Scandinavian Oncophorus ( Bryopsida , Oncophoraceae ) : species , cryptic species , and intraspecifi c variation

Abstract. Scandinavian members of the acrocarpous moss genus Oncophorus were revised after fi eld observations had suggested unrecognized diversity. Based on molecular (nuclear: internal transcribed spacers 1 and 2, ITS; plastid: trnGUCC G2 intron, trnG, rps4 gene + trnS-rps4 spacer, rps4) and morphological evidence, four morphologically distinguishable species are recognized, Oncophorus elongatus (I.Hagen) Hedenäs, O. integerrimus Hedenäs sp. nov. (syn. O. virens var. elongatus Limpr.), O. virens (Hedw.) Brid., and O. wahlenbergii Brid. (O. sardous Herzog, syn. nov.). Oncophorus elongatus was earlier recognized, but much of its variation was hidden within O. wahlenbergii. Its circumscription is here expanded to include plants with long leaves having mostly denticulate or sharply denticulate upper margins and with long and narrow marginal cells in the basal portion of the sheathing leaf lamina. The new species O. integerrimus sp. nov. diff ers from O. virens in having more loosely incurved leaves and entire or almost entire upper leaf margins. Besides these characters, the species in the respective pairs diff er in quantitative features of the leaf lamina cells. Several cryptic entities were found, in several cases as molecularly distinct as some of the morphologically recognizable species, and phylogeographic structure is present within O. elongatus and O. virens.

Twenty µl of each amplified fragment were cleaned using a mixture of 20 units of Exonuclease I from E.coli and 4 units of FastAP TM Thermosensitive Alkaline Phosphatase (Fermentas LIFE SCIENCE), mixed and incubated at 37°C for 30 min and inactivated at 80°C for 15 min.Cycle sequencing was performed using the ABI BigDye Terminator Kit (Applied Biosystems) according to the instructions on the kit (BDT ver.3.1), and the sequencing products were cleaned using the DyeEx® 96 Kit (QIAGEN).
The same primers as for the initial PCR were used.Sequencing products were resolved on an ABI3130xl automated sequencer.Double stranded sequencing was performed.

Sequence editing and analysis of molecular data
Nucleotide sequence fragments were edited and assembled for each DNA region using PhyDE® 0.9971 (http://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 & Ochoterena 2000), using SeqState (Müller 2005).The sequence alignments used in the analyses are available on request.European Nucleotide Archive (EMBL-ENA) accession numbers are listed in Table 1.
Paralogous ITS haplotypes are rarely 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 & Rosselló 2007).The revealed ITS variation is thus interpreted as being among homologous haplotypes.
The program TCS (Clement et al. 2000) with a cut-off level of 0.95 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 4.12.6 (Huson & Bryant 2006) to visualize 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 both visual inspection of the split networks, Jacknife trees, and the ILD test (Farris et al. 1995;200 replicates, p = 0.005) indicated that the two are incongruent.

