A new species of Brevianthus (Brevianthaceae, Marchantiophyta) from New Caledonia with unusual underleaf production

Abstract Brevianthus is a distinctive genus of leafy liverwort in its succubously inserted, entire leaves, lack of underleaves, restriction of sexual organs to lateral-intercalary branches, scattered rhizoids and dense leaf-surface ornamentation. The sole species, Brevianthus flavus, is divided into two subspecies, one in Tasmania the other in New Zealand. A second species, Brevianthus hypocanthidium, is described as new and is the first record of the genus for New Caledonia. Among its distinguishing characters are its shallowly bilobed leaves, and triangular underleaves present on small to medium-sized shoot sectors, the lack of a hyaline leaf margin, and the crenulate leaf margin formed by heavily thickened external cell walls. The most unusual features of the new species are the presence of underleaves between lateral leaf insertion lines that reach the ventral stem mid-line, and the absence of underleaves from larger shoots. To explain these features we propose a competitive model of shoot formation wherein the ventral merophyte progressively loses vigor as its relative stature decreases, and its derivative cells become discontinuous and isolated along the ventral stem surface, with intervening areas occupied by derivatives of the more vigorous lateral merophytes.


Introduction
Brevianthus J.J.Engel & R.M.Schust. possesses a highly distinctive suite of morphological characters incongruent with its phylogenetic relationships. Th e restriction of sexual organs to abbreviated lateral-intercalary branches, the lack of underleaves, and the scattered rhizoids were thought to immediately remove the plant from the Lophocoleaceae De Not. (Engel and Schuster 1982), whereas the combination of succubously inserted, undivided leaves, with coarse trigones, lack of underleaves, undiff erentiated stems, and short androecial branches suggest the isolated genus Jackiella Schiff n., within which the type species was originally described by Grolle (1970) on the basis of sterile and androecial individuals. Th e discovery of fertile, perianth-and sporophyte-bearing material prompted the species' removal from Jackiellaceae R.M.Schust. due to the well-developed perianth and the comparatively large and undiff erentiated seta. Th e female structures in combination with the minute androecia with strongly ventricose and monandrous bracts, the uniseriate antheridial stalks suggested relationships with the Adelanthaceae Grolle. However, the exclusively lateral intercalary branching (both vegetative and sexual), lack of stolons, lack of secondary pigmentation, and the 3-4 stratose capsule were all inconsistent with placement within the Adelanthaceae (Engel and Schuster 1982).
Th e wide-mouthed obscurely trilobed perianths, the isophyllous gynoecium, the 1-phase development of the outer capsule layer, and the seta anatomy all suggested affi nity with the old Geocalycineae R.M.Schust. (including the Lophocoleaceae and Plagiochilaceae Müll. Frib. & Herzog). However the spherical capsules, scattered rhizoids and apparent absence of a ventral merophyte were anomalous with that suborder so Brevianthus was placed, with the Chonecoleaceae R.M.Schust. ex Grolle, into an independent new suborder, Brevianthineae J.J.Engel & R.M.Schust. by Engel and Schuster (1982). Th ese authors also proposed a monogeneric family Brevianthaceae J.J. Engel & R.M.Schust. Molecular phylogenetic studies led to considerable changes in the classifi cation of liverworts (Crandall-Stotler et al. 2009) and supported the reinstatement of Lophocoleaceae (Hentschel et al. 2007) for the perianth-bearing elements of Geocalycaceae Endl. sensu Crandall-Stotler and Stotler (2000) and others.
Th e fi rst molecular phylogenetic study including Brevianthus, on the basis of a New Zealand specimen, resolved the genus sister to the Lophocoleaceae element Tetracymbaliella Grolle, then Plagiochila (Dumort.) Dumort. in a monophylum also containing Chiloscyphus Corda (He-Nygren et al. 2006), placing it fi rmly within the Lophocoleaceae-Plagiochilaceae familial complex in contradiction to much of the morphological evidence. Subsequent molecular phylogenetic studies seem to corroborate the sister relationship of Brevianthus and Tetracymbaliella, and resolved this clade as sister to the rest of the Lophocoleaceae (Feldberg et al. 2014).
Once believed endemic to Tasmania, Brevianthus was discovered on South Island (Blackball), New Zealand in 1998 (Glenny 2000), and has since been collected from a small number of sites on the West Coast of the South Island and in the upper North Island. Th e fi rst New Zealand collection was made in the North Island in 1990 but went unrecognized. New Zealand plants diff er morphologically from Tasmanian, and were given subspecifi c status by Engel (2011).
