Spore morphology of Taenitis , Syngramma and Austrogramme species ( Pteridoideae , Pteridaceae ) from South-Eastern Asia

A comparative study of spores of seven species: Taenitis blechnoides (Willd.) Sw., T. hookeri (C. Chr.) Holttum, T. pinnata (J. Sm.) Holttum, Syngramma alismifolia (Presl) J. Sm., S. lobbiana (Hook.) J. Sm., S. quinata (Hook.) Carruth., and Austrogramme boerlageana (Alderw.) Hennipman from South-Eastern Asia was performed using the method of scanning electronic microscopy (SEM). Spores of all examined species are trilete, tetrahedral or tetrahedral-globose. Spores of Syngramma and Austrogramme are very similar to each other in shape and ornamentation. Ornamentation of both sides of spore is similar, verrucate (microverrucate), surface covered by rodlets and granulate deposits. Spores of Taenitis species with conspicuous cingulum (Taenitis blechnoides) or without it, ornamentation of both faces of spore could be tuberculate or baculate. Spores of Taenitis hookeri and Taenitis pinnata demonstrate tendency to forming of comissural ridges. Spore size of all studied species is close: equatorial diameter of spores of Taenitis species varies within 24–43 μm, those for Syngramma species is 29.8–35.5 μm, spores of Austrogramme boerlageana are smallest, their equatorial diameter varies within 22.5–29.4 μm.

The representatives of Austrogramme occur in New Caledonia, New Guinea, the Moluccas, and the New Hebrides. Species of Syngramma inhabit Malaya to the Philippine Islands, New Guinea, and east to Fiji and the Caroline Islands (Palau), seven species occur in Borneo. The Taenitis species are distributed from southern India and Ceylon to Hainan, through Malesia to northern Queensland and Fiji, nine of them occur in Borneo (POWO. URL: http://powo.science.kew.org/).
The aim of this work is a comparative morphological study of the spores of Taenitis, Syngramma, and Austrogramme using scanning electron microscopy (SEM) and evaluation, whether the spore characteristics have the value for phylogeny of these genera.
The spelling of the names of taxa and authors is given according to "The International Plant Name Index" (IPNI, http://www.ipni.org).
The SEM studies were conducted in the Institute for Water and Environmental Problems (Barnaul, Russia) using a "Hitachi S 3400 N" electronic scanning microscope and in Tomsk State University (Tomsk, Russia) with the scanning electronic microscope "Mini-SEM SNE-4500M". Spores were applied on double-sided carbon adhesive tape, fastened on the object table with a diameter of 10 mm. The spore surface was coated in the sputtercoaters "Emitech SC 7620/QT S" (with the goldpalladium mixture) and "Quorum Q150R S" (with gold). The spore surface was scanned in a high vacuum at a voltage of 20 kV through 2000× and 15000× of magnification.
Equatorial diameter (as seen in the polar position of spore), polar axis (as seen in the equatorial position of spore), length and width of laesura arms (on the proximal position of spore), the width of cingulum (as seen in the polar position of spore) were measured for 10-20 spores of each species. All measurements were made on SEM-micrographs of spores using the computer program "Image J".

Results and discussion
Spores of all examined species of Austrogramme, Syngramma, and Taenitis are trilete, tetrahedral or tetrahedral-globose. In equatorial position, the distal side is convex to hemispherical, proximal side is flat, convex or conical. In polar position, spores are triangular, roundish-triangular or nearly roundish with straight or concave sides and rounded corners. Spores with cingulum or without it. Ornamentation of both sides of the spore may be verrucate, tuberculate, baculate, or rugate, spore surface covered by rodlets and granulate deposits. In spores of some species, the laesura arms are flanked by laesural ridges that can be well-defined entire or interrupted. The main morphological and biometrical characteristics of spores are shown in tables 1 and 2.
