The resurrection of Neohattoria Kamim. (Jubulaceae, Marchantiophyta): a six decade systematic conflict resolved through a molecular perspective

Abstract The systematic placement of Frullania herzogii has been contentious since its description six decades ago. Over the years it has been interpreted as either a member of the genus Frullania or segregated into its own genus, Neohattoria, due to morphological similarities with both Frullania and Jubula. Here we provide molecular evidence that supports the recognition of the genus Neohattoria and its inclusion within the Jubulaceae, together with Jubula and Nipponolejeunea. Jubulaceae are placed sister to Lejeuneaceae rather than to the monogeneric Frullaniaceae.


Introduction
Th e liverwort Frullania herzogii S.Hatt. was originally described by Hattori (1955) from a poor, sterile specimen collected on Mt. Hayachine in Iwate Prefecture, northern Honshu, Japan. Since that time the generic and even familial placement of the species has remained controversial. Th e species also has remained poorly known partially because of its seemingly limited distribution in the subalpine coniferous forest zones of Honshu and Hokkaido, Japan, and the Kuril Islands (Inoue et al. 1981, Stotler andCrandall-Stotler 1987). Hattori (1955) remarked that the leaf morphology, with acute teeth along the margin, diff ered from all the other Japanese Frullania Raddi species known by him. A few years later, in his monograph of Japanese Frullaniaceae, Kamimura (1961) erected the new genus Hattoria Kamim. to separate this taxon from other Frullania species. He stated that although his new genus superfi cially resembled species of Cololejeunea (Spruce) Schiff n. or Frullania, there was an important similarity between the branching patterns of Hattoria herzogii (S.Hatt) Kamim. and species in the genus Jubula Dumort. In both Jubula and Hattoria, the branches replace the lobule of the leaf at the point of insertion, and the leaf lobes are attached to both the main stem and to the branch. Although Kamimura (1961) noted the similarity of cell shape between Hattoria and Frullania, he considered the combination of branching architecture and leaf denticulation suffi cient to recognize Hattoria as a distinct genus. A year later he had to give a new name, Neohattoria Kamim., to his recently described genus (Kamimura 1962), because of the almost simultaneous although earlier description of Hattoria by Schuster for a liverwort in the Lophoziaceae (Schuster 1961).
Later Schuster (1963), in a key for the Southern Hemisphere genera of liverworts, expanded the circumscription of Neohattoria to include two more species, Frullania microscopica Pearson from New Caledonia, and F. parhamii (R.M.Schust.) R.M.Schust. ex von Konrat, L.Söderstr. & A.Hagborg from Fiji. He based his taxonomic decision on the morphology of the reduced leaves on branch bases, the subfl oral innovations, and the sharply delimited bracts and bracteoles of F. microscopica, and on the toothed leaf lobes of this species. Schuster (1963) did not provide any argument for placement of the Fijian F. parhamii in Neohattoria, other than the hyaline margins of the leaves that can be seen in this species and in F. microscopica (as inferred from the key). However, his key is restricted to the Southern Hemisphere and did not include the type of the genus, which completely lacks a hyaline border in leaf lobes. Schuster (1970) Hentschel & Heinrichs (von Konrat et al. 2010), while the rest of the taxa are currently included in Frullania subg. Microfrullania (R.M.Schust.) R.M.Schust. Th e current taxonomic placement of these taxa is based on both morphological Mizutani 1982, Schuster 1992) and molecular evidence (Hentschel et al. 2009, von Konrat et al. 2012. Asakawa et al. (1979) demonstrated, based on chemical compound diff erences, that Jubulaceae sensu lato should be divided into three families, i.e. Jubulaceae, Frullaniaceae and Lejeuneaceae. Th is view has been confi rmed by most molecular phylogenies published to date (e.g., Forrest et al. 2006, Heinrichs et al. 2005. Asakawa et al. (1979) listed 11 morphological characters that support the separation of Frullaniaceae and Jubulaceae, and placed Neohattoria together with Jubula in the Jubulaceae. Hattori (1982Hattori ( , 1984Hattori ( , 1986 and Hattori and Mizutani (1982) also accepted the separation between Jubulaceae and Frullaniaceae and argued that Amphijubula R.M.Schust., a genus formerly considered by Schuster (1970Schuster ( , 1980 as intermediate between Jubula and Frullania, should be placed within Frullania. Th is view was fi rst held by Engel (1978), who had earlier reduced Amphijubula to a synonym of Frullania.