In 2006 the late Elizabeth Brown made a relatively copious collection of Brevianthus at Mont Kouakoué in New Caledonia, suffi cient to facilitate the identifi cation of several distinctive morphological characters warranting its assignment as a separate species. We outline this proposition below, and provide additional observations on the ecology and distribution of the two subspecies of B. fl avus (Grolle)  Description. Plants closely prostrate, creeping, shoots sinuous, dull whitish green, opaque, water repellent, axes cylindrical, vermiform, to 2 mm wide and 30 mm long, the axes slightly laterally fl attened. Branches sporadic, lateral-intercalary. Stolons and fl agellae absent. Stems wiry, narrow for plant size, densely papillose, cortical cells oblong, all walls heavily thickened, in cross section 7-8 cells high, cortex undiff erentiated, cells same size as medulla, with massive, nodular thickenings either confl uent or separated by short stretches of unthickened primary wall. Rhizoids scattered on ventral side of stem, colourless, non-septate, tips often branched. Leaf insertion strongly succubous, nearly horizontal at postical end, not recurved at antical end, extending to stem mid-line on ventral side of large, but not small shoots, not extending to dorsal stem midline, leaving 1-2 cell rows leaf-free. Leaves strongly dorsally assurgent, not connivent over the dorsal stem surface, the axis appearing channeled in dorsal view with the stem partly or completely visible, the leaves unistratose throughout, densely imbricate, concave, ovate-rotund, lacking a hyaline border; apex shallowly but distinctly bifi d; margins crenulate by thickened cell walls; dorsal margin rounded, not or slightly decurrent, ventral margin rounded, the base weakly auriculate on small leaves and not auriculate on large leaves, not overlapping the ventral stem surface, not totally obscuring the stem in ventral view. Leaf cells not tiered, polygonal but typically hexagonal, isodiametric, with massive coarse, nodular trigones, confl uent or separated by narrow stretches of unthickened primary wall, primary walls visible within trigones, 39-50 μm diameter; marginal cells thick walled, external wall heavily thickened, especially medially, trigones coarse, not confl uent, consistently separated by unthickened primary wall, lumena not reduced, cells only slightly smaller than median cells, quadrate, 40-50 μm long and 27-36 μm wide, long axis parallel with margin. Intramarginal cells on abaxial surface covered with dense circular and confl uent to bacilliform anastomosing ornamentation continuous over cell junctions; urceolate to clavate 'papillae' over cell junctions absent. Cells, both marginal and intramarginal, on adaxial surface with similar ornamentation, comprised circular and confl uent to weakly anastomosing ornamentation. Underleaves present on small-to medium-sized shoot sectors, triangular, 4-6 cells wide at base and 8-9 cell tiers high, apex acute, formed by a single cell; 230-300 μm long by 9-140 μm wide at base, margins crenulate; ventral merophyte 0-2 cells wide. Asexual reproduction by leaf-borne regenerants arising from the adaxial leaf surface. Recognition. Th e genus Brevianthus is highly distinctive among leafy-liverworts in the white or nearly white, water-repellent, cylindrical shoots with dorsally assurgent and succubously inserted leaves and no or inconspicuous underleaves, and scattered rhizoids. Th e shoots are typically sinuous in growth, either down or across the substrate, and lay closely appressed to it. Th ey do not often overlap one another. Th is combination of macro-morphological characters facilitates fi eld identifi cation.
Th e three Brevianthus taxa recognized here all share these features, and are similar in their gross morphology. Th ey diff er primarily in micromorphological, microstructural, and anatomical details. However, characters vary in their manifestation with the stage of shoot stature and maturity, such that diagnostic diff erences must be sought within shoots of the appropriate age or size.
Th e triangular underleaves found only in B. hypocanthidium (Fig. 1L) are a case in point. Not only are these partly obscured by adjacent leaves, they are produced only on small and medium sized shoot sectors. Th ey are absent from the largest stature shoot sectors. As such, they are inconsistently present along a shoot, and may be entirely absent if the shoot examined is uniformly large. Th e other two Brevianthus taxa never produce underleaves, regardless of shoot stature.
