Abstract
The ultrastructure of the mature spore in four Japanese species of the acrocarpous moss genus Ptychomitrium is presented. In all species the spores have a similar pattern: there is no recognisable aperture nor sporoderm polarity, exine and perine are poorly developed, cytoplasm only occasionally shows polarity, and plastids have a well developed inner membrane system. The presence of frequent intine protrusions is a remarkable feature of this genus. A multilaminar structure of the exine, already observed in Grimmia, occurs also in these species although here it is less pronounced. The significance of these features is discussed within these species, as well as in comparison to other taxa, especially the genus Grimmia.
Keywords:
Ultrastructural observations on spore characters using TEM have proved useful to separate bryophyte taxa of any rank. According to Brown and Lemmon Citation(1990), the presence of perine separates mosses from liverworts and hornworts, and both Sphagnum and Andreaea show distinctive features regarding the exine stratification, enabling each of them to be differentiated from Bryiidae. In addition, the perine elements and stratification of the intine could be useful for taxonomic purposes at the generic or specific levels (Carrión et al. Citation1995; Carrión et al. Citation1993; Guerra et al. Citation1991; Estébanez et al. Citation1997). Nevertheless, moss spore ornamentation is much less conspicuous than in vascular plants and in recent years just a limited number of papers have been published on moss spores (Luizi‐Ponzo & Barth, Citation1999; Du, Wang & Zhang, Citation2000). None of these include TEM observations.
The taxonomic position of the genus Ptychomitrium Fürnr. is controversial. This genus is sometimes placed in the family Grimmiaceae and sometimes in its own family, the Ptychomitriaceae. Its relationships with other families, such as the Erpodiaceae, are unclear. Japanese authors (Noguchi & Iwatsuki, Citation1988; Iwatsuki, Citation2001) still include the genus Glyphomitrium in the latter family, although this genus is more commonly placed in Ptychomitriaceae (cf. Smith, Citation2004; Buck & Goffinet, Citation2000).
Our results using chloroplast rbcL sequences (Tsubota et al. Citation2003) place the families Seligeriaceae, Ptychomitriaceae and Grimmiaceae in a well supported clade, with the Seligeriaceae as a sister group of a Grimmiaceae s. l. clade, including both the families Ptychomitriaceae and Grimmiaceae s. str.
In a previous ultrastructural study on six species of the genus Grimmia (Estébanez et al. Citation1997) we reported a remarkable interspecific variability and some particular features, such as a multilamellar structure of the mature exine. No data on spore ultrastructure are available for the genus Ptychomitrium. This study presents a detailed analysis of mature spores in four species of Ptychomitrium, in order to assess the taxonomical position of this genus, especially its relationships with the potentially related genus Grimmia.
Material and methods
Mature capsules have been collected in the field from populations of the following species: Ptychomitrium dentatum (Mitt.) A. Jaeger, P. linearifolium Reimers & Sakurai, P. gardneri Lesq., and P. sinense (Mitt.) A. Jaeger.
Voucher specimens of the material are deposited at HIRO (see Specimens Investigated).
The capsules were briefly rehydrated in water (1 hour) before fixation.
For SEM observations, spores were fixed in 2.5% glutaraldehyde buffered in 0.1 M Na‐cacodylate, pH 7.2; then rinsed in 3% sucrose – 0.1 M Na‐cacodylate buffer, and dehydrated in an ethanol series. The fixed spores were critical‐point dried after substituting liquid CO2 for ethanol, and then sputtered with a thin layer of gold (ca. 30 nm), and observed with a JEOL‐JSM‐T‐330A microscope.
For TEM observations, spores were fixed in 2.5% glutaraldehyde buffered in 0.1 M Na‐cacodylate, pH 7.2; post‐fixed in 1% OsO4 in the same buffer; dehydrated in an ethanol series and embedded in Spurr's resin. Ultra‐ and semithin sections were made either with a LKB 2188 Ultrotome NOVA or a Reichert Jung ultramicrotome. Ultrathin sections were stained with 3% aqueous uranyl acetate for twenty minutes, followed by lead citrate (Reynolds, Citation1963) for five minutes, and observed with a Zeiss 902 or a JEOL‐JEM‐1200‐EX, operating at 80 Kv. Semithin sections were stained with 0.5% toluidine blue and observed with light microscopes. Size and number of plastids were assessed with light microscopy observations of whole spores.
