Oncophorus demetrii, a fifth Scandinavian species of Oncophorus (Musci) possible to recognize by morphology

One of the three lineages found within Oncophorus wahlenbergii Brid. in a recent revision of Scandinavian Oncophorus is explored in detail. Consistent and well-supported nuclear ITS and plastid trnG and rps4 signals for a total of eight specimens belonging to this lineage, and the possibility to recognize its members by morphology, support recognition of Oncophorus demetrii (Renauld & Cardot) Hedenäs, comb. nov. (Dicranum demetrii Renauld & Cardot). This species differs from members of the other two O. wahlenbergii lineages by its more longly and narrowly acuminate leaves, projecting cell walls in the upper leaf margins, and the excurrent costa being rough from projecting cells or cell walls. Contrary to members of the other two O. wahlenbergii lineages, O. demetrii seems to prefer base-rich habitats. Outside Scandinavia, it is at present known from Labrador in Canada. Oncophorus compactus (Bruch & Schimp.) Kindb. is considered a synonym of O. wahlenbergii since Dicranum homannii Boeck, a synonym of O. wahlenbergii, was cited as a synonym of Dicranum virens var. compactum Bruch & Schimp. when the latter was described.

One of the three lineages found within Oncophorus wahlenbergii Brid. in a recent revision of Scandinavian Oncophorus is explored in detail. Consistent and well-supported nuclear ITS and plastid trnG and rps4 signals for a total of eight specimens belonging to this lineage, and the possibility to recognize its members by morphology, support recognition of Oncophorus demetrii (Renauld & Cardot) Hedenäs, comb. nov. (Dicranum demetrii Renauld & Cardot). This species differs from members of the other two O. wahlenbergii lineages by its more longly and narrowly acuminate leaves, projecting cell walls in the upper leaf margins, and the excurrent costa being rough from projecting cells or cell walls. Contrary to members of the other two O. wahlenbergii lineages, O. demetrii seems to prefer base-rich habitats. Outside Scandinavia, it is at present known from Labrador in Canada. Oncophorus compactus (Bruch & Schimp.) Kindb. is considered a synonym of O. wahlenbergii since Dicranum homannii Boeck, a synonym of O. wahlenbergii, was cited as a synonym of Dicranum virens var. compactum Bruch & Schimp. when the latter was described.
The predominantly northern temperate genus Oncophorus (Dicranales: Oncophoraceae; Frey and Stech 2009, Goffinet et al. 2008, Stech and Frey 2008 was until recently supposedly well-known (Frahm et al. 1998). However, as shown by Hedderson and Blockeel (2006) and Hedenäs (2005), and especially in a recent review of the genus in Scandinavia based on both molecular and morphological information (Hedenäs 2017), the genus has actually been poorly understood. Four Scandinavian species that can be distinguished by morphology were accepted by Hedenäs (2017) Hedenäs (2017) and further molecular data for such specimens are therefore required to decide how to treat them. As a follow-up to Hedenäs (2017), five specimens having a morphology similar to that of O. wahlenbergii B are here selected to test the idea that a fifth species could potentially be recognizable also by morphology. All the additional specimens turned out to belong to the same molecular lineage as O. wahlenbergii B of Hedenäs (2017), confirming that this lineage can be recognized by morphology and that it should be recognized as a fifth, morphologically recognizable Scandinavian Oncophorus species. Among the numerous old names existing for members of the Oncophoraceae and similar-looking Dicranaceae one species level taxon that is identical with O. wahlenbergii B, was discovered, Dicranum demetrii Renauld & Cardot. According to Van der Wijk et al. (1962), this taxon is a synonym of O. wahlenbergii Brid. (s.l.), but as it agrees with Oncophorus wahlenbergii B of Hedenäs (2017) the latter is from here on called Oncophorus demetrii (Renauld & Cardot) Hedenäs. This paper presents the results of the molecular evaluation, the species' morphological circumscription, and describes its Scandinavian distribution and habitat.

