DON MARIANO MARCOS MEMORIAL STATE UNIVERSITY North La Union Campus Bacnotan, La Union TERMINAL REPORT (PUBLISHED IN: COLLEGE OF EDUCATION RESEARCH JOURNAL) THE EVOLUTIONARY TREND, BIOGEOGRAPHICAL DISTRIBUTION AND PHYLOGENETIC PLACEMENT OF THE THREE MAJOR ANEURACEAE GENERA (ANEURA, LOBATIRICCARDIA, RICCARDIA) WITH THE GENUS METZGERIA JOMAR L. ABAN 2015 HOW TO CITE: Aban, J. L. (2015). The evolutionary trend, biogeographical distribution and phylogenetic placement of the three major Aneuraceae genera (Aneura, Lobatiriccardia, Riccardia) with the genus Metzgeria. College of Education Research Journal. 3(1): 138-164. 1 THE EVOLUTIONARY TREND, BIOGEOGRAPHICAL DISTRIBUTION AND PHYLOGENETIC PLACEMENT OF THE THREE MAJOR ANEURACEAE GENERA (ANEURA, LOBATIRICCARDIA, RICCARDIA) WITH THE GENUS METZGERIA 1 Jomar L. Aban1 Don Mariano Marcos Memorial State University-North La Union Campus Email: jomar_d2@yahoo.com Abstract— Many previous systematic treatments of Aneuraceae and Metzgeria were based on morphological and molecular characters. However, some species on the family Aneuraceae still needs further taxonomic reclassification. This study is therefore necessary to support both the existing morphological and molecular data that has paved way to the recent placement of the family Aneuraceae and the genus Metzgeria under the order Metzgeriales. Using 32 trnL gene sequences obtained from GenBank, representatives from the three genera under Aneuraceae (Riccardia, Lobatiriccardia, and Aneura) and the genus Metzgeria were investigated metaanalytically and five phylogenetic trees (MP, ML, ME, NJ, and UPGMA) were reconstructed to strengthen the evolutionary trend, biogeographic distribution phylogenetic placement of the previously mentioned taxa using MEGA6 software. It was found that the morpho-anatomical evolutionary trend conforms to the ML tree constructed. The monophyletic sister clade of Aneura and Lobatiriccardia was also proven by MP. Four of the five constructed trees show three major clades (Riccardia, Aneura-Lobatiriccardia, and Metzgeria) but the MP tree shows four clades separating Aneura to Lobatiriccardia. Following this split, it was observed that Aneura alterniloba is more closely related to Lobatiriccardia and therefore needs taxonomic transfer. Keywords— Aneuraceae, phylogenetic inference, trnL, tree reconstruction, Metzgeria, biogeography ——————————  —————————— 1. INTRODUCTION The Marchantiophyta, commonly referred to as liverworts are considered to be the first plant to make the transition from the sea to land, some 400 million years ago, and share a common ancestry with Chlorophyta. Like other bryophytes, they have a gametophyte-dominant life cycle (Crandall-Stotler, 2000; Pain, 2010; & Walker, 2010). It is estimated that there are about 9000 species of liverworts. The most familiar liverworts consist of a prostate, flattened, ribbon-like or branching structure called a thallus; these liverworts are termed thallose liverworts. However, most liverworts produce flattened stems with overlapping scales or leaves in two or more ranks, the middle rank is often conspicuously different from the outer ranks; these are called leafy liverworts (Taylor et al., 1993; & Kendrick et al., 1997). Liverworts are typically small, usually from 2-20 mm wide with individual plants less than 10 cm long, and are therefore often overlooked. Today, liverworts can be found in many ecosystems across the planet except the sea and excessively dry environments, or those exposed to high levels of direct solar radiation (Heinrichs, 2005). 2 Traditionally, the liverworts were grouped together with other bryophytes, the mosses and hornworts, in the Division Bryophyta. However, since this grouping makes the Bryophyta paraphyletic, the liverworts are now given their own division, Marchantiophyta (Schofield, 1985; Crandall-Stotler et al., 2000; & Goffinet, 2000). Although there is no consensus among bryologists as to the classification of liverworts above family rank, the Marchantiophyta may be subdivided into three classes: (1) the Jungermanniopsida includes the two orders: Metzgeriales (simple thalloids) and Jungermanniales (leafy liverworts); (2) the Marchantiopsida which includes the three orders: Marchantiales (complex-thallus liverworts), Sphaecarpales (bottle hepatics), the Blasiales (previously placed among the Metzgeriales); and (3) the problematic genus Monoclea which is sometimes placed in its own order Monocleales (Jones, 2004; Forrest, et al., 2006; Heinrichs, et al., 2005; He-Nygren et al., 2006; Renzaglia, et al., 2007; & Forrest et al., 2004). The greatest impact of liverworts is through the reduction of erosion along streambanks, their collection and retention of water in tropical forests, and the formation of soil crusts in deserts and Polar Regions (Stern, 1991; Raven, 2005). Despite its known environmental impacts, there are only a few studies that are conducted as far as liverworts biogeography, evolution and phylogenetic placement is concerned. It is assumed that among the simple thalloid liverworts in the order Metzgeriales as described by Schuster (1984), Aneuraceae are the largest family in species number and taxonomically, probably the least well understood. There were early molecular-morphological classifications as in the case of Aneura pinguis. The questionable species delimitation due to high levels of morphological plasticity of the different taxa of Aneuraceae is nicely illustrated