PHYCOLOGIA 10 (2/3), 1971. Chemotaxonomic studies of Pleurastrum Chodat by means of pyrolysis-gas-liquid chromatography. DOUGLAS C. SPRUNG AND ...
PHYCOLOGIA 10 (2/3), 1971
Chemotaxonomic studies of Pleurastrum Chodat by means of pyrolysis-gas-liquid chromatography
DOUGLAS C. SPRUNG AND DANIEL E. WUJEK Department of Biology, Central Michigan University, Mt. Pleasant, Michigan 48858, U.S.A. Accepted May 7, 1971
ells of four s ecies of Pleurastrum Chodat and one each of Leptosi�a Borzi and Oe qogonil!m Link were . r n in modifi d Bold's basic medium. Desiccated sam ples were s�bJ.ected. to p�rolysls-gas-hqUld chrom� ography analysis and the resulting pyrograms were exammed for vanatIons m elution patterns for systematic treatment.
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Phycologia, 10(2/3): 251-254, 1971
Introdnction
Algal systematics have traditional�y b�en based on morphological and physlOlo�lcal characteristics. Smith (1950) and FrItsch (1935) discussed the cl�s�ification .of algae based on these characterIstics, stressmg mor phology and limiting physiological considera tions to pigmentation, storag� p�oducts, and cell wall constituents. Such CrItena frequently make species identification difficult. De�son and Bold (1960) indicated that morphological fea tures were not adequate for delimiting species of soil algae, and presented te�hni.ques for the establishment of additional cntena based on physiological response to various growth fac tors. Even with data obtained from growth studies, species identification remains a tim�-consU1:ning and difficult procedure. The analysIs of mIcro organisms by pyrolysis-gas-liquid chromato� raphy (PGLC) has recently becom� a . rapid and reliable method for the characterIzatIOn of potential pathogens (Reiner, 1965, 1967; Reiner and Ewing, 1968). Even closely re�a�ed strains of the same species have been distm guished under controlled conditions. The meth od has also been extended to the diagnosis of viral and fungal diseases in plants (Myers and Watson 1969). Workers have also reported the suc�essful use of this technique for rapid identification of algal and bacteria species (Cone and Lechowich, 1970; Nichols et al., 1968; Oyama and Carle, 1967).
The purpose of this study was to investigate the chaetophoracean alga Pleurastrum. Chodat (1894) established the genus Pleurastrum and described P. insigne. Since then, four taxa of Pleurastrum have been described whose taxo nomic affinities are uncertain. The taxa in ques tion, P. paucicellulare Vischer (1933), P. ter restris Fritsch and John (1942), P. erumpens Deason and Bold (1960), and P. terrestris var. indicum Mitra, nomina nuda,l have also been called Rhexinema paucicellulare, R. erumpens, Leptosira terrestris, and L. terrestris var. in dicum (Printz, 1964). Pyrolysis-gas-liquid chromatography was selected as the method of study to further estab lish the usefulness of the technique in taxo nomic studies of algae, and to clarify, if pos�i ble, the taxonomy of the Pleurastrum species in question. Materials and Methods
Cultures obtained from the Indiana Algae Culture Collection (Starr, 1964) included four species of Pleurastrum (P. pau�icellula�e-=H:332, P. terrestris-#333, P. terrestns var. mdlcum #334, P. erumpens-#979, Leptosira obovata Vischer-#979, and Oedogonium sp.- #849 .. All
taxa were grown in Bold's (1967) modIfied basic medium to which soil extract ( 100 ml/L) had been added. The algae were cultured in 250 ml Pyrex flasks at 22 C, 12/12 light/dark 1 A formal description of this ta:cO? as a species, in . cluding its electron microscopy, IS III preparation.
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SPRUNG AND WU.JEK: Chemotaxonomic studies of
cycle at approximately 100 ft-c for 180 days. Aliquots of three randomly selected cultures of each taxa were incubated in thioglycolate broth to test for the presence of aerobic or anerobic microbial contamination. Suspended cells were harvested by centrifugation; following centri fugation, the cells were removed with a sterile spatula to a sterile Millipore filter (HA WP 047 00 HA 0.45 fL, 47 mm white, plain) for vacuum filtration. The algae were then transferred to sterile Pyrex depression dishes for dessication. Dessication time varied from one to ten days. Analyses were performed on a Beckman GC-4 gas chromatograph equipped with a fragmentation module and hydrogen flame ionization detector. The dual columns were 8 ft X 0.25 in 00 copper tubing packed with 15 per cent carbowax 20M coated on 80/90 mesh HAnakran ABS". The carrier gas was argon. (flow rate 20 cc/min) and the flame ionization detector burned hydrogen (42 cc/min) and air (360 cc/min). The module pressures were: air 42 psi, argon 30 psi, and hydrogen 40 psi. Prior to analysis, the samples were quanti fied to 1 mg. The sample was loaded into a cartridge, placed in the fragmentation unit, and purged for 15 min. Fragmentation was for 15 sec at 70-80 milliamperes AC. At the com pletion of the fragmentation period, the prod ucts were introduced into the carrier stream. The oven was programmed linearly at 12 C/min from 55 to 190 C. Isothermal limit (190 C) was reached in 12 min and was main tained for the remainder of the analysis. Each program was 24 min in duration. A minimum of 8 samples of each taxon were analyzed. Samples were analyzed consecutively and at 2, 4, and 48 hr intervals. Chart speed was 0.5 in/min with attenuations from 1 X 103 to 5 X 104• Column conditioning was at 215 C for three days.