Morphological study and analysis of measurements
After the molecular relationships among the studied O. elongatus, O. wahlenbergii, and O. virens s. lat.specimens had been clarified, the morphology of selected specimens belonging to the distinguished molecular entities was studied.Earlier studies (e.g., Frahm et al. 1998) based only on morphology had clearly separated O. elongatus-O.wahlenbergii from O. virens s. lat., but had failed to correctly identify the further morphological entities distinguished by the molecular study.Therefore, an approach including both standard comparisons of qualitative and quantitative characters and the quantification of vegetative leaf size and especially leaf cell size and shape was used.Since vegetative leaves are invariably present in a plant it was expected that the latter approach, which had not been explored previously within Oncophorus, would reveal quantitative differences supporting the molecularly recognized entities.
Among molecularly identified O. virens s. lat., the two main entities O. virens s. str.and O. integerrimus Hedenäs (sp.nov.; see below) were sampled with 10 specimens each.The seemingly more heterogeneous O. elongatus was sampled with 18 specimens and O. wahlenbergii with 17 specimens.These 17 specimens included those belonging to the two small, molecularly well-supported O. wahlenbergii clades B and C in Fig. 1.The morphologically sampled specimens are indicated with an asterisk (*) in Table 1.For each of these specimens, three vegetative leaves were sampled from two shoots (2 leaves from one stem and 1 from the other, to avoid sampling all leaves from an untypical shoot for the specimen).For each leaf, length and maximal width was measured, and the length, width, and length-towidth 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 1.13 software (Olympus Corporation) for automatic and continuous image stacking.Measurements were then made from these leaf and cell images, using the Olympus cellSens Standard 1.13 software.
Comparisons among the four entities within O. elongatus-O.wahlenbergii and between the two within O. virens s. lat., respectively, are based on two approaches.First, the cell measurements were compared between the entities within each of the two groups.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 (normality) showed that the data do not meet the criteria of normality and homogeneity of variance.Thus, for O. elongatus-O.wahlenbergii the nonparametric Kruskal-Wallis test for multiple comparisons and for O. virens s. lat. the nonparametric Mann Whitney-U-test for comparing two groups were used to compare the cell measurements among or between the entities, respectively.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) to see whether the combined information corresponds with the molecularly identified entities.All statistical calculations were made in STATISTICA 12 (StatSoft 2013).

Herbarium data
Herbarium acronyms follow Index Herbariorum (Thiers continuously updated).For specimens citations, double quotes "…" indicates that citation is exactly as on label; square brackets […] indicates that information is interpreted from label data.

Molecular relationships
The total number of aligned ITS sites in the 100 studied Oncophorus specimens, and outgroup of two Cynodontium strumiferum and two Rhabdoweisia fugax specimens, after deletion of regions at the beginnings and ends that were incomplete for some specimens, was 1022.

Morphological evaluation
No differences could be found between O. wahlenbergii A and C (not shown), and these are therefore merged in the following (O.wahlenbergii AC).The PCA based on leaf and leaf cell measurements revealed that O. elongatus differs from O. wahlenbergii AC, with some overlap between the two (Fig. 2A).The differentiation is along PCA axis 2, showing that leaf length and width, as well as the width of cells in the apical and middle portions of the leaves, are most important in explaining this separation (Fig. 2A:1).
Oncophorus elongatus and O. wahlenbergii AC differ from each other in five out of nine individual leaf lamina cell sizes or length-to-width ratios, whereas O. wahlenbergii B is sometimes most similar to either O. elongatus or O. wahlenbergii AC, sometimes intermediate between these, or its mean values are larger or smaller than those of both the other two (Table 2; cf., Fig. 2A).Also O. virens and O. integerrimus sp.nov.differ from each other in the PCA based on these parameters, again with a small overlap (Fig. 2B).The main differentiation is along PCA axis 1, and leaf length, length of the apical leaf lamina cells and width of the middle ones, as well as the length-to-width ratio of the apical and middle lamina cells contribute most to this pattern (Fig. 2B:1).These two species differ in all cell measurements (Table 3).Finally, despite the found statistical differences, which are based on large numbers of measurements from molecularly identified groups, the overlap among  S2).

Geographical distribution and habitat
The distribution of the four Scandinavian Oncophorus species is mapped in Fig. 3, based on the material in S. All species can grow in several habitat types, but each species is most commonly found in one or two of these (Fig. 4).Three members of the genus (O.elongatus, O. wahlenbergii, O. integerrimus sp.nov.)         have between 40 and 55% of their recorded occurrences in fens, springs, wet meadows and similar habitats.In addition, 35% of the finds of O. elongatus are from decomposing wood, an unimportant habitat for the other species, and for O. wahlenbergii and O. integerrimus sp.nov.20-25% of the records are from shores of brooks and lakes, wet rocks, and similar habitats.Shores and wet rocks are the most important habitats for O. virens, which has more than 45% of its records there.This species is the only one with a significant number of occurrences in late snow-beds.
Within O. elongatus, the molecular differentiation corresponds with a geographic pattern.Samples from the lowlands and along the Bothnian coast mostly belong to O. elongatus A, whereas samples from the mountain range and the Norwegian coast are predominantly O. elongatus B (Fig. 5A).No such geographic pattern was found within the other species.Within O. virens, on the other hand, we find a very clear differentiation between low (O.virens A) and high (B) elevation haplotypes (Fig. 5B).

