﻿Re-assessment of type material of Plagiotheciumnovae-seelandiae Broth. and descriptions of four new Plagiothecium taxa (Bryophyta, Plagiotheciaceae) from Australasia

﻿Abstract A re-examination of the original collection of Plagiotheciumnovae-seelandiae described by Brotherus in 1916 indicated that this material is not homogeneous. Re-examination of the diagnosis of this species and morphological analysis supports that two separate taxa should be distinguished – Plagiotheciumnovae-seelandiaevar.novae-seelandiae and P.novae-seelandiaevar.brotherivar. nov. Also, comparisons with the original collection of Hypnumlamprostachys (= P.lamprostachys) showed differences, which supported their treatment as separate taxa. Revision of the genus Plagiothecium from Australasia (CANB, CHR, HO, MEL, WELT) and types of other species described from this part of the world (P.funale and P.lucidum) supported by the study of their diagnoses, qualitative and quantitative characteristics as well as mathematical analyses (PCA, HCA) allowed the division of the examined material into six separate groups – six separate taxa. Thereby, three distinct taxa are proposed – P.cordatumsp. nov., P.semimortuumsp. nov., and P.semimortuumvar.macquariensevar. nov. All taxa mentioned above are described in detail, their current known distribution and ecological preferences are also included. In addition, images illustrating their most important taxonomic features, as well as an original key to distinguish individual taxa are presented.


Introduction
In terms of species richness within the genus Plagiothecium Schimp., Australasia, comprised of Australia and New Zealand (Deverson 2005), is the most depauperate region in the world.Since the beginning of bryological research in this region of the world, only eleven names related to the described genus have a result of relatively few revisions and the many morphological complexities associated with the genus (Ireland 1992;Ochyra 2002).Moreover, this fact may probably be influenced by geology, geography, biotic and abiotic factors, so it is difficult to assess this fact at this stage.
In the first decades of the 20 th century, the perception of the genus Plagiothecium in Australasia was greatly influenced by the publications of Dixon (1929) who recognized P. denticulatum and P. novae-seelandiae for New Zealand.At the same time, he treated P. lamprostachys as a synonym of P. denticulatum and in relation to the latter indicated that "the (...) status of P. novae-seelandiae therefore, is open to question."Probably for this reason, in this part of the world P. lamprostachys was forgotten for many decades, and in later studies P. novae-seelandiae was reduced to a synonym of P. denticulatum (e.g., Sainsbury 1955).Thus, for the ensuing years, P. denticulatum was reported as the only representative of this genus (e.g., Sainsbury 1955;Scott and Stone 1976;Ramsay 1984;Streimann and Curnow 1989;Beever et al. 1992) for Australasia.However, Sainsbury (1955) did indicate the remarkable variability of this taxon.By the end of the 20 th century Plagiothecium laetum Schimp.was also recognized for the area (Vitt 1974), but later the presence of this typical Northern Hemisphere taxon was questioned by Fife (2019), and subsequently excluded from the flora of Australasia.
The end of the 20 th century sees the revision by Ireland (1992), which shed new light on the perception of the genus Plagiothecium in Australasia.This researcher stated that there is a significant difference between P. denticulatum and P. novae-seelandiae, proposing the resurrection of the latter, as a separate species and deletion of P. denticulatum from the moss flora of Australasia.Additionally, Ireland (1992) published the first occurrence of Plagiothecium lucidum (Hook.f.& Wilson) Paris from Australia and New Zealand.Ten years later, the presence of P. lucidum in Australasia was confirmed by Ochyra et al. (2000).
The beginning of the 21 st century brings Ochyra's (2002) publication in which he indicated that plants in the original collections of P. lamprostachys, in terms of leaf shape and general habit, match perfectly the type collections of P. novae-seelandiae.Thus, he proposed to synonymize the latter with P. lamprostachys (Ochyra 2002).This point of view was almost immediately adopted by all Australasian bryologists (e.g., Streimann and Klazenga 2002;Klazenga 2012;Seppelt et al. 2013;Fife 2019).Streimann and Klazenga (2002), like Ireland (1992) and Ochyra et al. (2000), also additionally reported P. lucidum.A few years later appears a review by Wynns (2015) and Wynns et al. (2018).Wynns (2015) not only understood P. lamprostachys and P. novae-seelandiae as separate taxa, but also described two new species from this area in his Ph.D. thesis -P.funale J.T.Wynns and P. humile J.T.Wynns.However, in a later study (Wynns et al. 2018), P. humile was no longer distinguished and was omitted.We have been unable to find material which formed the basis of P. humile, nom.inval.
The complicated taxonomic history and relatively small number of species was the impetus to provide a revision of the genus for Australasia with the aim of testing the assumptions and taxonomic concepts presented by previous researchers.