by a multigene analysis of Aneura pinguis which reveals that such particular species to be paraphyletic, while other members of the complex appear to be polyphyletic (Wickett & Goffinet, 2008). The studies of Baczkiewicz & Buczkowska (2005) and Wachowiak et al. (2007) that uses isoenzymes and PCR-RFLP markers of Aneura pinguis populations in Europe suggested multiple cryptic speciation events. The salient findings of the study of Long et al. (2007) revealed that Aneura pinguis is characterized by a complex evolutionary history, and suggests that the currently recognized morphological entities require extensive re-evaluation. It is notable to consider the unique case of family-wide relationship of the family Aneuraceae to fungi. Aneuraceae form a mycorrhiza-like interaction that is unique among liverworts. An extreme example of this is the monotypic, non- photosynthetic genus Cryptothallus. This genus was recently merged to the genus Aneura by Wickett & Goffinet (2008). Furthermore, this genus is characterized by the phenomenon of mycoheterotrophy wherein a life history strategy unknown elsewhere among bryophytes. According to Kottke & Nebel (2005), it appears that the mycoheterotrophy phenomenon is a unique case of the familywide, tight relationship with tulasnelloid fungi, which, among liverworts, is restricted to Aneuraceae. On the other hand, Nebel et al. (2004) and Duckett & Ligrone (2008) mentioned that from the ultrastructure of typical Aneuraceaen mycobionts, which is having imperforate parenthesomes, alone, it would be impossible to assign the fungus to the genus Tulasnella versus another heterobasidiomycete. In contrary to the aforementioned restrictions in the morphological classification of tulasnelloid fungi, Bidartondo et al. (2003) said that molecular methods have enabled the identification of Tulasnella as a mycobiont of Cryptothallus. This is also in congruence to the findings of Kottke et al. (2003) and Preubing et al. (2010) wherein they also utilized molecular methods to identify Tulasnella as mycobiont of Aneura and Riccardia. These investigations reveal a narrow spectrum of fungal partners of Aneuraceae, which seem to be 3 restricted to Tulasnellales. In some cases however, sequences were detected that show high similarity to members of Sebacinales which were shown in the results of the study of Bidartondo & Duckett (2008) and Preubing et al. (2010). According to Kottke & Nebel (2005), among hepatics, Tulasnellales as mycobionts have been exclusively detected in Aneuraceae thus far. Kottke & Nebel (2005) further reiterated that the extent of fungal colonization is not equally distributed among the members of Aneuraceae, and ranges from obligatory (Cryptothallus) to a complete lack (many Riccardia species) of dependence on fungal colonisation, which can be explored in a phylogenetic context. Looking into the phylogenetic relationship of different genera in the family Aneuraceae, Gradstein (2001) found liverwort species in southern Ecuador which resembled the genus Aneura, but microscopic studies revealed the presence of oil bodies typical of the genus Riccardia. Moreover, preliminary phylogenetic analysis of the liverwort found in southern Ecuador (which can either be Aneura or Riccardia) indicated a close relationship to Lobatiriccardia through its branching pattern, a genus unknown in South America at that time, according to Gradstein (2001). Gradstein (2001) further mentioned that the several Lobatiriccardia -like individuals were detected in paramo vegetation near Cuenca, Ecuador. Wickett & Goffinet (2008) has currently recognized four genera in the family Aneuraceae: Aneura Lobatiriccardia, Riccardia, and Verdoornia. A fifth genus, Cryptothallus, was recently reduced to synonymy under Aneura. Wickett & Goffinet (2008) further mentioned that the genus Aneura has a worldwide distribution and comprises 20 species. However, due to the above mentioned questionable species definitions, the taxonomy and distribution of species and genus as a whole remain unclear. Lobatiriccardia (5 species) was first described by Mizutani & Hattori (1957) as a subgenus of Riccardia and subsequently transferred to Aneura (Schuster, (1985). Furuki (1991) elevated the subgenus to generic rank, a treatment that is not followed in the checklists of New Zealand (Glenny, 1998) and Australia (McCarthy, 2006). In spite of a lack of strong molecular evidence for its generic status as mentioned by Stech & Frey in 2001; by Forrest & Crandall-Stotler in 2004 and 2005; and by Forrest et al in 2006, Lobatiriccardia was accepted at the generic level in several recent liverwort classifications (Crandall-Stotler & Stotler, 2000; Frey & Stech, 2005). The species-rich genus Riccardia, with approximately 280 taxa according to www.tropicos.org has a cosmopolitan, though predominantly Southern Hemispherical, distribution; only five species occur in North America, Europe and Northern Asia according to Grolle & Long, 2000. Schuster (1992) and Gradstein (2001) estimate that a thorough worldwide revision will reduce the number of valid species to approximately 100. The relatively high number of species in the genus, in combination with morphological and ecological plasticity, will undoubtedly complicate revisionary efforts. Another group of simple thalloid liverworts under the order Metzgeriales is the genus Metzgeria in the family Metzgeriaceae. Approximately 120 to 200 species of Metzgeria have