Integrative Taxonomy Reveals a New Species of the Genus Lejeunea (Marchantiophya: Lejeuneaceae) from Peninsular Malaysia

Prior to the advent of molecular work, the observable variation in vegetative reproduction has been used to classify Lejeunea into subgenera and sections. Thereby, the ability of developing caducous leaves was regarded as major factor. A reexamination of several Lejeunea specimens revealed that L. cocoes with caducous leaves shows considerable morphological differences with non-caducous leaved plants of L. cocoes. Phylogenetic analyses based on a three-marker dataset (rbcL, trnLF and the nuclear ribosomal ITS region) indicated two independent and robust lineages of the morpho-species L. cocoes. We consider both clades as two distinct species and therefore describe the new species, L. malaysiana for L. cocoes morpho-species with caducous leaves. Lejeunea malaysiana is characterized by its caducous leaves with ribbon-like and plantlet regenerants, strongly reduced leaf lobules, distant and deeply bilobed underleaves, long-keeled obovoid perianth, and autoicy and ranges from tropical Asia to the Pacific region.


Introduction
Malaysia has a very rich flora of liverworts with about 800 recorded species and infraspecific taxa [1]. The liverwort flora of Malaysia, particularly from the states of Sabah and Sarawak, has been much explored since the 19th century; however, many mountains and forests in Malaysia remain largely unexplored and many taxa are completely unknown [2]. Papers dealing with Malaysian liverworts are being published each year, including new floristic liverwort explorations, e.g., [3][4][5], descriptions of new species, e.g., [6][7][8], and integrative morphological and molecular studies, e.g., [9][10][11]. Here, we describe another new species of Lejeunea from the state of Pahang in Peninsular Malaysia based on morphological and molecular evidence.
A comprehensive taxonomic revision of the genus Lejeunea in Malaysia was published by Lee [12], leading to the recognition of 30 species and two varieties in this country. New records of Lejeunea continue to be discovered, and currently, 37 species and two varieties are reported from Malaysia, of which three are endemic, viz., L. contracta Mizut., L. gradsteinii G.E. Lee et al. and L. pectinella Mizut. [13]. The genus Lejeunea is one of the largest genera of the family Lejeuneaceae with about 200 to 300 species [14,15]. The molecular dating results have unveiled the origin and biogeographic history of Lejeunea in which the genus attained its greatest diversity during the Miocene period [16]. The genus is well known for its extensive morphological homoplasy and therefore morphology alone is inadequate to classify the genus. Only an integrative taxonomical approach combining molecular and morphological data can help to untangle species complexes and establish more natural species circumscriptions, e.g., [17].
In this study, we incorporated molecular data based on sequences of two chloroplast regions (rbcL, trnLF) and the nuclear ribosomal ITS region of multiple accessions morphological circumscribed species of L. cocoes Mitt. Our phylogenetic analyses (RAxML, MrBayes), in combination with extensive morphological reevaluations of the specimens, discovered two independent monophyletic lineages of the morpho-species L. cocoes: three accessions of L. cocoes with caducous leaves grouped together, whereas two accessions of L. cocoes without caducous leaves formed another independent lineage. We consider both clades as two biologically different entities and not closely related and therefore describe a new species for L. cocoes morpho-species with caducous leaves.

Morphology
Morphological study of 10 specimens initially assigned to L. cocoes from Peninsular Malaysia, Indonesia, Chagos Archipelago (Indian Ocean) and the Fiji Islands revealed that they have important differences in comparison to the typical L. cocoes, a tropical Asian species, including the vegetative reproduction by means of caducous leaves with ribbonlike and plantlet regenerants, strongly reduced leaf lobules, long-keeled obovoid perianth (up to 3/4 of the perianth length), and autoicy. However, on the other hand, both species are morphologically very similar and vary profusely in the absence or presence of caducous leaves, which are so far only regarded as a means of vegetative reproduction.