Taxonomy
For all species, the total number of studied specimens is indicated in Fig. 3, and a selection of studied specimens is found in Table 1.

Description
Plants up to ten cm high, mostly in loose tufts, green or yellow-green.Stem with large central strand, a cortex plus epidermis of 1-2 layers of incrassate cells, epidermis not differentiated as a hyalodermis; axillary hairs with 2-8-celled, hyaline upper portion, 8-10 µm wide, basal 1-2 cells rectangular, brown; rhizoids strongly branched, red-brown, smooth, in leaf axils or shortly above.Leaves 2.5-6.4 × 0.4-1.2mm, when moist from strongly sheathing base erect, or erect-patent to spreading and straight or slightly curved, when dry with middle leaf spreading and gradually curved upwards or upwards-inwards and curled or slightly curled, from oblong-obovate or ovate sheathing portion (narrowed or strongly narrowed towards insertion) with long and almost linear, gradually narrowing upper portion, apex long-or very long-acuminate; leaf margin plane throughout, below entire, in upper 20-25(-35)% partly irregularly dentate or denticulate with sharp forwards-directed teeth, or sometimes relatively weakly denticulate, or some leaves almost entire and other leaves with scattered teeth along upper margin, teeth single or occasionally double, sometimes with cell walls projecting along upper margin, margin varying from unistratose to bistratose; costa 53-125 µm wide near base, with dorsal and ventral epidermis cells slightly widened but incrassate, one layer of large guide cells, 1-3 layers of ventral stereids and 2-3(-4) layers of dorsal stereids, sometimes with mammillae, or erect to forwards-directed teeth on upper back; lamina cells in acumen incrassate or strongly so, 10-21 × 9-14 µm, 0.9-2.0 times as long as wide, in mid-leaf incrassate or strongly so, 11-29 × 8-13 µm, 1-3 times as long as wide, and in sheathing lamina strongly incrassate and eporose or indistinctly porose, 36-88 × 8-14 µm, 3.5-9.1 times as long as wide, transition between mid-leaf and basal cells gradual, due to relatively long mid-leaf cells and relatively short basal cells, lamina of basal, sheathing portion of leaf in middle and below with long and narrow cells also along margin; alar cells undifferentiated or a few cells slightly wider than other basal cells and sometimes brownish, unistratose, not or shortly and narrowly decurrent.Perigonia lateral on stem, not or shortly stalked, antheridia protected by oblong-triangular perigonial leaves having obtuse to narrowly acuminate acumen.Inner perichaetial leaves 5.36-7.14mm long, lower 26-42% oblong and broadly sheathing, above suddenly narrowed to long, narrow acumen.Seta tall, (6-)12-25 mm; capsule obloid, slightly curved, with distinct struma, 0.9-1.4× 0.5-1.0mm, 1.3-2.1 times as long as broad, more or less orthogonal; exothecial cells slightly incrassate, mostly slightly collenchymatous; stomata ovate-pored, surrounded by indistinctly radially arranged cells, near base of capsule; peristome orange-red, teeth cleft or perforated to one-or two-thirds down, with longitudinal rows of pits on outside; spores 23-30 µm, very finely rough.

