On the spore ornamentation of the microsoroid ferns (microsoroideae, polypodiaceae)

Microsoroideae is the third largest of the six subfamilies of Polypodiaceae, containing over 180 species. These ferns are widely distributed in the tropical and subtropical regions of the Old World and Oceania. We documented the spore ornamentation and integrated these data into the latest phylogenetic hypotheses, including a sampling of 100 taxa representing each of 17 major lineages of microsoroid ferns. This enabled us to reconstruct the ancestral states of the spore morphology. The results show verrucate ornamentation as an ancestral state for Goniophlebieae and Lecanoptereae, globular for Microsoreae, and rugulate surface for Lepisoreae. In addition, spore ornamentation can be used to distinguish certain clades of the microsoroid ferns. Among all five tribes, Lecanoptereae show most diversity in spore surface ornamentation. Electronic supplementary material The online version of this article (10.1007/s10265-020-01238-4) contains supplementary material, which is available to authorized users.


Introduction
The microsoroid ferns (Microsoroideae) are one of the largest subfamilies of Polypodiaceae, distributed mainly in the tropical and subtropical regions of the Old World. The generic classification of some genera nested in this lineage has been controversial, in particular the generic delimitation of Leptochilus Kaulf., Microsorum Link, and Phymatosorus Pic. Serm. that have been treated in previous taxonomic studies (e.g. Bosman 1991;Nooteboom 1997). The use of sequence-level data has further advanced studies on the phylogeny of the microsoroid ferns (Chen et al. 2020;Kreier et al. 2008;Testo et al. 2019;Wang et al. 2010;Zhang et al. 2019;Zhao et al. 2019). Based on the latest classification (Chen et al. 2020;PPG I 2016;Testo et al. 2019;Zhang et al. 2020), there are 16 currently accepted genera: Bosmania Testo, Dendroconche Copel., Ellipinema Li Bing Zhang and Liang Zhang, Goniophlebium (Blume) C. Presl, Lecanopteris Reinw. ex Blume, Lemmaphyllum C. Presl, Lepidomicrosorium Ching and K.H.Shing, Lepisorus (J.Sm.) Ching, Leptochilus, Microsorum, Neocheiropteris H. Christ, Neolepisorus Ching, Paragramma (Blume) T. Moore, Thylacopteris Kunze ex J. Sm., Tricholepidium Ching, and Zealandia Testo and A. R. Field. The number of genera may be reduced by expanding the definition of Lepisorus to also include Ellipinema, Lemmaphyllum, Lepidomicrosorium, Neocheiropteris, Neolepisorus, Paragramma, and Tricholepidium (Zhao et al. 2019). In total this group includes over 180 species but the species number may be underestimated in the species rich lineages such as Goniophlebium, Leptochilus and Lepisorus (Chen et al. 2020;PPG I 2016;Testo et al. 2019). In addition to the generic rank, authors have also proposed different ranks above and below genus, for example tribes (Chen et al. 2020), and subclades of the larger genera such as Leptochilus and Lepisorus Zhang et al. 2019;Zhao et al. 2019). These latest studies have clarified such relationships, but there are still uncertainties that need further examination. For example, there seems Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1026 5-020-01238 -4) contains supplementary material, which is available to authorized users.

3
to be an inconsistency in obtained results based on nuclear versus chloroplast genes (Nitta et al. 2018).
Spore morphology provides valuable information that helps to clarify taxonomy of the many lineages of ferns (Wagner 1974). Numerous studies using these characters have been carried out, such as the landmark publication by Tryon and Lugardon (1991) integrating information obtained using scanning (SEM) and transmission electron microscopy (TEM). Such studies have also been undertaken in Polypodiaceae focusing on morphological variation and sporogenesis (e.g., Giudice et al. 2004;Lloyd 1981; Morbelli and Giudice 2010;Van Uffelen and Hennipman 1985;Van Uffelen 19921997Wang 2001). Despite the limited taxon sampling in these studies, some general trends have been observed. For example, Tryon and Lugardon (1991) pointed out that the spore ornamentation of Colysis ampla Copel. (= Dendroconche ampla (F. Muell. ex Benth.) Testo, Sundue, and A.R. Field) differed from the other species assigned to Colysis C. Presl (= Leptochilus). Phylogenetic analyses showed this species to belong Dendroconche, and not to Leptochilus (Chen et al. 2020;Testo et al. 2019). In addition, spore ornamentation of the broadly defined Phymatosorus Pic.Serm has been found to be heterogenous (Tryon and Lugardon 1991), which is consistent with the polyphyly of the genus in the latest phylogenetic analyses (Chen et al. 2020).
Using the most recent robust phylogenetic hypotheses, it is now possible to re-evaluate the taxonomic value of the spore wall ornamentation. To achieve this, we integrated the spore surface data of the microsoroid ferns from previous, and our own studies, into the latest phylogenetic hypotheses with the aim to reconstruct the ancestral spore type for each clade/genus, as presented in Chen et al. (2020), and to assess possible trends of the spore surface development in the microsoroid ferns.