The terminology used for the spore sculptural relieves follows Erdtman Citation(1969).
Results
General remarks
The spores of the examined species are always unicellular. With SEM, the sporoderm in all species appears with a rugose relief, consisting of irregular, sometimes fusionated, small‐sized verrucae (Figs , , , ). In all four species, three layers in the sporoderm: intine, exine and perine, as defined by McClymont and Larson Citation(1964) for mosses are recognized by TEM. The intine is fibrillar and consists of two continuous layers (Figs ; ; ; ), often with an irregular, discontinuous third layer forming protrusions affecting the spore relief. The exine is usually very thin, and always unbroken, following the outline of the intine. This outline is often very irregular, due to the presence of the intine protrusions covered by the external layers (exine and perine: Figs ; ; ). The outermost layer, the perine, is composed of discrete primary elements very opaque to electrons, with variable density, sometimes accumulated on a thin, continuous basal layer. A more electron‐translucent material, henceforth designated as secondary elements, is also deposited on the primary elements of the perine (Figs ; ; ; ). Both have seemingly originated extrasporally from a secretory tapetum, since the cell layer lining the spore sac is covered with many globules of materials similar to the primary and secondary elements of the perine (Fig. ).
Figure 1 Ptychomitrium dentatum. A. SEM micrograph. B–G. TEM micrographs: (B) Section with cytoplasm not polarised, showing large plastids full of starch; (C) Sporoderm: bilayered intine, exine (arrow – locally bilaminar) and electrondense perine elements; (D) Characteristic intine protrusions in two spores; (E) Irregularities in the intine second layer and a protrusion formed by a local third layer; (F) Sharp protrusion of the intine, covered with a very thin intine and with perine elements. Arrow – exine in two laminae; (G) Spore with polarised cytoplasm. Scale bar – 5 µm (A); 2.5 µm (B); 0.25 µm (C); 2 µm (D & G); 1 µm (E); 0.4 µm (F).
Figure 2 Ptychomitrium gardneri. A. SEM micrograph. B–F. TEM micrographs: (B) Section with cytoplasm not polarised, showing large plastids full of starch and plastoglobuli; (C) Spore close to dead tapetal cells, lined with perine‐like elements; (D) Spore with eccentric nucleus, and sporoderm with two intine layers, thin exine (arrows – locally laminar exine), and scarce perine elements; (E) Sporoderm, showing exine intermingling with perine elements (arrows); (F) Plastid structure, showing well defined thylakoid stacks and plastoglobuli. Scale bar – 5 µm (A); 1 µm (B); 2 µm (C & D); 1 µm (E); 0.25 µm (F).
Although the verrucae observed with SEM seem to correspond to the perine sculpturing elements, the protrusions of intine material, often larger (Fig. ), also contribute to the external ornamentation.
The shape of the spore and the morphology of the sporoderm are rather homogeneous, showing little or no polarity, only the perine elements may have a diverse density (Figs ; ) or size (Figs ; ), throughout the spore surface. No aperture or specialized thickening of the intine recognizable as such (Mogensen, Citation1981) is distinguishable in any case.
Figure 3 Ptychomitrium linearifolium. A. SEM micrograph. B–G. TEM micrographs: (B) Spore section, showing cytoplasm with few organelles, and two plastids. Sporoderm abundantly covered by perine elements; (C) Another spore section showing two plastids; (D) Plastid structure, with almost no storage substances, and dense mitochondria with tubular cristae; (E) Sporoderm with two perine layers, intine, and perine composed of primary, electrondense elements, and very conspicuous, electrontranslucent secondary elements; (F) Sporoderm, showing exine divided into laminae (arrows); (G) Irregular outline of the intine due to irregular protrusions. Scale bar – 2.5 µm (A); 2 µm (B, C); 0.7 µm (D); 0.5 µm (E–G).
