1. Introduction
Living embryophytes mostly produce reproductive structures known as spore and pollen in the form of tetrads. The four haploid spores of the tetrad are formed as a result of meiotic division of a diploid spore mother cell. Each generally becomes dispersed in the environment as an individual spore following a dissociation process which is widespread in most of the non-angiospermous plant world. However, very rarely the tetrads do not dissociate to form a single spore (as seen in some of the modern hepatics) and are dispersed as permanent tetrads (Edwards, Wellman & Axe, Reference Edwards, Wellman and Axe1999).
The earliest evidence for the first land plants or the embryophytes comes from the microscopic dispersed spores generally known as cryptospores. They lack haptotypic features such as a trilete mark or a monolete mark (Steemans, Reference Steemans2000); however, they possess sporopollenin in their exine. Cryptospores are preserved in the form of monads, permanent dyads or as permanent tetrads. Permanent tetrads and dyads happen to be an important part of Ordovician – Early Devonian dispersed spore assemblages and were habitually dispersed intact in the environment with all four individual spores attached firmly (Edwards, Wellman & Axe, Reference Edwards, Wellman and Axe1999). The tetrads are either naked or enclosed within a thin cover. Naked tetrads are mostly laevigate; however, some ornamented forms also existed.
The existence of spore tetrads can be traced back to the Cambrian period based on a recent discovery of Cambrian palynomorphs from the Bright Angel Shale, Arizona, USA by Taylor & Strother (Reference Taylor and Strother2008). However, these palynomorphs (preserved as dyads and tetrads) are considered as protoembryophytes by these authors, rather than embryophytes, as their overall morphology is different from the definite embryophytes. The tetrad taxon Tetrahedraletes cf. Medinensis, identified in Dapingian deposits (early Middle Ordovician, c. 473–471 Ma) from the Zanjon and Labrado formations in NW Argentina, is the oldest tetrad record; it existed for c. 70 Ma on this land during Llanvirn (earliest Ordovician) – Lochkovian (earliest Devonian) times (Rubinstein et al. Reference Rubinstein, Gerrienne, Dela Puente, Astini and Steemans2010). The tetrads were abundant and diverse throughout the Ordovician and Silurian periods.
Until late Middle Ordovician time (463–461 Ma), the signatures of the earliest land plants are only recorded in the form of cryptospores and not as a complete plant. The earliest plant fragments (sporangia) that also possessed in situ preserved naked permanent tetrads and naked dyads were documented for the first time in Upper Ordovician (Caradoc) rocks of Oman. These spores, produced in large numbers inside very small sporangia, confirm their affinity with the land plants. Transmission electron microscopy (TEM) analysis of the spore walls of these tetrads further proves their affinities with the liverworts (Wellman, Osterloff & Mohiuddin, Reference Wellman, Osterloff and Mohiuddin2003). Oman fossils therefore represent the oldest sporangia and the oldest in situ tetrads and dyads on the Earth.
In comparison to a large number of reports on the dispersed spore tetrads, the data on in situ preserved tetrads is meagre. Edwards, Duckett & Richardson (Reference Edwards, Duckett and Richardson1995), Edwards et al. (Reference Edwards, Davies, Richardson, Wellman and Axe1996), Edwards, Wellman & Axe (Reference Edwards, Wellman and Axe1999), Wellman, Osterloff & Mohiuddin (Reference Wellman, Osterloff and Mohiuddin2003) and Edwards et al. (Reference Edwards, Morris, Richardson, Axe and Davis2012) recorded various types of in situ spore tetrads (Velatitetras, Synorisporites, Tetrahedraletes, Acontotetras and Cheilotetras from different kinds of mesofossils (sporangia, bifurcating axes or irregular spore mass) collected from a number of early Palaeozoic (Ordovician–Devonian) localities in the Welsh Borderland and in Oman.
In contrast to the abundance of lower Palaeozoic spore tetrads regarded as basal embryophytes the tetrads at the Permian–Triassic transition and from the Upper Triassic rocks are comparatively less represented and belong to the lycopods, whereas spore tetrads are scarce in Permian sequences (the only taxon is Jayantisporites from the Indian Permian stratum). The lower Palaeozoic spore tetrads have been assigned to the genera Synorisporites, Tetrahedraletes, Velatitetras, Acontotetras, Rimosotetras, Stegambiquadrella and Cheilotetras, whereas the tetrads of the Triassic period belong to the form genera Densoisporites, Lundbladispora, Uvaesporites, Verrucosisporites, Lapposisporites, Decisporis, Kraeuselisporites and Otynisporites (megaspore tetrad).