Materials and methods
All collections of the genus Plagiothecium deposited in CANB, CHR, HO, MEL, and WELT-almost 400 specimens -were examined.After the revision, only those specimens with symmetrical leaves were selected for further analysis.
Selected specimens were used not only for mathematical analyses, but also for the description of new taxa.The mathematical analyses were performed mainly on nomenclatural types of taxa previously known from Australasia and the similar but later-named P. semimortuum (Figs 1,2).
The selection of features for the following study was made on the basis of methodology adopted by Wolski (2017Wolski ( , 2019) ) and Wolski and Nowicka-Krawczyk (2020).Thus, the features include not only the most taxonomically important ones, but also other characteristics basic to the description of individual taxa: qualitative and quantitative features of gametophytes and sporophytes of the examined plants.Therefore, color, luster, and habit were tested first.From a uniform turf, one stem was chosen; the length of the whole stem was measured, and the arrangement of the leaves on the stem was evaluated.
Then, all the leaves were torn off from the central part of the stem, and six leaves were randomly selected for further measurements.For each of the examined leaves, the shape, symmetry, folding, and concavity were evaluated.They were also measured in terms of the length and the width at their widest points and the length of both costae.Additionally, the shape, curvature, and serration of the leaf apex were observed.
For each of the selected leaves, five groups of cells were measured: from the upper, the middle and the lower part of the leaf.Laminal cell shape was assessed, additionally, alar cells were measured, and their shape was assessed.Decurrent leaf base cells were measured, and the number of rows of cells was counted.The cross-section was taken from the central part of the stem and six cross-sections of the stems were randomly chosen.First, the diameter of the obtained stem cross-section was measured, then five epidermal cells and five parenchymal cells were randomly selected.
In addition, the length of the sporophyte was assessed, color of the seta, length and width of the capsules, its arrangement on the seta, shape and length of operculum -of course only if these elements were present in the material.Similarly, in the case of other features -they were omitted from the description when a given element was not present or the feature was impossible to determine.Due to the poor condition of specimens, this situation occurred in the case of some gametophytic features of P. lamprostachys.Moreover, sporophytes were missing for P. novae-seelandiae var.brotheri (PC0132644, CHR534780), P. cordatum (CHR538916) and P. semimortuum var.macquariense (HO610220).
All research in the presented manuscript was based on our own macroscopic and microscopic analysis of herbarium collections.Only in one case, and only for the purposes of the cluster analyses (Figs 1, 2), was data used based on P. funale literature (Wynns 2015;Wynns et al. 2018).However, when describing this species, only data collected from the analysis of herbarium specimens were taken into account (MO2408073, CHR267040).
On the basis of features recognized in the literature as the most taxonomically important -length and width of leaf, length and width of cells from midleaf (e.g., Wolski and Nowicka-Krawczyk 2020;Wolski et al. 2021) -grouping analyses of the studied taxa were carried out.Due to the incommensurability of the data (length and width of leaf to length and width leaf cell), Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were used to arrange the points in the ordering space.
These analyses are a basic tool that allows for grouping the examined specimens and thus showing the similarity between them.All mathematical analyses were performed in the PQSTAT v. 1.8.6 program.All other above-mentioned features considered representative of this genus were used to describe individual taxa (e.g., Wolski 2017Wolski , 2019;;Wolski, Nowicka-Krawczyk 2020;Wolski et al. 2022a, b).
Taking into account the above facts, it can be indicated that plants with complanate-foliate, asymmetric leaves, serrate apices, wide cells, making the cell areolation loose refer to P. novae-seelandiae which was described by Brotherus (1916) (Fig. 4).The second morphotype, refers to the new variety proposed here -P.novae-seelandiae var.brotheri (Fig. 5).
Narrow decurrencies are a feature that distinguishes Plagiothecium funale from other taxa of this genus with wide decurrencies, encompassing all those taxa currently known from Australasia.However, the analysis also indicated the presence of another taxon with narrow decurrencies, distinguished by julaceous stems, short and narrow (1.7-2.0 × 0.7-0.9mm), longitudinally folded, concave, lanceolate, symmetric leaves with heart-shaped leaf bases, entire, non-serrate leaf apices, and long and narrow cells (140-165 × 5-7 µm), making the cell areolation tight.Specimens with such features (Fig. 7) we propose to call Plagiothecium cordatum sp.nov.
The last two taxa are plants with a unique set of gametophytic qualitative and quantitative features (Figs 8,9).The unique feature, otherwise not found among taxa of this genus, is the absence of protoplast in the upper part of the leaf at maturity.