Remarks
Well-developed O. elongatus has very long leaves, which are narrow above their sheathing base.In oceanic portions of the Scandinavian mountain range and along the Norwegian coast the species often grows in open fens or wet heaths or meadows and then the leaves are mostly spreading and give the plants a very characteristic appearance.This is the kind of plants that made Hagen (1899) distinguish O. wahlenbergii var.elongatus and Hedenäs (2005) raise it to the species level.However, molecular data show that this species is variable both molecularly and morphologically and with the present circumscription, the species includes also plants with different leaf orientations and from different habitats than the plants originally referred to it.Morphologically, this species differs from O. wahlenbergii in that the middle and basal portions of the basal, sheathing lamina have long and narrow cells also along its margin.In Fig. 6.A-D.Oncophorus elongatus A (specimen P251; cf., Table 1).E-G. O. elongatus B (specimen P221, cf.addition, O. elongatus has longer vegetative leaves than O. wahlenbergii, and some or many leaves have almost always got denticulate or sharply denticulate upper margins, often with denticles on the back of the costa also.Rarely some plants have only a few leaves with denticles along the margin, and their denticles are in addition scattered and obtuse.In such cases the cell pattern of the sheathing lamina can be used to distinguish the two species.As noted by Hedenäs (2005) the spore capsules of O. elongatus mature somewhat before those of O. wahlenbergii.

Habitat and distribution in Scandinavia
This species is most frequent in open wetlands and humid forests, where it is found on humus-rich substrates and decomposing wood (Fig. 4).It occurs also on rocks, wet rocks, and on shores.Oncophorus elongatus is the most frequent and widespread member of the genus in Scandinavia (Fig. 3A), and is the only member of the genus that is relatively frequent also in large portions of the southern third of Sweden.-synonymized by Frahm et al. 1998).

Description
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 1-2 layers of incrassate cells, epidermis not differentiated as a hyalodermis; axillary hairs with (1-)2-4-celled, hyaline upper portion, 7-12 µm wide, basal 1-2 cells rectangular, hyaline or pale brown; rhizoids strongly branched, red-brown, smooth, in leaf axils or shortly above.Leaves 1.4-4.4× 0.3-0.8mm, when moist from sheathing base erect to spreading and straight or slightly curved, when dry with middle leaf spreading and gradually curved upwards-inwards and slightly curled or weakly curled, from oblong or slightly oblong-obovate sheathing portion (narrowed towards insertion) with narrowly triangular to almost linear upper portion, gradually narrowing upper portion, apex obtuse, acute, acuminate or narrowly acuminate; leaf margin plane throughout, below entire, above entire or with one or few occasional and indistinct, mostly obtuse teeth, especially close to leaf apex, sometimes with cell walls distinctly projecting along upper margin, margin varying from unistratose to bistratose; costa 31-88 µm wide near base, with dorsal and ventral epidermis cells slightly widened but incrassate, one layer of large guide cells, 1-2 layers of ventral stereids and 1-3 layers of dorsal stereids, sometimes with projecting cell walls or cell portions in upper costa; lamina cells in acumen incrassate, 10-33 × 10-15 µm, 0.9-2.4times as long as wide, in mid-leaf  incrassate, 10-33 × 8-17 µm, 0.9-3.0 times as long as wide, and in sheathing lamina strongly incrassate and eporose or indistinctly porose, 30-72 × 7-15 µm, 3.4-8.3times as long as wide, transition between mid-leaf and basal cells gradual, due to relatively long mid-leaf cells and relatively short basal 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 with obtuse or acute apex.Inner perichaetial leaves 2.73-4.24mm long, lower 34-67% oblong and broadly sheathing, above suddenly narrowed to long, narrow acumen.Seta tall, 9-22 mm; capsule obloid, curved or slightly curved, with distinct struma, 0.5-1.3× 0.3-0.7 mm, 1.2-2.5 times as long as broad, more or less orthogonal; exothecial cells incrassate, sometimes more strongly so in longitudinal walls, not or weakly collenchymatous; stomata sparse, ovate-pored or lacking pore, surrounded by radially arranged cells, near base of capsule; peristome red, teeth cleft or perforated to one-fourth or further down, with longitudinal rows of pits on outside; spores 21-29(-32) µm, finely rough.