Pleurastrum
TABLE 1 Elution time of peaks (minutes) in pyrograms of Pleurastrum, Leptosira, and Oedogonium
Results and Discussion
Pyrograms were interpreted by visual in spection. Retention times, the presence or ab sence of peaks, and the ratio of peak heights with relationship to each other were particular ly useful. No quantification or identification of ·peaks was attempted in these preliminary studies. For purposes of simplifying data pre sented in this paper, only a table is shown since results were consistent between both duplicate cultures and replicate pyrolysis determinations.
1.6 1.7 1.8 2.0 2. 1 2.4 3.1 3.3 3.5 3.8 4.5 4.8 5.3 5.5 5.8 6.0 6.2 6.4 7.1 7.4 7.7 7.9 8.4 8.8 9.2 9.5 9.8 10.0 10.7 10.9 1 1.5 11.8 12.3 12.5 12.7 12.8 13.1 13.5 13.6 13.9 15.1 15.5 16.4 16.7 17.2 J8.0 18.8 J9.2 19.8 20.2 21.0 22.2 23.4 23.8
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PHYCOLOGlA, VOL. 10(213), 1971
SPRUNG AND WUJEK: Chemotaxonomic studies of
The thioglycolate test for bacteria and fungal contamination of cultures was negative. Con siderable qualitative and quantitative differences were observed. The retention times listed in Table 1 were derived by an evaluation of all pyrograms of each taxon. The elution times listed in Table 1 indicated by the presence of peaks that chemical differ ences existed between the taxa. The most obvi ous difference was between Oedogonium and the other algae. The pyrograms of Oedogonium lacked peaks at 2.0, 8.4, 1 5.5, and 22.2 min, while having a unique peak at 1 9.8 min. The difference between L. obovata and the taxa of Pleurastrum were not as obvious. This suggests a closer affinity of the two genera than that which existed between Oedogonium sp. and the other algae. This would concur with classical methods of classification. The pyrograms of L. obovata had character istic peaks at 6.2, 7.7, and 23.4 min. In addi tion, it was possible to distinguish the pyrogram of L. obovata from pyrograms of Pleurastrum as follows: from P. paucicellulare by the pres ence of a peak at 5.8 min and the absence of peaks at 1 7.2, 1 8.8, and 20.2 min; from P. terrestris var. indicum by the presence of peaks at 5.8 and 7.7 min and the absence of peaks at 1 8.8 and 20.2 min; from P. terrestris by the absence of peaks at 1 0.0, 1 0.9, and 13. 1 min; and from P. erumpens by the presence of peaks at 1 7.2, 1 8.8, and 20.2 min. Taxonomic Implication of the Stndy
Pleurastrum was originally described as an unicellular alga reproducing by tetrads within the mother wall, tetrads capable of remaining in the Gleocystis state, or producing biflagel lated zoospores (Chodat, 1 894). Vischer ( 1 933) indicated that Chodat's de scription needed to be supplemented to obtain a complete characterization of the genus. He also stated that some of the phases described by Chodat may not have pertained to P. insigne. This would suggest that the original description was inaccurate. According to Vischer ( 1 933), Chodat dropped the genus after studying pure cultures, but later re-established it. Vischer ( 1 933) studied two cultures of algae which he described as P. paucicellulare. He in dicated that no differences could be observed between the two cultures, but if subsequent study revealed they were different, his culture
Pleurastrum
68 should retain the name P. insigne as the original species. This would suggest that Cho dat's description of P. insigne may have been based on what Vischer later was to call P. paucicellulare.
The pyrogram of L. obovata had an elution pattern similar to the pyrograms of Pleuras trum. From these observations we are of the opinion that these variations are no more than a species variation and indicate that Leptosira and Pleurastrum are one and the same genus. This is borne out in recent studies on Leptosira (Hirose and Akiyama, 1967; Tschermak Woess, 1 953; Wujek, 1 97 1 ) which indicated the presence of a pyrenoid. The two genera are now separated primarily on the presence (Pleurastrum) or absence (Leptosira) of a pyrenoid. The pyrenoid of L. obovata (Wujek, 197 1 ) closely resembles the pyrenoid of the hornwort Anthoceros. The combination and transfer of the species of Pleurastrum to the genus Leptosira is proposed and given below. Leptosira insigne (Chodat) comb. nov. Leptosira pauciceUulare (Vischer) comb. nov. Leptosira erumpens (Deason and Bold) comb. nov. Acknowledgements
This study was in part financed by National Science Foundation and C.M.V. Faculty Re search Committee grants. Literatnre Cited
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