Study species and material
Five specimens of Oncohorus demetrii were selected based on morphological criteria used in the key to the Scandinavian Oncophorus species in the Taxonomy section. Sequences generated for these specimens were added to those of the 100 Oncophorus specimens included in the earlier study (Hedenäs 2017), using Cynodontium strumiferum (Hedw.) Lindb. and Rhabdoweisia fugax (Hedw.) Bruch & Schimp. as outgroup based on their appearance in a clade sister to Oncophorus in Stech et al. (2012). The newly added specimens are listed in Table 1.
The subsequent morphological analysis was based on the eight specimens of O. demetrii that were included in the molecular analysis, whereas the geographical distribution of this species was mapped based on the Scandinavian material present in the Swedish Museum of Natural History (S).
Detailed label information for these specimens is available at  http://herbarium.nrm.se/ .

Molecular methods
The molecular methods employed to generate sequences of the nuclear internal transcribed spacers 1 and 2 (ITS) and the plastid trnG UCC G2 intron (trnG) and rps4 gene + trnS-rps4 spacer (rps4) are the same as in Hedenäs (2017), and the reader is referred to that paper for further information.

Sequence editing and analysis of molecular data
Nucleotide sequence fragments were edited and assembled for each DNA region using PhyDE 0.9971 ( www.phyde. de/index.html ). The assembled sequences were manually aligned in PhyDE. Regions of partially incomplete data in the beginning and end of the sequences were identified and excluded from subsequent analyses. Gaps were coded as informative by simple indel coding (Simmons and Ochoterena 2000), using SeqState (Müller 2005). The sequence alignments used in the analyses are available on request. European Nucleotide Archive (EMBL-ENA) accession numbers for the earlier sequences are found in Hedenäs (2017) and GenBank accession numbers for the newly generated ones are listed in Table 1. Locality information for specimens (P numbers) are provided in Hedenäs (2017) and Table 1. Paralogous ITS haplotypes are occasionally encountered in bryophytes (but see Košnar et al. 2012). However, the ITS chromatograms generated in this study did not show 'messy' patterns or noise that could suggest paralogy, and the 5.8S gene was invariable among the samples (cf. Shaw et al. 2002, Feliner andRosselló 2007). Therefore, the revealed ITS variation is interpreted as being among homologous haplotypes.
The program TCS (Clement et al. 2000) was used to evaluate relationships among specimens in a haplotype context. Reticulation was revealed in the haplotype networks based on either ITS or chloroplast data. Because reticulation occurs, a split network was computed with the NeighborNet (NN) method as implemented in SplitsTree ver. 4.12.6 (Huson and Bryant 2006) to visualise similarities or relationships among samples. A Jacknife analysis (1000 replications) was performed with the program TNT (Goloboff et al. 2003) to test whether supported lineages exist among the studied Oncophorus species in a phylogenetic tree context. ITS and chloroplast data were analysed separately, since Hedenäs (2017) found the two data sets to be incongruent.

Morphological study and analysis of measurements
The morphology of the five newly added O. demetrii specimens was studied as described by Hedenäs (2017), including standard comparisons of qualitative and quantitative characters and the quantification of vegetative leaf size, and leaf cell size and shape. For each specimen, three vegetative leaves were sampled from two shoots (two leaves from one stem and one from the other). For each leaf, length and maximal width were measured, and the length, width, and length to width ratio of 20 cells in the upper acumen, 20 in the lower acumen, and 20 in the sheathing basal lamina were recorded. Temporary images of the leaves were taken through an Olympus BX43 microscope using an Olympus SC50 digital camera and the Olympus cellSens Standard ver. 1.13 software (Olympus Corporation) for automatic and continuous image stacking. Measurements were made from these leaf and cell images, using the Olympus cellSens Standard ver. 1.13 software. In this way, measurements of all eight specimens of molecularly confirmed O. demetrii are now available.
Comparisons among O. elongatus, 'O. wahlenbergii A plus C' (two morphologically indistinguishable lineages), and O. demetrii (cf. Hedenäs 2017) are based on two approaches. First, the cell measurements were compared. Shapiro Wilks W-test (normality) and Levenes test (homogeneity of variance) were both statistically significant, and inspection of the distributions of residuals in preliminary Anovas showed that the data do not meet the criteria of normality and homogeneity of variance. Thus, the nonparametric Kruskal-Wallis test was employed to compare the cell measurements among the three. Second, the measurements of the individual leaves (length, width, and the mean cell length, cell width, and cell length to width ratio, at each of the three positions in the leaf; in total 11 parameters) were subjected to a principal component analysis (PCA)