Molecular Phylogeny
The Bayesian and Maximum Likelihood tree topology are identical and split into two well-supported main clades ( Figure 1). Species with caducous leaves and other means of vegetative reproduction, e.g., regenerants, cladia, and fragmenting stem and branches, are diffusely distributed and are found in both main lineages of Lejeunea. The three accessions were initially assigned to L. cocoes that have caducous leaves grouped with high support (BS 100, BPP 1), whereas two accessions of L. cocoes without caducous leaves formed another independent lineage (BS 100, BPP 1) in a well-supported clade including L. micholitzii Mizut. (BS 86, BPP 1). The latter two species are found in a sister relationship to a clade comprising autoicous species including L. anisophylla Mont., L. ibadana A.J. Harr. and E.W. Jones, L. subolivacea Mizut., L. adpressa Nees, L. dipterocarpa E.W. Jones, L. papilionacea Prantl, L. compressiuscula (Steph.) G.E. Lee and Heinrichs, and L. heinrichsii G.E. Lee et al. Based on our molecular phylogeny and morphological investigation, we describe the L. cocoes morpho-species with caducous leaves as a new species, L. malaysiana. The specific epithet 'malaysiana' refers to the type locality in Malaysia.
Diagnosis: Affinity to the tropical Asian L. cocoes Mitt. in the ovate leaves with narrowly rounded apices, smooth cuticles, laminal cells with indistinct trigones and without intermediate thickenings, and distantly arranged underleaves with divergent lobes but differs by its caducous leaves, strongly reduced leaf lobules, long-keeled obovoid perianths and autoicy.

