Morphological , Anatomical and Cytological Studies of some Moss Species from Nigeria

Morphological, anatomical and chromosome studies of Hyophila crenulata C, Mull. Ex Dus, Thuidium gratum (P. Beauv) Jaeg., Barbula lambarenensis P. Vard., Stereophyllum nitense Mitt. and Bryum coronatum Schwaegr from Nigeria, were carried out with a view to bridging some knowledge gaps that exist in their characterization and providing insightful information that could be useful in elucidating their taxonomic status. The morphological and anatomical studies revealed several gametophytic and sporophytic attributes which have not been previously reported and which were diagnostic for the moss species studied. The chromosome studies revealed the chromosome numbers to be Hyophila crenulata n = 4; Thuidium gratum n = 12(10+2 m); Barbula lambarenensis n=3; Stereophyllum nitense n = 9; and Bryum coronatum n = 10. From the results of the study, it could be concluded that the details of the morphological and anatomical descriptions as well as the chromosome numbers being reported for the first time in this study for the moss species studied could be very useful in their identification and taxonomic delimitation.


Introduction
Mosses are non-vascular plants that occur over a wide range of habitats such as rocks, soil, logs, tree trunks and concrete walls.They constitute a group in the division bryophyta, the other two groups in the division being Marchantiophyta (liverworts) and Anthocerotophyta also known as hornworts (Buck and Goffinet, 2000;Adebiyi et al., 2012;Oyesiku, 2012).
Much is still left to be done in the characterization of these speceies as information about their morphological and anatomical attributes is quite sparse and limited to a few information that emanated from the works of Egunyomi (1979Egunyomi ( , 1980Egunyomi ( , 1981Egunyomi ( , 1984)), Egunyomi and Vital (1984), Makinde and Odu (1993) and Fatoba and Odu, (1999) leaving knowledge gaps that has sometimes led to misidentification of some of these species by researchers.Many aspects of the morphological and anatomical attributes of species selected for this study are yet to be investigated and reported.These include aspects such as the morphological and anatomical characterization of the leaf cells, capsules, peristomes, operculum and spores.Also, there is no known record of the chromosome number of any of these moss species from Nigeria.
The objectives of this study are to characterize samples of some Nigerian moss species using morphological and anatomical parameters many of which have not been documented; and also established the chromosome numbers of these moss species for the first time.
documented with photomicrographs using an AmScope MT microscope camera version 3.0.0.1 attached to a light microscope.

Chromosome studies
Mitotic chromosome preparations were made by pretreating young gametophytes of the moss species studied with 0.004M colchicine for 2 hours and then fixing in 1:1:1 ethanol:glacial acetic acid:chloroform for 48 hours.After this, the shoot apices were excised and squashed on glass slides and then stained with FLP Orcein (2 gm of orcein in 100 cm 3 of solution containing equal parts of Formic acid, Lactic acid, Propionic acid and distilled water (Olorode, 1974) for 3 hours.Meiotic chromosome preparations were made by fixing freshly-harvested capsules at various stages of development in 1:3 acetic-ethanol for 48 hours.The capsules were then squashed on glass slides and stained with FLP orcein for 3 hours.Mitotic and meiotic cells with good spread representative of the species were documented by taking photomicrographs under phase contrast.

Results and Discussions
The mosses species that were investigated in this study are described below with respect to their general habit, habitat, qualitative and quantitative attributes of the gametophytes, sporophytes, leaf cells as well as chromosome as observed during the study.

Plant source and identification
The moss species investigated were Hyophila crenulata C, Mull.Ex Dus, Thuidium gratum (P.Beauv) Jaeg., Barbula lambarenensis P. Vard. and Stereophyllum nitense Mitt.Samples were collected from various locations in Ile-Ife, Nigeria (Table 1), during the raining seasons between April 2014 and November 2015) while the plants were in full bloom and in their optimal conditions.Samples collected were identified at the herbarium of the University of Ibadan, Nigeria.