Here we report and describe the biggest ever collection of unseparated naked spore tetrads from the lower Permian (Artinskian) Lower Barakar Formation of Bina Colliery, Singrauli Coalfield, Central India (Fig. 1), assignable to the dispersed microspore genera Indotriradites, Microbaculispora and Microfoveolatispora. Two types of tetrads, ornamented and zonate, are recognized in this study along with other pollen and spores. There are a few records of spore tetrads in the Indian Triassic sequences (Banerji & Maheshwari, Reference Banerji and Maheshwari1973; Maheshwari & Banerji, Reference Maheshwari and Banerjee1975; Tiwari & Meena, Reference Tiwari and Meena1989; Ram-Awatar, Reference Ram-Awatar2011); however, they have never been reported from any Permian strata in India apart from a solitary record of Jayantisporites recorded from the lowest Permian (Asselian–Sakmarian) Talchir Formation (Lele & Makada, Reference Lele and Makada1972). There is no evidence of any kind of sporangia or related plant parts that could help to determine the affinity of these tetrads. However, the presence of a trilete mark in some of our tetrads demonstrates their alliance with the pteridophytes. The study also includes a comparison of the present tetrad assemblage with the similar assemblages reported globally of Palaeozoic and Triassic age. The complete palynoassemblage of Bina Colliery is depicted in the frequency chart of Figure 2.
Location Map of the Singrauli Coalfield showing Bina Colliery (after Raja Rao, Reference Raja Rao1983).
Frequency chart showing all the palynomorphs and spore tetrads recovered from Bina Colliery.
We analysed the global records of fossil tetrads and the palaeoclimates prevailing during their deposition, and observed that they mostly occur in sediments which are deposited under extreme climatic conditions (extreme cold or extreme hot). This is supported by the fact that most of the occurrences of spore tetrads reported globally are found in Ordovician – Lower Devonian sediments; in the lower Permian Talchir (Asselian–Sakmarian) and the Lower Barakar (Artinskian) formations; at the Permian–Triassic boundary; and in the Lower Triassic Panchet (Induan–Olenekian) Formation. These occurrences have been correlated with extreme cold due to Late Ordovician (Finnegan et al. Reference Finnegan, Bergmann, Eiler, Jones, Fike, Eiseman, Hughes, Tripathi and Woodward2011) and Carboniferous–Permian glaciations, and extreme hot due to Early Triassic Siberian volcanic activity respectively.
Extreme cold conditions during the deposition of Talchir Formation (Asselian–Sakmarian) are inferred by the abundance of monosaccate pollen taxa (Parasaccites and Plicatipollenites) along with the spore taxa Callumispora and Jayantisporites in the Talchir palynoassemblages (Tiwari & Tripathi, Reference Tiwari and Tripathi1987). Similarly, a very cold to cool phase must have prevailed at the transition zone of the Karharbari and Lower Barakar formations in Bina Colliery, as is evidenced by the presence of pollen taxa Scheuringipollenites, Parasaccites, Faunipollenites and Striatopodocarpites along with the spore genus Callumispora in the present assemblage (Tiwari & Tripathi, Reference Tiwari and Tripathi1987; Vijaya et al. Reference Vijaya, Tripathi, Roy and Mitra2012).
The recent physiological and biochemical studies carried out on the extant plants Allium sativum and Petunia hybrida (Izhar & Frankel, Reference Izhar and Frankel1971; Winiarczy, Jaroszuk-Ściseł & Kupisz, Reference Winiarczy, Jaroszuk-Ściseł and Kupisz2012), correlating adequate specific pH values inside microsporangium, the release of callase enzyme, disintegration of the callose walls and the ultimate release of the single spore from the tetrads, are applied on the presently studied material from Bina Colliery. It is deduced that unfavourable conditions such as hot and cold extremes of climate might have induced changes in the plant physiological processes (i.e. non-functional tapetum/altered pH values in the microsporangium or the inactive callase) that prevented the dissociation of tetrads into individual spores. Such results imply that extreme climatic conditions might have affected the formation of spore tetrads in the past.
2. Geological setting
The Singrauli Coalfield (Fig. 1) lies at the northernmost boundary of the Son-Mahanadi Master Basin that stretches from the east coast to the centre of Peninsular India. This coalfield embodies the last deposits of Gondwana sedimentation. No Gondwana deposits occur beyond this coalfield area in the northern part of Peninsular India. The coalfield lies between latitudes 23°47′N and 24°12′N and longitudes 81°48′E and 82°52′E and is located in the drainage area of the Son and Rihand rivers. The total geographical area of this coalfield is c. 2200 km2, c. 80 km2 in the Sonbhadra District of Uttar Pradesh State and the remainder in the Singrauli District of Madhya Pradesh State.
The coalfield is structurally divided into two tectono-sedimentary sub-basins: (1) Moher sub-basin on the north-eastern side; and (2) the Singrauli main sub-basin to the west. There is no clear-cut demarcation between these two sub-basins because all the lower Gondwana formations are exposed uninterruptedly in these sub-basins. The only difference lies in the amount of coal reserves found in each. The coal reserves in the Moher sub-basin, covering an area of c. 220 km2, is c. 9×109 tonnes, of which 2.724×109 tonnes are proven reserves (Raja Rao, Reference Raja Rao1983). Singrauli main sub-basin covers an area of c. 1900 km2. All ten working opencast mines of Singrauli Coalfield (namely Dudhichua, Jayant, Kakri, Bina, Krishnashilla, Amlohri, Khadia, Block B, Nigahi and Jhingurdah) lie within the Moher sub-basin. The first nine collieries are in Barakar Formation and have three coal seams: the lowermost Turra seam; the middle Purewa bottom; and the uppermost Purewa top. Below Turra, a thin seam referred to as Kota also exists within the Karharbari Formation (Sakmarian–Artinskian). Jhingurdah Colliery is in the Raniganj Formation (Lopingian) and has the thickest coal seam (134 m) in India. It also has the deepest basinal area of all the collieries of this coalfield. The stratigraphic sequence found within the Singrauli Coalfield is described in Table 1.