Discussion
Integrative taxonomy includes information from different types of data and methodologies; in the present study, molecular data aimed to establish a more robust species hypothesis and circumscription. Particularly for the genus Lejeunea, integration of molecular and morphological data should therefore be encouraged, because only such combined molecular-morphological studies allow for a reexamination of morphological characters that were previously considered to be of diagnostic importance in the species circumscriptions. Recent studies have demonstrated congruence or conflict between molecular and morphological data in Lejeunea, e.g., molecular phylogenetic analyses resolved three morphologically similar species, i.e., the African Taxilejeunea pulchriflora Pearson and two Asian L. propagulifera Gradst. and L. tamaspocsii G.E. Lee, in a robust monophyletic lineage [9]. In comparison, morphologically similar accessions initially assigned to L. tumida Mitt. belong to four species nested in different clades [18]. Before molecular data, diagnostic characters such as toothed or eplicate perianths were used for generic distinction or subgenera delimitations. Such morphological data were often proved to be homoplastic in phylogenetic analyses [9,19]. In the present study, one such morphological character is caducous leaves which were regarded as one characteristic for the species L. cocoes. In our phylogenetic analyses, however, we discovered two independent monophyletic lineages that by reexamination of the plant material turned out to be linked to the presence or absence of caducous leaves on specimens of the morpho-species of L. cocoes. We thus consider both clades as two different species and introduce the new species L. malaysiana for the clade including plants with caducous leaves.
Generally, vegetative reproduction of Lejeunea is classified into five types, viz., plantlet regenerants, ribbon-like propagules, cladia, caducous, and fragmenting stem or branches [12,20]. Regenerants and propagules that grow on leaf margins are very common forms of vegetative reproduction and have previously been described in Lejeunea [9,[20][21][22][23][24]. The cladia type thus far produces exclusively on L. dimorpha T.Kodama, an obligate epiphyllous taxon reported from Vietnam, Peninsular Malaysia, Borneo, Papua New Guinea and the Philippines [25]. The caducous type may occur on the leaves, leaf lobes, underleaves, or branches, but caducous leaves is the most common and has been reported in many species of Lejeunea, e.g., in the Neotropics: L. deplanata Nees, L. phyllobola Nees and Mont., L. oligoclada Spruce, L. perpapillosa M.E.Reiner and K.C.Pôrto, L. parviloba Ångstr., L. flagellifera Bastos et al.; in tropical America: L. cochleata Spruce, L. ptosimophylla C. Massal., L. rionegrensis Spruce [20,26]; in Africa: L. kuerschneriana Pócs [27], and in tropical Asia: L. subacuta Mitt. [28,29]. The caducous leaves of the species mentioned above are occasionally with plantlet regenerants on the marginal leaf cells, e.g., [30] (p. 14), whereas in L. malaysiana, apart from the leaf margin, the plantlet regenerants are also seen growing on the top of long ribbon-like regenerants. Two different regenerants are developed on the caducous leaves of L. malaysiana: the young plantlets grow on top of the long ribbonlike regenerants (10-24(30) cells or 0.2-0.6 mm long and 4-5 cells wide) whereas other ribbon-like regenerants (4-6 cells long and 4-5 cells wide) developing on the marginal leaf cells and grow into young plantlets. Each caducous leaf of L. malaysiana may produce 2(-6) regenerants and the leaf lobes produced on the stem are hardly found. However, the gynoecial shoot is usually is present with all its leaves. Although vegetative reproduction plays a vital role in the range size of liverworts and increases the chances of successful long-range dispersal in species with unisexual populations, only a few species of Lejeunea reproduce frequently by vegetative reproduction. In the molecular phylogeny of Lejeunea, we included 24 species, including L. malaysiana, with vegetative reproduction and found them diffusely distributed throughout the two subgenera of Lejeunea (Figure 1). Out of 24 species, only seven (L. deplanata Nees, L. malaysiana, L. kuerschneriana Pócs, L. oligoclada Spruce, L. phyllobola Nees and Mont., L. ptosimophylla C. Massal, and L. tapajosensis Spruce) are with the occurrence of caducous leaves and are nested in the subg. Crossotolejeunea but they do not form a monophyletic group. All but L. malaysiana are nested in a robust monophyletic lineage with the Neotropics accessions of L. rotundifolia Mitt., L. trinitensis Lindenb., L. ruthii (A. Evans) R.M. Schust., and L. subplana (Steph.) C.J. Bastos. However, vegetative reproduction by caducous leaves is not known in the latter four species. In our molecular analyses, we also included L. cancellata (Steph.) C.J. Bastos, which can produce caducous branches, but this species is nested in the largely neotropical clade, subg. Lejeunea.
Lejeunea malaysiana and L. cocoes share similar characteristics, including the cells with relatively small or poorly developed trigones and without intermediate thickenings, the smooth cuticle, the small and bilobed underleaves with lanceolate lobes, and the obovoid perianths. Indeed, L. malaysiana closely approaches L. cocoes in these features and plants of L. malaysiana not showing vegetative reproduction can be mistaken for L. cocoes. The development of caducous leaves is often abundant in L. malaysiana and is usually evident even on casual examination, unlike in other Lejeunea species (except for L. dimorpha [25]) where the propagules are more sporadic and infrequent. Vegetative reproduction of L. cocoes is sparsely produced and exclusively by means of ribbon-like regeneration, never by caducous leaves. Equally distinctive is the autoicy, which separates L. malaysiana from L. cocoes. In our molecular phylogenetic analyses, both species are resolved in different monophyletic lineages where L. cocoes forms a sister relationship with L. micholitzii. Lejeunea malaysiana is perhaps most closely related to L. subacuta reported from India and China, sharing with it a strong ability to produce caducous leaves; however, L. subacuta differs from L. malaysiana in the dioicy, oblong leaf lobe with occasionally marginal rhizoids, and presence of male bracteoles throughout the androecial shoot [28,29].

Plant Materials and Morphological Investigation
Morphological characteristics of L. malaysiana were studied based on herbarium specimens from the Eszterházy Károly College herbarium (EGR), the Conservatoire et Jardin Botanique de la Ville de Genève (G), the Göttingen University herbarium (GOET), the herbarium of Universiti Kebangsaan Malaysia (UKMB), and the herbarium of Universiti Malaysia Terengganu (UMTP). The holotype is deposited at the herbarium UMTP under the management of South China Sea Repository and Research Centre, Institute of Oceanography and Environment, Universiti Malaysia Terengganu. Plant material was examined by light microscopy and the drawing of the type specimen was produced using an Olympus BX43 microscope equipped with a drawing tube.