Taxon sampling and the chloroplast DNA sequencing
Based on the latest hypothesis of phylogeny, and the available spore surface data, we chose species from each of the 15 out of 16 currently accepted genera, plus two Microsorum groups MG4 and MG5 (Chen et al. 2020;Testo et al. 2019;Zhang et al. 2020), with at least one species per genus/group, but recently described Ellipinema (Zhang et al. 2020) was not included because we did not have access to any material of this new genus. We also considered the subclades of larger genera, such as Lepisorus, and sampled them as thoroughly as possible Zhao et al. 2019). In total, 98 out of 183 microsoroids species and two outgroup species, Aglaomorpha meyeniana Schott and Pyrrosia polydactyla (Hance) Ching, were included.
The chloroplast sequences (rbcL, rps4 + rps4-trnS, trnL + trnL-trnF, atpA, atpB and matK) for molecular analyses were mostly those used in previous studies (e.g., Chen et al. 2020), but several previously unpublished sequences were added to the analyses here. Voucher information and Genbank accession numbers are provided in Table 1. DNA extraction, amplification, and sequencing methods are described in Chen et al. (2020).

The spore data
Spore data were compiled by incorporating the results of previously published studies (Bosman 1991;Dai et al. 2006;Devi 1981;Hennipman 1990;Huang 1981;Jiang et al. 2010;Kholia et al. 2012;Large and Braggins 1991;Large et al. 1992;Mitui 1971Mitui 1977Nayar and Devi 1964;Nooteboom 1997;Pal and Pal 1970;Qi and Zhang 2009;Rödl-Linder 19901994Shalimov et al. 2013;Shi 2002;Shi and Zhang 1998;Sugong et al. 2005;Tryon and Lugardon 1991;van Uffelen 1993;Wang 2001;Zhang et al. 2006;Zink 1993) and novel observations partially based on the MSc thesis of the first author (Chen 2011). Spore samples were obtained from specimens recently collected in Taiwan, and from herbarium specimens of the National Sun Yat-Sen University of Taiwan (SYSU), and Taiwan Forestry Research Institute (TAIF). The new collected specimens were preserved as vouchers and deposited mainly in the SYSU (Table S1).
Spore surface ornamentation was observed and the size was measured using both light microscopy (LM) and SEM. For the size measurements, 10-20 untreated spores per accession were chosen randomly and measured using the program ImageJ (Schneider et al. 2012). The perispore was included in the measurements, and the data of spore size was described including both polar and equatorial diameter. For studies of the ornamentation, untreated spores were fixed on aluminum stubs, coated with ca. 15 nm of gold with the ion sputter (Hitachi E-101), and examined using SEM (Hitachi S2400 and TM3000) at 12-18 kV. Spores treated in this way remain suitable for examination with the SEM for at least one month (Van Uffelen and Hennipman 1985). Magnification of 1000-3000 X was used for the micrographs of the whole spores and 4000-8000 X for the surface details.
To integrate the spore ornamentation data, we chose to use the most common descriptions if there was conflict between published studies. The main spore surface ornamentation types were illustrated using the software Gimp (gimp.org). Table 1 List of material used for obtaining sequences given as taxon name, voucher specimen with collecting locality, collector, or specimen number and the herbarium where deposited, followed by Gen-Bank accession numbers for six plastid regions : rbcL, rps4 and rps4-trnS, trnL and trnL-trnF, atpA, atpB and rbcL-atpB, matK