General stratigraphic succession of Singrauli Coalfield (after Vijaya et al. Reference Vijaya, Tripathi, Roy and Mitra2012).
3. Materials and methods
Morphological studies have been performed on the microspores recovered from the macerals belonging to a lower Permian Gondwana exposure in central India. Five samples were collected from each of three grey-carbonaceous fine-grained shale horizons designated A, B and C (24°9′36.3″N; 82°44′39.1″E) from the lowermost Turra coal seam exposed in the Bina Colliery located in the eastern part of Singrauli Coalfield, Son-Mahanadi Basin, Madhya Pradesh State (Fig. 1). These exposures belong to the Lower Barakar Formation. The spore tetrads have only been found in shale horizon A; B and C are completely devoid of them (Fig. 3). Other palynomorphs have been recovered from all three horizons. The shale sediments are fluvial in origin. Microspores including the spore tetrads are extracted by digesting the shale samples following standard palynological processes using HF (40%) and HNO3 (63.01%) for different time durations. The potential maceral specimens are permanently mounted on slides using polyvinyl and Canada balsam for light microscopic analysis. The morphological characters of the microspores are examined under an Olympus (BX-61) high-power binocular microscope and photography was performed using Olympus DP-20 digital camera.
(a) Photograph showing measured section of a part of Turra Seam, Bina Colliery, Singrauli Coalfield. (b) Litho-column depicting different litho-units of a part of Turra Seam. Asterisk indicates the spore-tetrads-yielding horizon.
In all, 34 spore tetrads have been isolated in the present investigations which are assignable to the dispersed microspore genera Indotriradites, Microbaculispora and Microfoveolatispora. Tetrads form a sizeable chunk, about 17% of the total palynoassemblage, consisting of 15 spore/pollen genera (Fig. 2). Plant megafossils comprising Noeggerathiopsis hislopi and ten species of Glossopteris (namely G. gigas, G. tenuifolia, G. browniana, G. cf. G. feistmanteli, G. communis, G. recurva, G. nakkarea, G. pantii, G. leptoneura and G. searsolensis) have also been recovered from the same shale horizons of the Turra seam of Bina Colliery in which the present palynomorphs were recorded.
All the slides containing spore tetrads described in this paper are deposited in the repository of Birbal Sahni Institute of Palaeobotany, Lucknow, vide statement no. 1366 and Museum Slide no. 15134–15136.
4. Results
4.a. Observations of the present spore tetrads
Complete palynoassemblage recovered from the Bina colliery consists of 15 spore/pollen genera with a dominance of non-striate bisaccate pollen Scheuringipollenites (20%) and spore tetrads (17%) (Fig. 2), and a subdominance of girdling monosaccate pollen Parasaccites (14%) and striate bisaccate Faunipollenites (9%). Other associated palynomorphs are Indotriradites (6%), Verticipollenites (6%), Rhizomaspora (5%), Striatopodocarpites (4%), Microbaculispora (3%), Microfoveolatispora (3%), Densipollenites (3%), Callumispora (3%), Weylandites (2%), Tiwariasporis (2%), Arcuatipollenites (1%) and Distriatites (1%) (Fig. 2).
The occurrence of pollen taxa Scheuringipollenites, Parasaccites and Striatopodocarpites and spore taxon Callumispora in abundance along with other species (namely Indotriradites, Microfoveolatispora, Rhizomaspora and Densipollenites) in the present palynoassemblage place these beds at the transition of the Karharbari Formation and Lower Barakar Formation (Artinskian).
All the spore tetrads recovered from the Turra seam are permanent, naked and tetrahedral in shape. The individual microspores of the tetrads appear to be morphologically mature as they possess well-developed ornamentation and are almost of equal size to that of the dispersed microspores of concerned tetrad genera. Some tetrads are compactly arranged (Fig. 4g, h), whereas a few are very open (Fig. 4a, d). They are mostly fused; however, a distinct cleavage can be noticed between the individual spores in some of them (Fig. 4a, d, g). They range from 59 to 66 μm in diameter with an average of 62.50 μm along the x axis (horizontal). Individually, the tetrad genus Indotriradites has an average diameter of 62.50 μm, Microbaculispora 61 μm and Microfoveolatispora 62 μm.
