J. Phycol. 52, 131–134 (2016) © 2015 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1111/jpy.12363 NOTE CONJUGATION MORPHOLOGY OF ZYGOGONIUM ERICETORUM (ZYGNEMATOPHYCEAE, CHAROPHYTA) FROM A HIGH ALPINE HABITAT1 Rosalina Stancheva Department of Biological Sciences, California State University San Marcos, San Marcos, California 92096, USA Klaus Herburger Institute of Botany, University of Innsbruck, Sternwartestraße 15, Innsbruck A-6020, Austria Robert G. Sheath Department of Biological Sciences, California State University San Marcos, San Marcos, California 92096, USA and Andreas Holzinger2 Institute of Botany, University of Innsbruck, Sternwartestraße 15, Innsbruck A-6020, Austria Reproductive characteristics are important for distinguished from other zygnematophytes by (i) defining taxonomic groups of filamentous irregular plate-like chloroplasts; and (ii) purple Zygnematophyceae, but they have not been fully cytoplasmic residual content left in sporangia out- observed in the genus Zygogonium. Specimens of side of the fully developed aplanospores (Stancheva Z. ericetorum previously studied and used to clarify et al. 2014). Cytoplasmic residue outside of the the generic concept lacked fertile material, which zygospores was postulated, but could not be demon- was obtained recently. This study illustrates for the strated as no zygospores were observed in the previ- first time, using color light microscopic and ous study. Such residues would imply similarities fluorescence images, a consequent conjugation stage between asexual aplanospore and sexual zygospore in Z. ericetorum, including completely developed formation. The main features of Z. ericetorum conju- zygospores and purple cytoplasmic residue content gation were described and illustrated by drawings left outside the zygospores, similar to aplanospore (De Bary 1858, West and Starkey 1915, Hodgetts formation. Structures confirmed earlier reports and 1918, Transeau 1933, 1951, Kadlubowska 1984, Run- provided new observation informative regarding dina 1998), but uncertainties exist. The only light phylogenetically relevant reproductive characters of microscopic (LM) documentation of conjugation in Z. ericetorum. Z. ericetorum filaments (Alston 1958) did not allow one to draw conclusions, as neither color micro- Key index words: alpine habitat; conjugation; cytoplas- graphs nor a description of the conjugation process mic residue; green algae; Zygnematophyceae; Zygo- were provided. gonium In additional sampling of the Z. erictorum popula- Abbreviations: BP, band pass; LM, light micro- tion from the same location in Austria, we collected scopic; LP, long pass sexually reproducing filaments, and conducted LM and fluorescence microscopic observations of its conjugation morphology. Detailed LM observations Recently, a phylogenetic and morphological study and color images of conjugation in Z. ericetorum of Zygogonium ericetorum Kutzing,€ the type species of complete the understanding of its reproductive biol- the genus, was conducted on a natural population ogy and the taxonomic significance of reproductive from Austria, providing new data on the taxonomic features. The implications of this knowledge can be importance of morphological characteristics of the used in determining the phylogenetic position and genus (Stancheva et al. 2014). Zygogonium can be ancestral character state distribution within the wider group of closely related filamentous genera Zygnema and Zygnemopsis. 1Received 30 July 2015. Accepted 13 October 2015. 2 For this study, we obtained algal material from a Author for correspondence: e-mail Andreas.Holzinger@uibk. ac.at. natural population of Z. ericetorum growing in the Editorial Responsibility: J. Raven (Associate Editor) same Austrian habitat in Mt. Schonwieskopf€ 131 132 ROSALINA STANCHEVA ET AL. (46°500998 N, 11°000903 E), at 2350 m a.s.l. near rated to discharge the zygospores due to distinct rup- Obergurgl, Tyrol, which has been previously sam- ture along the contact line between them, which is pled during the summer seasons 2007, 2008 (Hol- visible as an equatorial line in the surface of the zinger et al. 2010), 2009, 2010, 2012 (Aigner et al. zygospores (Fig. 1, A and B). Zygospore germination 2013), and 2013 (Stancheva et al. 2014). The chemi- was not observed. cal and physical conditions of the collection site Taxonomically, reproductive characteristics are have been described previously (Holzinger et al. very important for defining groups of filamentous 2010, Aigner et al. 2013); for this study, no new data Zygnematophyceae, but they are infrequently of the collection site were recorded. We think that observed