been described morphologically in general (Schuster, 1992). Phylogenetic analyses revealed distinct lineage of Metzgeria in Europe and North America. Metzgeria furcata and Metzgeria conjugata’s molecular divergence among taxa occurred in the absence of morphological change. In addition, all Metzgeria from Eastern North America were both morphologically uniform and genetically homogeneous although not identical (Fuselier, 2005). This aspect provides significant insight into Metzgeria that has complex taxonomy. It also indicates that these liverwort taxa have wide distributions, extreme sex ratios, and continental disjunctions harbor cryptic lineages. Many previous systematic treatments of Aneuraceae and Metzgeria were based solely on morphological characters. However, recent studies already have incorporated molecular data to 4 strengthen the existing taxonomic classification, phylogenetic placement and biogeographic distribution of Aneuraceae and Metzgeria. Statement of the Problem This study is necessary to support both the existing morphological and molecular data that has paved way to the recent placement of the family Aneuraceae and the genus Metzgeria under the order Metzgeriales. Specifically the study performed (1) a meta-analysis of evolutionary trends within Aneuraceae based on available GenBank sequences can be conducted; (2) strengthening the relationships among Aneura, Riccardia, Lobatiriccardia and Metzgeria; (3) testing the phylogenetic placement of the available GenBank sequences of the chosen genera under Aneuraceae and the genus Metzgeria using MP, ML, UPGMA and Minimum Evolution Tree and suggest possible generic reclassification based on the tree constructed; and (4) strengthening backbone phylogeny of Aneuraceae through tree construction supported with existing research studies; (5) support Aneuraceae and Metzgeria’s biogeographic origin through tree construction of available GenBank sequences. 2. METHODOLOGY Research Method and Design This is a combined descriptive-meta-analytic type of research which utilized existing GenBank sequences in understanding the evolutionary, biogeographical and phylogenetic placement of Aneuraceae in relation to the genus Metzgeria. This study is particularly important since Aneuraceae is the largest family under the order Metzgeriales as far as species number is concerned. Likewise, taxonomically speaking, it is probably the least well understood. The metaanalysis may also lead to a better understanding to the present classification of the Family Aneuraceae and the genus Metzgeria both under the order Metzgeriales. Taxon Selection and Retrieving Sequences Using GenBank GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences. Its database is designed to provide and encourage access within the scientific community to the most up to date and comprehensive DNA sequence information (GenBank Home: http://www.ncbi.nlm.nih. gov/genbank/). GenBank was therefore used as a primary tool in obtaining gene sequences of the organisms to be meta-analyzed. In the selection process of taxa, the researcher conducted previous investigation to determine problematic group of organisms with at least thirty GenBank sequences of species belonging to the Family or Order of interest. The questionable species delimitation due to high levels of morphological plasticity of the different taxa of Aneuraceae and its relation to Metzgeria has led to the conduct of this research. The accession numbers of all the chosen organisms were recorded. Gene Selection for the Retrieved Sequences The sequences 32 species which belonged to the family Aneuraceae and genus Metzgeria of the Order Metzgeriales were retrieved from GenBank. The retrieved nucleotide sequences of the species in the order of interest came from one specific gene that is available for all of these organisms in GenBank (NCBI). Since the partial trnL gene was found to be available to all the 32 sequences, it was used as the gene of interest. The partial tRNA-Leu (trnL) intron gene in the 5 chloroplast was used in the study because it is one of the most commonly used marker in many previously published taxonomic and phylogenetic studies on liverworts (Schaumann et al., 2005; Preubing, et al., 2010). Furthermore, according to the study conducted by Taberlet et al. (2007) the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in wide array not only as an important molecular marker in lower plant forms (such as mosses, liverworts, and ferns). It also has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies. Determining the Outgroup The selection of outgroup for the chosen GenBank sequences were facilitated through the use of the Basic Local Alignment Search Tool (BLAST) of the NCBI. BLAST is a tool used to compare an unknown sequence against the database sequences to determine likely matches. BLAST also tallies the differences between sequences and assigns a score based on sequence similarity. BLAST is a powerful bioinformatics and can be helpful in determining an outgroup. In the study, BLAST was performed to one randomly selected sequence (Aneura pinguis) in the ingroup. The organisms with an E-value of > 1.0 were noted, only Jensenia decipiens with an accession number of AY547517 was found to have an E-value of 1.1 and was therefore chosen as an outgroup. The genus Jensenia is under the family Pallaviciniaceae in the same order as Aneuraceae and Metzgeria, Metzgeriales. The six or seven species