Spore tetrads recovered from the A horizon of Turra Seam, Bina Colliery. (a) Microbaculispora tetrad showing individual spores arranged tetrahedrally, prominent cleavages (signs of splitting) seen between spores (CL – cleavage; TM – trilete mark). Scale bar = 10 μm (Q46/2–4). (b) Upper spore of the Microbaculispora tetrad in (a), showing faint trilete mark and baculae on the exine (BAC – Baculae). Scale bar = 5 μm (Q46/2–4). (c) Dispersed spore of Microbaculispora showing trilete mark, fold and baculae on the exine (FD – Fold). Scale bar = 5 μm (S50/1). (d) Microfoveolatispora tetrad showing tightly adhered individual spores having trilete mark, spores forming a prominent tetrahedral. Scale bar = 10 μm (M42/2). (e) Individual spore (upper) of Microfoveolatispora tetrad in (d) showing foveolae structure on the exine with distinct trilete mark (FS – foveolae structure). Scale bar = 5 μm (M42/2). (f) Dispersed spore of Microfoveolatispora showing trilete mark with foveolate exine. Scale bar = 10 μm (W33/1–3). (g) Indotriradites tetrad showing firmly attached individual spores, sign of cleavage seen between two lower spores (CL), well-preserved flange (FL) seen on the periphery of the spores, faintly preserved trilete mark on the exine. Scale bar = 10 μm (N31/3). (h) Indotriradites tetrad showing tightly adhered spores with faintly preserved trilete mark, coni and flange on the periphery of individual spores. Scale bar = 10 μm. (i) Dispersed zonate spore of Indotriradites showing well-preserved flange (FL) on the periphery and a distinct trilete mark. Scale bar = 5 μm (T35/1).
The spore tetrads found in the present study are divided into two groups of ornamented and zonate forms.
4.a.1. Ornamented forms
Ornamented forms are represented by the genera Microbaculispora and Microfoveolatispora.
Microbaculispora Bharadwaj Reference Bharadwaj1962: Individual spores in this tetrad (Fig. 4a–c) are subtriangular in shape, have broadly rounded corners and unequal axes; exine thin, baculate, measures ±1.5 μm in thickness; bacula small ±1 μm high and 2–3 μm broad at base with flat to sharp tip, densely distributed on the exine; trilete mark distinct, rays extend up to corners, faint secondary folds exist along the rays. Based on these morphological characters, this spore tetrad is assigned to the microspore genus Microbaculispora Bharadwaj Reference Bharadwaj1962.
Microfoveolatispora Bharadwaj Reference Bharadwaj1962: Individual spores in Microfoveolatispora (Fig. 4d, e) are triangular to broadly rounded, angled, sides are convex; trilete mark distinct, thick lipped, ending just before the corner; rays are associated with folds, secondary fold prominent; exine thin and foveolate, foveolae uniformly distributed, foveolae measures ±1 μm. All these morphological characters confirm that this spore tetrad belongs to the microspore genus Microfoveolatispora Bharadwaj Reference Bharadwaj1962.
4.a.2. Zonate forms
Zonate forms are represented by the genus Indotriradites.
Indotriradites Tiwari Reference Tiwari1964: Individual spores in this zonate tetrad, Indotriradites (Fig. 4g–i), are roundly triangular to subrounded in shape, show central body and inner body; exine 2–3 μm thick in the central body, uniformly micropunctate and coniate, coni 1–2 μm high, broad at base; trilete mark distinct, rays extend up to margin of the flange, flange broad with a smooth to slightly wavy margin. Based on these characters this tetrad compares well with the miospore genus Indotriradites Tiwari Reference Tiwari1964.
4.b. Observations of the global distribution of spore tetrads
As compared to the substantial representation of the early Palaeozoic (Ordovician – Early Devonian) spore tetrads (cryptospores, mostly of bryophytic origin) recorded globally (Richardson & Lister, Reference Richardson and Lister1969; Strother & Traverse, Reference Strother and Traverse1979; Gray, Reference Gray1985; Johnson, Reference Johnson1985; Richardson, Reference Richardson, El-Arunati, Owns and Thusu1988, Reference Richardson1996; Vavrdová, Reference Vavrdová1988, Reference Vavrdová1990; Burgess, Reference Burgess1991; Burgess & Richardson, Reference Burgess and Richardson1991; Strother, Reference Strother1991; Wellman & Richardson, Reference Wellman and Richardson1993; Edwards, Duckett & Richardson, Reference Edwards, Duckett and Richardson1995; Edwards et al. Reference Edwards, Davies, Richardson, Wellman and Axe1996, Reference Edwards, Morris, Richardson, Axe and Davis2012; Strother, Al-Hajri & Traverse, Reference Strother, Al-Hajri and Traverse1996; Wellman, Reference Wellman1996; Wang, Li & Wang, Reference Wang, Li and Wang1997; Edwards, Wellman & Axe, Reference Edwards, Wellman and Axe1999; Steemans Reference Steemans2000, Reference Steemans2001; Steemans, Higgs & Wellman, Reference Steemans, Higgs, Wellman, Al-hajri and Owens2000; Wellman, Higgs & Steemans, Reference Wellman, Higgs, Steemans, Al-hajri and Owens2000; Wellman, Osterloff & Mohiuddin, Reference Wellman, Osterloff and Mohiuddin2003; Steemans & Wellman, Reference Steemans, Wellman, Webby, Paris, Droser and Percival2004; Taylor & Strother, Reference Taylor and Strother2008; Rubinstein et al. Reference Rubinstein, Gerrienne, Dela Puente, Astini and Steemans2010), their occurrence has never been recorded in the rocks of similar age in India.