Remarks
Oncophorus wahlenbergii is the smallest Scandinavian species.Contrary to O. elongatus it has quadrate or rectangular marginal cells in the middle and basal portions of the basal, sheathing lamina.In addition, its vegetative leaves are shorter than in O. elongatus, and in their upper potions the margins are entire or have occasionally one or a few indistinct teeth, especially close to the leaf apex.In some plants the cell walls project distinctly along the upper margin, and sometimes on the uppermost back of the costa.

Habitat and distribution in Scandinavia
Oncophorus wahlenbergii is most often found in open wetlands, but occurs also on soil, rocks, wet rocks, and on shores (Fig. 4).The species is widespread, but compared with O. elongatus it is much less common outside the mountain range and the far north (Fig. 3B), and has only been collected a few times in the southern third of Sweden.

Nomenclatural notes
The isotype of Oncophorus sardous Herzog (Herzog 1910) in S has plane leaf margins and no or few marginal teeth near the leaf apex, and several rows of short cells along the leaf margin in the sheathing lamina.Despite an earlier synonymisation with O. virens Hedw.(Frahm et al. 1998), the material unambiguously belongs to O. wahlenbergii Brid.The partly mammillose apical costa back and few marginal teeth or projecting cell walls remind about the molecularly identified O. wahlenbergii B (Fig. 1).However, since the geographical origin of the O. sardous type is distant from Scandinavia, further information is required to confidently judge its relationship with other O. wahlenbergii specimens.

Description
Plants up to 8 cm high, usually in loose tufts, green or yellow-green.Stem with large central strand, a cortex plus epidermis of 1-2 layers of incrassate cells, epidermis not differentiated as a hyalodermis; axillary hairs with 2-4-celled, hyaline upper portion, 10-11 µm wide, basal 1-2 cells rectangular, hyaline; rhizoids strongly branched, red-brown, smooth, in leaf axils.Leaves 2.1-4.2 × 0.6-1.0mm, when moist from sheathing base patent to spreading, straight or screwed, when dry above sheathing base erect and tightly incurved to spreading, above strongly twisted, from ovate or rounded oblong base with narrowly triangular acumen, apex acuminate to longly acuminate; leaf margin at least partly distinctly recurved in lower leaf, below entire, upper margin in all or many leaves regularly to irregularly dentate or coarsely and somewhat irregularly denticulate, sometimes only close to leaf apex but mostly in at least some leaves down to mid-leaf or almost so, teeth single or double, mostly sharp and when strong often directed forwards, margin varying from unistratose to bistratose; costa 83-120 µm wide near base, with dorsal and ventral epidermis, one layer of large guide cells, 1(-2) layers of ventral stereids and 2-3 layers of dorsal stereids; lamina cells in acumen incrassate, 6-30 × 6-18 µm, 0.5-3.3times as long as wide, in mid-leaf incrassate, 6-33 × 6-15 µm, 0.6-3.8(-4.2) times as long as wide, and in sheathing lamina incrassate and slightly porose, 20-109 × 5-19 µm, 1.6-15.8times as long as wide, transition between mid-leaf and basal cells relatively sudden, due to relatively short upper lamina cells and relatively long basal cells; alar cells differentiated, rectangular, slightly to strongly inflated and forming a diffusely delimited group of 3-4 cells wide and 2-4(-5) cells long, partly bistratose, decurrent.Perigonia lateral on stem, not stalked, antheridia protected by oblong perigonial leaves with "cut-off" or acute apex.Inner perichaetial leaves 3.27-6.18mm long, lower 29-50% oblong and broadly sheathing, above suddenly narrowed to long, narrow acumen.Seta tall, 12-29 mm; capsule cylindric or shortly so, curved or slightly curved, with distinct struma, 0.9-1.5 × 0.5-0.6 mm, 1.6-2.9times as long as broad, more or less orthogonal to homotropus; exothecial cells incrassate or longitudinally incrassate, slightly collenchymatous or not; stomata ovate-pored, surrounded by radially arranged cells, near base of capsule; peristome red, teeth cleft or perforated to one-third or further down, with longitudinal rows of pits on outside; spores 23-38 µm, very finely rough.