Relationships among Oncophorus wahlenbergii s.l. specimens
The five newly sequenced specimens of Oncophorus demetrii group with the three that had been studied earlier, both based on ITS and plastid data (Fig. 1B, D). In the ITS analysis they form a clade with a Jacknife support of 99, sister to O. wahlenbergii A, in a clade with Jacknife support of 100 (Fig. 1B). ITS data place O. wahlenbergii C in a different branch (cf. Fig. 1A). In the plastid analysis all three O. wahlenbergii entities are found together (Jacknife support: 100), but the three form three separate and well-supported lineages (Jacknife support: 94, 98, 98), and within this main  (Fig. 2B). Even if there are differences between some or all species and we find a relatively clear separation in the PCA, the species overlap considerably in all the measured characters (Fig. 3). 2. Lamina of basal, sheathing leaf portion in its middle and lower portions with long and narrow cells along margin; margin and often back of costa in upper (10)25-35(50)% of many leaves denticulate or dentate, often sharply so, rarely with only a few leaves having scattered obtuse teeth above. Vegetative leaves 2.5-6.4  0.4-1.2 mm O. elongatus -. Lamina of basal, sheathing leaf portion with quadrate to elongate-rectangular cells extending down along margin (from spreading lamina); margin and back of costa smooth or with one or a few indistinct and mostly obtuse teeth, especially close to apex, or with projecting cell walls along upper margin and costa rough on back from projecting cells or cell walls. Vegetative leaves 1.4-4.8  0.3-0.8 mm 3 3. Leaf margin entire or with an occasional indistinct denticle near apex, if cell walls slightly projecting as obtuse denticles in upper margin, then excurrent costa smooth on back; leaf apex obtuse to narrowly acuminate O. wahlenbergii -. Leaf margin above often with one or a few indistinct and mostly obtuse teeth, especially close to apex, in many or all leaves cell walls distinctly projecting in upper 5-25% of leaf margin, and excurrent costa rough from projecting cells or cell walls; leaf apex narrowly acuminate or occasionally acuminate O. demetrii 4. Leaf margin in acumen and often down to mid-leaf regularly to irregularly dentate or coarsely denticulate with mostly sharp and often some double teeth, strong teeth often directed forwards, occasional leaves less distinctly dentate. Dry leaves with acumen from above sheathing base erect and tightly incurved to spreading, above strongly twisted O. virens -. Leaf margin in acumen entire or indistinctly and obtusely denticulate, occasionally distinctly denticulate close to leaf apex. Dry leaves with acumen from above sheathing base erect-patent to spreading and loosely incurved or curved upwards, upper acumen loosely but relatively strongly Plants from a few mm to several cm high, in loose tufts or dense cushions, green or yellow-green. Stem with large central strand, a cortex plus epidermis of 12 layers of incrassate cells, epidermis not differentiated as a hyalodermis; axillary hairs with 27(8)-celled, hyaline upper portion, 69 µm wide, basal 12 cells rectangular or elongate-rectangular, hyaline or pale brown; rhizoids strongly branched, red-brown, smooth, in leaf axils or shortly above. Leaves 2.6-4.8  0.4-0.8 mm, when moist from sheathing base almost erect to patent or sometimes spreading and straight or slightly curved, when