Palynology of Amphidium Schimp . ( Amphidiaceae M . Stech ) : can spore morphology circumscribe the genus ?

Amphidium Schimp. is characterized by cushion-forming erect primary stems, linear-lanceolate leaves, and gymnostome capsules. The phylogenetic position of Amphidium is uncertain, with the genus having been variously included in Zygodontaceae Schimp., Rhabdoweisiaceae Limpr., Orthotrichaceae Arn. and Amphidiaceae M. Stech. A palynological investigation was performed of the three species of the genus that occur in the Americas: Amphidium lapponicum (Hedw.) Schimp., Amphidium mougeotii (Bruch & Schimp.) Schimp., and Amphidium tortuosum (Hornsch.) Cufod. Spores were observed and measured for greatest diameter under light microscopy both before and after acetolysis. Non-acetolyzed spores were observed under scanning electron microscopy to assess surface ornamentation of the sporoderm. All spores observed were smaller than 20 μm and heteropolar, with surface ornamentation reflecting a combination of different elements, such as gemmae, rugulae and perforations. The palynological characteristics observed here suggest that the genus Amphidium, and thus its contained species, be placed in their own family.


Introduction
The moss genus Amphidium comprises plants that grow as cushions and possess an erect primary stem; linear-lanceolate leaves with rounded and papillose cells; a longitudinally-furrowed sporophyte with gymnostomous capsules that are emergent or exserted, and a cucullate calyptra (Rooy 1992;Frahm et al. 2000).The species are generally rupicolous, growing directly upon rocks or in rock cracks (Rooy 1992;Frahm et al. 2000;Sim-Sim et al. 2017).
Amphidium lapponicum and Amphidium mougeotii were first described by Schimper (1856) as belonging to the family Zygondontaceae due to their papillose leaf cells and cucullate calyptra.Other studies have suggested alternate systematic positions for this genus and even its species circumscription.Brotherus (1924) included Amphidium in the subfamily Rhabdoweisioideae of Dicranaceae, which was followed by Walter (1983) and Allen (2002).Many authors have included Amphidium in Orthotrichaceae (Vitt 1984;1994;Rooy 1992;Vitt et al. 1993;Gradstein et al. 2001) due to similarities with Orthotrichum, particularly regarding the development of young capsules, as pointed out by Lewinsky (1976).Ochyra et al. (2003) suggested that species of Amphidium be placed in the subfamily Amphidioideae of Dicranaceae.
The present study aimed to investigate the palynology of the three species of Amphidium that occur in the Americas, as recognized by Frahm et al. (2000) -Amphidium lapponicum, Amphidium mougeotii, and Amphidium tortuosum -in order to evaluate the importance of spores to the taxonomic circumscription of Amphidum and to compare the spore morphology of this genus to that of other haplolepidous mosses.
Spores were observed under light microscopy (LM) and scanning electron microscopy (SEM).Spores for LM were prepared and analyzed following the methods of Wodehouse (1935) and Erdtman (1960), as modified by Luizi-Ponzo & Melhem (2006a) for bryophytes.Spores for SEM analysis were dispersed upon stubs covered with double-sided tape and coated with a 20-µm layer of gold.
Spore largest diameter was measured under LM with an ocular micrometer.A single sample was established as the standard for each species, while other samples were used for comparison.Fifty acetolyzed spores from three different microscope slides were measured for the standard material, while 30 acetolyzed spores from three different microscope slides were measured for the comparative material.Measurements were taken in polar view.Arithmetic mean (X), standard deviation (S), standard error (Sx), coefficient of variability/variation (CV %), and the 95 % confidence interval (CI), as well as minimum and maximum spore size (Xmin-Xmax), are reported for each of the analyzed samples.Mean polar diameter and equatorial diameter were obtained by measuring, always in equatorial view, 30 randomly selected acetolyzed spores.Since data for maximum diameter, polar diameter and equatorial diameter did not meet the assumptions of normality, they were analyzed using the non-parametric Kruskal Wallis test.Dunnet's a posteriori test was then used to test differences between standard and comparative material for each species and among standard material of all three species.Statistical analyses were performed in R version 3.4.2(2017-09-28) (R Core Team 2017).
Due to the thinness of the perine and exine, they were measured together as the sclerine (Luizi-Ponzo & Barth 1998;1999).Thus, the layers that compose the sporoderm -sclerine and intine -were measured from 10 randomly selected spores, which were prepared following the method of Wodehouse (1935) (with modifications by Luizi-Ponzo & Melhem 2006a), and arithmetic means calculated.Spore descriptions follow Punt et al. (2007) for terminology and Erdtman (1952) for spore size classes.