In 1987, Stotler and Crandall-Stotler published a thorough treatise of the taxonomic history of Neohattoria herzogii (S.Hatt.) Kamim. in the context of a detailed re-evaluation of its morphology, including the discovery of immature female infl orescences. In that contribution they came to the conclusion that this taxon should be considered within the circumscription of Frullania, although in its own subgenus, F. subg. Dentatilobi Stotler & Crand.-Stot. Th eir conclusion was based on both vegetative and reproductive characters, including the morphology of the bracts surrounding the female gametangia, lobule anatomy, leaf cell pattern, and the morphology of regenerants. Although they recognized that leaf-lobe insertion, branch morphology, and morphology of stylus are more similar to Jubula than to Frullania, they concluded that on the basis of the Frullania-like infl orescences and regenerants, Neohattoria should be synonomized with Frullania. Th is synonomy was adopted by Grolle and Meister (2004) who described a morphologically similar plant from Oligocene amber from Bitterfeld (Germany) as Frullania (subg. Dentatilobi) hamatosetacea Grolle. However, this fossil species appears morphologically closer to F. subg. Microfrullania than to Neohattoria, and this issue will be explored in detail in a forthcoming monograph of the latter subgenus.
Lack of useable specimens has previously precluded inclusion of Neohattoria in molecular phylogenetic studies. As a result of recent collecting activities, fresh material became available that allowed for successful DNA extraction and amplifi cation. In the present study, we use molecular sequence data to investigate the phylogenetic position of Neohattoria. We investigate whether the genus should be placed in the Frullaniaceae or the Jubulaceae and evaluate whether molecular evidence supports the recognition of Neohattoria as a distinct genus.

Microscopy
For the production of microscopic images an Olympus BX51 microscope was used, equipped with both a QICAM Fast1394 camera from QIMAGING (Surrey, Canada), and a slide scanner (moving platform stage attached between the objectives and the condenser) from Objective Imaging Ltd. (Cambridge, UK). Th e software "Surveyor" from the latter company was used for the digitally rendered images.

DNA extraction, PCR amplification and sequencing
We worked with two independent datasets to address two diff erent questions, (1) what is the position of Neohattoria relative to the Frullaniaceae, Jubulaceae and Lejeuneaceae, and once we obtained results from these analyses, we asked (2) what is the position of Neohattoria within the Jubulaceae. For dataset 1 sequences were generated for two mitochondrial (nad1, rps3), and two chloroplast loci (psbA, rbcL), following DNA extraction, amplifi cation and sequencing methods described by Shaw et al. (2003), and using primer sequences provided in Cooper et al. (2011). For dataset 2 we used the aforementioned plastid regions (psbA and rbcL) together with the nuclear ITS region following the methods described by Shaw et al. (2003), and the chloroplast trnL-trnF region, amplifi ed and sequenced as described in von Konrat et al. (2012). All sequences were edited and manually aligned in PhyDE v0.9971 (www.phyde.de) following the alignment rules and hotspot defi nitions presented in Kelchner (2000), Olsson et al. (2009), and Borsch and Quandt (2009).

Taxon sampling and outgroup selection
For dataset 1 seven species of Radula were selected as outgroup taxa following the results already published in recent liverwort phylogenies (Davis 2004, Forrest et al. 2006, Feldberg et al. 2014, Heinrichs et al. 2005. Th e same criteria were undertaken for dataset 2, including all taxa with sequences available in GenBank for Jubula and Nipponolejeunea S.Hatt. (Ahonen 2006, Ahonen et al. 2003, Konstantinova and Vilnet 2011, Pätsch et al. 2010, Wilson et al. 2004, using selected taxa of the Lejeuneaceae and species of Frullania as outgroup based on results from dataset 1. GenBank accession numbers for both newly generated sequences and for already published sequences are provided in Appendices 1 and 2 for datasets 1 and 2 respectively.