of the genus, including Jensenia decipiens belong to a southern Gondwana element and is characterized by their thallus that grows erect, and branches tree-like rather than trailing the ground (Engel, 1990; and Perold, 1993). Seen below are the 32 retrieved sequences from GenBank with their corresponding accession numbers: Table 1. Retrieved Gene Sequences from GenBank and their Accession Numbers Species Family / Order Accession Number Aneura alterniloba Aneuraceae / Metzgeriales AY763562 Aneura latissima Aneuraceae / Metzgeriales FM210482 Aneura maxima Aneuraceae / Metzgeriales KJ400415 Aneura mirabilis Aneuraceae / Metzgeriales FM210481 Aneura pinguis Aneuraceae / Metzgeriales KJ400414 Aneura sp. Aneuraceae / Metzgeriales AY763563 Lobatiriccardia alterniloba Aneuraceae / Metzgeriales FM210493 Lobatiriccardia coronopus Aneuraceae / Metzgeriales FM210494 Lobatiriccardia lobata Aneuraceae / Metzgeriales AY507553 Lobatiriccardia Aneuraceae / Metzgeriales FM210497 oberwinkleri Lobatiriccardia Aneuraceae / Metzgeriales FM210505 verdoornioides Lobatiriccardia Aneuraceae / Metzgeriales FM210506 yakusimensis Riccardia amazonica Aneuraceae / Metzgeriales FM210510 Riccardia andina Aneuraceae / Metzgeriales FM210511 Riccardia bogotensis Aneuraceae / Metzgeriales FM210512 6 Riccardia chamedryfolia Riccardia eriocaula Riccardia fucoides Riccardia incurvata Riccardia multifida Riccardia pallida Riccardia palmata Riccardia prehensilis Riccardia smaragdina Riccardia trichomanoides Metzgeria agnewiae Metzgeria aurantiaca Metzgeria ciliate Metzgeria claviflora Metzgeria conjugate Metzgeria decipiens Jensenia decipiens (outgroup) Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Aneuraceae / Metzgeriales Metzgeriaceae / Metzgeriales Metzgeriaceae / Metzgeriales Metzgeriaceae / Metzgeriales Metzgeriaceae / Metzgeriales Metzgeriaceae / Metzgeriales Metzgeriaceae / Metzgeriales Pallaviciniaceae / Metzgeriales FM210514 AY007627 FM210515 FM210516 FM210517 FM210518 FM210519 AY763565 FM210520 FM210521 HQ342634 HQ342635 HQ342656 HQ342624 AY507541 GQ336159 AY547517 Multiple Alignment of DNA Sequences In phylogenetic tree reconstruction based on DNA sequences, there is a need to align sequences of organisms under study. The purpose of alignment is to determine homology and informative sites. ClustalW is the program used to perform multiple alignment of sequences. In using ClustalW, different weights were given to sequences and parameters in different parts of the alignment. BioEdit was the software used in performing the ClustalW program. On the working page of BioEdit, a new alignment page was selected and the Taxonomy Browser of the Entrez page on GenBank was opened to search for the species of interest. Once the species were shown, the accession number of one of the chosen species was typed and its Fasta file was copied to the alignment page of BioEdit. The rest of the chosen species were imported to the alignment page of BioEdit. After importing all the gene sequences of interest, the accessory application ClustalW Multiple Alignment was run to start the alignment of the sequences. The loose edges of the sequences were cut and the partial trnL intron chloroplast gene was retained. Phylogenetic Inference and Tree Reconstruction Inferring evolutionary relationships based on morphological traits is sometimes not reliable because of convergent evolution. A more reliable method of inferring phylogenetic trees is through sequence comparison. Reconstruction of the evolutionary history of genes and species is currently one of the most important subjects in molecular evolution. If reliable phylogenies are produced, they will shed light on the sequence of evolutionary events that generated the present day diversity of genes and species and help us to understand the mechanisms of evolution as well as the history of organisms (Nei and Kumar 2000). Phylogenetic relationships of genes or organisms usually are presented in a treelike form. The branching pattern of a tree is called a topology. The phylogenetic tree reconstruction of available GenBank sequences was performed using MEGA6. Using this software, there are numerous methods for constructing phylogenetic trees from molecular data (Nei and Kumar 7 2000). Five phylogeny tests were performed and five corresponding phylogenetic trees were reconstructed including Maximum Likelihood (ML), Maximum Parsimony (MP), Minimum Evolution (ME), Neighbor-Joining (NJ) and UPGMA. To test the reliability of the ML, MP, ME, NJ and UPGMA tree, the Felsenstein's (1985) bootstrap test was used, which is evaluated using Efron's (1982) bootstrap resampling technique. As a general rule, if the bootstrap value for a given interior branch is 95% or higher, then the topology at that branch is considered "correct". In this study, bootstrap method was used as a test of phylogeny using 1000 bootstrap replications. Maximum Likelihood. In this method, an initial tree is first built using a fast but suboptimal method such as Neighbor-Joining, and its branch lengths are adjusted to maximize the likelihood of the data set for that tree topology under the desired model of evolution. Then variants of the topology are created using the NNI (nearest neighbor Interchange) method to search for topologies that fit the data better. Maximum-Likelihood branch lengths are computed for these variant tree topologies and the greatest likelihood retained as the best choice so far. This search continues