dry with middle leaf spreading and gradually curved upwards-inwards and more or less curled, from oblong or slightly oblong-obovate sheathing portion (often narrowed towards insertion) with gradually narrowed, usually long and narrow upper portion, apex acuminate or mostly narrowly acuminate; leaf margin plane throughout, below entire, above often with one or a few indistinct, mostly obtuse teeth, especially close to leaf apex, cell walls projecting along upper 5-25% of margin, margin unistratose or partly bistratose; costa 39-88 µm wide near base, excurrent, with dorsal and ventral epidermis cells slightly widened but incrassate, one layer of large guide cells, 1-2 layers of ventral stereids and 1-2 layers of dorsal stereids, upper back and often sides of excurrent costa rough from projecting cell walls or cell portions, at least close to apex; lamina cells in acumen incrassate, 10-20  9-12 µm, 0.9-2.0 times as long as wide, in mid-leaf incrassate, 15-27  9-13 µm, 1.6-2.8 times as long as wide, and in sheathing lamina strongly incrassate and porose or indistinctly so, rarely eporose, 44-84  7-12 µm, 4.8-10.2 times as long as wide, transition between mid-leaf and basal cells gradual, due to relatively long lower mid-leaf cells, lamina of basal, sheathing portion of leaf with quadrate or rectangular cells extending down along margin from spreading lamina; alar cells undifferentiated or a few cells wider than other basal cells, unistratose, not or shortly and narrowly decurrent. Perigonia lateral on stem, not or shortly stalked, antheridia protected by oblong-triangular perigonial leaves, sometimes with broad acumen to as long as basal leaf, apex obtuse or acute. Inner perichaetial leaves 3.57-4.68 mm long, lower (25)30-50% oblong and broadly sheathing, above suddenly narrowed to long, narrow acumen. Calyptra cucullate, smooth, naked. Seta tall, 8-22 mm, yellowish when young, orange-red when mature; capsule obloid or cylindrical, curved or slightly curved, rarely almost straight, with distinct struma, when dry 1.0-1.4  0.3-0.5 mm, 2.0-3.3 times as long as broad, more or less orthogonal or occasionally homotropous, when empty and dry furrowed or strongly so; exothecial cells incrassate, sometimes more strongly so in longitudinal walls, not or weakly collenchymatous, sometimes alternating with zones having thin-walled or slightly incrassate cells; stomata few, ovate-pored or lacking pore, surrounded by radially arranged cells, near base of capsule; annulus not separating; operculum conical-rostrate; peristome red, teeth cleft or perforated to one fourth or further down, with longitudinal rows of pits on outside; spores 21-27 µm, finely papillose. sharply denticulate or dentate (Hedenäs 2017). Oncophorus demetrii has acuminate or mostly narrowly acuminate leaves, whereas in O. wahlenbergii the leaves frequently end in a clearly obtuse point. In O. wahlenbergii specimens with narrowly acuminate leaves, the leaves can have very obtusely denticulate uppermost margins (high magnification), where the denticles are formed by projecting cell walls. In O. demetrii, on the other hand, many leaves have the leaf margins uneven due to projecting cell walls in their upper 5-25%, which was nicely illustrated in Plate 3A by Renauld and Cardot (1896) in their description of the species, and the excurrent costa is at least partly rough from projecting cells or cell walls.