Characters of the leaf apex and margins are useful in distinguishing the taxa of Brevianthus. Th e leaf apex of B. hypocanthidium (Fig. 1O) is shallowly but distinctly bifi d, and this is a consistent feature of leaves of all sizes, though on the smallest leaves of leaf-borne propagules this is obscure. In B. fl avus subsp. crenulatus small leaves are bifi d (Fig. 5O, P), while medium and large leaves have an undivided apex ( Fig. 5G-L). In B. fl avus subsp. fl avus the leaf apex is always undivided and entire ( Fig. 3E-J).
Th e leaf margin provides several diagnostic diff erences between the three taxa that are of more consistent manifestation. In B. hypocanthidium the leaf margin (Fig. 1F) is crenulate by virtue of its heavily thickened exterior cell wall, and the marginal leaf cells are chlorophyllous and similar in size to the medial cells. In B. fl avus subsp. crenulatus the leaf margin ( Fig. 5F) is crenulate by virtue of bulging marginal cell lumena, and the marginal cells are colourless and smaller than medial cells. In B. fl avus subsp. fl avus the leaf margin ( Fig. 3L) is entire, the marginal cells are again colourless and smaller than medial cells.
Trigones in leaf-cells diff er between species. In B. hypocanthidium (Fig. 1E) they are block-like and angular with truncate ends and straight sides. In both subspecies of B. fl avus (Figs 3L, 5F) they are coarse to bulging but with curved sides, and never as large or angular as observed in B. hypocanthidium. Leaf surface ornamentation may exhibit species-specifi c diff erences though there is intra-individual variation; our interpretation, however, may suff er from the relatively small number of observations we have made via SEM. Individuals of Brevianthus fl avus subsp. fl avus (Figs 3K; 4C, D) possess urceolate to clavate 'papillae' over the cell junctions on the abaxial leaf surface, at least between cells in the median-basal to basal portions of leaves, and at least sporadically on leaves along a single shoot. Th ese 'papillae' have not been observed in individuals of B. fl avus subsp. crenulatus (Figs 5E; 6C, D) or the type of B. hypocanthidium (Figs 1E; 2C, D). Parts of these structures are removable with chloroform, providing evidence that they partly consist of surface waxes (Heinrichs and Reiner-Drehwald (2012)).
Other leaf characters diff erentiate the taxa. Th e interstices between cells also appears to exhibit species-specifi c diff erences. In B. fl avus subsp. crenulatus (Fig. 6) leaf cell junctions appear recessed within the leaf such that the upper and lower parts of the cell appear surrounded by a narrow trench, which is less pronounced or absent in both B. hypocanthidium (Fig. 2) and B. fl avus subsp. fl avus (Fig. 4).
Leaf shape, orientation and imbrication also diff er. In both B. fl avus subsp. crenulatus (Fig. 5D, M) and B. fl avus subsp. fl avus (Fig. 3D) the leaves are imbricate over the ventral stem surface, obscuring stem tissue in ventral view, while in B. hypocanthidium (Fig. 1D, L, M) the ventral stem surface is often partially visible between the leaves. Th e leaves of B. hypocanthidium (Fig. 1A, G-K) are ovate-rotund, and when viewed in situ laterally, have their antical margin orientated more or less perpendicular to the stem. Brevianthus fl avus subsp. fl avus (Fig. 1A, E-I) has ovate to oblate leaves whose antical margin is inclined in lateral view, with the lowest part of the margin closest to the shoot apex. Th e same is true of B. fl avus subsp. crenulatus (Fig. 5A, H-L) though the angle of inclination is not so steep.
Conservation status. Th at B. hypocanthidium is known from two gatherings precludes inference of its likely distribution and abundance, we therefore recommend the species be considered Data Defi cient.
Additional For a full description of B. fl avus see Engel and Schuster (1982). Distribution and ecology. Brevianthus fl avus subsp. fl avus is widespread on the wetter western, south-western and southern sectors of Tasmania, between 300 and 900 m altitude where it occurs in a wide variety of habitat types encompassed by this altitudinal range. Brevianthus fl avus occurs within or on the margins of a wide variety of forest types, for example riparian scrub dominated by Leptospermum J.R.Forst. & G.Forst., Acacia Mill. and Banksia J.R.Forst. & G.Forst., with dense thicket of Bauera Banks ex Andrews on alluvial terrace in a gully with south-easterly aspect at Condominium Creek, or montane forest of Nothofagus cunninghamii (Hook.) Oerst., Eucryphia Cav., Arthrotaxis Endl., Atherosperma Labill. and Richea dracophylla R.Br. with broken canopy to 8 m tall on a ridge with southerly aspect on Mt Dundas. Th e species also occurs in subalpine and alpine scrub such as that dominated by Eucalyptus L'Hér., Sprengelia Sm., Leucopogon, Banksia, Orites R.Br. and Gymnoschoenus Nees as at Mount Eliza, and dominated by Astelia Banks & Sol. ex R.Br., Richea and Nothofagus Blume at Mount Hesperus. Brevianthus fl avus is

Conservation status.