In the cytoplasm of all four species, plastids are large and with well developed thylakoids. There are usually many vacuoles with osmiophilic, very electron‐opaque deposits, that appear partially dissolved (Figs ; ; ; ). Olesen and Mogensen Citation(1978) interpreted these deposits as lipid droplets, partially solubilized during the fixation and embedding process. There is also another kind of droplet more electron‐translucent and well preserved in the sections (Figs ; ; ; ). Both kinds of lipid droplets are referred here as the “soluble” and the “insoluble” types respectively. The cytoplasm may or may not show polarity, even in the same species.
Description of each species
Ptychomitrium dentatum (Fig. )
Shape and size
– Subsphaerical to ellipsoidal, 15–25 µm (Fig. ).
Sporoderm
– Intine: Two continuous layers, the inner one more electrondense and granular, ca. 0.1 µm thick, the outer one fibrillar, and extremely irregular in thickness, with an undulate external border (Fig. ). A third discontinuous layer of amorphous texture forms very prominent protrusions of the sporoderm (Fig. ).
Exine: Less than 10 nm thick, unbroken (Fig. ), sometimes locally intermingled with perine‐like material (Fig. ), or dividing into laminae (Fig. , arrows).
Perine: Consisting of a continuous basal laminar layer, as thin as the exine, and sculpturing elements covering it unevenly (Fig. ). In one hemisphere, the primary elements are more abundant and consist of clavae and baculi, 0.25–0.6 µm tall, sometimes fused at their tops, occasionally branched (Fig. ). The other hemisphere presents sparse, small verrucae (Fig. ). The primary sculpturing is covered by papillar secondary elements (Fig. ).
Cytoplasm
Dense, granulose, filled by both soluble and insoluble lipid droplets (Fig. ). Plastids mostly fusiform, 5–6 µm long and 3 µm wide, densely filled with granal stacks of 3–10 thylakoids and large starch grains (Fig. ). Mitochondria 0.6 µm long, with tubular cristae (Fig. ). Nucleus with a prominent nucleolus (Fig. ). The distribution of the organelles is usually concentric, with lipid droplets close to the sporoderm, and plastids tangentially crowning the central nucleus (Fig. ). Occasionally, the distribution gets polarised, with lipid droplets at one pole, and plastids and nucleus at the other (Fig. ).
Ptychomitrium gardneri (Fig. )
Shape and size
– Subsphaerical, 15–20 µm (Fig. ).
Sporoderm
– Intine: Two continuous layers of fibrillar texture (Fig. ), the inner one more electron‐translucent, ca. 0.4 µm thick, the outer one looser in structure, more electron‐opaque, and irregular in thickness (0.1–0.3 µm thick).
Exine: 10–20 nm thick, electron‐translucent, sometimes intermingled with perine secondary material (Fig. , arrows).
Perine: Sparse, irregular verrucae, sometimes on an underlying thin layer (Fig. ), of irregular presence throughout the spore surface (Fig. ). Distribution slightly polarized, as in P. dentatum. Secondary elements papillose are abundant (Fig. ).
Cytoplasm
– Granular, densely filled with organelles, and many lipid droplets of both kinds. Plastids usually 4 (sometimes up to 6), largely fusiform, ca. 6 µm long and 2.5–3 µm wide, densely filled with thylakoids in stacks of 3–10 (Fig. ). Plastoglobuli and starch present (Fig. ). Mitochondria close to the plastids, up to 0.6 µm, with tubular cristae (Fig. ). Nucleus with prominent nucleolus (Fig. ). Cytoplasm distribution often polarised (Fig. ).
Ptychomitrium linearifolium (Fig. )
Shape and size
– Subsphaerical to ellipsoidal, 7–11 µm (Fig. ).
Sporoderm
– Intine: Two continuous layers, the inner one less than 0.05 µm thick, electron‐opaque, the outer up to 0.2 µm thick, both fibrillar (Fig. ). A third, amorphous outermost one, appears irregularly filling wide, but not prominent protrusions (Fig. ).