Terminology
We compared our observations with previously reported descriptions and images using the established descriptive terminology (Lellinger 2002;Punt et al. 2007;Shalimov et al. 2013;Tryon and Lugardon 1991). We studied and compiled data of two spore features: surface ornamentation and type of projections. Distinction of exospore and perispore requires more precise estimates using transmission electron microscopy (TEM). Numerous studies have tried to understand the spore wall structure of Polypodiaceae (e.g., Hennipman 1990; Tryon and Lugardon 1991;van Uffelen 1993), but the TEM data is still insufficient and thus, in this study, we treat the visible surface ornamentation as one character. Some species may show variation between the samples and this also influenced our use of the terms. For example, terms retate and rugate indicated muri with or without anastomosing respectively (Lellinger 2002), but these ornamentation types can be observed in the different specimens of the same species, especially the species within the tribe Lepisoreae. To minimize these effects, the ornamentation was classified using general macro-characteristics. Surface ornamentation was scored (as illustrated in Fig. 1): (0) verrucate: width of surface projections greater than height (Punt et al. 2007) (Fig. 1a); (1) psilate or almost psilate: with a smooth surface (Lellinger 2002;Punt et al. 2007) (Fig. 1b); (2) verrucate with longitudinal crest (Shalimov et al. 2013) (Fig. 1c); (3) (9) cable-like filamentous (Tryon and Lugardon 1991) (Fig. 1j).
BI analysis was implemented using MrBayes 3.2.7a (Ronquist et al. 2012) on the CIPRES, with the partitioned regions used. Markov chain Monte Carlo was run independently twice with one cold and three hot chains. In each run, chains were sampled every 1000 cycles. A total of 10 million generations were run and a majority rule consensus tree was calculated based on all trees sampled, excluding the first 25% of the sampled trees, which were discarded within the burn-in phase. This was examined using Tracer v. 1.6 (Rambaut and Drummond 2007) to ensure convergence of chains and sufficient sampling of generations. The posterior probabilities (PP) were calculated and presented using the majority rule consensus tree.
The parsimony analyses were conducted using the heuristic search algorithms of NONA 2.0 (Goloboff 1998) with the WinClada (Nixon 2002) shell under the following settings: maximum trees kept (hold) = 100,000; number of replications (mult*N) = 1000; starting trees per rep (hold/) = 100; random seed = time; search strategy = multiple TBR + TBR (mult*max); unconstrained search. The obtained trees were examined and analysed under different optimizations using WinClada. Bootstrap value was calculated using 1000 replications and 10 search replications with one starting tree per replication and without tree bisection-reconnection (TBR). All character states were treated as unordered and equally weighted, and gaps were treated as missing data.

Ancestral state reconstruction of spore characters
We calculated probabilities of ancestral states in Bayes-Traits version 3.0 (Pagel and Meade 2006), and mapped on the consensus tree obtained from MrBayes. To incorporate phylogenetic uncertainty, we used R to choose, at random, 100 post burn-in trees from the MrBayes analysis, with the information of branch-length included. Ancestral states were reconstructed for 22 nodes (a-v in Figs. 3a, 4) for each character. We used the "Multistate" model. A reversible-jump hyperprior with an exponential prior was used to reduce uncertainty of choosing priors in the MCMC analysis. The option "AddNode" was used to find the proportion of the likelihood associated with each of the possible states at each node. The MCMC run was performed with 10 million iterations. Chains were sampled every 1000 iterations with a burn-in of 5 million iterations.

Phylogenetic analyses
In general, the consensus trees obtained from the ML analyses (Fig. 3a) and BI analyses (Fig. 4) were congruent except the MG4 (Microsorum commutatum clade), IV-V subclades of Lepisorus, and Tricholepidium normale (D. Don) Ching. The former two are part of the polytomy in BI topology (Fig. 4), the latter, T. normale located in the basal position of Neocheiropteris-Lepidomicrosorium-Neolepisorus in ML topology (Fig. 3a), but in the basal position of Neocheiropteris-Lepidomicrosorium in BI topology (Fig. 4). In order to simplify presentation of the results, the values of posterior probabilities of the BI analyses were illustrated on the ML topology (Fig. 3a). In the parsimony analyses, the molecular dataset had 5814 characters, with 1459 of those being parsimony-informative. Thirty equally parsimonious trees of length 5535 (CI = 50, RI = 72) were obtained. The strict consensus tree included several polytomies: subclades within Leptochilus, clades Tricholepidium-Neolepisorus, and subclades of Lepisorus (Fig. 3b).