Brevianthus fl avus subsp. fl avus appears widely distributed in western and southern Tasmania, however collecting and survey eff ort to date provides insuffi cient basis for an accurate appraisal of the species' threat status. We therefore recommend B. fl avus subsp. fl avus be regarded as Data Defi cient pending a more informed assessment.   At Craigieburn Road on the West Coast of the South Island, B. fl avus subsp. crenulatus is an occasional corticol on Manoao colensoi trunks in low forest with uneven, broken canopy comprised of Leptospermum scoparium J.R.Forst. & G.Forst. with Nothofagus solandri var. cliff ortioides, N. menziesii (Hook.f.) Oerst., and emergent Manoao colensoi, on saturated soil of an old alluvial terrace. Here the species was common on Manoao trunks, occasional on Nothofagus solandri var. cliff ortioides, but apparently absent from trunks of N. menziesii, and grew in association with Acrochila biserialis, Acromastigum cavifolium, Heteroscyphus menziesii, Heteroscyphus sp., Schistochila tuloides, Radula multiamentula, Radula tasmanica Steph., Frullania ptychantha Mont., Frullania sp., Macromitrium longipes (Hook.) Schwägr., Dicnemon calycinum (Hook.) Schwägr., and Hymenophyllum armstrongii.

Discussion
Th e presence of underleaves in Brevianthus hypocanthidium reduces the morphological distance between this isolated genus and more typical Lophocoleaceae species including those belonging to Tetracymbaliella, which was shown to be sister to Brevianthus in the analysis of He-Nygren et al. (2006). Th e retention of at least partial underleaf production in B. hypocanthidium is probably plesiomorpic and the complete absence of underleaf production in B. fl avus probably derived. Th e variable production of underleaves and a ventral merophyte by mature gametophyte shoots in B. hypocanthidium is unusual within species of the Lophocoleaceae-Plagiochilaceae complex, and perhaps all Jungermanniopsida Stotler & Crand.-Stotl. Only in broad phylogenetic terms is a parallel seen, in that across the Jungermanniales Hedw. as a whole, as stature of the lateral merophytes increases so does anisophylly, which is at the expense of the ventral merophyte (Schuster 1966 (Engel 2010), and many species of Heteroscyphus (J.J.Engel unpublished data); all of these taxa have a wide ventral merophyte. Underleaves in most species of the family, however, are bilobed in all stages of expression. In the related Plagiochilaceae, whose species for the most part have reduced ventral merophytes, unlobed and ciliform underleaves are known, for example, in Dinckleria fruticella (Hook.f. & Taylor) J.J. Engel & Heinrichs. In both D. fruticella and B. hypocanthidium the underleaves are probably derivatives of a reduced ventral merophyte, whose abbreviated morphology refl ects underlying developmental degeneracy.
What then of the variable underleaf production exhibited by B. hypocanthidium? Th at merophytes form continuous rows, two lateral and one ventral, all contributing stem and leaf tissue implies the leaf and underleaf insertions ought not exhibit overlap across shared cell rows along the stem. Th is generally holds across the Jungermanniopsida as a result of the orderly proliferation of cortical cells within each merophyte row.
Dorsal and ventral 'leaf-free' strips are readily reconciled via greater division of stemproducing derivatives of the merophyte initial. Th e converse, overlap of leaf and underleaf insertion lines across rows of cortical stem cells, is not so readily reconciled with a model of growth wherein each merophyte derivative contributes to discrete stem sectors.
In small stature shoots of B. hypocanthidium the leaf insertion lines do not reach the ventral stem mid-line, leaving a row or two of ventral cortical cell rows leaf-free, onto which the underleaves are inserted. In small stature shoots the merophyte rows appear both continuous and non-overlapping, and their growth is compatible with conventional liverwort development (Fig. 7).
