Andreaeobryum macrosporum Steere & B.M. Murray, Phytologia

Andreaeobryum macrosporum Steere & B.M. Murray, Phytologia View in CoL 33: 407. 1976.

Figs. 2–39 View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig View Fig .

Plants in moderately dense tufts, intense smaragdine green in recently formed parts, readily turning to dark brown. Stems ascending to erect, but as the species grows usually on inclined to vertical surfaces, stem tips face downwards (towards the substrate), with the longer leaves facing in the same direction; repeatedly branched by subterminal innovations; rather slender, red-brown, 2–3(–7) cm long (including old parts), 100–150(–200) mm in diameter, without hyalodermis and central strand, sclerodermis consisting of 1–2 layers of thick-walled cells, except the distal parts of fertile shoots, where stem is composed of completely homogeneous, moderately thick-walled cells; central strand absent; foliage dimorphous, some shoots vermicular, densely julaceously foliate with small orbicular leaves; other shoots moderately densely foliate with much longer leaves turned to one side, usually towards the substrate; julaceous shoots with age usually start to develop longer leaves, but sometimes remain thin and julaceous indefinitely and form a part of the tuft with shoots exclusively of this type; transitional zone from small-leaved to large-leaved part of shoot is most commonly quite abrupt, occasionally gradual; in upper fertile parts of shoots foliage becomes more variable, involving small primary leaves in addition to large leaves; rhizoids 1 copious at stem bases, forming brownish mat, transforming to secondary persistent protonema that produces new stems and terete protonemal leaves; axillary hairs beaked, few in leaf axils, becoming elevated one cell up on adaxial leaf surface with age, most numerous in the fertile zone of the female plants; usually two-celled, with upper cell beaked. Axillary hairs that appear maximally developed, judging from the most inflated apical cell, to 15–16 µm wide. Most beaked axillary hairs two-celled, 30–50×10–15 µm, but occasionally with basal cells up to five in number, and occasionally even more.

Leaves dimorphic in two types of shoots. Small orbicular to broadened orbicular leaves 0.2–0.3 mm long and 0.3–0.4 mm wide, ecostate, concave. Larger leaves erect, moderately falcate-secund, 1.2–1.7 mm long, 0.2– 0.3 mm wide, lanceolate, from ovate base gradually tapered to bistratose acumen 1/5–1/2 the leaf length, slightly concave-channeled; margins plane, entire to slightly sinuose; cells of unistratose lamina in lower part of leaves irregularly rounded, 10–25 µm in diameter, towards the base larger and longer, short rectangular, 20–40×15–28 µm, with thick, but with apparently soft walls; costa confluent with upper 2- to 5-stratose acumen, without apparent differentiation in transverse section, or central cells in middle with narrower lumens.

Dioicous. Perigonia bulbiform, terminal, but antheridia also occurring in axils of 1–2 leaves adjacent to perigonia; perigonial leaves broadly ovate, short apiculate, unistratose throughout; antheridia 250–350 µm long, to 150 µm wide when mature, on 2-seriate stalks to 80 µm long; up to 7, and often exhibiting different stages of development. Archegonia not surrounded by specialized perichaetial leaves, scattered along the uppermost part of stem, sometimes to 1 mm long, often associated with small ‘ primary leaves ’. Sporophytes appearing terminal, but as the lateral archegonia are sometimes fertilized, their position appears somewhat shifted from the apex, and rarely two capsules are developed on one stem. Seta reddish-black, of the same color as the capsule, flattened and twisted. Capsule reddish-black, shining, originally globose, then conic distally and trullate-shaped in side view; 0.5–0.7× 0.5–0.7 mm, dehiscent longitudinally by 4–8 slits; valves remaining connected for a long time; stomata absent. Calyptra large, enveloping the whole capsule, persistent. Spores orange to brownish with age, (50–)70–90(–100) µm, spheric to irregularly ovate, granulose, multicellular.

Rhizoids ( Fig. 6 View Fig ) occur as extensive light brownish mats at the shoot base and also near wounded parts of stem and at base of innovations (probably also appearing in injured parts of moss body).

In the former case, the mass of rhizoids was observed on rock surface, being intermingled with sand pieces and thus forming a specific adhesions upon rocks. These formations apparently follow paths of water flows on the inclined rock surface below moss cushions. Terete protonemal leaves often originate along these ‘paths’ ( Fig. 6B View Fig ). Rhizoids in these mats have extremely thick-walled cells, with lumen often unseen at all or seen as a ‘median line’ – a poorly discernible line along the cell middle ( Fig. 6A View Fig ). In a few places, however, the oblique walls between cells are seen, confirming identity of the rhizoids.

Many ends of cells in these mats are of irregular shape, swollen and sucker-shaped, facilitating an attachment to the substrate.

Rhizoids from innovations are even more irregular, changing their stature during maturation. At first they are thin-walled and hyaline ( Fig. 6D View Fig ), with walls between cells oblique at about 30°. Cell divisions in rhizoids occur at ca. 10° ( Fig. 6 View Fig Ca). Later rhizoid cell-walls may become brown and thick, with an unusually narrow angle between cells, ca. 10° ( Fig. 6D View Fig ). In another case we observed rhizoids branched at very narrow angle, thus at place of branching the daughter cell followed mother cell, making rhizoids bicellular in width ( Fig. 6 View Fig Cb); in these zones rhizoids are hyaline, not brownish, and include numerous small chloroplasts ( Fig. 6 View Fig Cb). In older parts within rhizoid bundles, some chloronemata filaments were terminated in beaked-shaped cells ( Fig. 6 View Fig Cc), while others ended in a swollen and coral-shaped cells ( Fig. 6 View Fig Cd). The beaked outlines were observed in rhizoid mass ( Fig. 6A View Fig ). This is still an opened question, if the beakedshape in some rhizoid cells correspond to any mucilage production in them.