Phylogenetic inferences
Both datasets were analysed with PartitionFinder v1.1.0 (Lanfear et al. 2012(Lanfear et al. , 2014 to develop best-fi t partitioning schemes and models of molecular evolution. Dataset 1 was partitioned setting one separate data block for each of the four genes used, each of them divided in three according to each codon position; introns and/or spacers were coded as extra partitions. Dataset 2 was partitioned in four parts, corresponding to the regions included only, without inner codon partition for the coding regions analysed. For dataset 1, phylogenetic reconstructions under maximum likelihood (ML) were performed in GARLI v2.01 (Zwickl 2006), setting up seven diff erent models for the eleven partitions determined by PartitionFinder. Two independent searches each with 100 bootstrap replicates were made, and the 50% majority-rule consensus tree from all obtained trees was obtained with SumTrees v3.3.1 included in the package DendroPy v3.12.2 (Sukumaran and Holder 2010). Bayesian Posterior Probabilities analyses (PP) were executed in MrBayes v3.2.2 (Huelsenbeck andRonquist 2001, Ronquist andHuelsenbeck 2003) also with the partitioned data set as given by PartitionFinder, and setting a diff erent model for the individual partitions from the available options in MrBayes, with all characters given equal weight and gaps treated as missing data. Th e default settings of the program for a priori probabilities were used. Four runs, each with four MCMC chains (one million generations each) were run simultaneously, with the temperature of the heated chain set to 0.2 (default setting). Chains were sampled every 100 generations. Calculation of the consensus tree and posterior probabilities of clades was based on the set of trees sampled after the chains had converged, as observed graphically using Tracer v1.5 (Rambaut and Drummond 2007). For dataset 2, phylogenetic reconstructions under ML were performed in GARLI v2.01 and Bayesian analyses were executed with MrBayes v3.2.2 following the protocols as described above. For this dataset only three diff erent partitions were suggested by PartitionFinder, and the models given by this software for each partition were incorporated into the settings of both the ML and the Bayesian analysis. Trees were edited and support values added using TreeGraph v2.0.54-364 beta (Stöver and Müller 2010).

Results
Th e complete alignment for dataset 1 including all four regions mentioned above, with fl anking areas pruned to avoid ambiguous readings, comprised 4818 characters for 54 accessions, of which 694 were parsimony informative. A total of 101 new sequences were generated for this study (Appendix 1). In the analysis of the Neohattoria sequences with accessions of the Frullaniaceae, Jubulaceae and Lejeuneaceae (dataset 1), Neohattoria is strongly supported (as defi ned by Pedersen et al. 2007) as one of three clades belonging to the Jubulaceae in both ML and Bayesian analyses, with accessions of Nipponolejeunea, resolved in a second clade and those of Jubula, in a third clade (Fig. 1), although the latter with low support (ML = 52, PP = 0.6). Th e Jubulaceae is resolved as sister to the Lejeuneaceae with strong support in both types of analysis. Th e position of the Frullaniaceae as sister to this latter clade (Jubulaceae + Lejeuneaceae) was strongly supported by the Bayesian analyses (PP = 1.0), but it was not recovered by the ML analyses. Th e Bayesian analyses also resolved Neohattoria as sister to the rest of the Jubulaceae (Nipponolejeunea + Jubula) with strong support (PP = 1.0).   Th e complete alignment for dataset 2 including all four regions included, and after pruning the fl anking areas to avoid ambiguous readings and deleting unalignable areas of the ITS region, comprised 3737 characters for 55 accessions, of which 548 were parsimony informative. Th e four diff erent regions were not equally represented in the matrix, as shown in Appendix 2. Th e results of the analyses (Fig. 2) confi rm with strong support the placement of Neohattoria within the Jubulaceae (ML = 100, PP = 1.0), and forming a sister clade to Nipponolejeunea, although recovered with strong support only by the Bayesian analysis (ML = 64, PP = 0.97). Jubula was resolved as the sister clade to the Neohattoria-Nipponolejeunea clade, although with low support (ML = 65, PP = 0.5).
Th e voucher of Neohattoria herzogii used for DNA extraction is illustrated in Figure 3.