Morphological studies
The gametophytic as well as sporophytic attributes of the different species of mosses collected were characterized by morphological description according to Smith (1978) and measurements were made with the aid of the light microscope, dissecting microscope and ocular micrometer.
Characters investigated include: plant habitat, plant colour when moist and when dry, perichaetial leaf shape, perichaetial leaf apex shape, perichaetial leaf margin shape, shape of lamina cells at the apex, middle and basal portion of the perichaetial leaves, shape of marginal cells of perichaetial leaves, seta colour, mature capsule shape, ornamentation of capsule, carriage of mature capsule on seta, shape of exothecial cells of the capsules, operculum type, type and shape of spores, spore ornamentation, peristome form, presence or absence of polysety and presence or absence of gemmae.
The morphological and anatomical parameters of the mature shoots representative of each of the species studied were described and documented with photomicrographs using an AmScope MT microscope camera version 3.0.0.1 attached to a light microscope.

Anatomical studies
Mature gametophytes from each moss taxon studied were washed with distilled water and cleared of dirt and other impurities.The chlorophyll content of the shoots was removed by soaking them in MacCartney bottles containing 10 ml of Dimethyl-Sulphuroxide (DMSO) for 24 hours at 67 ºC.The leaf cells and other anatomical parameters of the mature shoots were described and 405

Chromosomes of the moss species studied
The Hyophila crenulata studied showed a chromosome number of n = 4.The metaphase I cells (Fig. 4A) showed the occurrence of four bivalent chromosomes (4II) with callous walls surrounding the cells.The Thuidium gratum studied showed a chromosome number of n = 12 (10 + 2m).The metaphase I (Fig. 4B) revealed the presence of two chromosomes which were usually smaller and lightly stained (arrowed), but always seen as part of the chromosome complement.These were regarded as m-chromosomes.Barbula lambarenensis studied showed a chromosome number of 2n = 6 (Fig. 4C).Two of the chromosome complements were conspicuously large.The Stereophyllum nitense studied showed a chromosome number of 2n = 18 chromosomes (Fig. 3D).The Bryum coranatum studied showed a chromosome number of n = 10 (Fig. 4E).The chromosomes of the five moss species studied were usually sticky and tend to always clump together at metaphase I.This was more notable in Stereophyllum nitense, Thuidium gratum and Bryum coranatum.