Protonemal leaves occur on mats of rhizoids ( Fig. 6B View Fig ). They are cylindric and multistratose throughout. Their transverse sections were presented by Murray and they are nearly identical to those of primary leaf transverse sections, shown, e.g., in Fig. 14 View Fig .

Beaked axillary hairs ( Figs. 7–8 View Fig View Fig ). A peculiar structure of the axillary hairs of Andreaeobryum was pointed out by Murray (1987, 1988). They have a conspicuous beak, which provides the mucilage release through the apical pore, in a way somewhat similar to Takakia , and, supposedly, in none of other mosses. Murray (1988) also indicated that axillary hairs are occasionally not beaked and are closed distally. At the same time, they are still not the same as in Andreaea , which has axillary hairs of the same type as other mosses.

The polymorphism of axillary hairs has been reviewed by Hedenäs (1990) for pleurocarpous groups, while their variation in acrocarpous species was only briefly discussed by Zolotov & Ignatov (2001). Axillary hairs in mosses are formed by 1–2 short brownish basal cells and 1–5(–10) upper cells which are usually longer, thin-walled, hyaline, rounded at apex. The release of mucilage is not very well studied, but likely it never has any definite place of liberation.

Although both opened and closed axillary hairs occur in the Siberian plants, we think that the closed axillary hairs represent just an older stage, after an active period of the mucilage release, or can be simply underdeveloped ones. Moreover, the exact interpretation of a given individual axillary hair might be uncertain due to the fact that the ‘beaked’ pattern can be seen also in apical cells of rhizoids ( Fig. 6 View Fig Cc) and apical cells of paraphyses ( Fig. 12E View Fig ).

However, in the areas where axillary hairs are especially abundant, for example, near gametangia and beside the stem apical cell ( Fig. 13F, H View Fig ), the beak is always apparent. Near these places it is easy to find some older beaked axillary hairs, with broken apical nipple ( Fig. 7D View Fig ) and then their apical cells are approaching to the rounded shape of distal end, looking somewhat similar to ordinary moss axillary hairs, albeit with still thicker cell walls ( Fig. 7H View Fig ). Rounded apical cells of axillary hairs were also seen in longisections of plants ( Fig. 7G View Fig ), but such observation is misleading, as the beak in not always strictly apical and often turned to one side, thus the beak can be seen only in one of several 1–2 µm sections.

Murray (1988) indicated the similarity of Andreaeobryum with Takakia in apical mucilage release from the axillary hairs. This statement is confirmed by our observations, but there are also some differences ( Fig. 7M–N View Fig ). In Takakia , the release is explosive through the somewhat attenuate tip of the axillary hair, and a round droplet appears first at the torn tip, sometimes followed by abundant exudation, when droplet reaches in diameter two lengths of the axillary hair which produces it ( Fig. 3 View Fig in Schuster, 1966). The mucilage papillae in hepatic may have the shape similar to Takakia and Andreaeobryum ( Galatis & Apostolakos, 1977) , but no special structures near the tip of axillary hair were found by TEM studies ( Galatis & Apostolakos, 1977; Duckett et al., 1990).

However, Takakia is not the only moss other than Andreaeobryum , that has an apical mucilage release. The latter is characterisic also of Sphagnum , which it seems, has never been discussed before, despite of a special description, e.g., in Berthier et al. (1974). The axillary hairs in Sphagnum are two-celled, with an inflated apical cell and a rather inconspicuous apical structure ( Fig. 7R View Fig ). However, autofluorescence contrasts it enough to make certain that the apical pore occurs ( Figs. 7O–Q View Fig ) and we were able to see it in every sample of living plants of S. girgensohnii and S. magellanicum examined.

Compared to Takakia and Sphagnum , Andreaeobryum has an extraordinarily complicated axillary hairs ( Figs. 7–8 View Fig View Fig ).

The structure is difficult to see in the light microscope (e.g., in Figs. 7 View Fig H-I, 14B), but autofluorescence contrasts it. Figs. 7B, J and L View Fig illustrate sublongitudinal fibrils, which reach the apical pore mouth and connect to the circular cup-like cover of the pore. It seems that the shift of this cover allows the mucilage discharge ( Fig. 7L View Fig ). These sublongitudinal fibrils often make the cell somewhat angulose, so basal cells can be assumed as a quadrate-shaped in transverse section ( Fig. 7F View Fig ).

TEM images illustrate that the body of the axillary hairs are rich in endoplasmatic reticulum and dictyosomes ( Figs. 8 View Fig C-D). The beak area has abundant fibrillose structures that form a net shortly below the apical pore (or rupture) ( Figs. 8 View Fig A-B). Shortly below the apical pore a conic structure is seen ( Fig. 8B View Fig ). There are especially abundant fibril bundles around it, and this fact along with structure and position below pore allow to propose its function as a reservoir of mucilage, which release can be regulated.