Spore character evolution
The number of globular elements on the spore surface varied to great extent between species, only the species with high density globular (usually more than 150) were scored as globular state in Fig. 4a. In Bayesian analyses, most nodes showed significant posterior probability values in at least one character state (Table S2). The ancestral state for the spore surface ornamentation was verrucate for the microsoroid ferns, present in the basal nodes a-g, including Goniophlebieae and Lecanoptereae (PP = 0.8649 and PP = 0.6348, Table S2). For Microsoreae, psilate and globular ornamentations were reconstructed as the ancestral states, the former was specific for the node l (i.e. core Microsorum), and the latter at nodes j, k, m, n, o, corresponding to Microsoreae, MG4 plus core Microsorum, MG5 plus Leptochilus, MG5, and Leptochilus, respectively (Fig. 4a). For tribe Lepisoreae (nodes p-v), rugulate was the ancestral state at all studied nodes (Table S2, Fig. 4). Of all the microsoroid ferns clades, species of Lecanoptereae showed most variation in their spore ornamentation, with five types represented: vermiculate-papillate, verrucate, globular, sheath-like, and cable-like filaments (Fig. 4a).
For type of projections, the lack of spinose/baculate surface was the most common ancestral state at all nodes except for node o (i.e. Leptochilus), at which the longer spinose reconstructed as a synapomorphy (PP = 0.9996, Table S2; Fig. 4b).

Morphology and evolution of spore ornamentation
Our observations are mostly congruent with earlier reports about spore surface ornamentation of the microsoroids. Ten different types of spore surface ornamentations formed by spore walls were observed in this study ( Fig. 1; Fig. 4a). Some of the ornamentations are formed by exospore, such as verrucate of Goniophlebieae and Lecanoptereae (Large and Braggins 1991;Large et al. 1992;Tryon and Lugardon 1991); some ornamentations by perispore, such as sheathlike and cable-like filaments of Lecanopteris, and spinose of Microsoreae (Hennipman 1990;Tryon and Lugardon 1991;van Uffelen 1993van Uffelen 1997; with some determined by both exospore and perispore, such as verrucate with longitudinal crest of Goniophlebieae (Tryon and Lugardon 1991). This demonstrates the diversity and complexity of the microsoroid ferns, which is consistent with the classification regarding sporoderm by Tryon and Lugardon (1991). However, as already mentioned above, more complete comparison of exospore and perispore requires additional data using TEM, since TEM sections may provide more precise estimates than sections obtained via breaking of the spore wall during the preparation for the SEM. There have been numerous efforts to understand the spore wall structure of Polypodiaceae (e.g., Hennipman 1990;Tryon and Lugardon 1991;van Uffelen 1993), but the TEM observations of microsoroids are still insufficient. In order to understand and compare different species of the group also ontogeny of the spores should be studied in detail. This is why, also in our analyses, we treated the visible surface ornamentation as one character.
Reconstruction of the ancestral state shows that verrucate is most likely the ancestral state of the spore surface ornamentation of the microsoroid ferns, exhibited in the basal nodes (a-g), including tribes Goniophlebieae and Lecanoptereae (Table S2, Fig. 4a). All studied species of Goniophlebieae (Goniophlebium) have verrucate surface, with or without longitudinal crests, and present in different subclades (Fig. 4a). Clades Zealandia and Dendroconche of Lecanoptereae also have verrucate ornamentation, however, the shape and size of verrucae differ from those found in Goniophlebium. Verrucae of Zealandia are more irregular, while in Dendroconche ampla and D. scandens, they are relatively small micro-verrucae (Large et al. 1992;Tryon and Lugardon 1991). For the other two genera of Lecanoptereae, Lecanopteris exhibits cable-like filaments as the ancestral state, but with only low support value (PP = 0.4105, Table S2); Bosmania has vermiculate-papillate as the main ornamentation (Fig. 4a), which has been considered a special exospore type in the previous studies (Hennipman 1990;van Uffelen 1997). Among the studied genera/clades of the microsoroid ferns, spore ornamentation of Lecanopteris is relatively diverse and unique, including cable-like filaments, sheath-like, and globular elements (Fig. 4a). The former two ornamentation types are unique types found only in this genus (Tryon and Lugardon 1991), and likely autapomorphies in the microsoroid ferns (Fig. 4a). It is reasonable to suppose that spore diversity of Lecanopteris may be related to their relationship with ants, since some studies show that the spore of Lecanopteris may be transported and utilized by them (Tryon 1985;Tryon and Lugardon 1991).
Globular ornamentation is reconstructed as the ancestral state for tribe Microsoreae, except for core Microsorum, where psilate is the main ornamentation type (PP = 0.9316, Table S2). Unlike the relatively simple surface of core Microsorum, the other three genera/clades (MG4, MG5, and Leptochilus) exhibit numerous globular elements, with or without spinose on the surface, that might represent a synapomorphy (Fig. 4). For Leptochilus, not only globular but spinose are likely ancestral states, with posterior probabilities of 0.5215 and 0.4730, respectively (Table S2). Spinose projections of Leptochilus are usually larger and less uniform, which may be a synapomorphy. Spores in the clades of MG4 and MG5 also have spinose surfaces, but not in all species. Spinose projections of these two clades are smaller differing from species of Leptochilus (Fig. 4b).