Potonie & Lele (Reference Potonié and Lele1959) reported Quadrisporites horridus from the Talchir Formation (Asselian) near Goraia village in the South Rewa Gondwana Basin as the first spore tetrad from any locality in India. However, it was not a true tetrad as the four bodies in Q. horridus (Potonie & Lele Reference Potonié and Lele1959, plate 1, figs 26–36) lie close together in the bedding plane, forming a rounded square or rounded rhombus and not a tetrahedron as is found in tetrads of embryophytes and trachaeophytes. No germinal aperture (either monolete or other marks) can be seen in these spore tetrads. Pant & Singh (Reference Pant and Singh1990) kept this taxon in Bryophyta (Riccia), whereas Tiwari & Meena (Reference Tiwari and Meena1989) identified Quadrisporites as related to the acritarchs and not the spore tetrads of embryophytes.
The first ever true tetrad to be described from Indian Gondwana was by Lele & Makada (Reference Lele and Makada1972) who recorded the tetrads of the miospore genus Jayantisporites represented by three species (J. pseudozonatus, J. indicus and J. conatus) from the lowest Permian Talchir Formation (Asselian) in Jayanti Coalfield, Bihar State. They suggested that these tetrads were immature because of the smaller size of the spores they hold. A well-developed trilete mark in the individual spores of the tetrad of J. pseudozonatus (Lele & Makada, Reference Lele and Makada1972, plate 1, fig. 13) proves their affinity with the group lycopods. Jayantisporites therefore stands as the only spore tetrad taxon represented in Indian Permian strata prior to the present study. The genera Jayantisporites, Indotriradites, Microbaculispora and Microfoveolatispora are the only tetrad taxa found in the Permian sequences of India.
The records of the spore tetrads in the Triassic sediments of India are also meagre. The tetrads of the spore genus Decisporis are reported from the Panchet Formation of Auranga Coalfield and Maitur Formation of Raniganj Coalfield by Banerji & Maheshwari (Reference Banerji and Maheshwari1973) and Maheshwari & Banerji (Reference Maheshwari and Banerjee1975), respectively. Tiwari & Meena (Reference Tiwari and Meena1989) recorded Densoisporites, Lundbladispora and Verrucosisporites from the Panchet Formation of Raniganj Coalfield. Spore tetrads of the miospore genera Densoisporites, Lundbladispora, Verrucosisporites and Lapposisporites have recently been reported from the Upper Pali Formation (Lower–Middle Triassic) in Sohagpur Coalfield by Ram-Awatar (Reference Ram-Awatar2011).
A large number of unseparated tetrads belonging to definite trachaeophytes (lycopods) have been reported from many end-Permian and Triassic localities in the continents: (1) Sverdrup Basin in Arctic Canada (Utting, Reference Utting1994); Barents Sea, Arctic region (Mangerud, Reference Mangerud1994); and East Greenland (Looy et al. Reference Looy, Twitchett, Dilcher, Van Konijnenburg-Van Cittert and Visscher2001; Visscher et al. Reference Visscher, Looy, Collinson, Brinkhuis, Van Cittert, Kurschner and Sephton2004); (2) Europe, including Italy (Massari et al. Reference Massari, Neri, Pittau, Fontana and Stefani1994; M. Smit, unpub. MSc thesis, Utrecht University, 1996; B. Van de Schootbrugge, unpub. MSc thesis, Utrecht University, 1997; K. De Graaf, unpub. MSc thesis, Utrecht University, 1999); and Hungary (Góczán, Oravecz-Scheffer & Szabó, Reference Góczán, Oravecz-Scheffer and Szabó1986; Haas et al. Reference Haas, Góczán, Oravecz-Scheffer, Barabás-Stuhl, Majoros and Bérczi-Makk1986, Reference Haas, Tóth Makk, Oravecz Scheffer, Góczán, Oravecz and Szabó1988); (3) Asia, including Pechora Basin, Urals, Russia (Tuzhiakova, Reference Tuzhikova1985); Moscow Basin, Russia (Afonin, Reference Afonin2000); Jungar Basin, North China (Qu & Wang, Reference Qu, Wang, Yang, Hou, Qu and Sun1986; Ouyang & Norris, Reference Ouyang and Norris1999); Meishan, South China (Ouyang & Utting, Reference Ouyang and Utting1990); Sri Lanka (Dahanayake et al. Reference Dahanayake, Jayasena, Singh, Tiwari and Tripathi1989) and India, including Damodar Basin (Lele & Makada, Reference Lele and Makada1972; Banerji & Maheshwari, Reference Banerji and Maheshwari1973; Maheshwari & Banerji, Reference Maheshwari and Banerjee1975; Tiwari & Meena, Reference Tiwari and Meena1989) and Son Basin (Ram-Awatar, Reference Ram-Awatar2011 and present study); (4) Africa: Mombassa Basin, Kenya (Hankel, Reference Hankel1992); and (5) Madagascar (Wright & Askin, Reference Wright and Askin1987).
These end Permian – Triassic tetrads have been assigned to the form genera Densoisporites, Lundbladispora, Uvaesporites, Verrucosisporites, Lapposisporites, Decisporis and Otynisporites (megaspore tetrad).