Discussion
The genus Plagiothecium in Australasia has been misunderstood, and perceptions have changed considerably.First, practically all specimens from this part of the world were identified as P. denticulatum, then P. novae-seelandiae, and later P. lamprostachys.Thus, a single taxon name was replaced by successive names without a careful and detailed revision of the group (e.g., Mitten 1856Mitten , 1882;;Wilson 1859;Brotherus 1916;Ireland 1992;Ochyra 2002).This is one of the reasons for the low number of taxa reported so far from Australasia.The second important contributing factor to the low number of taxa was the relatively small number of studies on this genus (Ireland 1992;Ochyra 2002;Wynns 2015;Wynns et al. 2018).Thus, for such a huge and diverse continent, the number of taxa recorded until the beginning of the 21 st century is extremely low, especially in comparison with other parts of the world, not just the relatively well-studied Northern Hemisphere (e.g., Nyholm 1965;Buck and Ireland 1989;Smith 2001;Wolski et al. 2021).Dixon (1929) was a big influence on the perception of this genus, for example, his synonymization of P. lamprostachys with P. denticulatum led to the loss of the concept of that species for decades.A similar influence was Sainsbury (1955), who synonymized P. novae-seelandiae with the aforementioned P. denticulatum.On the other hand, the confusion of Australian and New Zealand specimens with P. denticulatum is not so surprising.Because both the habit and the most important microscopic features (e.g., serrate leaf apex, loose areolation of cells, distinct decurrency composed of inflated cells) resemble this most common Northern Hemisphere taxon (Wolski et al. 2021).However, as in many other cases (e.g., Plagiothecium schofieldii G.J.Wolski & W.R. Buck and P. lamprostachys), despite the morphological similarity, geographical and molecular differences between them are indisputable (Wynns et al. 2018;Wolski et al. 2021), as are subtle morphological differences.
Although P. denticulatum has been reported from Australasia for decades, Ireland (1992) rightly excluded it from the local moss flora.As in the case of P. denticulatum, the same was with P. laetum (Vitt 1974).This species was most likely confused by Vitt with the similar P. lucidum, in terms of leaf symmetry and cell dimensions.However, as in the case of P. denticulatum, we now know that P. laetum is a taxon occurring only in the Northern Hemisphere, and thus was excluded from Australasia by Fife (2019).Ochyra (2002) indicated that the types of P. lamprostachys match perfectly to the type collections of P. novae-seelandiae.This synonymization further influenced the understanding and perception of this genus in Australasia.Current research indicates that indeed one of the morphotypes of P. novae-seelandiae is similar to P. lamprostachys, but they are not identical, which is indicated by the statistical analyses.Treating these species separately was already proposed by Wynns (2015), and it is supported here.
Interestingly, none of the earlier researchers (e.g., Ireland 1992; Ochyra 2002; Fife 2019) indicated that the original collection of P. novae-seelandiae consisted of two separate morphotypes.Only Wynns (2015) mentioned it, but generally ignored the issue.However, even a cursory analysis signifies that the two previously mentioned morphotypes of P. novae-seelandiae differ in many important taxonomic features -e.g., habit, foliage, symmetry, concavity of leaf, serration of apex, cell dimensions.These differences are supported by the statistical analyses presented above.Morphological studies combined with the analysis of the diagnosis indicate that the taxon mentioned by Brotherus (1916) is material characterized by, for example, complanate-foliate habit, domination of asymmetrical leaves, serrate leaf apices, wide cells, which makes the cell areolation loose (100-130 × 12-17 µm).Specimens with such a set of features were named Plagiothecium novae-seelandiae [var.novae-seelandiae] (CHR534781!, CHR534780p.p.!, PC0132644p.p.! PC0132645!, PC0132646!).But, the morphotype with julaceous stems, dominance of symmetrical leaves, entire, non-serrate apices, narrow cells, which makes the cell areolation tight (100-140 × 7.5-10 µm) is here named Plagiothecium novae-seelandiae var.brotheri.Wynns (2015), in his doctoral thesis, described two new species from Australasia -Plagiothecium funale and P. humile.However, in his publication based on his thesis (Wynns et al. 2018), he does not mention the latter.On the other hand, the former -P.funale can be distinguished easily from the other taxa recorded currently from Australasia by asymmetrical, concave, undulate leaves, short and smooth apex, long and narrow cells and wedge-shaped, and narrow decurrencies composed of rectangular cells.The last feature -wedge-shaped decurrencies, composed of rectangular, non-inflated cells -is a very important and the most unique feature compared to other species.Decurrent angular rounded cells forming distinct auricles are characteristic of all previous species (e.g., Sainsbury 1955;Scott and Stone 1976;Ireland 1992;Fife 2019;Ochyra 2002).