Some axillary hairs are composed of more than two cells ( Fig. 7I View Fig ), and in these cases the basal cell is apparently divided, producing up to five basal cells that form a uniseriate stalk. In extreme, the beaked axillary hair may sit on the top of a small leaf. The latter case was nicely illustrated by Murray (1988). In Siberian plants, we saw rather a hyaline apical leaf cell, with a beak-like knob ( Fig. 13A View Fig ), however the leaves terminated with developed axillary hairs were not seen simply because of less observation comparatively with those done in American specimens.

Young axillary hairs, obviously prior to mucilage release ( Fig. 7C View Fig ), have an inflated round beak, covered by another outer cover. Their position close to the stem apical cell assures that this type comprises the youngest, only recently developed axillary hairs. Within the middle part of its apical cell, a complicated heterogeneity (likely due to numerous vacuoles) is seen ( Fig. 7C View Fig ).

After the release of their mucilage, axillary hairs have their pores uncovered ( Fig. 7B View Fig ), or partly covered ( Figs. 7J, L View Fig ). A nipple-like tip above the cup-like cover ( Fig. 7J View Fig , and see also Fig. 13F View Fig ) is still not a fully understood structure. A possible interpretation could be that it is attached to the ovate body inside the axillary hair near its top, and thus keeps the pore cover in a position of ajar lid. Another analogy from moss structure could be a systilious operculum in mosses, which may potentially regulate spore release by changing its relative position to urn depending on moisture.

It is still indefinite if the axillary hairs from the sterile parts of shoots are obtuse from the beginning and may be treated as underdeveloped, or, alternatively, they became closed after active period of functioning with an open apical pore, which later became invisible ( Fig. 7G View Fig , 13H).

Sometimes axillary hairs in Andreaeobryum appear branched under light microscope, but additional examination reveals that in these cases just the protoplast falls off the apical cell wall, surprisingly retaining its shape ( Fig. 7E View Fig ).

In the older part of shoots, the axillary hairs are displaced from the axil and are situated on leaf adaxially ( Fig. 7F View Fig ). This position is in agreement with the fact that axillary hairs remain attached to the leaf bases after their detachment (thus the study of axillary hairs on stem can succeed only in exceptional zones, for example in distal part of female shoots with archegonia).

Stem ( Figs. 9– 10 View Fig View Fig ). Stem apical cell is relatively small and narrow, 30–35×13–16 µm ( Fig. 10 View Fig ), and does not differ appreciably from that in other mosses.

Shape of the apical cell is approximately triangular in transverse section, but with unequal sides ( Fig. 9 View Fig ). Hence the angle of the first division within the apical cell, as is seen in longisection, differs from 60° to almost 0° (i.e., being parallel to apical cell length). It seems, that such a plasticity allows development of leaves of quite different stature ( Fig. 10 View Fig ).

Shortly below its apex, the stem is unusually narrow ( Figs. 9 View Fig , 14 View Fig ), which is difficult to assume from plants with leaves. Sections usually reveal its diameter being only 90–150 µm. The cortex is one-layered, and no central strand is seen under the observation in light microscope. However, the series of sections in Fig. 14 View Fig indicate a certain differentiation of cells in the center of the stem. The most conspicuous are the intracellular spaces, which have no fluorescence from berberin staining. It indicates the presence of cellulose-free space, which conducting capacity remains unclear.

Stem leaves ( Figs. 5 View Fig , 11 View Fig , see also 9) in Andreaeobryum are in general dimorphous, including (1) orbicular small leaves ( Figs. 5 View Fig L-N) and larger lanceolate leaves ( Figs. 5 View Fig H- K) on ‘normally’ foliate stems ( Fig. 6G View Fig ). One shoot usually has one leaf type, although many julaceous shoots transform to ‘normally’ foliate ones distally ( Figs. 3D View Fig , 4D View Fig , 6F).

Subterminal innovations in their proximal parts have leaves of one type, either orbicular or lanceolate, retaining the leaf uniformity further up to a considerable distance ( Figs. 6G View Fig ).

Orbicular leaves have fully unistratose lamina, originated through the divisions of bifacial apical cell in a way common for almost all mosses.

Lanceolate leaves differ from orbicular ones since early development. They become multistratose due to oblique divisions of leaf apical cell. Figs. 11E–H View Fig show two young leaves from different sides, where leaf #2 appears already bistratose at the level of the leaf uppermost cells.

An areolation pattern characteristic for Andreaea , with unifacial apical cell ( Kühn, 1874) was never observed in the leaf apical part of Andreaeobryum , where cell sectors are separated in left and right halves by zigzag line ( Fig. 10-b View Fig 26 View Fig ; 11F View Fig : leaf #3). However, in the proximal part of leaf the rectangular direction of cell divisions becomes prevalent ( Fig. 10 View Fig , 11D View Fig ), which may correspond to the basal cells of the leaf. Small leaves, similar to that shown in Fig. 11C View Fig , often have clavate shape, with obviously already ‘multstratose’ upper part (similar to that in leaves shown in Figs. 11E–H View Fig ), and the narrow basal part. Fig. 11C View Fig does not show the structure of basal cells, but in longisections in Fig. 10 View Fig , as well as in transverse section series in Fig. 9 View Fig , there is a number of views showing that the more distal, the more multistratose the leaf is (e.g., # 5 in series in Fig. 9 View Fig ).