Nomenclatural notes
The leaves in the isotype of Dicranum demetrii Renauld & Cardot (Fig. 4) are slightly worn, but the features of the type material agree well with those of the Scandinavian material (Fig. 5)

Habitat and distribution in Scandinavia
Oncophorus demetrii occurs in the Scandinavian mountain range from the sea level at the northern Norwegian coast to 1300 m a.s.l. (Fig. 6). In the lowlands to the east of the mountains, there is one find from the shore of the Swedish river Indalsälven, which sources are in the mountain range. It is the Scandinavian Oncophorus species with the most restricted geographical distribution; maybe this is why it has remained undetected until now. Outside Scandinavia, the species is known from Labrador in Canada (type locality for Dicranum demetrii), which suggests that it occurs also in other northern areas.
According to label information and field observations in the rich north-facing slopes between Björkliden and Vassijaure, west of Lake Torneträsk (northern Swedish Lappland) in 2017, the species often grows in base-rich or calcareous habitats. This includes meadows and wet meadows, heaths and wet mountain heaths, including Dryas-Cassiope heaths, rich fens, wet or periodically wet rocks and rock crevices, and brook and river shores, where it can occasionally grow on rotten wood. In its habitat, O. demetrii therefore deviates from O. wahlenbergii (A plus C) and O. elongatus, which occur predominantly in base-poor habitats.

Discussion
In the previous study of Scandinavian Oncophorus (Hedenäs 2017), three O. wahlenbergii lineages were identified by molecular data. Two lineages included only three or four specimens, respectively, and these were included per chance thanks to a sufficiently large sample size. To reveal such unexpected species-level diversity, it is necessary to study a relatively large number of representative specimens from different geographical regions. The three specimens of the poorly represented lineage that corresponds with O. demetrii differ from those of the other two lineages in a few morphological traits. A molecular examination of additional specimens with O. demetrii morphology shows that this species is distinguishable also by morphology. Within the genus Oncophorus in Scandinavia, we have thus five morphologically distinguishable species, and an additional cryptic one which morphology is similar to that of O. wahlenbergii.
Oncophorus occurs mainly in arctic to temperate regions of the Northern Hemisphere (Frahm et al. 1998), also in the high-arctic (Frisvoll and Elvebakk 1996), and diversification within the genus therefore likely occurred in environments and climates similar to what we find in these areas today.
Oncophorus species are present in the fossil record from the late Pliocene and Quaternary periods (Janssens 1983, Hedenäs 1994a, Hedenäs and Bennike 2009, showing that its diversification has a history of at least 2.5 MY. For northern, cold-adapted taxa like Oncophorus an important driver for diversification could therefore have been the glacial fluctuations that repeatedly segregated independently evolving populations in isolated refugia, also small northern ones, during the glacial periods. However, to establish if this is likely as an explanation or partial explanation, the timing of the diversification events needs to be established, which requires a better fossil record for Oncophorus and related taxa than is available at present. Compared with the two European species accepted by Frahm et al. (1998) the increase to six, including O. dendrophilus Hedd. & Blockeel (Hedderson and Blockeel 2006), or with the cryptic O. wahlenbergii one, even seven species, is highly significant. During the last decades, the number of accepted species has increased in several European moss groups. This was first a result of classical taxonomic studies (Frisvoll 1983, 1988, Hedenäs 1994b, Blom 1996, and this trend continues in the age of integrative taxonomy (Köckinger et al. 2010, Köckinger and Kučera 2011, 2016, Medina et al. 2013, Buchbender et al. 2014, Hedenäs et al. 2014, Caparrós et al. 2016, Ignatova et al. 2016, Köckinger and Hedenäs 2017. In addition, cryptic species diversity is revealed, as in O. wahlenbergii (Hedenäs 2017) and Hamatocaulis vernicosus (Mitt.) Hedenäs (Hedenäs and Eldenäs 2007). It now seems very likely that diversity losses at the intraspecific level have been significant (Hedenäs 2016), and based on the just cited information on species we may ask 1) how much of the European species level diversity that still hides among synonyms or undescribed species and 2) how much of this is threatened or has already been lost? Bryophyte taxonomy and systematics are declining disciplines in many European countries today, despite the obvious lack of knowledge regarding even which species actually exist on the continent.