Discussion
Although Egunyomi (1980) had described some aspects of the morphology of some of the moss species studied, additional details which have not been previously reported are being reported in this study.These include aspect of the morphological and anatomical attributes of the leaf cells, capsule, peristome, operculum and spore ornamentation.Also the chromosome numbers of these species are being reported for the first time.
The anatomical description of the perichaetial leaves of the species studied (Fig. 3 and Table 3) showed that they are clearly distinct species as the lamina cells varied across the species in all the three regions studied (i.e upper, middle and basal regions of the perichaetial leaves) with respect to their lamina cell shapes, sizes and arrangement within the leaves.Lamina cell length increased from the apex to the basal region in Hyophila crenulata and Barbula lambarenensis, while they were longest at the middle region in Thuidium gratum, Stereophyllum nitens and Bryum coronatum.Lamina At the apices and upper regions of the leaves, the lamina cells were generally irregular in all the species studied, but their sizes and arrangement within the leaves varied (Fig. 3 and Table 3).While the lamina cells were much longer than broad in Thuidium gratum (37.79 ± 2.44 µm long, 7.57 ± 0.16 µm broad), Stereophyllum nitens (43.29 ± 2.52 µm long, 6.57 ± 0.27 broad) and Bryum coronatum (56.86 ± 2.56 µm long, 12.50 ± 1.04 µm broad), they were shorter in Hyophila crenulata (8.29 ± 0.25 µm long, 7.36 ± 0.10 µm broad) and Barbula lambarenensis (8.21 ± 0.36µm long, 5.79 ± 0.20 µm broad).
At the basal region of the leaves the lamina cells were all irregular though the shapes, lamina cell sizes and arrangement differed from one species to the other.The lamina cell length was much longer than broad at the basal region in all the species studied.
The exothecial cells of the capsules of the species studied also varied in shape, sizes and arrangement (Fig. 3 and Table 3) and are diagnostic for the species studied.Although the shapes were irregular in all the species studied, the shapes, sizes and arrangement varied across the species.
The anatomical characters of the leaves and capsules of the moss species studied were diagnostic hence, can be taxonomically employed to delimit the species from each other.Anatomical parameters of different plant parts have been used as aids in the taxonomic recognition of species (Kathiresan et al., 2011).Schofield (1985) noted that cell shapes and arrangement within leaves of moss species usually differ remarkably and they provide some of the most reliable characters that could be used to distinguish them.
The sporophytic features of the moss species studied also varied distinctly across the species studied.Though they were all stegocarpous, the capsule shapes, capsule carriage, operculum type, peristome type and spore varied distinctly across the species studied.While the capsules were cylindrical and smooth in Thuidium gratum and Barbula lambarenensis, they were cylindrical and striate in Hyophila crenulata, cylindrical and furrowed in Bryum coronatum, ovoid and furrowed in Stereophyllum nitense.The capsule carriage was erect in Hyophila crenulata and Barbula lambarenensis while they were reflexed in Thuidium gratum and Stereophyllum nitense and pendulous in Bryum coronatum.The operculum was snouted in all the species studied except in Bryum coronatum where they were blunt.The peristomes were spirally twisted in Hyophila crenulata, deeply forked and paired in Thuidium gratum, deeply forked and unpaired in Barbula lambarenensis, Stereophyllum nitense and Bryum coronatum though their shapes varied.
Capsule carriage and texture, peristome form, spore ornamentation of mature sporophytes are reliable characters that can be useful in delimiting the species studied since they are usually genetically determined.
The occurrence of gemmae noticed on only Barbula lambarenensis (Table 2) out of the five species studied, is an indication of an asexual strategy of propagation and spread; while the occurrence of polysety in Hyophila crenulata is an indication of strategy for population spread by sexual means.According to Schofield (1985), gemmae serve as diaspores that engage in vegetative reproduction and are important in the expansion of local populations.Oyesiku (2012) speculated that the mechanism behind polysety is not fully known, but may be because of simultaneous fertilization of two or more individuals of archegonia, aided by sugary exudates from the mature archegonia.
The occurrence of sporophytes that were stegocarpous and operculate with spores that were relatively small in size and numerous, in all the species studied, suggest the likelihood of long distance dispersal of their spores and thus possibility of their occurrence over a wide range of geographical location provided the habitat conditions are conducive with respect to moisture, pH and other environmental conditions.
The chromosome numbers of Hyophila crenulata, Thuidium gratum, Barbula lambarenensis, Stereophyllum nitense and Bryum coronatum from Nigeria are revealed for the first time in this study.Hyophila crenulata has a chromosome count of n = 4; Thuidium gratum has a chromosome count of n = 12 (10 + 2 m); Barbula lambarenensis has a chromosome count of n = 3 (i.e.2n = 6); while Stereophyllum nitense has a chromosome count of n = 9 (2n = 18).There is no known previous record of chromosome numbers of Hyophila crenulata, Thuidium gratum, Barbula lambarenensis and Stereophyllum nitense being reported in this study.However, there are known records of Bryum coronatum from other parts of the world.Kumar et al. (1988) reported the chromosome number of Bryum coronatum as n =10.
The occurrence of chromosomes which were usually sticky and tend to always clump together at Metaphase I is also noteworthy and being reported in this study.Its notable occurrence in Stereophyllum nitense, Thuidium gratum and Bryum coronatum than in the other species studied, could be because of the relatively larger chromosome numbers in their complements (i.e.n = 9, n = 12 and n = 10 respectively).Stickiness of chromosomes was also reported in moss species in the family Brachytheciaceae by McAdam (1982).
In this study, the presence of m-chromosomes was found only in Thuidium gratum.Reports on the presence of m-chromosomes and accessory chromosomes in bryophytes have shown that in both cases, the chromosomes are smallest in size compared to the other members of the complement.However while m-chromosomes were always present in the complement, accessory chromosomes may or may not be present (Ramsay, 1964(Ramsay, , 1969;;Snider, 1973;Muntung, 1974).Also, light-staining and precocious segregation are diagnostic of m-chromosomes while accessory chromosomes usually present mitotic/meiotic irregularities resulting in numerical variation in chromosomes of species or populations (Ramsay, 1964(Ramsay, , 1969;;Snider, 1973;Muntung, 1974).

Conclusions
Some details of the morphological and anatomical attributes as well as chromosome numbers of Hyophila crenulata, Thuidium gratum, Barbula lambarenensis, Stereophyllum nitense and Bryum coronatum from Nigeria are being reported for the first time in this study and could be very useful in the identification and taxonomic delimitation of these species.However, more detailed morphometric and molecular studies are required to establish their taxonomic status and evolutionary relationship.

Table 1 .
Sources of moss species studied

Table 2 .
Comparative morphology of the moss species studied

Table 3 .
Comparative anatomy of the leaf and exothecial cells of capsules of the moss species studied