The spores of the Austrogramme species are the smallest, simplest in ornamentation and similar to each other. Spores are tetrahedral-globose in equatorial view, often with depressions on the proximal side between laesura arms. In polar position, spores are triangular-roundish or nearly roundish with straight, concave or convex sides. The proximal and distal sides are microverrucate, with verrucae 0.2-0.4 μm in diam. in A. boerlageana, or verrucate, with verrucae 0.4-1.2 μm in diam. in A. decipiens, A. francii, and A. marginata. The surface of the spores of three species is covered by granular deposits, dense in A. boerlageana, scattered in A. francii and rare in A. marginata. The spore surface of A. decipiens is covered by rodlets rather than granular deposits. Laesura arms are straight, 0.6-1.1 μm in length. In spores of A. marginata, laesura arms immersed in the narrow laesural ridges that are reached to the spore corners and covered by scattered verrucae (Fig. 1, Table 1). The equatorial diameter ranges between 22.5 μm and 36.7 μm and the polar axis -between 14.8 μm and 20 μm. The smallest spores are characteristic of A. francii and A. marginata ( Table 2).
The spores of most Syngramma species are very similar to spores of the Austrogramme species in shape and surface sculpture. In equatorial view, spores of Syngramma alismifolia, S. borneensis, S. cartilagidens, and S. wallichii are tetrahedralglobose with depressions between laesura arms on the proximal side. In polar position, spores are triangular, roundish-triangular or nearly roundish with straight, slightly concave or convex sides and wide-rounded corners. Distal and proximal surfaces are microverrucate in S. quinata, S. lobbiana,   Table 1 The main spore features in Austrogramme, Syngramma, and Taenitis    Note: '-' means lack of the feature; ND -no data; an asterisk indicates the species with published data for spore size (Vaganov et al., 2018 T. cordata) proximal part and mostly hemispherical distal part; spores of T. hookeri and T. pinnata are nearly globose. Three of the most noticeable features are characteristic for the Taenitis spores: cingulum, laesural ridges and tuberculate surface (Fig. 4-5). Seven of nine studied species have spores with cingulum: T. blechnoides, T. cordata, T. diversifolia, T. interrupta, T. luzonica, T. obtusa, and T. requiniana. Cingulum in spores of most species is continuous, variable in width, sometimes narrower at the corners, undulate (T. obtusa), or entire (remaining species). The widest cingulum is characteristic for T. luzonica (8.0-9.4 μm), cingulum in T. cordata spores is wide (6.5-9.2 μm) and thick (in equatorial view) (Fig. 4-5, Table 2). Spores of T. hookeri and T. pinnata are non-cingulate. Spores of three species have the prominent laesural ridges, entire in T. cordata and interrupted, formed by Fig. 2. SEM-micrographs of the spores of Syngramma J. Sm.: 1 -Syngramma borneensis (Hook.) J. Sm.: a -distal side; b -proximal side; c -fragment of proximal side of spore and laesura arms; 2 -Syngramma cartilagidens (Baker) Diels: a -distal side; b -spore in proximal-equatorial position; c -fragment of proximal side of spore and laesura arms; 3 -Syngramma grandis (Copel.) C. Chr.: a -distal side; b -proximal side; c -fragment of proximal side of spore and laesura arms; 4 -Syngramma quinata (Hook.) Carruth.: a -distal side; b -proximal side; c -fragment of proximal side of the spore and laesura arms. 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b -20 μm; 1c, 2c, 3c, 4с -5 μm. arranged in the rows fused tubercles in spores of T. hookeri and T. pinnata. Ornamentation of distal and proximal surfaces in spores of most species is densely tuberculate, in spores of T. hookeribaculate, in spores of T. interrupta and T. pinnatatuberculate-rugate, and in T. cordata -rugate ( Fig. 4-5). The most conspicuous character of the ornamentation of spore surfaces is the presence of rodlets associated with tubercles: rodlets are mostly fused in tufts and situated on the top of tubercles.
T. requiniana, rodlets situated also on the cingulum margin. Laesura arms are prominent in spores of T. interrupta and T. luzonica and are depressed and obscured by dense rodlets in spores of T. blechnoides,T. diversifolia,T. obtusa,. Spores of Taenitis species are larger than spores of Austrogramme and Syngramma, equatorial diameter ranges in most species between 40 and 51 μm on average, polar axis -from 24 to 40 μm. Two species -Taenitis hookeri and T. pinnata -have small spores, 26-31 × 26-28 μm on average (Table 2).