Laminal cells are originally quadrate closer to leaf margin and more rectangular in the middle, multistratose part of leaf, becoming round to ovate with age, occasionally oblate, unistratose except along the margin at base ( Fig. 14 View Fig ). Older cells have incrassate cell walls, formed by cellulose and have strong berberin-fluorescence. An unusual character is great variation of cell wall thickness, as seen in transverse sections (cf. Fig. 14 View Fig ).

SEM observation without special preparation revealed a surprisingly regular papillosity on both surfaces of the Andreaeobryum leaf ( Figs. 5O–P View Fig ). After high vacuum collapse, many moss leaves display hollows upon cell lumen and ridged cell walls, but in case of Andreaeobryum , the situation is more complicated. Cell walls on the dorsal leaf surface became not solid, but beaded by chains of round papillae, and closer to the leaf margin 1–2–3 shallow papillae occur above the lumen as well. Ventral surface has even more puzzling pattern, which can be explained by a highly uneven internal structure of cell walls.

Crandall-Stotler & Bozzola (1990) described the develpment and ultrastructure of leaf papillae in Andreaeobryum , finding in cell wall a microfibrillar network, a reminiscent of hygrophilic surface polysachharides. They conluded that such papillae are important for water absorption. The present observations on difference is leaf surface sculpture between dry ( Fig. 5O–P View Fig ) and wet ( Figs.9–11 View Fig View Fig View Fig ) state support this, indicating the malleability of the cell wall surface in Andreaeobryum .

Perigonial leaves and Antheridia

As perigonia are arranged on the stem at approximately equal distance, one may assume that they appear once a year, and, if this is true, the age of a plant in Fig. 6F View Fig can be evaluated as no less than 10 years old. Julaceous parts of plants may likely grow faster. The latter conclusion is based on the fact, that among the subterminal innovations, julaceous shoots are much more variable in length, while ‘normally’ foliate shoots with elongate leaves have perigonia at not more than upper 2(–2.5) mm.

Murray (1988) admitted that more than one generation of perigonia might originate on one shoot within one growing season, and this agrees with our observation of two nearby perigonia including still unopened antheridia at different stages of maturation. However, this is not a common case and considering that the habitat is covered by snow for no less than nine months a year, we can not also exclude a possibility that Andreaeobryum does not successfully produce gametangia every year.

Perigonial leaves are broadly ovate, ecostate ( Figs. 4K– L View Fig ). Although the majority of antheridia are grouped in a quite compact male inflorescences, in many cases we observed solitary antheridia (usually bigger and more mature in axils of leaves next to perigonia. One of possible variants with additional antheridium beside the perigonium is shown in Fig. 12K View Fig . The photo is taken through the leaves, as their detaching may result in breakage and possible misplacement of antheridia. Paraphyses are abundant both within the perigonium and around solitary antheridia.

Antheridia are long-stalked ( Figs. 12A, D, I View Fig ), similar in this respect to those of Andreaea , Sphagnum (especially of Eosphagnum and Ambuchanania ), and hepatics. Elongate and acute when premature ( Fig. 12A View Fig ), they become ovate later, at time of gamete release. Schiff staining provides color differentiation of antheridia ( Figs. 12B–D View Fig ) and paraphyses ( Figs. 12E–G View Fig ) along with their maturation.

Spermatozoids are released through a small pore surrounded by a massive thick-walled cells ( Figs. 12I–J View Fig ). It seems that the discharging of antheridia is fairly gradual, at least in the beginning of the male gamete liberation. In mosses and liverworts, as far as we know, the antheridium opening is explosive. After a more or less complete spermatozoid release, the pore region is strongly colored by Schiff staining, indicating thicker walls, as compared with the rest of antheridium walls. Although opened antheridia in Andreaeobryum are often darker near their apical pore, the darker area includes mostly 1–2 cell rows.

Perichaetial leaves, Primary leaves and Archegonia

Murray (1988) considered both perichaetial leaves and primary leaves as a very unusual structures of Andreaeobryum , and the present observation revealed even more their peculiar characters. Primary leaves were defined as a much smaller leaves, located in leaf axils by one or two, and considered as having a very unusual structure. In Siberian plants, the upper part of female plants is full of such structures, and interpretation is not easy.

Archegonia appear terminal, although in every series of sections we never saw them in a really terminal position. When an archegonium is formed, stem apical cell continues its growth ( Fig. 9 View Fig ). Thus, archegonia appear to be spread along the distal part of stem and sometimes up to 10 mm below stem apex.

It is still unclear if archegonium may substitute for the apical cell and thus stop stem growth, but the parallel observation of Schuster (1971) would be suggestive. In discussing the anacrogynous species of Haplomitrium , where numerous archegonia also develop and are displaced downwards the stem along with the growth, Schuster admitted, that after production of a sufficient number of archegonia, the plant may ‘exhaust itself’ and finally produce terminal archegonium. However, real evidence for this was not obtained.

Murray (1988) underlined a probably unique case of anacrogyny in mosses in Andreaeobryum , which is similar to hepatics. Fig. 9 View Fig shows a series of transverse sections, where the subapical archegonium is cut to its base, showing that it originates from one merophyte cell, developing into archegonium and leaf. This explains numerous cases where a small leaf is sitting side by side with archegonium ( Figs. 13F View Fig ; 14B–C View Fig ), in our specimens mostly to the right from it.