Exine: 20–30 nm, medium electron‐opaque, apparently slightly separated from the intine (Fig. ), sometimes locally dividing into laminae (Fig. , arrow).
Perine: Slightly polar (Fig. ), in one hemisphere with clavae or baculi which are ca. 0.25 µm tall, regularly distributed, in the other hemisphere, more sparse, irregular verrucae (Fig. ), sometimes fused at the base (Fig. ). No perine laminar layer underlining these sculpturing elements. Many secondary elements of different sizes, most of them papillose (Fig. ).
Cytoplasm
– Granular, with low density of organelles. Lipid droplets scarce, mostly of the “soluble” type (Fig. ). Plastids always two. In section, the two plastid profiles are different in length, one is long ellipsoidal, ca. 6 µm thick, the other shorter, that could correspond to sections of two plastids of similar size oriented perpendicularly (Fig. ). Thylakoids in disperse stacks of 5–12, with little or no starch nor plastoglobuli (Fig. ). Mitochondria ca. 0.6 µm thick, with dark matrix and tubular cristae (Fig. ). Nucleus eccentric, with prominent nucleolus. Organelle distribution always asymmetrical, but not showing polarity (Fig. ).
Ptychomitrium sinense (Fig. )
Shape and size
– Subsphaerical, 15–22 µm (Fig. ).
Figure 4 Ptychomitrium sinense. A. SEM micrograph. B–E. TEM micrographs. (B) Spore with polarised cytoplasm; (C) Spore almost without polarisation, showing irregular deposition of perine elements; (D) Sporoderm with bilayered intine, thin exine (arrows – locally divided into laminae) and perine elements with conspicuous secondary material; (E) Plastid with thylakoid stacks and almost no storage substances, sporoderm with mushroom‐like intine protrusions. Scale bar – 5 µm (A); 2 µm (B); 2.5 µm (C); 1 µm (D, E).
Sporoderm
– Intine: Two continuous layers, both fibrilar and of medium electron‐opacity, the inner one ca. 0.4 µm thick (Fig. ), the outer irregular in thickness, often showing an undulate outline (Fig. ). A third, amorphous layer appears irregularly filling small but sharply prominent (sometimes mushroom‐like) protrusions of the sporoderm (Fig. ).
Exine: 20–30 nm, medium electrondense, sometimes dividing into laminae intermingled with some perine elements (Fig. , arrows).
Perine: Sparse verrucae (up to 0.5 µm tall) irregularly distributed but showing no clear polarity (Fig. ). Abundant material, similar to that of the secondary elements in other species, covering them (Fig. ). No continuous perine laminar layer underlining the verrucae.
Cytoplasm
– Dense, granular (Fig. ). Abundant lipid droplets, mostly of the “insoluble” type (Fig. ). Plastids 4–6, usually largely fusiform, up two 7–8 µm long, 3–4 µm wide (Fig. ), with thylakoids in stacks of 4–10, and storage substances (starch and plastoglobuli) in small quantities. Mitochondria less than 0.6 µm, with tubular cristae (Fig. ). Nucleus with prominent nucleolus. Distribution of organelles often polarised, with the nucleus and plastids at one pole, and the lipid droplets opposite (Fig. ).
Discussion
Diverse authors have proposed the following spore characters as primitive: pluricellularity (Mogensen, Citation1983), lack of polarisation (Horton, Citation1982), thin, faintly sculptured spore wall (Mogensen, Citation1981, Citation1983, Carrión et al. Citation1993), and well developed chloroplasts (Horton, Citation1982). Generally, thin‐walled spores are most commonly found in bryophytes. As a recent example, Muñoz et al. Citation(2004) considers them all to be “green spores”. However, only some 50 species have been investigated with TEM, making it difficult to assess the actual evolutionary implication of these characters. Nevertheless, most of these species, representing a wide taxonomic spectrum, have well developed plastids.