Results
The spores of species of Amphidium are small, heteropolar, isomorphic, and have a subcircular amb (Tab. 1, Fig. 1).The sporoderm comprises the intine and the sclerine (exine plus perine) and exhibits various ornamentation elements.These elements are similar to gemmae and are connected with each other and associated with thick rugulae on the proximal spore face; the sporoderm is perforated on the distal spore face (Fig. 2  ornamentation comprising connected gemmae associated with thick rugulae and a delicate sulcus.The three analyzed species of the genus Amphidium possess similar sporoderm ornamentation (Fig. 2), while they vary in morphometric characteristics (Tabs.1-4), as explained below.
The spores of Amphidium lapponicum had the lowest mean largest diameter (Tab.1).In addition, the mean largest diameters for the two comparative samples [Schmith s/n° (R 80442) and R. F. Cain 243 (CANM)] did not fall within the 95 % confidence interval of the standard material (Tab.1), but were not significantly different (p = 0.17 and p = 0.7, respectively).The mean polar diameter for the species was 9.54 µm while the mean equatorial diameter was 13.08 µm.
For spores of Amphidium mougeotii, the mean largest diameter of the comparative sample M. Lewis 88005 (SP) did not fall within the 95 % confidence interval of the standard material, but was not significantly different (p = 0.48).The mean polar diameter for the species was 10.74 µm while the mean equatorial diameter as 14.95 µm.The thicknesses of the sporoderm layers of this species were similar to that of the sporoderm of A. lapponicum (Tab.2).
Spores of the Amphidium tortuosum had the greatest largest diameter among the studied species (Tab.1).The largest diameter of the comparative sample W. J. Hoe 35070 did not fall within the 95 % confidence interval of the standard material, and was significantly different    (p < 0. 001) (Tab.1).The mean polar diameter for this species was 16.10 µm while the mean equatorial diameter was 18.54 µm.The intine of the sporoderm of A. tortuosum was found to be thinner than the strata (sclerine and intine) of the other species (Tab.2).

Discussion
Spore size for the genus Amphidium is unimodally distributed, and there is an absence of aborted spores.This, according to Mogensen (1981), is indicative of isomorphic spores.
The coefficient of variability found here for spore size is comparable to that reported by previous studies of bryophyte spores, which were around 10 % (Luizi-Ponzo & Barth 1998;Caldeira et al. 2009), due to the size amplitude observed in bryophyte spores.
Few studies have provided palynological observations for Amphidum, and those that did only provided superficial information on spore morphology.Hirohama (1977) studied the spores of a variety of different species including two species of Amphidium, using SEM and recognized distinct patterns of spore ornamentation among the genera analyzed.Gradstein et al. (2001) cited the spores of some species of Amphidium as being 'apparently ornate', while Allen (2002) described them as being 'slightly papillose'.van Rooy (1992) reported the spores of A. lapponicum and A. tortuosum as being small, 'sub-rounded', and with largest diameters of around 11-15 µm and 12-22 µm, respectively, which are consistent with the results of the present study.
Sim-Sim et al. (2017) described the spores of Amphidium asiaticum, and reported a largest diameter of 14 µm, which is smaller than that found for the species of the present study.Sim-Sim et al. (2017) also reported A. asiaticum as having a smooth sporoderm, however, this may be a result of their observations being restricted to LM, which precludes a more complete sporoderm analysis.
The ornamentation observed for the spores of the species of the present study was found to be similar to that reported for Zygodon obtusifolius (Luizi-Ponzo 2001), a species of Orthotrichaceae, which is a diplolepidous moss lineage (Goffinet et al. 1998).Molecular analyses have suggested that the group formed by Orthotrichales, Splachnales and Bryales sensu lato is monophyletic and the sister-group of haplolepidous mosses (Goffinet et al. 1998).
Thus, the palynological descriptions presented here provide important information for future studies dealing with the phylogeny of haplolepidous mosses, which to date have remained inconclusive about the placement of Amphidium (Farge et al. 2002;Tsubota et al. 2003;Hedderson et al. 2004;Stech 2004;Stech et al. 2012).
Our results support the taxonomical circumscription proposed by Stech and Frey (2008), who proposed the family Amphidiaceae to segregate Amphidium.

Conclusion
Species of Amphidium possess palynological characters that distinguish them from other taxa of Rhabdoweisiaceae, and from the spores of other species of the haplolepidous lineage.The spores are heteropolar with an apertural area on the proximal face that is clearly distinguishable by its remarkable ornamentation: connected gemmae associated with thick rugulae.The distal face, on the other hand, possesses a perforated sporoderm.The results of the present palynological study support the segregation of Amphidium in its own family.

Figure 1 .
Figure 1.Line graphs representing the size distribution of the spores of the studied species of Amphidium Schimp.

Table 1 .
Morphometric data of largest diameter of the spores from Amphidium Schimp.species.Data presented in micrometers (*standard material; other data: comparison material).X min -X max = Sample minimum and maximum values; X = arithmetic mean; S X = standard deviation; S = standard error; CI = confidence interval; VC = variation coefficient.

Table 2 .
Mean values of sporoderm layers thickness (in µm) of spores of Amphidium Schimp., and mean size value of aperture area (in µm) (*standard material).

Table 3 .
P values resulting from the Dunnet test for the

Table 4 .
P values resulting from the Dunnet test for the largest diameter of the three Amphidium Shimp.species analyzed