With the exception of a few examples of the spore tetrads – Tetrahedraletes medinensis from Upper Ordovician Southern Ohio (average diameter 28.1 μm; Taylor, Reference Taylor1995) and Velatitetras 17–38 μm and many in situ tetrads contained in the sporangium or as spore masses recovered from the upper Silurian rocks at Ludford Lane, Shropshire, UK (30–35 μm) and from the Early Devonian strata located at north Brown Clee Hill locality, Welsh Borderland (17–20 μm; Edwards, Wellman & Axe, Reference Edwards, Wellman and Axe1999; Edwards et al. Reference Edwards, Morris, Richardson, Axe and Davis2012) – most of the lower Palaeozoic tetrads (Cheilotetras 48–53 μm, Tetrahedraletes 49–67 μm, Rimosotetras 49 μm, Stegambiquadrella 52 μm and Acontotetras 39–46 μm; Edwards, Wellman & Axe, Reference Edwards, Wellman and Axe1999; Edwards et al. Reference Edwards, Morris, Richardson, Axe and Davis2012; Steemans et al. Reference Steemans, Petus, Breuer, Mauller-Mendlowicz, Gerrienne and Talent2012), Permian tetrads (Indotriradites 59–66 μm, Microbaculispora 61 μm and Microfoveolatispora 62 μm, present study) and Triassic tetrads (Densoisporites 57–67 μm, Lundbladispora 54–62 μm, Uvaesporites 55.29 μm, Verrucosisporites 68 μm, Lapposisporites 50–57 μm, Decisporis 55–64 μm and Otynisporites megaspore tetrad 800 μm; Banerji & Maheshwari, Reference Banerji and Maheshwari1973; Tiwari & Meena, Reference Tiwari and Meena1989; Looy et al. Reference Looy, Collinson, Van Konijnenburg-Van Cittert, Visscher and Brain2005; Ram-Awatar, Reference Ram-Awatar2011) range from 39 μm to 68 μm in diameter.
5. Discussion
Two successive cleavages are responsible for the formation of a tetrad from spore mother cell. Dyad is formed in the first stage when the wall formed during meiosis I divides the spore mother cell. The subsequent division of this dyad during meiosis II gives rise to the tetrad. Callose (β-1, 3-glucan, a polysaccharide) is deposited on the external walls of these developing meiocytes during the prophase stage of meiosis. The callose wall isolates meiocytes from other tissues and keeps them separated by preventing their underlying walls from fusing (Li, Gong & Wang, Reference Li, Gong and Wang2010). The quantity of callose increases during meiosis; however, at the end of microsporogenesis the callosic walls are disintegrated due to the hydrolytic activities of an enzyme called callase (β-1,3-d-glucanase) (Stieglitz, Reference Stieglitz1977; Chen & Kim, Reference Chen and Kim2009) that hydrolyses the β-D glycosidic linkages present in the callose. Ultimately, the spores of the tetrad are separated from each other and released as mature pollen grains/spores (Xie, Wang & Hong, Reference Xie, Wang and Hong2010; Wan et al. Reference Wan, Zha, Cheng, Liu, Lv, Liu, Wang, Du, Chen, Zhu and He2011). Stieglitz & Stern (Reference Stieglitz and Stern1973) and Pacini, Franchi & Hesse (Reference Pacini, Franchi and Hesse1985) suggested that the tapetum layer in the sporangium produces as well as releases the callase enzyme necessary for tetrad dissolution. If the activity of the callase enzyme diminishes or ceases for some reason, the dissolution process of the callose walls is also affected, thereby preventing the release of the spores of the tetrad.
In a significant study carried out on the anthers of sterile Allium sativum (garlic), a species producing sterile pollen and propagating only through vegetative methods and fertile Allium atropurpureum, Winiarczy, Jaroszuk-Ściseł & Kupisz (Reference Winiarczy, Jaroszuk-Ściseł and Kupisz2012) demonstrated the significance of pH values inside the microsporangium in regulating the activities of callase. Callase activity was monitored in both the species during microsporogenesis, and A. atropurpureum (which possesses functional male gametophytes) was used as a control. The existence of callose in the microsporocyte was detected using fluorescence microscopy and aniline blue staining. In this microscopic observation a callose wall is seen encircling the microsporocyte in prophase I, whereas during prophase II each dyad showed a thin intersporal callose wall and a thicker external callose wall (Winiarczy, Jaroszuk-Ściseł & Kupisz, Reference Winiarczy, Jaroszuk-Ściseł and Kupisz2012, fig. 1). Complete separation of the four microspores involved the development of a callose wall between them. The detailed studies carried out on callase confirmed the existence of three isoforms of this enzyme that are active at specific pH values. Winiarczy, Jaroszuk-Ściseł & Kupisz (Reference Winiarczy, Jaroszuk-Ściseł and Kupisz2012) believed that at least one of the callase isoforms might be responsible for the degradation of the callose walls. As the callose wall is not degraded and it stays around the tetrads in Allium sativum for more than 2 weeks, gametogenesis could not take place and as a result no functional male gametophyte is formed. The degeneration of the microspore cytoplasm is also observed, which results in the abortion of the microspore (Winiarczy, Jaroszuk-Ściseł & Kupisz, Reference Winiarczy, Jaroszuk-Ściseł and Kupisz2012). They further demonstrated that the callose wall stays around meiotic cells in A. sativum when an acidic environment of pH value 5.0 or less prevails in the microsporangium, and only one callase isoform is active with optimum activity at pH 4.8. However, disintegration of the callose wall takes place only when the pH rises up to 6.0 and simultaneously the two other callase isoforms that are active at pH 6.0 and 7.3 appear.