This ability to transform one part of leaf into archegonium explains as well the series shown in the Fig. 14 View Fig . This series illustrates four elements in leaf axil arranged in one row, although deriving from stem at an interval of 70–80 µm. Three of them are interpreted as leaves due to homogeneous cells in transverse section, while one with tubulose distal part can not be anything but the archegonium, as it has an empty cylindrical part in its centre. It still has a homogeneous structure at base, as it is seen in transverse section, interpreted as an archegoium pedestal (its length of ca. 50 µm agrees with the pedestal length in other archegonia in Figs. 13 View Fig and Fig. 14 View Fig ). As a whole, this four-element structure, occupying about 1/3 of the stem circumference, has maximal similarity with the tetra-filamentose leaf of Takakia , where one of lobes is substituted by an archegonium.

Organ substitution in mosses is usually not considered, as there is no evidence. However, in another bryophyte lineage, the Hepaticae, the substitution of a halfleaf with the branch is a fact, basic for any discussion on numerous types of branching (Crandall-Stotler, 1972). In Haplomitrium half of a leaf can be transformed into an antheridium ( Schuster, 1966 b). Among mosses, in addition to Andreaeobryum , the ability for substitution occurs in Takakia , where one lobe of bilobate leaf may be substituted by lateral branch.

The ability to metamorphose also explains perichaetial leaves of strange shape. Most of them look similar to archegonia, but with the attached piece of leaf lamina, often just on one side. It is noteworthy, that such structures are especially common at base of epigonium, where they more likely represent unsuccessful archegonia, which, instead of implementation of their main function, started to transform into a leaf.

An interpretation of the position of small primary leaves as axillary may be erroneous, at least partly, due to very dense spiral arrangement of leaves and their derivative organs near the female stem apex.

Archegonia are pedestaled in Andreaeobryum ( Fig. 13 View Fig ), as well as in Andreaea ( Fig. 13I–J View Fig ). The central part of the pedestal looks colored, hampering localization of egg-cell. However, all searches resulted in finding egg-cell at 30–50 µm above the stem, shortly above the constriction well seen in most archegonia.

The putative young archegonia (assumed as such by the position near stem apex and by comparison with archegonia at later stages of development) have paired round cells at base ( Fig. 13C View Fig ), divided by constriction from cells above. It seems likely that these pairs further develop into pedestals. The latter have a structure quite similar to that seen in leaves in transverse sections (series in Fig. 14 View Fig ).

Placenta and epigonium ( Figs. 15–21 View Fig View Fig View Fig View Fig View Fig View Fig View Fig ).

After fertilization, young capsule starts to develop, and at first stages, up to 0.5–0.7 mm long, a spindleshaped sporophyte remains totally embedded in the mucilage-like medium formed within the epigonium. Epigonium at this stage is filled with the semi-decomposed cells, separated from the inner surface of its wall (subsequently calyptra) and also surrounded by vaginular tissues. Hence, within the epigonium, an extraordinary broad space is formed; it surrounds the sporophyte to the end of the foot and even beyond it; this space is continuous and will be called below ‘placental space’, although it is not restricted to the zone of contact of foot with vaginula, but stretches between the whole young sporophyte body and epigonium inner wall.

The maceration of cells from epigonium wall is observed in all directions. The solvent-like medium penetrates ahead of the foot first to the volume of archegonium pedestal, and then in the vaginula, building a space for the growing foot. In the series in Fig. 23 View Fig , the modification of gametophytic cells is seen at the distance of no less than 20 µm from the haustorial cells. The modification includes shrinking of cytoplasm volume, deposition of soft polysaccharide compounds along the cell walls, and maceration of cells, so that they appear to be separated by intracellular space, also rich in polysaccharide compounds.

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The foot is covered by inflated cells from its tip and sometimes by elongate and occasionally multicellular outgrowths ( Figs. 17E–F View Fig ), as in Diphyscium ( Ligrone et al., 1993) . Transfer cells are well developed in sporophyte part in 2–3 layers. Moreover, the outer surface of the foot bears labyrinth-like structures facing the placental space ( Figs. 18A–D View Fig ). At the same time, cells of the gametophyte have no ingrowths, excepting small zone of immediate contact with the haustorial cells, where the surrounded vaginular cells have fluorescence similar to that of placental space. No inward projections were observed in the gametophyte cells ( Fig. 19C View Fig ). At the same time, the vaginular cells immediately adjoining the placental space near the foot tip have a number of differences: they are strongly elongate ( Fig. 16 View Fig , series) and differentiated in color ( Fig. 19A View Fig ).

Placenta in the developed capsule has a narrower placental space, cells from the sporophyte are dark, with poorly discernible inner structure, and labyrinths are seen only in some cells. Gametophytic cells closer to placental space ( Figs. 18E–F View Fig ) have softer cell walls, although organelles and nuclei look normally developed, at least in some of them. Further cells with numerous osmiophilic globules and with non-modified cell-walls follow.

Placental space material is obviously of a gametophytic origin. The especially strong decomposition of gametophyte cells is seen around the haustorial cell ( Figs. 19D, F View Fig ) and near the sporophyte apex, which is the same place as the base of the archegonium neck ( Fig. 21E View Fig ).

