using of them germinated. In the intragroup crosses of micropipette. Each well of the Nunc plate (micr- Japan (Japan x Japan), although O-11(+) x Is- owell 96F) was innoculated with a zygote and 2(-) did not produce zygote, the other all crosses incubated at 25C under the continuous light. produced zygote. However, only zygote from a Intraspecific crosses of Gonium pectorale 37

Table 1. Zygote formation, maturation and germination test

+, Positive reaction; -, Negative reaction 38 SHRESTHA et al.

Fig. 1. Correlation between similarity coefficient and sexual affinity based on electrophoresis and inter- population crosses of G. pectorale. Similarity coefficient A0.80-1.00, B0.60-0.80, C0.60>. Sexual affinity in terms of zygote number O O many, O few, without symbol No reaction.

pair of Ir-7(+) O-11(-) germinated. In the of plus (Tables 1 and 3). Genetic and physiologi- intergroup crosses of Japan x Tibet only one pair cal mechanisms accounting for the survival of only Ir-7(+) x T-1(-) produced zygotes but they did mating type plus progeny in these intergroup cros- not germinate at all. ses will be presented in the following paper. In In the four crosses in which zygotes germinated, N-8(+) x N-8(-), 16 of 148 F1 progenies iso- from several wells containing germinated gones we lated survived, designated as N8N8F1. The via- isolated several single colonies originated from bility was 10%. A ratio of plus mating type to germinated gones and allowed them to grow in the minus one was 7: 9 (Tables 2 and 4). In Ir- culture conditions as described in culture for stock 7(+) x O-11(-), 9 of 57 F1 progenies isolated culture in materials and methods. In the cross, survived, designated as Ir7O11F1. The viability N-8(+) x T-3 (-), 10 of 80 F1 progenies iso- was 16%. A ratio of plus mating type to minus lated survived, designated as N8T3F1. The viabil- one was 7: 2 (Tables 2 and 4). ity was 12% (Table 2). In N-8(+) x T-1(-), 15 of 72 F1 progenies isolated survived, designated as Discussion N8T1F1. The viability was 20%. In both cros- ses, all F1 progenies obtained showed mating type Previously we reported that there was a high Intraspecific crosses of Gonium pectorale 39

Table 2. Viability and mating type of F1 in intraspecific crosses

Table 3. Mating type + and - designation test of F1 progeny of G. pectorale

A=Zygote absent, P=Zygote present 40 SHRESTHA et al.