The correlation between pH values and the callase activity is also established in an extant Petunia hybrida plant. Due to higher pH values (6.8–7.0) prevailing in the anthers of the sterile Petunia hybrida during meiosis, the callase remained inactive and the spore tetrads therefore remained enclosed within the callose wall for a longer time. However, slightly lower pH values (5.9–6) at the tetrad stage in fertile genotype of this plant enabled the microspores to disperse (Izhar & Frankel, Reference Izhar and Frankel1971). Working on the reproductive biology of the extant plant Arabidopsis (family Brassicaceae), Fei & Sawhney (Reference Fei and Sawhney1999) proposed that the existence of callose wall in the tetrads for longer periods may lead to the degeneration of microspore cytoplasm. They also postulated that the tapetum, which produces enzymes to be used in the development of the male gametophyte, remained non-functional and therefore callase could not be synthesized; as a result the callose wall was not degraded and remained intact.
Since the present study is related to fossil tetrads, it is impossible to postulate the pH values in the microsporangium of the fossil plant to which these belonged. However, it is convincing from the above studies on the extant plants Allium and Petunia that the pH values were not adequate. Either the callase could not be produced by the tapetum or it did not work optimally; ultimately the callose walls could not be dissolved, which is required to facilitate the release of microspores.
The tetrad genera reported from the Triassic sequences are mostly aligned to the orders Selaginellales and Isoetales (Balme, Reference Balme1995) on morphological details. The ultrastructural analysis carried out on the tetrad genera Densoisporites, Lundbladispora, Otynisporites and Uvaesporites collected from the Permian–Triassic transition of Italy and Greenland also confirm Isoetalean (Pleuromeiaceae) affinity to the first three spores and selaginalean to Uvaesporites (Looy et al. Reference Looy, Collinson, Van Konijnenburg-Van Cittert, Visscher and Brain2005). The tetrads Verrucosisporites, Lapposisporites and Decisporis are broadly affiliated to the pteridophytes as they possess trilete marks. The affinities of Permian tetrads (namely Jayantisporites, Indotriradites, Microbaculispora and Microfoveolatispora) could not be determined due to the absence of any sporangia or related plant parts in the assemblage; however, the presence of well-developed trilete marks in them at least demonstrate their alliance with the pteridophytes.
Different hypotheses have been proposed to evaluate the reasons for the release of intact spore tetrads from the sporangia. Most of the workers who reported tetrads from the Permian–Triassic transition and from Triassic strata advocated extreme hot conditions as being responsible for the tetrad formation. While investigating the Lower Panchet (Induan–Olenekian) palynomorphs from the Raniganj Coalfield, Tiwari & Meena (Reference Tiwari and Meena1989) observed a high incidence of tetrads in the palynoassemblage; however, they were missing from the upper Permian (Lopingian) Raniganj palynoflora. They opined that warmer conditions due to high temperature and low rainfall at the beginning of the Triassic period might be responsible for the inertness of the callase enzyme. They further stated that it could also be an adaptive strategy of certain taxa to produce tetrads instead of monads so that they could withstand the changing climatic conditions and protect individual spores from perishing in such disastrous conditions.
Looy et al. (Reference Looy, Collinson, Van Konijnenburg-Van Cittert, Visscher and Brain2005) also linked the enormous production of tetrads during the Triassic period to environmental changes that affected the regular sporogenesis cycle in some specific taxonomic groups. Foster & Afonin (Reference Foster and Afonin2005) opined that the formation of such unusual palynomorphs around the Permian–Triassic boundary is the result of deteriorating atmospheric conditions. Visscher et al. (Reference Visscher, Looy, Collinson, Brinkhuis, Van Cittert, Kurschner and Sephton2004) referred to the permanent tetrads found in the end-Permian rocks as unusual mutated palynomorphs, where mutations were brought about by the worldwide increase in near-surface ultraviolet-B radiation (UV-B, 280–315 nm). Extensive volcanic eruptions over a large area of Siberia at the beginning of the Triassic period resulted in the release of huge amounts of organo-halogens (CH3Cl) into the atmosphere. These organo-halogens depleted the stratospheric O3 globally, allowing the harmful ultraviolet-B radiation into the troposphere, thereby changing the nucleotide sequencing of the genome of the plants in question and causing mutations to occur (Visscher et al. Reference Visscher, Looy, Collinson, Brinkhuis, Van Cittert, Kurschner and Sephton2004; Looy et al. Reference Looy, Collinson, Van Konijnenburg-Van Cittert, Visscher and Brain2005). While applying the Cambridge two-dimensional chemistry–transport model to assess the specific causes of O3 depletion, Beerling et al. (Reference Beerling, Harfoot, Lomax and Pyle2007) projected that both HCl released during eruption of the Siberian Traps, as well the release of massive amounts of CH3Cl by the combustion of dispersed organic matter entrapped in the Siberian rocks, were responsible for the O3 depletion. They also concluded that the combined effect of these chemicals vigorously depleted the O3 of the stratosphere and the resulting high-intensity UV-B radiation destabilized the plant genomes.