Discussion
Our molecular analyses support recognition of the genus Neohattoria as distinct from the genus Frullania, as fi rst proposed by Kamimura (1961) almost 55 years ago. Moreover, our molecular analysis strongly supports its inclusion within the Jubulaceae, together with Jubula and Nipponolejeunea. A close relationship with Jubula, based on similarities in branch morphologies, was fi rst suggested by Kamimura (1961, p. 94), and also accepted by Hattori et al. (1972). Inoue et al. (1981) provided new karyological, chemical and ecological data on N. herzogii and concluded that the biosystematic evidence collected suggested distance between Jubula and Neohattoria, but, nonetheless, retained Neohattoria in the Jubulaceae. While morphologically closer to Jubula than Nipponolejeunea to which it is sister, it is clearly not nested in the Jubula clade. Th is combination of molecular and morphological evidence, in fact, supports its recognition as a distinct genus in the Jubulaceae.

Circumscription and relationships of the Jubulaceae
Our results strongly support the position of the Jubulaceae (containing Jubula, Nipponolejeunea and Neohattoria) sister to the Lejeuneaceae, and the Frullaniaceae as sister of the latter clade, although without signifi cant support (Fig. 1). Th ese results agree with several molecular phylogenies (e.g. Ahonen 2004, Forrest et al. 2006, Heinrichs et al. 2005. Th us the traditional view of a widely circumscribed Jubulaceae including Frullania is further rejected in this study. Th ese three families (Frullaniaceae, Jubulaceae and Lejeuneaceae) share several morphological characters, including the leaves divided into two (or three) parts [lobe, lobule (and stylus)], the beaked perianths, the sporophyte enclosed in a stalked true calyptra, the bistratose capsule wall, and the vertically aligned elaters that are attached to the valve apices (Crandall-Stotler et al. 2009, Gradstein et al. 2001, Schuster 1992. However, these characters need to be carefully evaluated to understand their evolution and their role in demonstrating the history of these lineages. In the past, Jubula, Frullania and members of the Lejeuneaceae were placed in a single taxonomic group (the subtribe Jubuleae), based largely on the similarities among their sporophytes (e.g. Müller 1915). Verdoorn (1930) argued that based on most characters (e.g., number of archegonia, seta form, and lobule ontogeny) Jubula belongs nearest to Frullania, which later lead Schuster (1992, p. 6) to describe Jubula as a "bona-fi de genus of Jubulaceae [= Frullaniaceae]". Mizutani (1961) was the fi rst to propose that, except for the lobule structure, Jubula had no alignment with Frullania, and subsequently placed Jubula into the Lejeuneaceae. However, Asakawa et al. (1979) concluded that chemically, both Jubula and Frullania are quite diff erent from Lejeunea Lib. species. Interestingly, the phylogenetic analysis by Crandall- Stotler and Stotler (2000) of 40 gametophyte and 21 sporophyte characters distributed among 34 liverwort families, resolved F. asagrayana Mont. as sister to a clade containing J. hutchinsiae (Hook.) Dumort. subsp. pennsylvanica (Steph.) Verd. and L. cavifolia (Ehrh.) Lindb. However, in the systematic treatment of the same work (Crandall-Stotler and Stotler 2000) Jubulaceae is presented as including both Jubula and Frullania, whereas the Lejeuneaceae is presented as a separate family, following accepted classifi cations of the time. Th e revised version of that classifi cation, incorporating some recent molecular data, presents the Frullaniaceae, Jubulaceae and Lejeuneaceae as three separate families within the suborder Jubulineae (Crandall-Stotler et al. 2008, which is accepted here but with the transfer of Neohattoria from the Frullaniaceae to the Jubulaceae. Assessing the importance of diff erent morphological characters in circumscribing Frullaniaceae, Jubulaceae and Lejeuneaceae has been a diffi cult problem, but there are several characters that are consistent with the molecular phylogenetic results presented here. In most Lejeuneaceae a true stylus does not develop, but instead a single, unstalked slime papilla is formed at the junction of the lobule base and the stem, while in Jubula and Neohattoria there is a one-or two-celled fi