Remarks
This species is on the average weaker than O. integerrimus sp.nov.In addition, it differs by its usually strongly dentate leaf margins and in that dry leaves are more tightly incurved.Plants from high elevations, which can be molecularly differentiated from lowland ones, are often relatively small.However, neither this nor other morphological features consistently distinguish plants from different elevations.

Habitat and distribution in Scandinavia
Oncophorus virens occurs on rocks, especially wet rocks, and on brook and lake shores (Fig. 4).It is the only member of Oncophorus that is frequent in late snow-beds, and it grows also on soil and in open wetlands.This species is frequent mainly in the mountain range and the base-or calcium-rich lowland regions of northern Sweden (Fig. 3C).Like O. wahlenbergii, it has only been collected a few times in the southern third of Sweden.

Etymology
The epithet 'integerrimus' refers to the mostly entire leaf margin.

Remarks
When well developed this is a large species, with on the average more longly and narrowly acuminate leaves than in O. virens.When dry, the leaves are loosely incurved or curved upwards, and the upper acumen is loosely but relatively strongly twisted.The leaf margin in the acumen is mostly entire, but sometimes slightly uneven or has a few and usually low and irregular obtuse teeth.Occasionally one or a few sharp teeth may occur, especially close to the leaf apex.

Habitat and distribution in Scandinavia
Oncophorus integerrimus sp.nov. is found most often in open wetlands, but occurs also on soil and wet rocks, often on shores (Fig. 4).The distribution of O. integerrimus sp.nov. is similar to that of O. virens, but geographically more restricted (Fig. 3D).Of the four Scandinavian Oncophorus species it is the only one that has not been found in southern Sweden, although there are few finds from southern Norway.

Nomenclatural note
The synonymy of Oncophorus virens var.elongatus Limpr. is based on protologue information (Limpricht 1886) and on material collected by Gander at the type locality two years after the description of the taxon (S-B232585).The name of the variety cannot be used at the species level, since it is blocked by Oncophorus elongatus (I.Hagen) Hedenäs.