However, brown or yellow spores are present in several groups, including Andreaea and Sphagnum, which are among those regarded as possible basal groups to all mosses (Cox et al. Citation2004). Brownish colours could be related with plastid reduction, but at least in some cases it is due to the spore wall opacity (Horton, Citation1982; Estébanez et al. Citation1997). Plastid reduction in mature spores has been proved only in Funaria, Sphagnum, Dawsonia, Riella (cf. Neidhart, Citation1979), Ceratodon (Olesen & Mogensen, Citation1978) and three species of Grimmia (Estébanez et al. Citation1997). All of these species have a thick sporoderm. Green spores have been reported for closely related species (belonging to the same genus or family) in all cases. Bearing in mind the scarcity of studies, green spores could be in fact the basic character state in bryophytes, and plastid reduction (and probably the presence of a thick wall) a specialization achieved in several clades of the phylogenetic tree.
In the Grimmia/Seligeria clade resulting from the analysis of rbcL sequences, the genus Ptychomitrium stands as a monophyletic group, basal to a Grimmiaceae s. str. clade that would include the genera Grimmia, Racomitrium, Schistidium, Coscinodon and Campylostelium (Tsubota et al. Citation2003).
The spores in the four species of Ptychomitrium studied here, although generally unicellular, have symmetrical shapes (very rarely concave‐convex), show little or no polarity in the sporoderm (only occasionally in the cytoplasm), a relatively inconspicuous perine accumulation, and plastids always with a well developed thylakoid system. Therefore, they have a relative undifferentiated state, probably related with immediate germination after spore dispersal. However, they also have some particular and distinctive features, such as the irregular protrusions of the intine.
The observations of Grimmia, on the contrary, usually show a more developed exine (Fig. ), and different degrees of specialization according to the species. G. orbicularis, G. pulvinata spores are polarised, with a thick perine cover and plastids with reduced internal membrane system (Estébanez et al. Citation1997). Nevertheless, many sporal characters of Grimmia funalis, including symmetry, thylakoid development, and even occasional presence of irregular intine protrusions, link both genera. It is possible to imagine a common ancestor with spores similar to those of Ptychomitrium, from which the diversity in Grimmia would have originated through progressive adaptation to stronger environmental restrictions. This would support the phylogenetic relationship of both genera according to rbcL sequences (Tsubota et al. Citation2003).
Figure 5 Grimmia elatior. TEM micrographs of the sporoderm. A. Multilaminar structure of the exine. B. Thick, non laminar exine. Scale bar – 0.2 µm.
The exine in Ptychomitrium is thinner than in Grimmia (Fig. ), and it shows locally a laminar structure (see arrows in the figures), although not so frequently nor so clearly marked as in Grimmia species (Fig. ). In mosses this structure is usually observed only in immature spores (Brown & Lemmon, Citation1990; Matsuda et al. Citation1999), but at least in Ptychomitrium and Grimmia, as well as in Pottia (Carrión et al. Citation1993), this character apparently persists also in mature spores. These lamellae of the exine are often intermingled with an opaque material similar to that of the perine. This is striking, as the perine is assumed to be of extrasporal origin and exine intrasporally synthesized (Neidhart, Citation1979; Brown & Lemmon, Citation1990). Our observations of perine‐like globules, apparently including material of both primary and secondary elements, lining the tapetum cells in these species (Fig. ), would confirm the centripetal deposition of this layer. The intermingling of both layers could reflect almost simultaneous deposition. On the other hand, the texture and electrondensity of the secondary elements of the perine are very similar to the exine. It would be interesting to check whether their composition is also similar. It is known that in both species sporopollenin is present, predominantly in the exine, but also, intermingled with carbohydrates and other lipoids, in the perine (Neidhart, Citation1979). A detailed study of the differential composition of the perine elements is lacking. Should it be “exinous”, a partially extrasporal origin of the exine would be supported, and this laminar structure of the exine could be explained through the fusion of perine globules of secondary material into an “exinous” layer.