lament terminated by a slime papilla in this position Guerke 1980, Stotler andCrandall-Stotler 1987). Both types of structures are clearly diff erent from those of the Frullaniaceae, where the stylus is always formed by more than two cells and is usually very conspicuous. Th e Jubulaceae and Frullaniaceae can be clearly diff erentiated from the Lejeuneaceae by the lobule, which is almost free from the larger dorsal lobe, and typically modifi ed into an infl ated, balloon-like to helmet-shaped sac whose aperture is directed either toward the shoot base or toward the stem, with the exception of Nipponolejeunea which has Lejeuneaceae-like lobules. Guerke (1978) hypothesised that Jubula was more advanced than Frullania on the basis that Jubula has many specialized characteristics e.g., a highly reduced stylus, seta, and foot, and features associated with the sporeling. In contrast, Schuster (1992, p. 9) stated that taxa such as Amphijubula microcaulis (Gola) R.M.Schust. (≡ F. microcaulis Gola), with a 16 + 4 seriate seta and monogynous gynoecia, diminish the distinctions between the two groups such that he prefers not to attempt a "subfamilial separation" at all. However, revision of the chemical, morphological, and ecological data provided support for the recognition of two subfamilies in the Jubulaceae (Guerke 1978, von Konrat 2004. Alternatively, Asakawa et al. (1979), on the basis of biochemical and morphological evidence, proposed two families: Jubulaceae (Jubula, Neohattoria) and Frullaniaceae (Frullania, Steerea S.Hatt., Amphijubula, and Schusterella S.Hatt.). Hattori (1982Hattori ( , 1984Hattori ( , 1986 and Hattori and Mizutani (1982) also accepted two families. Th is approach has been adopted in most recent hepatic fl oras and classifi cations (Paton 1999, Damsholt 2002, Casas et al. 2009, Crandall-Stotler et al. 2009, Frey and Stech 2009). Schuster (1980Schuster ( , 1992 questioned the division into two families and argued that only the single family Jubulaceae should be recognized, but commented that this area of classifi cation remains replete with ambiguities and contradictions. Interestingly, he also suggested that there was a possibility that Neohattoria might share a closer affi nity to Jubulopsidaceae (= Lepidolaenaceae) than to Jubulaceae (Schuster 1996), a view fi rst expressed when Grolle (1966) transferred Jubula novae-zelandiae E.A.Hodgs. & S.W.Arnell, which is the generitype of Jubulopsis R.M.Schust., to Neohattoria. However, recent molecular analyses (e.g., Heinrichs et al. 2005, Forrest et al. 2006) have demonstrated that Jubulopsis (= Lepidolaena) is far removed from the Jubulaceae.
Morphologically, the monogeneric Frullaniaceae can be diff erentiated from the Jubulaceae by: (1) plants usually with conspicuous secondary pigmentation, often reddish; (2) initial leaves of branches either trifi d or bifi d; and (3) spores with rosette-like protrusions. Conversely, in the Jubulaceae the plants are: (1) soft and without secondary pigmentation (thus usually dull green to pale brown); (2) the initial leaves of branches are small, subtriangular, and never tri-or bifi d; and (3) the spores without rosettelike protrusions. Th e fi rst two of these characters support the placement of Neohattoria within Jubulaceae rather than Frullaniaceae (spores remain unknown in Neohattoria).
Interestingly, Schuster (1996) suggested that there was a possibility that Neohattoria might share a closer affi nity to Jubulopsidaceae (= Lepidolaenaceae) than to Jubulaceae. Th is view was fi rst expressed when Grolle (1966) transferred Jubula novae-zelandiae E.A.Hodgs. & S.W.Arnell, which is the type species of Jubulopsis R.M.Schust., to Neohattoria. However, preliminary unrooted trees made for this contribution including Ascidiota C.Massal., Gackstroemia Trevis., Goebeliella Steph., Lepidogyna R.M.Schust., Lepidolaena Dumort. (= Jubulopsis) and Porella L. together with representatives outside the Porellales, showed Neohattoria far away from Lepidolaenaceae but within Jubulaceae (results not depicted). Th ese results are basically the same as the ones observed in recent molecular phylogenies (e.g. Heinrichs et al. 2005, Forrest et al. 2006, demonstrating that these groups are only distantly related to either the Jubulaceae or the Frullaniaceae.