Table 4. Mating type + and - designation test of F1 progeny of G. pectorale

A=Zygote absent, P=Zygote present similarity value in isozyme band patterns between in their genes from the strains of Nepal and the most strains from Nepal and all strains from the Ryukyu Islands. However, in the strains from Ryukyu Islands (Japan), whereas there was a Nepal and the Ryukyu Islands, despite of their relatively low similarity value between the Tibetan long distance of isolation, high sexual affinity was strains and the strains from Nepal and the Ryukyu shown in many crosses and most of crosses has Islands8). In the present study, it was found that high similarity coefficients in isozyme pattern. Tibetan strains had extremely low sexual affinity Conclusively our observation demonstrated that not only with the Nepalese strains but with the the strains from Nepal and the Ryukyu Islands Ryukyu islands' strains. That may be due to the which had high sexual affinity for each other had reason that Tibetan strains are isolated by moun- the high similarity coefficients (0.80-1.00 and 0.60- tain barrier and have developed a different feature 0.80) in isozyme pattern, while Tibetan strains Intraspecffrc crosses of Gonium pectorale 41 which had low sexual affinity with the strains from shown in G. pectorals. In either case, however, Nepal and the Ryukyu Islands had low similarity we think that the alterations of sex competence are coefficients below 0.60 in most crosses with them. ecologically or evolutionally meaningful events. Compared with results shown by Saito7), there was a decreasing tendency in the gametic compati- Acknowledgement bility and in the ability to produce F1 progenies. Especially, most zygotes formed in many crosses This work was supported in part by a Grant-in- did not germinate in the present experiment. The Aid for Scientific Research (Nos. 02760112 and alterations of sex competence over time during 03660192) from the Ministry of Education, Science routine culture maintenance have been observed by and Culture of Japan. The authors are grateful to several investigators in , Gonium Dr. S.Saito, Hirosaki University, for providing and other algae1-3,7). Coleman4) emphasized that many strains of Gonium pectorale. the alterations are not trivial. Based on the fact that gamete contact in Chlamydomonas depends References on carbohydrate ligand on one gamete type inter- rating with a trypsin-sensitive, sugar-binding com- 1) Cain, J. 1979. Survival and mating behaviour of progeny and germination of zygotes from ponent on the other, Wiese and Wiese11) proposed intra- and interspecific crosses of the following model about speciation by evolution Chlamydomonas eugametos and C. moewusii on gametic incompatibility: 1) Formation of (Chlorophyceae, Volvocales). Phycologia. 18: these contact substances is necessarily subjected to 24-29. 2) Cain, J. and F. Trainor. 1976. Regulation of mutation alterations; 2) Any alteration of proper gametogenesis in Scenedesmus obliguun (Chlor- functional structure will result in mutant which ophyceae). J. Phycol. 12: 383-390. appear as a sterile strain or be classified as strain 3) Chiang, K, J. Kates, R. Jones and N. Sueoka. without known sex partner, because in 1970. On the formation of a homozygous zygotic population in Chlamydomonas reinhar- Chlamydomonas and other organisms with vegeta- dtii. Dev. Biol. 22: 655-669. tive or sporulative reproduction the mutant will 4) Coleman, A. 1975. Long-term maintenance of continue to exist by uninhibited asexual propaga- fertile algal clones: experience with Pandorina tion; 3) In the opposite sex the functional struc- (Chlorophyceae). J. Phycol. 11: 282-286. 5) Meins, F. 1983. Heritable variation in plant ture of the mating type substance is likewise sub- cell culture. An. Rev Plant Physiol. 34: 372- jected to mutative alteration; 4) With a certain 346. 6) Saito, S. 1984. Growth and differentiation of probability a mutated component may be synthes- Gonium. Sci. Pap. Inst. Alg. Res. Fac. Sci. ized which is, in a new manner, complementary to Hokkaido Univ. 7: 195-26. the mutated one of the first strain; 5) A new pair 7) Saito, S. 1990. Distribution and sexual com- of sexual strains has then arisen which is incompat- patibility in mating clones of colonial green alga Gonium pectorale (Volvocales, Chloro- ible with the ancestor of strains. However, phyceae) in Nepal, Tibet (China) and Ryukyu Trainor10) propose that gradual loss of fertility Islands (Japan). In Watanabe and Malla occurrs in research laboratories not by way of (Eds.) Cryptogams of Himalaya. 2: 49-58. selection of mutants, but as a result of culture 8) Sako, Y., K. Shrestha, A. Uchida, Y. Ishida and S. Saito. 1991. Isozyme analysis of mating procedure. Such alteration could be accounted population of Gonium pectorale (). for by epigenetic phenomena, that is, the alteration J. Phycol. 27: 309-315. is not mutations but changes brought by the activa- 9) Stein, JR. 1965. Sexual population of Gonium pectorale (Volvocales). Am. J. Bot. tion of a master switch5). 52: 379-388. At present we cannot decide which propositions 10) Trainor, FR. 1985. On the restoration of fer- are available for the alteration of sex competence tility of sexually inactive, heterothallic clone of 42 SHRESTHA et al.

Chlamydomonas (Chlorophyta). B. Phycol. J. by evolution of gametic incompatibility: a 20: 1-4. model case in Chlamydomonas. Am. Nat. 111: 11) Wiese, L. and W. Wiese. 1977. On speciation 733-347.