As for the intine, its stratification could have some taxonomical value (Miles & Longton, Citation1992; Estébanez et al. Citation1997). In Ptychomitrium, the presence of two continuous layers is constant, although their relative thickness and electrondensity vary in the different species. The frequent presence of intine protrusions is also remarkable. It depends on the formation of a third, irregular, amorphous layer, that contributes to the ornamentation of the spore surface in three of the four species: P. dentatum (Fig. ), P. linearifolium (Fig. ), and P. sinensis (Fig. ). Similar protrusions were observed in Grimmia funalis (Estébanez et al. Citation1997), but have not been recorded in any other moss species, and therefore could be distinctive for Grimmiaceae and Ptychomitriaceae. The function of the intine protrusions is unclear. They might be a wall material reservoir, allowing some plasticity of the sporoderm in order to expand the spore cell. On the other hand, in these species where perine sculpturing is scarce, the protrusions could also provide some necessary relief in order to increase the superficial water tension and thus control the water uptake in insufficient humidity conditions, preventing precocious germination.
In general, the spore characters support the taxonomical separation of Grimmia and Ptychomitrium, but nevertheless some significant particularities (such as the presence of intine protrusions and of an exine multilaminar structure) suggest a link between them that would be worthwhile to confirm with other characters. Those features have not been observed in species of the presumed allied genus Glyphomitrium or any genera in the family Erpodiaceae (Estébanez, Yamaguchi & Deguchi, in preparation).
The intrageneric diversity shown by the spores of these species is not very high, in contrast to that observed in Grimmia (Estébanez et al. Citation1997). Only P. linearifolium is readily distinguishable due to the simplification of its cytoplasm content. Usually, the use of several spore characters is needed to differentiate each species. In Table the main diagnostic characters have been selected:
Table I. Summary of the main sporal features in each species of Ptychomitrium.
According to the above criteria, P. linearifolium would represent a higher degree of specialization (reduction in plastid and organelles, thicker exine and slightly polar perine), followed by P. sinense. On the other hand, P. gardneri, and P. dentatum, in this order, would be closer to the less differentiated type. The size in all of them is generally in accordance with previous descriptions (Noguchi & Iwatsuki, Citation1988).
There are other variable characters such as the presence or absence of cytoplasmic polarity (in particular, the more or less eccentric nucleus position), lipid droplets electron‐opaque or electron‐translucent and the plastid storage substances. None of these are, however, sufficiently stable intraspecifically to be used to distinguish the various species. The possibility of initial germination stages influencing the observations (especially in the case of cytoplasmic polarity) cannot be discarded.
Conclusions
The spores of the four species of Ptychomitrium studied here show overall similarity in shape, size, wall sculpturing and ultrastructure, so that, although they may be distinguishable by ultrastructural features, no single character is sufficient to separate them from each other. The main diagnostic characters are the intine, exine and plastid morphology.
The spores have thin sporoderm, no reduction in the plastid structure, and are apparently ready for germination shortly after release.
Some features that are otherwise uncommon in moss spores have been observed: a locally multilaminar exine and the presence of intine protrusions. Both have been observed also in Grimmia (in this genus the multilaminar exine is more developed, and the exine protrusions less).
Acknowledgements
This study was partly supported by the Grant‐in‐Aid of the JSPS to B. Estébanez (no. 97411) and KAKEN (no. 1640698) to H. Deguchi.
References
- Brown , R. C. and Lemmon , B. E. 1990 . “ Sporogenesis in bryophytes. ” . In Microspores, evolution and ontogeny , Edited by: Blackmore , S and Knox , R. B . 55 – 94 . London : Acad. Press . In
- Buck , W. R. and Goffinet , B. 2000 . “ Morphology and classification of mosses. ” . In Bryophyte biology , Edited by: Shaw , A. J and Goffinet , B . 71 – 123 . Cambridge : Cambridge Univ. Press . In
- Carrión , J. S. , Cano , M. J. and Guerra , J. 1995 . Spore morphology in the genus Pterygoneuron Jur. (Pottiaceae). . N. Hedwigia , 61 : 481 – 496 .
- Carrión , J. S. , Guerra , J. and Ros , R. M. 1993 . Spore morphology of European species of Phascum Hedw. (Pottiaceae, Musci). . N. Hedwigia , 51 : 411 – 433 .
- Carrión , J. S. , Ros , R. M. and Guerra , J. 1993 . Spore morphology of Pottia starkeana (Hedw.) C.Muell. (Pottiaceae, Musci) and its closest species. . N. Hedwigia , 56 : 89 – 112 .