Discussion
Although the monophyly of Oncophorus was not an issue in the present study, the positions of its species in relation to Cynodontium strumiferum and Rhabdoweisia fugax do not suggest otherwise.Neither in the ITS-nor chloroplast-based analyses, O. crispifolius, O. dendrophilus, and O. rauei were found in positions that suggested that they are especially closely related to any of the Scandinavian species.The molecular evidence presented here supports the recognition of the recently described O. dendrophilus (Hedderson & Blockeel 2006).The molecular information available for Scandinavian Oncophorus suggests that species additional to the ones so-far accepted exist in the area.Four molecularly supported entities can also be distinguished based on their morphology, by a few qualitative characters, statistically significant differences in leaf cell measurements or on PCA scores based on these and leaf size.Four is therefore the minimum number of species to be recognized in the area.However, additional evolutionary lineages and in some cases most likely also biologically distinct cryptic species occur among the Scandinavian Oncophorus, considering the number of mutational differences between lineages as well as the lack of intermediates between these lineages.Cryptic species are here understood as lineages that are molecularly as distinct as those of the morphologically recognized species but which lack (known) morphological distinguishing features.
For semi-cryptic species, weak quantitative morphological differentiation towards other molecularly identified entities is presently suggested by the data.
The two species Oncophorus elongatus and O. wahlenbergii are easily distinguished from O. virens and O. integerrimus sp.nov.by their plane rather than partially recurved leaf margins.Oncophorus elongatus differs from O. wahlenbergii in the shape of the marginal cells of the sheathing leaf portion, the denticulation or dentation of the upper leaf margins and costa, and overall leaf size, and except for O. wahlenbergii B in the quantitative leaf characters included in the PCA (Fig. 2A).Within O. elongatus one specimen shifted between O. elongatus A and B depending on whether ITS or chloroplast data were analysed.Such incongruence could be a result of several processes, such as incomplete lineage sorting or hybridization, but to determine the cause additional evidence is required (cf., Wendel & Doyle 1998;Harris 2008).On the other hand, there appears to be a phylogeographic signal distinguishing O. elongatus B as a western, oceanic or sub-oceanic entity that was mostly collected in fens and had usually got the long leaf portions above the sheathing base widely spreading, in contrast with the predominantly more eastern O. elongatus A that was mainly collected on dead wood or rocks and mostly presented less or not spreading leaves.Combined with the incomplete congruence between the two molecular data sets, this could be interpreted as two lineages that are still in the process of diverging.The distinct habit of O. elongatus B made Hedenäs (2005)  Oncophorus integerrimus sp.nov.includes no jacknife-supported intraspecific variation.The leaf margins of O. integerrimus are entire, or occasionally denticulate near leaf apex, whereas O. virens has predominantly dentate or coarsely denticulate upper leaf margins; the species also differ in the quantitative leaf characters that were included in the PCA (Fig. 2B).Oncophorus virens displays intraspecific variation that is mainly incongruent between ITS and chloroplast data (cf., O. elongatus above), except that O. virens B is distinct from O. virens A in both data sets.All seven specimens of O. virens B came from higher elevations than any of the 15 O. virens A specimens (Fig. 5B).This is highly unlikely as a chance result and therefore suggests that O. virens B is restricted to the cold climate of higher mountains.Since the temperature in the mountains is expected to increase with several degrees until the year 2100 (Berglöv et al. 2015a(Berglöv et al. , 2015b;;Nylén et al. 2015), it is urgent to examine the distribution of O. virens B and similarly distributed species and intraspecific entities in more detail.Organisms restricted to cold environments, such as O. virens B and the northern haplotypes of Scorpidium cossonii (Schimp.)Hedenäs (Hedenäs 1989) (Hedenäs 2009), will likely decline when the areas of suitably cold habitats shrink.
All four species that can be clearly distinguished by morphology have relatively northern distributions and are especially common in the mountain range and the lowlands of the far north (Fig. 3).The mapped distributions clearly underestimate the frequency of the species in Norway, since the mapped occurrences are based on S material only and the distribution of members of the genus Oncophorus is much wider according to 'Artskart' (https://artskart.artsdatabanken.no/;accessed 9 Feb. 2016).
All four species occur with different frequencies in several humid to wet habitats (Fig. 4).Oncophorus elongatus obviously prefers organic substrates, such as decomposing wood and peaty soil in mires, whereas O. virens is clearly a species of brook and lake shores, and wet rocks, and is the only species that is often found in late snow-beds.The two remaining species are most frequently found in mire habitats, but are relatively abundant also on brook and lake shores, and wet rocks.Oncophorus elongatus and O. wahlenbergii occur in relatively base-poor environments, whereas O. virens and O. integerrimus sp.nov.are mostly found in base-rich to calcareous habitats.Whether entities within species that display clear intraspecific molecular variation also differ from each other in habitat preferences should be explored when molecular information exists for additional specimens.
From a biodiversity and conservation point of view the cryptic diversity in Oncophorus deserves serious attention (Hedenäs 2016).A fuller sampling of O. virens, O. elongatus, and O. wahlenbergii is required to understand their cryptic and semi-cryptic diversities, as well as intrinsic geographic and habitat components.Additionally, this would possibly allow the discovery of morphologically stable characters to distinguish at least O. wahlenbergii B. However, at the present state of knowledge, and considering the wish by most biologists to be able to distinguish species by morphology, four 'species' of Oncophorus are here recognized for Scandinavia.