However, in medium-sized shoot sectors where underleaves are produced, the insertion lines of the two lateral merophytes reach the ventral stem midline. Th e underleaf insertion line is completely overlapped by the insertion line of the non-adjacent leaf (Figs 1L, M; 7B). Th is is incompatible with a conventional growth model involving three continuous and discrete merophyte rows. How might the underleaves come to be completely nested within the overlapping leaf insertion lines? One explanation could be that the ventral merophyte row is not straight but perhaps zig-zags between the leaf insertion lines. Observation of ventral cortical cell rows is hampered by their surface ornamentation, rhizoids, and their overlap by both underleaves and lateral leaves. We have not undertaken destructive sampling of underleaf-bearing shoots to confi rm this due to the relative paucity of the type material. So while we cannot categorically exclude this possibility by direct observation of cortical cell rows, it fails to explain why underleaves are not produced on large shoots.
Th e stature-correlated pattern of underleaf production in B. hypocanthidium provides clues to development, and there are a number of observations a developmental model must explain: 1) Shoot stature increases and underleaf size remains constant on small and medium sized shoots. 2) Underleaves are absent from large shoot sectors. 3) Lateral leaf insertion does not reach the ventral stem midline on small shoots, therefore a discrete and continuous merophyte row is present. 4) Lateral leaf insertion reaches the ventral stem midline on medium sized and large shoots, therefore a discrete and continuous merophyte row is lacking. 5) Underleaf insertion is completely overlapped by the leaf insertion lines on mediumsized shoots. 6) Underleaf position relative to the adjacent leaf on medium-sized shoots varies.
Sometimes the underleaf is next to the adjacent leaf on a continuation of the same insertion line. At other times the underleaf is behind and a little more apical in position to the leaf on a separate line of insertion that overlaps the insertions of the adjacent leaf as well as the opposing leaf.
Assumption of a helical segmentation sequence seems reasonable given the apparent invariance across the Jungermanniales and its manifestation on at least small and medium-sized shoots, as evidenced by the sequence of merophytes. Th e most unusual and counter-intuitive features are the presence of underleaves when lateral leaf insertion lines reach the ventral stem mid-line, and the absence of underleaves from the larger shoots. Here we posit a competitive model of shoot growth to explain the fi ve observations above, wherein merophytes vie for occupancy of the mature shoot. An increase in shoot stature can be achieved by an increase in stature of the lateral merophytes only, or shoot stature can be increased by increasing the size of the lateral merophytes at the expense of ventral merophyte stature (Schuster 1966).
In Brevianthus hypocanthidium both may contribute, and our competitive model combining changes in both lateral and ventral merophytes is postulated as follows. Th e stature of lateral merophytes increases with shoot stature, while the stature of ventral merophytes does not, resulting in a decrease in relative stature of the ventral merophyte. With reduced stature comes a reduction in relative vigour of ventral merophyte derivatives, resulting in proportionally fewer cell divisions particularly those that contribute to the stem cells. Th e derivative cells fail to occupy the complete length of the potential ventral stem surface, they become localized to the region of initial deposition only. In such cases the 'vacant' ventral stem surface is occupied by tissue derived from lateral merophytes. Th ese lateral merophyte derivates carry their leaf insertion lines to the ventral stem midline. Th e ventral merophytes become "marooned" or isolated at the sites of deposition. Growth of opposite and adjacent lateral merophytes causes the ventral merophyte to appear both laterally displaced and enveloped by the lateral merophytes. Perhaps eventually the ventral merophyte initials lose vigour to the extent that no divisions resulting in leafy tissue are completed. Developmental studies, perhaps including selective sequential sectioning, might test this model when more material becomes available.
Perianths and bracts. In Plagiochila shoot stature increases prior to gynoecium production. A female bracteole is often produced, as may be underleaves associated with one or two of the subtending gyres. In some members of this genus increase in shoot stature results in the re-expression of leafy appendages on the ventral merophyte. Th e same ought be true in B. hypocanthidium, given that in B. fl avus bracteoles are expressed in the two cycles of female bracts on gynoecium-bearing branches. Th ese bracteoles are either broadly connate or free from the adjacent bracts (Engel and Schuster 1982). Th e brevity of the lateral intercalary branches upon which gynoecia are borne in this species precludes assessment of transformation from normal leafy shoots. Location of fertile material of B. hypocanthidium would confi rm the consistency of gynoecium position and associated characters within the Brevianthus lineage.