The contents of the surface foot cells includes in addition to labyrinths, endoplasmatic reticulum, and numerous small chloroplasts, also a whorl-shaped structure, similar to ER-phagous ER-whorls in yeast ( Schuck et al., 2014). This is likely not just a superficial similarity, as the foot cells have to utilize their membranes, along with the cell wall ingrowth progress. Inner cells of the epigonium wall also contribute to the placental space with macerated cells: innermost of them appear transitional in their structure to ‘cells’ of the placental space ( Fig. 21D View Fig ).

Two extensive overviews of the moss placenta structure did not illustrate Andreaeobryum , although they mentioned that it has one layer of transfer cells with ingrowths in the sporophyte, and no modification of the cell wall in gametophyte ( Frey et al., 2001; Ligrone & Gambarella, 1988). In general, the placenta at the stage of mature capsule has such a structure, although the mucilage content of the placental space allows only poor impregnation of this zone, resulting in crumbling of the cuttings.

Placental structure is similar to Andreaea , Takakia and Polytrichaceae by the absence of cell wall ingrowths in gametophytic cells.

The extensive mucilage-filled placental space seems to be unique in Andreaeobryum . As this character relates to multistratose calyptra, it can be an additional evidence for the relationship of Andreaeobryum with Takakia .

Seta

Murray (1988) noted the flattened seta in Andreaeobryum . Her explanation referred to the thin-walled cells filling almost the whole seta and easily collapsing upon drying.

Although all this is true, we found that the seta of Andreaeobryum is dorsiventral in transverse sections (series in Fig. 15 View Fig ) since the whole sporophyte is only 200 µm long, and far from any drying and living in comfortable liquid volume inside the mother plant body. The cause for such asymmetry can be in unequal halves of the endothecium since its original differentiation (discussed below under ‘Columella’).

One almost mature sporophyte, including foot, seta and part of capsule, was transversally sectioned ( Figs. 23–24 View Fig View Fig ), confirming the dorsiventral structure of seta. In the seta middle, the medullar thin walled central part is broader than high in 20–30%, and cell orientation and outlines are also keeping orientation of the overall dorsiventrality (e.g., in 23-1020).

Seta of Andreaeobryum is described as having no central strand. The present observation confirms this statement, if consider central strand as a narrow structure formed by cells with narrow lumen. Such structure is definitely absent in Andreaeobryum , but there is another one. It starts at about 0.35–0.4 mm from the foot tip, e.g., at the level where the foot is transforming into seta (surface cells losing their ingrowth structure and outer foot cells loose their regular, radially orientated arrangement, cf. Fig. 23 View Fig -258). At first, the conducting tissue of seta looks like a central group of cells with open lumens and somewhat thicker cell walls ( Fig. 23 View Fig -462). They differ in contrast from the surrounding cells, being full of highly refractive material, perhaps starch.

Shortly after its differentiation, this ‘central strand’ has already obtained a diameter about a half that of the seta ( Fig. 23 View Fig -540) and soon reaches the diameter of the whole seta excepting two or three outer cell layers. Cells of the latter are still rich in starch, while thin- but firm-walled cells in the middle look as numerous guide cells, confirming conductive capacity of the ‘central strand’, which can be probably assumed as such despite a rather unusual structure.

Immediately below the capsule base, the seta layers receive additional stratification: the innermost cells become still larger, forming a lighter central part, and still further, at the level of columella base, cell structure is even more complicated: some cells become narrower, being flattened in tangental direction, while central cells are larger and conspicuously firm-walled ( Fig. 23 View Fig -1430).

In dry state, the seta is strongly flattened and usually also conspicuously twisted.

The development of conducting tissues in young sporophyte proceeds at a rather early stage. Two series of sections in Figs. 16 View Fig and 20 View Fig indicate much longer cells at the level of seta, although the latter one represents only a slightly older stage (sporophyte is 135 µm in diameter vs. 100 µm in Fig. 16 View Fig ). Attempts to obtain a longitudinal section of seta at the stage about the same as in Figs. 23 View Fig and 24 View Fig were not successful, as cells of the central part of the seta were crumpling in a way similar to that of the foot-vaginula junction in Fig. 23 View Fig -462.

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Archesporium and Columella

Endothecial cells in the central part of the young capsule are surrounded by 3–4-layered amphithecial cells ( Figs. 25 View Fig and 26 View Fig ). The former reach the top of the future spore chamber, being at the same time arranged in a series of alternating sectors, similar to that in Andreaea ( Figs. 26B–C View Fig ).

Archesporial cells were found in the series of longisections at the stage of well-developed capsule, while at the earlier stages they were rather indistinct (cf. series in Figs. 20 View Fig and 26 View Fig ). However, similarity of the pattern with clearly expressed sectored structure of endothecium (cf. Figs. 27 View Fig and 26C View Fig ) indicates a great overall similarity between Andreaeobryum and Andreaea .

Archesporial cells are at the beginning of their differentiation in Figs. 20 View Fig and 26 View Fig . They are only slightly differentiated in shape, thus their status is deduced from their position in the outermost layer of endothecium.

A rather regular structure of cell arrangement in amphithecium is notable ( Fig. 26 View Fig ), corresponding to the peristomal formulae 4:2:2. characteristic for mosses of both nematodontous ( Polytrichum , Tetraphis ) and double-opposite arthrodontous ( Encalyptaceae ) groups.

Some sections demonstrate a somewhat later differentiation in the distal endothecial segments ( Fig. 20 View Fig ) as compared with the proximal ones, and this pattern well corresponds with sporogenesis in Andreaea ( Fig. 26B View Fig ), where the archesporial differentiation in the upper part of the capsule is also late.