- Cox , C. J. , Goffinet , B. , Shaw , A. J. and Boles , S. B. 2004 . Phylogenetic relationships among the mosses based on heterogeneous Bayesian analysis of multiple genes from multiple genomic compartments. . Syst. Bot. , 29 : 234 – 250 .
- Du , G.‐S. , Wang , M.‐Z. and Zhang , Y.‐L. 2000 . Observations of spore morphology of some acrocarpous mosses in China. . Acta Bot. Yunn. , 22 : 277 – 281 .
- Erdtman , G. 1969 . Handbook of palynology , New York : Hafner Publ. Co .
- Estébanez , B. , Alfayate , C. and Ron , E. 1997 . Observations on spore ultrastructure in six species of Grimmia (Bryopsida). . Grana , 36 : 347 – 357 .
- Guerra , J. , Martínez , J. J. , Ros , R. M. and Carrión , J. S. 1991 . El género Phascum (Pottiaceae) en la Península Ibérica. . Cryptog., Bryol., Lichénol. , 9 : 343 – 352 .
- Horton , D. G. 1982 . The evolutionary significance of superficial spore characters in the Bryidae. . J. Hattori Bot. Lab. , 53 : 99 – 105 .
- Iwatsuki , Z. 2001 . Mosses and liverworts of Japan , Tokyo : Heibonsha .
- Luizi‐Ponzo , A. P. and Barth , O. M. 1999 . Spore morphology of some Dicranaceae species (Bryophyta) from Brazil. . Grana , 38 : 42 – 49 .
- Matsuda , S. , Estébanez , B. and Deguchi , H. 1999 . Sporogenesis in Oncophorus crispifolius (Dicranaceae, Bryopsida). . Bryobrothera , 5 : 139 – 151 .
- McClymont , J. W. and Larson , D. A. 1964 . An electron‐microscope study of spore wall structure in the Musci. . Am. J. Bot. , 51 : 195 – 200 .
- Miles , C. J. and Longton , R. E. 1992 . Spore structure and reproductive biology in Archidium alternifolium (Dicks. ex Hedw.) Schimp. . J. Bryol. , 17 : 203 – 222 .
- Mogensen , G. S. 1981 . The biological significance of morphological characters in bryophytes: the spore. . Bryologist , 84 : 187 – 207 .
- Mogensen , G. S. 1983 . “ The spore. ” . Edited by: Schuster , R. M . Nichinan : Hattori Bot. Lab . New manual of bryology (vol. 1, pp. 325–342)
- Muñoz , J. , Felicísimo , A. , Cabezas , F. , Burgaz , A. R. and Martínez , I. 2004 . Wind as a long‐distance dispersal vehicle in the Southern hemisphere. . Science , 304 : 1144 – 1147 .
- Neidhart , H. V. 1979 . “ Comparative studies of sporogenesis in bryophytes. ” . In Bryophyte systematics , Edited by: Clark , C. G. S and Duckett , J. G . 251 – 280 . London : Acad. Press . In
- Noguchi , A. and Iwatsuki , Z. 1988 . “ Gen. Ptychomitrium Fuernr. ” . Nichinan : Hattori Bot. Lab . – In An illustrated moss flora of Japan (vol. 2, pp. 373–383)
- Olesen , P. and Mogensen , G. S. 1978 . Ultrastructure, histochemistry and notes on germination stages of spores in selected mosses. . Bryologist , 81 : 493 – 516 .
- Reynolds , E. S. 1963 . The use of lead citrate at high pH as an electron‐opaque stain in electron microscopy. . J. Cell Biol. , 17 : 208 – 212 .
- Smith , A. J. E. 2004 . Cambridge : Cambridge Univ. Press . The moss flora of Britain and Ireland (2nded.)
- Tsubota , H. , Ageno , Y. , Estébanez , B. , Yamaguchi , T. and Deguchi , H. 2003 . Molecular phylogeny of the Grimmiales (Musci) based on chloroplast rbcL sequences. . Hikobia , 14 : 55 – 70 .