in natural populations. The scarcity of con- general climate data available for the collection site jugation in Z. ericetorum combined with common do not contribute to the understanding of the trig- abnormalities (Fig. 1A) due to its strong tendency ger for conjugation, an event that occurs only very toward encystment, whenever habitat conditions locally at a specific-habitat scale. Z. ericetorum sam- change (Transeau 1951), contributed to misinterpre- ples were collected from several different areas in a tations of this process in the early studies (De Bary spring pool on June 9, 2015 by the authors Stan- 1858, Hodgetts 1918). This study confirmed the pos- cheva and Holzinger, and kept cool until next day tulation that characteristics of the aplanospore for- when they were processed in the laboratory in Inns- mation described in our previous work (Stancheva bruck for light microscopy. Fresh conjugating fila- et al. 2014) are applicable to the zygospore forma- ments were observed and photographed with a Zeiss tion, which is generally valid for the whole family Axiovert 200M (Carl Zeiss AG, Jena, Germany) Zygnemataceae (Transeau 1951). We demonstrated inverted fluorescence microscope equipped with a that during the conjugation process in Z. ericetorum, 639 Neofluor 1.4 NA objective lens. Images were similar to the aplanospore formation, the colored captured with a Zeiss Axiocam MRc5 digital camera. cytoplasmic vacuolar residue is left outside the zygos- To visualize chlorophyll autofluorescence, filaments pore. Thus, the spore lacks purple pigmentation, were excited with a Zeiss filter set 09, excitation but contains cell organelles and storage products band pass (BP) 450–490, emission 515 nm long pass (Fig. 1, F–H). Excretion of the purple pigment dur- (LP). Blue autofluorescence of the cell walls was ing both zygospore and aplanospore formation generated by Zeiss filter set 01 (excitation 365/ might be a protective strategy to avoid toxic pigment 12 nm, emission LP 397 nm). concentrations in the newly formed spore. The Z. ericetorum vegetative morphology was iden- Furthermore, the color LM and fluorescent tical to the previous description by Stancheva et al. microscopic images of fertile material revealed that (2014). Cells of the filaments were 15–31 lm wide, the zygospore wall is smooth, multilayered, colorless containing two plate-like green chloroplasts, thick to yellowish. The previous descriptions of this spe- multilayered cell walls, and frequent H-shaped wall cies typically omit the color of the zygospore wall structures. Cell content was light purple. Several fila- (e.g., Transeau 1933, 1951, Alston 1958), or it was ments formed zygospores or akinetes, but aplanos- reported as colorless or yellow-brown (Kadlubowska pores were not observed. 1984), yellow to brown (Rundina 1998), or dark The conjugation was scalariform, involving two, brown (Johnson 2011). It is possible that in Z. erice- three or four filaments in irregular fashion (Fig. 1, torum, the color of the zygospore wall is variable, A–E). Only a few cells in conjugating filaments took but our observations showed that the aplanospores part in the reproduction (Fig. 1D), and abnormal and zygospores are most likely lacking special spore- gametangia with uncompleted conjugation were com- wall pigment and they are typically colorless (Stan- mon (Fig. 1A). The conjugation process started with cheva et al. 2014, this study). This observation is in compaction of the chloroplasts, nucleus, and other contrast to the closely related genus Zygnema, which cell organelles into the gamete, which migrated was phylogenetically divided into two main groups toward the conjugation tube formed between two cor- based on the blue versus brown color of the mesos- responding cells in the adjacent filaments. The two pore wall layer (Stancheva et al. 2012), indicating gametes united directly in the tube and formed a the importance of the spore wall color as an evolu- zygospore separated by a gametangial wall from the tionary character in Zygnemataceae. The genus Zyg- purple cytoplasmic residue, consisting of a large vac- nema includes many species that form zygospores in uole remaining in the conjugating cells (Fig. 1, A, B, the conjugation tubes; some of these species appear D, E). The gametangial wall became the wall of the similar to Zygogonium in their mode of gamete zygospore. The zygospores were ovoid or ellipsoid, fusion, as well as zygospore and gametangial wall 15–26 lm wide, 19–38 lm long (Fig. 1, A, D, F–H) formation. Since the formation of zygospores in containing four compacted green chloroplasts, each tubes versus in gametangia in Zygnema