Developed archesporial cells are situated in the peripheral parts of endothecial sectors, as the central part is building the columella.

The columella in Andreaeobryum has been described as stellate in cross section ( Murray, 1988). Our observations are fully congruent with this, showing details of columella structure ( Figs. 24 View Fig , 27 View Fig , 28 View Fig , 30 View Fig ), and especially of projections, joining at places with the outer spore sac, reminiscent of Takakia ( Renzaglia et al., 1997) .

Although the columella connections to the outer spore sac are fairly irregular, it seems possible to recognize in general the four-pointed star ( Fig. 24 View Fig ), which corresponds to some sections of the capsule.

The most surprising observation on the sporophyte development is that the columella on its top is connected to the outer spore sac. The latter case is observed in some views, but the complete series of sections ( Figs. 27–28 View Fig View Fig ) demonstrates that the Andreaeobryum case is not at all like that of Sphagnum . Hence the description of Andreaeobryum columella as overarched by sporogeneous tissue has to be challenged. Having connection to the outer spore sac, columella in Andreaeobryum has some similarity with Bryalean mosses, albeit in the latter columella is much broader above.

At the same time, a great similarity with Andreaea is also seen, in which archesporium is also sectored (cf. Figs. 26 View Fig and 29 View Fig ). Thin membranaceous connectives are poorly developed in Andreaea ( Fig. 29C View Fig ), hence Andreaea was also considered as a moss with columella not reaching the top of the spore chamber. However, the uppermost sectors of the the endothecium in Andreaea can be late in development, thus the ‘young columella + undifferentiated archesporial tissue’ may join the top of spore chamber ( Fig. 29C View Fig ). Similar cases are shown in Fig. 29E, G, I View Fig : above the archesporial cells, there is a continuation formed by cells, which cannot be anything else but the ‘young columella + undifferentiated archesporial tissue’.

Spores

Sporophytes with spore mother cells and early stages of sporogenesis were not found in available material. Spores in a still closed capsule are shown in Figs. 24 View Fig and 30 View Fig . They are of bright orange color when observed under stereomicroscope, however in transmitted light of the compound microscope their green chloroplasts are well seen. A number of abortive spores were seen in most of the studied capsules ( Fig. 24 View Fig , 30 View Fig ), although in some capsules they were almost absent. Abortive spores are darker in color in osmium-staining sections ( Fig. 24 View Fig ), and have more intense color under upper light of halogen lamp ( Fig. 30 View Fig ). Ultrastructurally they are variable, but with invariably present perine layer ( Fig. 37 View Fig ). The flattened and angulose shape of abortive spores is likely a result of pressing within the spore mother cell wall during the maturation.

Spores have an uneven granulose surface, making the spore look rough, like a diseased skin, under the light microscope ( Figs. 30–31 View Fig View Fig , 33–34 View Fig View Fig , 37–38 View Fig View Fig ). SEM observation of spores from herbarium material without additional preparation shows them often strongly collapsed. The granulose surface ruptures at places, and perfectly smooth layer underlaying the granulose layer becomes exposed ( Fig. 32 View Fig ).

The spore wall structure of Siberian plants is shown in SEM ( Fig. 32 View Fig ), TEM ( Figs. 33–37 View Fig View Fig View Fig View Fig View Fig ) and LSCM photographs ( Fig. 38 View Fig ), and LM of acetolyzed spores ( Fig. 38 View Fig : D–G).

The main part of spore wall is electron-translucent and not ( Fig. 34A View Fig ) or weakly ( Fig. 35D View Fig ) stratified to two or three layers slightly different in color in TEM images. Sometimes the innermost layer at the contact with osmiophilic drops is lighter ( Fig. 34A View Fig ), but this is far from often. LSCM fluorescence images illustrate a rather complex structure and gradual transitions from innermost layer (blue fluorescence) to outer part of cell wall that has yellow fluorescence of cellulose after berberine staining ( Fig. 38A–D View Fig ). Outside the homogeneous part of spore wall two granulose layers occur. The inner one comprises the electron-dark osmiophilic small granules, (0.01–) 0.1–0.5 µm, embedded in electron-transparent medium. The outer layer is usually not continuous and is formed by only osmiophilic globules of larger size, mostly 0.5– 2.0 µm ( Figs. 34–35 View Fig View Fig ). Spore wall of acetolized spores breaks into irregular fragments, indicating only a moderately continuous sporopollenin layer ( Figs. 38E–H View Fig ).

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In general the spore wall structure in Andreaeobryum is similar to that in Andreaea . Two studies of the development of spore wall were done in A. rothii ( Brown & Lemmon, 1984) and in A. rupestris (Filina & Filin, 1984) . Their results are principally similar, despite their authors followed different terminology. This difference mainly concerned the origin of material forming the outer granulose layer, which is usually called perine ( Brown & Lemmon, 1984, 1988; Brown et al., 2015), while Filina & Filin (1984) considered it to be of the same origin as exine.