Circumscription and relationships of Neohattoria
Our results place Neohattoria in the Jubulaceae with strong support, together with Nipponolejeunea and Jubula. Within the Jubulaceae, Neohattoria is resolved as sister to Nipponolejeunea, and this latter clade sister to Jubula, although this relationship is sen-sitive to taxon sampling (cf. Figs. 1 and 2), and not strongly supported in the analyses. When describing the genus Hattoria (later renamed Neohattoria), Kamimura (1961) conceived it as a monotypic genus containing only the Japanese endemic N. herzogii. Th e singularity of this taxon was well described and illustrated, highlighting its closer affi nities to Jubula instead of Frullania, mostly because of its branching pattern and leaf insertion: "[…] the branch replaces the lobule of leaf in origin and the lobe is inserted partly to the stem and partly to the branch. Th e fi rst leaf and underleaf of branches are much deformed, being the "Vorblätter" of Verdoorn (1930)." (Kamimura 1961, p. 94). Th e characteristic combination of traits that led Kamimura to describe this new genus vanished when Schuster (1963Schuster ( , 1970 added more species in the circumscription of Neohattoria as explained above. Schuster (1970) still recognized the taxonomic singularity of N. herzogii when placing it in its own subgenus within Neohattoria, but failed to see the relationships of this taxon with other Jubula species, precisely because of his wide concept of Neohattoria that includes members of F. subg. Microfrullania and F. subg. Diastaloba.
Oil-bodies in Neohattoria are homogenous, usually more than ten per cell, and similar in size to chloroplasts (Hattori et al. 1972, Inoue et al. 1981. Hattori et al. (1972) reported 10-20 oil-bodies per leaf lobe median cell for N. herzogii and later Inoue et al. (1981, p. 25) reported a similar number "usually 7-15 per leaf-lobe cell (rarely up to 22)". Hattori et al. (1972) stated that oil-bodies of Neohattoria are hyaline and homogenous, and Inoue et al. (1981) recorded in their specimen of Neohattoria that the oil-bodies were completely colourless and homogenous. However, they noted that sometimes they were faintly papillose with a few distinct granules; Inoue et al. (1981) were uncertain if this was due to degeneration of the oil-bodies. Reports of oil-body numbers for Jubula are ambiguous: although Guerke (1979) and Paton (1999) suggested they range between 3-7 in all Jubula taxa, Schuster (1992) stated that the oil-bodies are numerous in the North American material of J. pennsylvanica (≡ J. hutchinsiae subsp. pennsylvanica), ranging from 6-16 per cell, and Mizutani (1961) reported 2-10 for Japanese Jubula. All authors agree that the oil-bodies in Jubula are faintly granular or homogeneous. In Nipponolejeunea, on the other hand, the oil-bodies range between 3-5(7) per cell, are hyaline to somewhat grayish, and are formed by 15-20 internal oil-globules (Mizutani 1961). In Frullania the oil-bodies are usually larger, fi nely to coarsely papillose rather than smooth, and few per cell, with their number generally increasing from the leaf-lobe marginal cells to the basal cells, except in the species that have basal ocelli; however, this number rarely reaches the number of oil-bodies seen in Neohattoria or Jubula. Th e average number of oil-bodies from the 22 species studied by von Konrat (2004) is 4.3 per median lobe cell. One remarkable exception is the North American species F. stylifera (R.M.Schust.) R.M.Schust., which has up to 16 oil-bodies per median cell (von Konrat 2004). A survey of over sixty species (including literature data) suggests that this is a rare condition in the genus (von Konrat 2004). Schuster (1992) described the oil-bodies of Frullania as formed of numerous oil-globules and usually appearing coarsely to fi nely papillose, the only exception being the oil-bodies of F. subg. Microfrullania, which are smooth and frequently appear as almost homogeneous oil-droplets (von Konrat 2004). Th e oil-bodies of Neohattoria then appear closer to the other Jubulaceae genera in appearence (although smooth, homogeneous oil-bodies are also seen in Frullania subg. Microfrullania) and number, notwithstanding the number reported for Nipponolejeunea and some reports of Jubula taxa with fewer oil-body numbers.