In the genus Plagiothecium, the decurrency is one of the most important taxonomic features (Wolski et al. 2021).Although this feature plays a fundamental role in the division of individual taxa into sections of this genus, unfortunately it is often overlooked when analyzing material -therefore, it is always necessary to analyze these structures, which in the case of this genus often remain on the stem after dissection.Without checking the decurrency, it is very easy to confuse some even distantly related species that are similar in some respects, e.g., P. denticulatum and P. nemorale (Mitt.)A.Jaeger (e.g., Wolski and Nowicka-Krawczyk 2020;Wolski et al. 2021).
Plagiothecium cordatum, like P. funale, is characterized by a unique set of gametophyte features, including, and most importantly, a wedge-shaped decurrency composed of uninflated cells (Wynns 2015;Wynns et al. 2018).This feature distinguishes these taxa from other taxa in this part of the world.The other characteristics, e.g., julaceous stem, short and narrow (1.7-2.0 × 0.7-0.9mm), concave, clearly and strongly folded leaves with a heart-shaped base, entire, non-serrate leaf apices, and long and narrow cells (140-165 × 5-7 µm) make it quite easy to distinguish P. cordatum from P. funale.
This research has also allowed the description of Plagiothecium semimortuum var.semimortuum and P. semimortuum var.macquariense.Both have a unique feature not found in any other species of the genus.The leaf cells are devoid of protoplasts occupying as much as 2/3 of the leaf length.The absence of the protoplasts in part of the leaf is unusual for the genus Plagiothecium (Smith 2001;Wolski et al. 2021), but not for some types of mosses.Many taxa, especially those growing in open, sunny habitats, are characterized by a lack of protoplasts in the leaf or part of the leaf, e.g., Bryum argenteum Hedw., Gigaspermum mouretii Corb., Orthotrichum diaphanum Brid., Polytrichum piliferum Hedw., Tortula muralis Hedw.and others (e.g., Noguchi 1995;Smith 2001).
Despite some similarities, P. semimortuum var.semimortuum and P. semimortuum var.macquariense differ in a number of qualitative and quantitative gametophytic features: the size and folding of the leaf, the serration of the leaf apex, the dimensions of the cells, but also the habitat -mountains versus lowlands.All these features confirm the validity of distinguishing the above-mentioned taxa.Description.Plants medium size, yellowish to yellow-green, with metallic luster, forming dense mats; stems 1.5-2.5 cm long, in cross-section rounded, the central strand well-developed; leaves asymmetrical to almost asymmetrical, ovate, concave, rather imbricate and closely arranged on the stem, those leaves from the middle of stem 2.5-2.6 mm long and the width measured at the widest point 1.1-1.2mm (Fig. 3); the apex acute, entire, not denticulate;  Description.Plants medium size, green, with metallic luster, forming rather dense mats, complanate-foliate; stems 4-6 cm long, in cross-section rounded, 300-350 μm in diameter, the central strand well-developed; leaves asymmetrical, not overlapping on the stem to slightly imbricate, rather flat to undulate, sometimes one side of the leaf flat or folded over the rest of the leaf, leaves from the middle of stem 1.7-2.2μm long and the width measured at the widest point 1.0-1.5 mm; the apex acute and denticulate; costae two, rather thick and strong, extending usually to ½ of the leaf length; laminal cells more or less symmetrical, the length and width variable but dependent on location: 110-140 × 10 μm at apex, 100-130 × 12-17 μm at midleaf, and 75-150 × 17.5-20 μm toward insertion; due to the wide cells, cell areolation loose; decurrency of 3-5 rows of rounded and inflated cells, forming distinct auricles, 200 μm long.Sporophytes 2.5-4.0 cm long, setae reddish-orange; capsules horizontal, 1.7-2.8× 0.7-1.0mm (Fig. 4); sexual condition unknown.
Etymology.The name of this taxon -Plagiothecium cordatum refers to the heart-shaped (Latin: cor -heart) base of leaves of this species.
Etymology.The name of this species -Plagiothecium semimortuum (Latin: semi -half; mortum -dead) refers to the leaves without protoplasts; they are dead even up to half the leaf.
Etymology.The name of this variety -Plagiothecium semimortuum var.macquariense -refers to Macquarie Island (Australia, Tasmania), from which the plant was first recorded, and where the holotype (HO610220) was collected.