In any case, for Andreaea both studies revealed the absence of a lamellate layer, common to other moss groups and important as a starting point for the exine formation ( Brown et al., 2015; Schuette et al., 2009). Contrary to this, the exine development in Andreaea starts upon the plasmalemmas by accumulation of osmiophilic globules at various scattered points. These globules grow and join each other, forming a spongy mass. The intine develops later, forming the layer inwards of the exine and also filling the ‘caves’ and ‘holes’ among exine material. In the cross sections the exine layer is seen as a mixture of darker bodies of irregular shape (exine) interlayered with the lighter ones (intine) (cf. Brown et al., 2015, plate 7, fig. 1). Intine and perine in Andreaea have a structure similar to that in other mosses, including Andreaeobryum .

Brown & Lemmon (1988) provided one image of mature spore of Andreaeobryum , where outside a thick intine a scanty exine was recognized, although its segregation was not very clear. Our observations on mature spores do not make possible to delimit exine more clearly. Although the stratification is seen in some images ( Figs. 35D–F View Fig , 38A–D View Fig ), it is absent in others, and fluorescent images provide a rather variegated picture, indicating complexity of the spore wall. However, being strongly decomposed in acetolysis, the presence of continuous exine in Andreaeobryum is not confirmed, thus the main content of the homogeneous spore wall in Andreaeobryum should be referred to intine. Some stratification occurring in few TEM and most of LSCM images thus is interpreted as a two layers of intine, I1 and I2 correspondingly. In most sections, however, intine is marked by just as I, representing undifferentiated electron-translucent major part of the cell wall. Interestingly, occasional occurrence of osmiophilic globules within the intine layer was seen ( Figs. 34F View Fig , 35F View Fig , 36C View Fig ), and also osmiophilic globules along the inner surface of intine may make its surface wavy ( Fig. 34B View Fig ) or penetrate within intine ( Fig. 34C, E View Fig ).

Upon intine as it defined here, two granulose layers occur ( Figs. 33–38 View Fig View Fig View Fig View Fig View Fig View Fig ), both being quite similar to published picture from American specimen by Brown & Lemmon (1988, Fig. 37 View Fig ). The outer granulose layer, i.e., formed by globules large in size and looser in arrangement, is similar to perine in other moss groups, while the inner granulose layer requires more attention to understand its homology.

Within the inner granulose layer, the osmiophilic globules occur at least in some places not immediately on the intine surface, but at a certain distance. For example, Fig. 34B View Fig illustrates osmiophilic globules scattered in the electron-transparent medium, but at places this electron-light layer lacks dark globules. In other images, e.g. Fig. 35B View Fig , the mass of osmiophilic globules is confluent with the outer parts of spore wall.

It is possible to admit that this translucent thin layer corresponds to exine, while osmiophilic globules develop within it. Fig. 37 View Fig in publication of Brown & Lemmon (1988) showed that in tetrad stage, the space between proximal parts of still not separated spores includes numerous dark globules. Their origin is not yet known, but most likely they start to develop within the spore mother cell wall. Filina and Filin (1984) discussing a similar globules in Andreaea (l.c., Fig. 3a View Fig ) at the stage when tetrad is still within the spore mother cell concluded that they have to be referred to exine rather that perine, since they definitely did not originate from tapetum.

The final conclusion of the homology of layers of the spore wall of Andreaeobryum will be possible only after all the stages of its development have been studied. And likely even after that, the problem with terminology will not disappear, as the places of beginning of globule formation are scattered unlike other mosses. And even if this can be called exine or exine with embedded perine particules (by analogy of exine globules embedded in intine in Andreaea ), it would be a very unusual sort of exine. Considered to be one of the most stable structures among living organisms, exine should not be so easily decomposed. At the same time, these is a correlation between the two granulose layers and spore size: the outer granulose layer (P2) looks denser in smaller spores, becoming more spaced in larger spores, where the inner layer (P1) is continuous, but further, with the increase of diameter of multicellular spores, both P1 and P2 appear to be split into polygonal groups and outer intine is exposed, as is seen both in SEM ( Fig. 32J View Fig ) and TEM images ( Fig. 36B View Fig ).

Therefore for present we retain the teminology of Brown & Lemmon (1984, 1988), denoting two granulose layers as inner perine (P1), a mixture of electron-dark osmiophilic and electron-light material, and outer perine (P2), is formed by only osmiophilic globules ( Fig. 34 View Fig ).

Spores of Andreaeobryum are large and have the endosporic germination. In some of them up to eleven cells were seen ( Fig. 36 View Fig ).

The most unusual fact about spores has been found in an eleven-celled spore ( Fig. 36 View Fig ). In its middle, a structure similar in shape to an axillary hair is seen. Its outlines do not perfectly fit, but there is also two cases of indirect evidence supporting its interpretation as an axillary hair. First, Murray’s (1988) drawing of the spore germination illustrates simultaneous liberation from the sporoderm of the flat protonema and an axillary hair side by side. Second, the axillary hair in Fig. 36 View Fig has a continuation to a channel and then to a pool. The latter does not seem to belong to any cell of the spore, as it lacks any cellular organelles. A putative alteration of the intine occurs at the edge of the pool, marking a place where it is most likely protonema will come out of the spore.

Multicellular spores without trilete mark differentiate Andreaeobryum from Takakia , Sphagnum , and Oedipodium ( Brown et al., 2015; Polevova, 2015), but are similar to Andreaea . The similarity with the latter genus includes nonpersistent and more or less two-layered perisporium. The perisporium is usually not persistent also in Sphagnum where it may fall off as a whole envelope, releasing the naked spore body with exine as a surface. The perisporium of Oedipodium is poorly developed and falls off the spore surface mostly inside the capsule.