until no greater likelihoods are found. This method can be used for both nucleotide and protein data. Maximum Parsimony. This method originally was developed for morphological characters, and there are many different. In MEGA, we consider both of these methods for nucleotide and amino acid sequence data (Eck and Dayhoff 1966; Fitch 1971). For constructing an MP tree, only sites at which there are at least two different kinds of nucleotides or amino acids, each represented at least twice, are used (parsimony-informative sites). Other variable sites are not used for constructing an MP tree, although they are informative for distance and maximum-likelihood methods. MEGA estimates MP tree branch lengths by using the average pathway method for unrooted trees (see Nei and Kumar [2000]. To search for MP Trees, MEGA provides four different types of searches: the max-mini branch-and-bound search, min-mini heuristic search, Subtree-Pruning-Regrafting (SPR), and Tree-Bisection-Reconnection (TBR) heuristic search. Only the branch-and-bound search is guaranteed to find all the MP trees, but it takes prohibitive amount of time if the number of sequences is large (>15). Minimum Evolution. In the ME method, distance measures that correct for multiple hits at the same sites are used, and a topology showing the smallest value of the sum of all branches (S) is chosen as an estimate of the correct tree. While the NJ tree is usually the same as the ME tree, when the number of taxa is small the difference between the NJ and ME trees can be substantial (reviewed in Nei and Kumar 2000). In this case if a long DNA or amino acid sequence is used, the ME tree is preferable. Neighbor-Joining. This method (Saitou and Nei 1987) is a simplified version of the minimum evolution (ME) method (Rzhetsky and Nei 1992). The ME method uses distance measures that correct for multiple hits at the same sites; it chooses a topology showing the smallest value of the sum of all branches (S) as an estimate of the correct tree. UPGMA. This method assumes that the rate of nucleotide or amino acid substitution is the same for all evolutionary lineages. An interesting aspect of this method is that it produces a tree that mimics a species tree, with the branch lengths for two OTUs being the same after their separation. Because of the assumption of a constant rate of evolution, this method produces a rooted tree, though it is possible to remove the root for certain purposes. 8 3. RESULTS AND DISCUSSIONS Using available GenBank sequences, this research aimed to support existing taxonomic classification, phylogenetic placement and biogeographic distribution of the three member genera in the family Aneuraceae with the genus Metzgeria. Phylogenetic inferences through MP, ML, ME, NJ and UPGMA tree reconstruction were performed using MEGA6. Evolutionary Trend of Morpho-anatomical Traits based on Phylogenetic Framework The results of phylogenetic reconstruction presented in this study are largely consistent with the results of previous work by Crandall-Stotler et al. (2005); Forrest et al. (2006) and the results of molecular analyses by Preubing et al. (2010) in that Aneura and Lobatiriccardia comprise a clade (Figure 1) and this relationship is consistent with the presence of a highly developed mycothallus in parenchyma cells found only in the genera Verdoornia, Lobatiriccardia and Aneura. In addition, the phylogenetic analyses resolve Lobatiriccardia and Aneura as two monophyletic sister clades that are maximally supported in the ML analysis. Figure 1. Molecular Phylogenetic Analysis by Maximum Likelihood Method. (Highlighted are the two monophyletic sister clades, Aneura and Lobatiriccardia.) Figure 1 above shows the evolutionary history inferred using the Maximum Likelihood method based on the Tamura-Nei (1993) model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the 9 bootstrap test (1000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The analysis involved 32 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 186 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). As mentioned by Preubing et al. (2010) and based also in the result of the phylogenetic reconstruction, it can be argued that both genera should be treated as distinct entities, given that morphological characters such as mycothallus in different thallus layers, position of sporophyte, and shape and position of scales on the thallus that were evaluated by Preubing et al. (2010) as shown in Figure 2 are consistent with the MP phylogenetic split presented in Figure 3. Figure 2 below shows the ancestral character state reconstruction in the three genera of the family Aneuraceae. The character state reconstruction for the presence of mycothallus in different thallus layer; Figure 2. Ancestral character state reconstruction for the presence of mycothallus in different thallus layers (above), position of sporophyte (below-left), and shape and position of scales on the thallus (below-right) (Preubing et al., 2010) 10 Figure 3. Molecular Phylogenetic Analysis by Maximum Parsimony Method. (Highlighted are the three genera under the family Aneuraceae.) position of the sporophyte; and shape and position of the scales of the thallus anchors the placement of the three genera of the family Aneuraceae as far as the evolutionary trend of morpho-anatomical traits of the three genera is concerned. There was an observable ancestral split between Riccardia and the monophyletic sister clade of Lobatiriccardia and Aneura having an ancestral central sporophyte with broad central scales and with no mycothallus for the former and having a ventral sporophyte with small scales and parenchymal mycothallus for the latter. The split was later showed that the majority the members of the genus Riccardia (highlighted in blue) have absent mycothallus (black pie) and a few members have mycothallus only in epidermal cells (grey pie). Likewise, in the genus Lobatiriccardia, (highlighted in pink) and the genus Aneura, (highlighted in green) all members have mycothallus evenly present in parenchymal cells (white pie) since the two genera form a monophyletic sister group. Figure 3 on the other hand presents the evolutionary history inferred using the Maximum Parsimony method. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (Kumar-Nei, 2000) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). Figure 3 shows its congruence with the ancestral character state reconstruction in Figure 2. Although, with few low bootstrap value (below 50%), 11 the figure still clearly confirm that the Riccardia group (highlighted in blue) forms a monophyletic clade where majority of its species have no mycothallus. On the other hand, Lobatiriccardia and Aneura forms two monophyletic sister clades where members of the genus Lobatiriccardia (highlighted in pink) and the members of the genus Aneura (highlighted in green) have mycothallus evenly present in parenchymal cells. Figure 3 also conforms to Figure 2’s additional character states such as having marginal sporophyte and lateral on margin scales for the members of the genus Riccardia (highlighted in blue); having marginal sporophyte and broad marginal scale for Lobatiriccardia; and having ventral sporophyte with small ventral scales for the genus Aneura. The non-highlighted taxa in Figure 3 (Metzgeria) seem to form its own monophyletic clade which is presumed to retain the more ancestral trait of any of the three enumerated taxa earlier. Preubing (2010) mentioned that majority of the members of Metzgeria may have possessed parenchymal mycothallus, central sporophyte and broad central scales. Biogeographic Distribution In the biogeographical aspects and distribution of the genera under Aneuraceae, Furuki (1991) described the genus Lobatiriccardia as being restricted to East Asia, South East Asia and Australia. Particularly, its distribution comprises Japan, Philippines, Malayan Peninsula, Yunnan China, Indonesia, Papua New Guinea, New Caledonia, Australia, Tasmania, and New Zealand. Preubing et al. (2010) showed that the genus Lobatiriccardia can also be found in South America which further expands its distribution considerably. The genus Aneura which forms a monophyletic sister group with Lobatiriccardia as presented in the phylogenetic tree that was constructed earlier, has a worldwide distribution. Despite being cosmopolitan, majority of its members are also found in the southern hemisphere, particularly in New Zealand, in South East Asia and in Oceania. Unlike Lobatiriccardia, Aneura is absent in Australia. Preubing et al. (2010) suggests a strong and perhaps complex biogeographic pattern and indicate that the distribution of the genus Aneura is still poorly known. On the other hand, the species-rich genus Riccardia also has cosmopolitan, though predominantly Southern Hemispherical, distribution. There were only five species that occur in North America, in Europe and in North Asia. The separate monophyletic group of the genus Metzgeria have a Northern Hemispherical distribution unlike the three genera under Aneuraceae that were presented earlier. The currently known distribution of the members of this genus is North America and Europe, although there are some members which can be found in South America. Figure 3 shows the distribution of the different genera in the family Aneuraceae (Riccardia, Lobatiriccardia, and Aneura) and the genus Metzgeria. It can be gleaned from the figure that majority of the member species in the family Aneuraceae are have Southern Hemispherical distribution. On the other hand, the members of the genus Metzgeria have a Northern Hemispherical distribution. Despite many studies having presented the different biogeographical distribution of the different members of the order Metzgeriales (Aneuraceae, Metzgeriaceae), the taxonomy and distribution of each species and genus under each respective families as a whole remains unclear due to the previously mentioned questionable morphological species definition. Further molecular and phylogenetic circumference is therefore needed to strengthen the analysis of the biogeographic distribution of the different members of Metzgeriales. 12 Figure 4. Biogeographic distribution of the different genera in the family Aneuraceae and the genus Metzgeria. (Black spots = Lobatiriccardia; Red spots = Aneura; Green spots = Riccardia; Yellow spots = Metzgeria) Figure 5. UPGMA tree constructed for the members in the genus Lobatiriccardia to show their biogeographic relationship. 