Fig. 2 .
Fig. 2. The positions of three leaves from each of 18 molecularly identified specimens of O. elongatus, 14 of O. wahlenbergii A and C, and 3 of O. wahlenbergii B (cf. Fig. 1) (A), and of 10 specimens of O. virens and 10 of O. integerrimus Hedenäs sp.nov.(B), along the first two axes in a PCA.The PCA is based on each leaf's length (LL), width (LW), and leaf lamina cell length, width, and length/width ratio in the apical (AL, AW, AR), middle (ML, MW, MR), and sheathing basal (BL, BW, BR) lamina.Cell measurements are the mean values of 20 cells per position in each leaf.Axes 1 (x) and 2 (y) explain 30.69% and 25.24% of the variation in A, and 32.56% and 22.01% of the variation in B. Insert diagrams A:1 and B:1 depict explanatory factors in the plane of these two axes.

Fig. 10 .
Fig. 10.Oncophorus integerrimus Hedenäs sp.nov (holotype). A. Stem leaves.B. Upper leaf acumen.C. Portion of leaf at widest point, showing the broadly recurved margin.D. Lower part of sheathing leaf portion, showing differentiated alar region (margin to the right).

Table 2 .
Means plus standard errors for cell measurements in the acumen (20 cells per leaf), in mid-leaf (20), and in the sheathing base (20) from three leaves in each of 18 specimens of Oncophorus elongatus, three specimens of O. wahlenbergii B, and 14 specimens of O. wahlenbergii A and C (AC).The number of measured cells at each position, n, is indicated after the species.Significant pair-wise differences between species revealed by the Kruskal-Wallis test are indicated by different letters appended after the values in the respective columns, for the Bonferroni corrected p values corresponding with p < 0.05.

Table 3 .
Means plus standard errors for cell measurements in the acumen (20 cells per leaf), in mid-leaf (20), and in the sheathing base (20) from three leaves in each of ten specimens of Oncophorus virens and O. integerrimus Hedenäs sp.nov.The number of measured cells at each position, n, is indicated after the species.Significant differences between species revealed by the Mann-Whitney U-test are indicated by different letters appended after the values in the respective columns, for the Bonferroni corrected p values corresponding with p < 0.05.
). A. Stem leaves.B. Upper leaf acumen.C. Portion of leaf at widest point, showing the broadly recurved margin.D. Lower part of sheathing leaf portion, showing differentiated alar region (margin to the left).
believe that only this kind of plants belongs to O. elongatus, which is now shown to be incorrect.Oncophorus elongatus C is clearly distinct from O. elongatus A and B by 5-6 mutational changes according to both ITS and chloroplast data.Because only two of the sampled specimens belong to this lineage and no morphological feature was found that supports it, O. elongatus C is best treated as a potential cryptic species that requires further study.Within O. wahlenbergii several well-supported molecular lineages that are differentiated by numerous mutations and have a high jacknife support exist.It seems likely that the three distinct lineages that are present in O. wahlenbergii represent cryptic species, or in the case of O. wahlenbergii B, which in some quantitative morphological traits overlaps more with O. elongatus than with O. wahlenbergii (Fig.2A, Table2), semi-cryptic species.The sampling of specimens from two of these three lineages occurred by pure chance, since neither O. wahlenbergii B nor C was clearly distinguishable by morphology alone.It is possible that a fuller sampling of O. wahlenbergii B, that allows comparing additional specimens with O. wahlenbergii A and C, would make it possible to refer at least a significant proportion of O. wahlenbergii B specimens to this lineage by morphology.According to ITS data, O. wahlenbergii C appears on another side of a major split than O. wahlenbergii A and B, O. crispifolius, O. dendrophilus, and O. rauei, which suggests that this lineage is more isolated than O. wahlenbergii B from O. wahlenbergii A, despite that O. wahlenbergii B is the one that may deviate morphologically from the latter.