Taxonomic considerations
Spore surface types of the microsoroid ferns are generally congruent with the phylogenetic relationships obtained using plastid DNA sequence data. There are five tribes currently accepted within the microsoroid ferns (Chen et al. 2020). Tribe Thylacoptereae has only one species and it shows globular ornamentation, tribe Lecanoptereae shows the most diversity in spore surface ornamentation with six types. Of the other three tribes, Microsoreae has four, Lepisoreae three, and Goniophlebieae two types, respectively (Fig. 4a).
Lecanoptereae contains four genera: Bosmania, Lecanopteris, Dendroconche, and Zealandia. The vermiculatepapillate ornamentation of Bosmania is unique and can be distinguished from other Polypodiaceae (Hennipman 1990;Van Uffelen 1997). Spores of Lecanopteris show diversity, especially the cable-like filaments of L. mirabilis are distinct, and have not been reported in other species (Hennipman 1990;Tryon and Lugardon 1991). The four species of Lecanopteris studied differ from each other in their spore ornamentation. It would be important to explore this unusually labile nature of the ornamentation more in detail, and how it relates to the possible functional adaptation of spores (Tryon and Lugardon 1991). Genera Dendroconche and Zealandia have species found mostly in Oceania, with verrucate as the main spore ornamentation, except for D. linguiforme and Z. powellii. The former has globular spore surface, while the latter has psilate ornamentation (Fig. 4a). The position of Z. powellii varies, as it has been proposed to belong to both Microsoreae and Lecanoptereae (Chen et al. 2020;Nitta et al. 2018;Testo et al. 2019). In our analyses Z. powellii (sample from Solomon Islands) belongs to core Microsorum of Microsoreae, and its psilate ornamentation is similar to most species of core Microsorum also highlighting close relationship (Fig. 2 k1; Fig. 4a). However, this difference of position may also be caused by misidentification. The sequence data show differences between the specimens from Solomon Islands and Moorea respectively (Chen et al. 2020;Nitta et al. 2018). Further study is needed for reliable identification of these specimens and the type. In the same way, different ornamentations observed for the spores of M. scolopendria may be due to misidentification, specimens confused with M. grossum. Both species are morphologically similar and have overlapping ranges, with the former species can occur further north (Possley and Howell 2015).
In addition to core Microsorum, Microsoreae also includes Leptochilus, MG4 and MG5 clades (Chen et al. 2020). Of these four genera/clades, species of Leptochilus consistently have long spinose and globular elements as the main surface ornamentation (Fig. 1 g) of their spores, but the number of the spinose and globular elements differs between species. For example, L. pteropus and L. macrophyllus have more globular than spinose elements (Fig. 2 g3) (Tryon and Lugardon 1991, Figs. 116.3-4). Leptochilus pteropus has previously been placed in various genera (Microsorum, Kaulinia, and Colysis) based on the macromorphology (e.g., Bosman 1991;Fraser-Jenkins 2008;Nayar 1964;Nooteboom 1997). Leptochilus has recently been confirmed as the genus where this species belongs on the basis of molecular data , and our spore data are consistent with this placement. Unlike Leptochilus, the spore ornamentation of core Microsorum is mainly psilate with a few globular, and without spinose elements. The phylogenetic position of both MG4 and MG5 clades has been studied recently (Chen et al. 2020). Our results show a similar topology except for the location of M. hainanense, which is in MG4 clade in our study with weak support value. Unfortunately, spore data cannot differentiate the two clades. Species within both MG4 and MG5 clades have globular elements as surface ornamentation, with or without spinose elements. When spines are present they are smaller than those seen in Leptochilus (Figs. 1 h, 4b). Based on the spore data these two clades differ from Microsorum and Leptochilus.