Figure 3 .
Figure 3. Selected taxonomic features of P. lamprostachys A shape and dimensions of the leaf B leaf apex C cells from the middle part of the leaf D leaf basal cells (from the type of material of H. lamprostachys BM000677528!, photo.G. J. Wolski, 01 August 2023).

Figure 4 .
Figure 4. Selected taxonomic features of Plagiothecium novae-seelandiae var.novae-seelandiae A shape and dimensions of leaves B serrate leaf apex C dimensions and shape of cells from middle part of the leaf D decurrency (from the type material of P. novae-seelandiae PC0132644p.p.!, photo.G. J. Wolski, November 21, 2021).

Figure 5 .
Figure 5. Selected taxonomic features of Plagiothecium novae-seelandiae var.brotheri A shape and dimensions of leaf B dimensions and shape of cells from middle part of the leaf C stem cross-section D decurrency (from the type material of P. novae-seelandiae PC0132644p.p.!, photo.G. J. Wolski, November 22, 2021).

Figure 6 .
Figure 6.Selected taxonomic features of Plagiothecium funale A, B shape and dimensions of leaves C folding of the apex of the leaf D dimensions and shape of cells from middle part of the leaf E narrow decurrency composed of rectangular cells (from the type of material of P. funale CHR267040!, MO2408073!, photo.G. J. Wolski, November 2022 and July 2023).

Figure 7 .
Figure 7. Selected taxonomic features of Plagiothecium cordatum A, B shape and dimensions of leaves C leaf apex D dimensions and shape of cells from middle part of the leaf (from the type of material of P. cordatum CHR538916!, photo.G. J. Wolski, November 12, 2022).

Figure 8 .
Figure 8. Selected taxonomic features of Plagiothecium semimortuum var.semimortuum A, B shape and dimensions of leaves C decurrency on the stem D dimensions and shape of cells from middle part of the leaf (from the type of material of Plagiothecium semimortuum var.semimortuum MEL1016042 and WELT-M28128, photo.G. J. Wolski, November 13, 2022).

Figure 9 .
Figure 9. Selected taxonomic features of Plagiothecium semimortuum var.macquariense A, B shape and dimensions of leaf C julaceous turf D dimensions and shape of cells from middle part of the leaf (from the type of P. semimortuum var.macquariense HO610220, photo.G. J. Wolski, November 13, 2022).

Figure 12 .
Figure 12.Selected taxonomic features of Plagiothecium lucidum A shape and dimensions of leaf B leaf apex C shape and arrangement of capsule (from the type of P. lucidum PC0132689!, PC0132690!, photo.G. J. Wolski, November 18, 2021).