DNA Extraction, PCR Amplification, Sequencing and Alignment
Gametophytic plant tissue was isolated from five dried herbarium specimens initially identified as L. cocoes. Total genomic DNA was isolated using the Invisorb Spin Plant Mini Kit (Stratec Molecular GmbH, Berlin, Germany) prior to amplification. Amplification of the three molecular genetic markers, rbcL, trnLF, and ITS was carried out with the PCR protocol [9,31]. The newly generated sequences were assembled and edited using PhyDE v0.9971 and then compared to Genbank sequences using nucleotide BLAST search [32] to evaluate their assumed belonging to the genus Lejeunea. The new sequences were then aligned manually and integrated into the Lejeunea alignment of Lee et al. [16] using BioEdit 5.0.9 [33]. Taxa used in the study, including GenBank accession numbers and voucher details, are listed in Table S1. Ambiguous hotspot positions were excluded and the reverse complement of one inverted repeat in the trnLF region was aligned with the other sequences prior to molecular phylogenetic analyses [34].

Phylogenetic Analyses
Maximum Likelihood (ML) analysis was conducted with RAxML-HPC 8.2.8 [35]. Appropriate DNA partitions and corresponding substitution models, rate of invariable sites, and gamma rate heterogeneity were identified using JModelTest 2 [36] and the Akaike information criterion (AIC). This resulted in TVM + I + G for rbcL, TVM + G for trnL-trnF, and TIM1 + I + G for nrITS. Since these models were not available in RAxML, the best fitting overparameterized model GTR + G was used. Trees were generated by selecting 10 independent runs and the multi-parametric bootstrap option autoMRE, resulting in 300 bootstrap replicates to compute node support as bootstrap values at each node. The compatibility of the chloroplast and nuclear regions was explored by comparing the trees obtained from independent ML analyses for each region. The trees were visually compared to identify conflicting nodes with BV higher than 70% [37], and an ML analysis was conducted with two chloroplasts (rbcL, trnLF) partitions and a nuclear (ITS) partition.
Bayesian Inference (BI) was undertaken in MrBayes 3.2.6 [38]. The same dataset, nuclear substitution models, and partitions as those in the ML analysis were used. Two metropolis-coupled Markov chain Monte Carlo (MCMC) analyses, including three heated chains and one cold chain, were run for two million generations and sampled every 200 generations. Convergence and stationarity of the runs were checked in TRACER 1.7 and an average standard deviation (SD) of split frequency below 0.01 indicated a sufficiently long run. The first 25% of the trees were discarded as burn-in and the remaining trees were summarized by TreeAnnotator 1.8.3 [39] using median node heights. The resulting maximum clade credibility (MCC) tree was visualized in FigTree 1.4.3. Nodes with posterior probability values ≥ 0.95 were regarded as good support [40].

Conclusions
Our present study shows a strong conflict between molecular phylogenies and morphologybased classification in Lejeunea and its lineages. The apparent incongruence between the molecular variation and the morphologically circumscribed taxa is primarily demonstrated by extensive homoplasy in Lejeunea where most previously assumed morphological characters appear to be highly homoplastic. Hence, the natural classification of Lejeunea will require integrative taxonomical studies, including comprehensive taxonomic revision and DNA sequence data. These data are essential for evaluating different species concepts and exploring the morphological and genetic variation in reconstructing relationships and the circumscriptions of Lejeunea species.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11131642/s1, Table S1: Taxa used in the present study with information about the origin of the studied material, vouchers, as well as GenBank accession numbers. Acknowledgments: The first author is indebted to the Alexander von Humboldt Foundation for sponsoring her stay in Germany, and to the late Jochen Heinrichs for accepting her to carry out her Lejeunea/Taxilejeunea research at the University of Munich. Thanks are due to the directors and curators of the herbaria EGR, G, GOET, JE, and UKMB for the loan of Lejeunea specimens.

Conflicts of Interest:
The authors declare no conflict of interest.