Lepisoreae contains seven genera: Lemmaphyllum, Lepidomicrosorium, Lepisorus, Neocheiropteris, Neolepisorus, Paragramma, and Tricholepidium (Chen et al. 2020), with Lepisorus divided into ten subclades (Fig. 3a). The spore ornamentation is mainly rugulate and seems to be quite consistent in this tribe, with only a few species showing the other two types (Fig. 4a). For example, L. accedens has tuberculate spore surface and is located in the Lemmaphyllum, according to our study (Fig. 3), however, with only weak support value based on molecular data (aLRT = 4.5%/ aBayes = 0.57/UFBoot = 57.0%). The location of L. accedens differs from those found in previous studies (e.g., Chen et al. 2020;Zhao et al. 2019), this may be due to smaller sampled sizes in this study (Wei et al. 2017). The other species having tuberculate type are mixed with rugulate type, all of these can be found in the Lepisorus clade (Fig. 4a). Three species, Neocheiropteris palmatopedata, Lepisorus soulieanus (Christ) Ching and S.K. Wu and L. waltonii (Ching) S.L. Yu have a relatively smooth spore surface (Fig. 4a). The former is one of two species in the small genus Neocheiropteris (PPG I 2016), and the latter two species belong to clade II of Lepisorus. Another species of the clade II, L. clathratus, has slightly rugulate exospore (Fig. 2), and has been described also as psilate/smooth in some studies (Devi 1981;Kholia et al. 2012). Rugulate spore surface ornamentation is common in Lepisorus with different rugulate levels between subclades or species, these spore types are not a synapomorphy for this genus. Among ten subclades, species of the subclade X (i.e., L. accedens) have only tuberculate ornamentation; another nine subclades include rugulate plus tuberculate type in clades I, IV, VII, and VIII (e.g., Figure 2f4), those with slightly rugulate or psilate type are found in subclades II, III, and VI (e.g., Fig. 2 f1-f2), and subclades V and IX have moderately rugulate surface (e.g., Fig. 2 f3, f5). Descriptions of the spore surface of the species of Tricholepidium vary between different studies. The spore ornamentation of T. normale from Yunnan, China is psilate (Tryon and Lugardon 1991, under name Microsorum normale), or granulate (Wang 2001, under name T. angustifolium), but material from India shows baculate structure (Nayar and Devi 1964, under name Microsorum normale). The specimen we studied is from India, showing a rugulate surface (Fig. 2 j1). The subclades of Lepisorus and the genera of Lepisoreae, cannot be clearly distinguished based on their spore ornamentation. Zhao et al. (2019) recently treated species of Lepisoreae as Lepisorus sensu lato, and our observations of the spore ornamentation are not in conflict with this.
Classification of Goniophlebieae has varied in the past. It has either been treated as one genus (Kreier et al. 2008;PPG I 2016), or has been divided into several smaller genera, including Goniophlebium sensu stricto, Metapolypodium, Polypodiastrum, and Polypodiodes (Zhang et al. 2013). The spores of all species of Goniophlebieae have verrucate ornamentation, with or without the membraneous crest. Verrucate with membraneous crest is found in two subclades, one subclade contains G. persicifolium, G. pseudoconnatum, and G. subauriculatum, while another subclade contains G. argutum and G. mengtzeense (Fig. 4a). The former subclade belongs to Goniophlebium sensu stricto in the classification using small segregate genera, while the latter subclade belongs to Polypodiastrum, respectively.

Conclusions
Spore surface ornamentation has been shown to be informative and useful also for phylogenetic studies (Schneider et al. 2009). Here we explored spore ornamentation of the microsoroid ferns and its taxonomic value, and based on our analysis, the ancestral state of the microsoroids spores surface appears to be verrucate. This surface ornamentation can be found in the genera of Goniophlebieae and Lecanoptereae. For the tribes Microsoreae and Lepisoreae the ancestral states of the spore surface ornamentation seem to be with globular elements and rugulate, respectively. Spore surface ornamentation types generally seem to be congruent with the clades found in the phylogenetic analyses based on molecular data, and this character can be used to distinguish genera and tribes of the microsoroid ferns, or even species in some cases, such as Lecanopteris mirabilis. Tribe Lecanoptereae shows most diversity in spore surface ornamentation, with three of the five ornamentations, vermiculate-papillate, sheath-like, and cable-like filaments, unique in the microsoroid species. The latter two ornamentations types are found in particular in Lecanopteris. This diversity of spore ornamentations types might prove to be useful in studies exploring the possible functional adaptation of microsoroid spores.