In our opinion, the spore tetrads are simply the unseparated miospores stacked together due to non-dissolution of the callose walls and not the mutated palynomorphs as stated by Visscher et al. (Reference Visscher, Looy, Collinson, Brinkhuis, Van Cittert, Kurschner and Sephton2004) and Looy et al. (Reference Looy, Collinson, Van Konijnenburg-Van Cittert, Visscher and Brain2005). If the mutations had occurred in the plants inhabiting the Early Triassic palaeoforests, they would have transformed genetically and not produced normal spores during Middle–Late Triassic time. This assumption is based on the fact that some of the miospore genera (namely, Lundbladispora and Densoisporites), whose spore tetrads existed at the Permian–Triassic boundary or in the Lower Triassic rocks, are found to occur again in the Middle and Upper Triassic rocks. The miospore taxa Lundbladispora raniganjensis, Densoisporites contactus and D. playfordi are recorded in the borehole RD-1/4, depth 532.48 m, near Durgapur City, Raniganj Coalfield (Tiwari & Rana, Reference Tiwari and Rana1981). These authors correlated this section as belonging to the Mahadeva Formation (lower Middle Triassic–Anisian) on the basis of the palynoassemblage. Hermann et al. (Reference Hermann, Hochuli Peter, Bucher and Roohi2012) recently reported Densoisporites spp. (as dispersed miospore) from the Landa Member (Anisian) of the Salt Range and Surghar Range sections in Pakistan. Similarly, Ram-Awatar et al. (Reference Ram-Awatar, Tewari, Agnihotri, Chatterjee, Pillai and Meena2014) reported miospore genera Lundbladispora and Densoisporites from the C Member of Lashly Formation (Middle–Upper Triassic) of Allan Hills, central Transantarctic Mountains, South Victoria Land, Antarctica. If some kind of mutagenic activity occurred in the plants that produced the genera Lundbladispora and Densoisporites during Early Triassic time, they would have changed genetically and would not have produced the normal miospores of these genera during Middle–Late Triassic time; instead, they would have produced the so-called mutated tetrads. Similarly, the theory of mutagenesis does not apply to the tetrads reported from Ordovician–Lower Devonian rocks and early Permian strata as no volcanic activity has been reported during these time scales, despite the presence of a large number of tetrads in the rocks of these periods.
6. Conclusions
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1. A good assemblage of naked, permanent tetrad spores has been reported for the first time from a cold-regime depositional stratum, that is, the Lower Barakar Formation of early Permian (Artinskian) age in Singrauli Coalfield, Son-Mahanadi Basin, central India. This is also the first record of tetrads from any early Permian locality in the world. The affinity of these tetrads could not be ascertained due to the absence of sporangia or related plant parts in the assemblage; however, the presence of well-developed trilete marks in the tetrads at least demonstrates their alliance with the pteridophyte group.
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2. The study also supports the hypothesis that the formation of tetrad spores is somewhat linked to extreme climatic conditions as evidenced by the global records of their occurrences in Ordovician – Lower Devonian sediments, in lower Permian (Asselian–Artinskian) rocks, at the Permian–Triassic boundary and thereafter in Lower Triassic sediments, all correlated with extreme cold (due to Ordovician–Silurian and Carboniferous–Permian glaciations) and hot (due to Early Triassic Siberian volcanic activity) climatic conditions.
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3. It is proposed that the extreme climatic conditions prevailing during the above mentioned regimes might have triggered some sort of malfunction in the sporogenesis process, which altered the pH values inside the microsprangium and inhibited the release of callase enzyme from the tapetum or diminished its optimal activity. As a consequence, large number of tetrads could not be dissociated into individuals or monads and remained intact.
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4. The hypothesis of mutagenesis based on the identification of spore tetrads as the mutated palynomorphs is not justified, as similar spore tetrads also occur in the Ordovician – Lower Devonian sediments and in early Permian rocks where there is no evidence of any kind of volcanic activity that might have induced the mutations as has been established for the tetrads of the Triassic period. We prefer to refer to them as the unseparated miospores, adhered together due to the non-dissolution of the callose walls.
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5. The occurrence of spore tetrads in the sediments can be linked to the prevalence of intense climatic conditions during the geological past.
Acknowledgements
The authors are grateful to Professor Sunil Bajpai, Director of Birbal Sahni Institute of Palaeobotany, for providing necessary facilities and permission to publish this work (BSIP/RDCC/50/2014–15). With sincere gratitude we acknowledge Professor Dianne Edwards of the School of Earth and Ocean Sciences, Cardiff University, Wales and Dr John F. Rigby of Queensland University, Queensland, Australia who read an early version of the manuscript and provided valuable comments. This work greatly benefited from the helpful suggestions provided by anonymous referees. Sincere thanks are also due to Mr T.K. Nag, CMD and Mr Niranjan Das, Director (Technical/Project and Planning), Singrauli Coalfield, and also to the Chief General Manager and General Manager of Bina Colliery for their help and permission to collect the plant fossils. We also appreciate the help from Mr V.P. Singh of BSIP during sample collection in the field.