On the whole, our examination of four species of Austrogramme, seven species of Syngramma, and nine species of Taenitis showed that Syngramma and Austrogramme are very similar to each other in the spore shape and ornamentation and very different in these spore characteristics of Taenitis species. Syngramma and Austrogramme have simple spores with a weakly expressed ornamentation (microverrucate or microrugulate sculpture). Only two species of Syngramma have spores with lowtuberculate ornamentation. In comparison with spores of Syngramma and Austrogramme, spores of Taenitis species have a more complex structure and noticeable ornamentation: the presence of cingulum, laesural ridges and a very clear tuberculate (as an option -baculate) or rugate sculpture.
Other authors who studied the spores of Austrogramme, Symgramma, and Taenitis indicated the same characters for the same and other species of these genera. E. Hennipman (1975) emphasized that spores of Taenitis are very different from those in Austrogramme and Syngramma, and are of two kinds either with or without a cingulum. A. Tryon and B. Lugardon (1991) described the similar features for spores of these genera. Furthermore, E. Hennipman (1975) considered that differences in chromosome number in Taenitis are associated with the presence or absence of a cingulum. On the basis of the spore morphology and chromosome number, E. Hennipman (1975) considered that Syngramma is distinguished from Taenitis and related with Austrogramme; A. Tryon et. B. Lugardon (1991) supported this disposition. Published data indicate a comparatively larger spore size than the ones we found. E. Hennipman (1975) indicated larger spore size for all studied genera: 45-70 μm for Taenitis, 40-50 μm for Syngramma, and 35-40 μm for Austrogramme. A. Tryon and B. Lugardon (1991) reported the spore size for the Taenitis and Syngramma species as 33-53 μm, for Austrogramme -30-40 μm. Spores of species not involved in our studies have features specific for the species of the corresponding genera. Judging by the published photos, Austrogramme asplenioides (Holttum) Hennipman has simple spores with microverrucate surface and granular deposits on it (Hennipman, 1975), spores of T. hosei (Baker) Holttum lack a cingulum. Based on the properties of the spores and chromosome numbers, E. Hennipman (1975) indicated the relationship between Austrogramme and Syngramma; the relationship between Austrogramme and Taenitis seems less obvious, also in view of the variation of the paraphyses in the three genera studied. Based on leaf morphology, M. Kato (1993) considered Taenitis pinnata as the most primitive; the other species represent different lines of reduction and specialization.
According to database "POWO: Plants of the world online" (POWO. URL: http://powo.science. kew.org/), all studied species except Taenitis luzonica are now accepted, disagreements exist only in relation to Taenitis luzonica: the name T. luzonica is considered as a synonym of the name Austrogramme luzonica (Alderw.) M. Kato. This species was described as Syngramma luzonica Alderw. (1920). R. E. Holttum (1975) transferred it from Syngramma to Taenitis (T. luzonica), and M. Kato (1988) considered it as belonging to Austrogramme (A. luzonica). On the whole, spore morphology of three studied genera is congruent with the result of molecular-phylogenetic studies (Sánchez- Baracaldo, 2004;Cochran et al., 2014;Zhang et al., 2015Zhang et al., , 2017, which shows the close relationship between Syngramma and Austrogramme and the position of Taenitis as a sister to Syngramma and Austrogramme (together) in the collective clade (Syngramma -Austrogramme -Taenitis). Taenitis luzonica was not involved in molecularphylogenetic studies, but by the morphology of spores, it is closer to other Taenitis species than to species of Syngramma or Austrogramme. So we support the opinion of R. E. Holttum (1975) that this taxon belongs to Taenitis (T. luzonica).

Conclusion
The results of this study confirm the diagnostic value of the spore morphology for the phylogeny and taxonomy of three fern genera -Austrogramme, Syngramma, and Taenitis. Spores of Austrogramme and Syngramma are the simplest in ornamentation and similar to each other, whereas spores of the Taenitis species are very different from spores of Austrogramme and Syngramma. This corresponds to molecular-phylogenetic studies according to which Austrogramme and Syngramma have a closer relationship to each other, and Taenitis takes a sister position to both of them. Differences in the spore morphology correlate with the division of genera into sections.