13 Figure 5 shows the UPGMA tree constructed for the members in the genus Lobatiriccardia to show their biogeographic relationship. Lobatiriccardia coronopus is found to be more closely related to Lobatiriccardia yakusimensis. According to Hattori (2006), Lobatiriccardia coronopus can be found in the Philippines, Malaysia, and Indonesia. Furuki (1996) described Lobatiriccardia yakusimensis to be found in Japan. These countries are found in South East Asia which reveals why they are closely related to one another. On the other hand, the UPGMA tree constructed also shows that Lobatiriccardia alterniloba is more closely related to Lobatiriccardia lobata. Hook et al. (1991) determined New Zealand as the main distribution area of Lobatiriccardia alterniloba. Likewise, Furuki (1996) also highlighted New Zealand as the area where Lobatiriccardia lobata can be found. The constructed UPGMA tree strengthens the biogeographic distribution pattern of the two Lobatiriccardia species. As previously mentioned, Furuki (1991) described the genus Lobatiriccardia as being restricted to East Asia, South East Asia and Australia. However, the records of Preubing (2010) shows that Lobatiriccardia can further be expanded to South America. Two newly discovered species Lobatiriccardia oberwinkleri and Lobatiriccardia verdoornioidess were found in Ecuador. The UPGMA tree constructed proves the close relationship of the two aforementioned species of Lobatiriccardia. Strengthening Phylogenetic Placement through Various Tree Reconstruction Preubing et al. (2010) provided new insights in the evolution of the liverwort family Aneuraceae (Metzgeriales, Marchantiophyta). The researchers used trnL as one of the markers to reveal the occurrence of the liverworts in Asia, Australia and new species from Ecuador. Their phylogenetic reconstructions support Lobatiriccardia and Aneura as monophyletic sister groups consistent to the mycothallus development in Aneuraceae. Furthermore, although high levels of genetic structure can be observed among members of the Aneuraceae complex, current member species are still questionable, and detailed analyses of cryptic speciation and biogeographic patters are needed to understand the evolution of Aneura. Ancestral state reconstructions also suggest an evolutionary trend of female gametangia, and subsequently the sporophyte, moving from a central position on the dorsal side of the thallus to a marginal position between thallus lobes to a more ventral position under the thallus margin. To strengthen the previously established phylogenetic placement of the family Aneuraceae and the genus Metzgeriales, this research utilized different statistical tree reconstruction tools such as ML, MP, ME, UPGMA, NJ as phylogenetic inferences on available GenBank sequences. These analyses will further reinforce the previously recognized placement under the order Metzgeriales. Figure 6a (topology only) and Figure 6b (original tree – radiation) shows the molecular phylogenetic placement using Maximum Likelihood method. The tree with the highest log likelihood (-1362.3186) is shown. The percentage trees in which the associated taxa clustered together is shown next to the branches. Initial tree for the heuristic search was obtained by applying the Neighbor-Joining Method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. Figure 7 shows the phylogenetic placement inferred using the Maximum Parsimony Method. Tree #1 out of 2 most parsimonious trees (length =262) is shown. The consistency index is 0.618557, the retention index is 0.859848 and the composite index is 0.616991 for all sites and parsimony-informative sites. The percentage of replicate trees in which the associated tax clustered together in the bootstrap test is shown next to 14 the branches. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 1 in which the initial trees were obtained by random addition of sequences. Figure 8 shows the evolutionary placement of Aneuraceae and Metzgeria using the Minimum Evolution method. The optimal tree with the sum of branch length = 1.98255251 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test of 1000 replicates is shown next to the branches. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1.The Neighbor-joining algorithm was used to generate the initial tree. Figure 9 shows the phylogenetic placement of Metzgeriales using the Neighbor-Joining Method. The optimal tree with the sum of branch length = 1.98255251 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to branches. Figure 6a. Phylogenetic Placement Using Maximum Likelihood (Straight View - Topology Only) 15 Figure 6b. Phylogenetic Placement Using Maximum Likelihood (Radiation View – Original Tree) 16 Figure 7. Phylogenetic Placement Using Maximum Parsimony (Straight View - Topology Only) 17 Figure 8. Phylogenetic Placement Using Minimum Evolution (Straight View - Topology Only) 18 Figure 9. Phylogenetic Placement Using Neighbor-Joining (Straight View - Topology Only) 19 Figure 10. Phylogenetic Placement Using UPGMA Method(Straight View - Topology Only) 20 Figure 10 shows the phylogenetic placement inferred using the UPGMA method. The optimal tree with the sum of branch length = 1.97111195 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test of 1000 replicates is shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method is in the units of the number of base substitutions per site. Out of the five constructed trees using the ML, MP, ME, NJ and UPGMA, four trees (MP, ME, NJ and UPGMA) were found to be similar in forming three major monophyletic clade consisting of: Riccardia clade; Aneura-Lobatiriccardia clade; and Metzgeria clade. This is well supported by the phylogenetic reconstruction of Preubing et al. (2010) using Bayesian Inference (BI and Maximum Parsimony (MP) where the Riccardia clade was found to be closely related to a clade comprising of Lobatiriccardia and Aneura. The monophyly of each genus is maximally supported in all analyses. Furthermore, the taxon sampling of Preubing et al. (2010) both Lobatiriccardia and Riccardia are well resolved and supported at the generic level which was also reflected by the current phylogenetic reconstruction in this study. In addition the phylogenetic analyses resolve Lobatiriccardia and Aneura as two monophyletic sister clades that are maximally supported in four of the five reconstructed trees which are largely consistent with the results of previous work of Crandall-Stotler et al. (2005) and Forrest et al. (2006) as well as the study of Preubing et al. (2010) where they argued that both genera should be treated as distinct entities. Likewise, all the five reconstructed trees place the members of Metzgeriales in a separate monophyletic clade. In contrary to the four congruent trees, the Maximum Parsimony (MP) tree (traditional straight view-topology only) showed that four distinct clades namely: Riccardia, Lobatiriccardia, Aneura, and Metzgeria where two members (Aneura sp. & Aneura alterniloba) of the genus Aneura were found to be more closely related to the genus Lobatiriccardia. However, using the same original Maximum Parsimony tree in its radiation view, it can be observed that, the three previously mentioned clades (Riccardia, Aneura-Lobatiriccardia, and Metzgeria) can still be highlighted. There were previously recorded transfers of some taxa to Lobatiriccardia. Furuki (1991) transferred Aneura alterniloba of Hooker & Taylor (1844) in Gottsche et al. (1846) to his new Lobatiriccardia genus. A molecular phylogenetic study of Preubing et al. (2010) confirmed this treatment. However, two recently recognized varieties from Australia and a species from New Zealand were not transferred as they do not occur in the area treated by the monograph (Furuki 1991). Similarly, the identified Aneura alterniloba and Aneura sp. found in Australia and New Zealand respectively as mentioned in the study of Schaumann et al. (2005) were still recognized under the genus Aneura but the MP phylogenetic reconstruction in this study reveals that they are more closely related to Lobatiriccardia rather than Aneura. It is therefore suggested that if Lobatiriccardia and Aneura retains their present generic classification level, these two species (Aneura alterniloba and Aneura sp.) be placed in the genus Lobatiriccardia and further studies be conducted to determine if the Aneura alterniloba by Schaumann et al. is the same species or a subspecies of the Lobatiriccardia alterniloba in the study of Preubing et al. (2010). 21 4. CONCLUSIONS The evolutionary trend based on morpho-anatomical traits deemed to be congruent to the MP tree constructed using MEGA6. There was an observable ancestral split between Riccardia and the monophyletic sister clade of Lobatiriccardia and Aneura where the majority the members of the genus Riccardia's mycothallus were absent, with marginal sporophyte, and with lateral margin scales while Lobatiriccardia and Aneura have mycothallus evenly present in parenchymal cells, with marginal sporophyte, and with broad marginal scale (Lobatiriccardia) or with ventral sporophyte with small ventral scales (Aneura). Metzgeria form its own monophyletic clade having the more primitive parenchymal mycothallus, central sporophyte and broad central scales. The biogeographic distributions of the member species in the family Aneuraceae are have Southern Hemispherical distribution. On the other hand, the members of the genus Metzgeria have a Northern Hemispherical distribution. The Aneura-Lobatiriccardia sister clade are found in the East and South East Asia including Australia while the Riccardia clade is dominant in South America. On the other hand, Metzgeria which forms a monophyletic clade is found to be dominant in North America. In terms of the phylogenetic placement, Out of the five constructed trees using the ML, MP, ME, NJ and UPGMA, four trees (MP, ME, NJ and UPGMA) were found to be similar in forming three major monophyletic clade consisting of: Riccardia clade; Aneura-Lobatiriccardia clade; and Metzgeria clade. In addition the phylogenetic analyses resolve Lobatiriccardia and Aneura as two monophyletic sister clades that are maximally supported in four of the five reconstructed trees. MP however showed four major monophyletic clades instead: Riccardia, Aneura, Lobatiriccardia, and Metzgeria. The identified Aneura alterniloba and Aneura sp. found in Australia and New Zealand respectively as mentioned in the study of Schaumann et al. (2005) reveal in the phylogenetic reconstruction that they are more closely related to Lobatiriccardia rather than Aneura. 5. RECOMMENDATIONS In light of the above stated conclusions, it can be recommended that further molecular studies be conducted to determine the true extent of distribution of the family Aneuraceae and the genus Metzgeria. In terms of the phylogenetic placement, the researcher suggests any of the two possible actions to be undertaken in terms of taxa reclassification: (a) Since four of the five reconstructed trees (MP, ML, ME, NJ, and UPGMA) formed three major monophyletic clade (Riccardia, Lobatiriccardia-Aneura, and Metzgeria), the genera Lobatiriccardia and Aneura are suggested to be placed in one genus as likewise suggested by Furuki (1991). 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