pISSN 1225-8318 − Korean J. Pl. Taxon. 48(1): 1 8 (2018) eISSN 2466-1546 https://doi.org/10.11110/kjpt.2018.48.1.1 Korean Journal of ORIGINAL ARTICLE Plant Taxonomy Divergence time estimation of an ancient relict genus Mankyua (Ophioglossaceae) on the young volcanic Jejudo Island in Korea Hee-Young GIL and Seung-Chul KIM* Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea (Received 15 December 2017; Revised 3 March 2018; Accepted 12 March 2018) ABSTRACT: Mankyua chejuense is the only member of the monotypic genus Mankyua (Ophioglossaceae) and is endemic to Jejudo Island, Korea. To determine the precise phylogenetic position of M. chejuense, two cpDNA regions of 42 accessions representing major members of lycophytes are obtained from GenBank and analyzed using three phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference). In addi- tion, the divergence time is estimated based on a relaxed molecular clock using four fossil calibration points. The phylogenetic position of Mankyua still appears to be uncertain, representing either the earliest diverged lin- eage within Ophioglossaceae or a sister to the clade containing Ophioglossum and Helminthostachys. The most recent common ancestor of Ophioglossaceae and its sister lineage, Psilotum, was estimated to be 256 Ma, while the earliest divergence of Mankyua was estimated to be 195 Ma in the early Jurassic. Keywords: Mankyua chejuense, monotypic genus, Jejudo Island, molecular dating, Ophioglossaceae The Korean peninsula, which is on the far east coast of Asia, Lee and Hong, 2011). Echinosophora, however, is currently has rich floristic diversity compared to its relatively small size treated as Sophora (Lee et al., 2004). Due to continuing decline (Park, 2005). It can be divided into three floristic regions with in quality and quantity of habitat, all endemic genera of Korea adjacent regions (i.e., China, Japan, and Far Eastern Russia) were classified as threatened categories of IUCN Red List and possess diverse habitats due to topographic and climatic Categories (Abeliophyllum, endangered [EN]; Pentactina, complexities (Park, 2005; Chang et al., 2011). Although large critically endangered [CR]; Hanabusaya asiatica, EN; number of taxa in the Korean peninsula are shared with Mankyua, CR; Megaleranthis, EN), except for Coreanomecon adjacent regions, seven genera are endemic to the Korean (Chang et al., 2016). Several phylogenetic and population peninsula, including Mankyua B.-Y. Sun, M. H. Kim & C. H. genetic studies have been conducted for these endemics but Kim (Ophioglossaceae), Megaleranthis Ohwi (Ranunculaceae), their divergence times from their sister lineages have not been Pentactina Nakai (Rosaceae), Echinosophora Nakai (Fabaceae), determined. This lack of estimated divergence time hinders us Abeliophyllum Nakai (Oleaceae), Hanabusaya Nakai to fully understand the origin and evolution of endemic genera (Campanulaceae), and Coreanomecon Nakai (Papaveraceae) in the Korean peninsula as well as to manage and develop (Park, 2005; Kim and Park, 2013). All these genera are conservation strategy of highly threatenened and valuable monotypic, which comprises single species, except for floristic members in Korea. Hanabusaya [H. asiatica (Nakai) Nakai, H. latisepala Nakai]. Mankyua chejuense B.-Y. Sun, M. H. Kim & C. H. Kim Recent molecular phylogenetic studies shed light on the origin (Ophioglossaceae) is the only member of the genus Mankyua of these Korean endemic genera and their status as endemic and is endemic to Jejudo Island, Korea. This monotypic genus genera (e.g., Abeliophyllum, Kim et al., 2000; Echinosophora, has unique combination of morphological characters which Lee et al., 2004; Hanabusaya, Roquet et al., 2008; Mankyua, differ from other genera of Ophioglossaceae (i.e., trophophore Sun et al., 2009; Megaleranthis, Kim et al., 2009; Pentactina, blade, trophophore venation, sporophore, and sporangium). *Author for correspondence: [email protected], [email protected] http://e-kjpt.org, © 2018 the Korean Society of Plant Taxonomists. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2 Hee-Young GIL and Seung-Chul KIM Since it has unique morphology and is rare and endemic to analyses using the same heuristic search options as above. Gaps volcanic island, considerable attention has been focused on this were treated as missing data. For the ML and BI analysis, the enigmatic species. Several studies about morphology, best-fit model was selected based on the Akaike information phenology, molecular phylogenetics, conservation, and criterion implemented in the program jModelTest version 2.1.6 population genetics were conducted (Chung et al., 2010; Hyeon (Darriba et al., 2012). For both ML and BI analysis, GTR + et al., 2010, 2011; Hyun et al., 2014; Kim et al., 2014; Stuessy I + G model was selected. ML analyses were conducted using et al., 2014). Although previous phylogenetic studies have RAxML 8.0.26 (Stamatakis, 2014) implemented in raxmlGUI examined the relationships within Ophioglossaceae (Sun et al., version 1.3.1 (Silvestro and Michalak, 2012). ML trees were 2009; Shinohara et al., 2013), phylogenetic position of calculated using 1,000 rapid bootstrap inferences. BI analysis Mankyua is still controversial. was performed with 5,000,000 generations initiated with a Jejudo island is of volcanic origin and 90km south off the random starting tree, sampling every 500 generations and coast of the Korean peninsula (Woo et al., 2013). While the allowing the program to estimate the likelihood parameters family Ophioglossaceae is early diverged basal lineage of required. We discarded 25% of the samples as burn-in. monilophytes group, Jejudo Island is very young with the age of approximately 2 million years old (Woo et al., 2013). Since Divergence time estimation Jejudo Island has recurrently connected to adjacent continents Divergence times of the family Ophioglossaceae and major during the glacial cycles in the Quaternary Period, the flora of lineages within the family were estimated by BI approach using Jejudo Island has been affected by the various floristic elements the program BEAST v.2.3.1 (Bouckaert et al., 2014). Huperzia from the Korean peninsula, China, and Japan, where habitats and Isoetes were excluded from the analysis to reduce the are diverse. Despite continuous interests on this highly computational burden. As earlier study of molecular dating of enigmatic old lineage of Ophioglossaceae, there has been no ferns (monilophytes (Pryer, 2014)), we used four fossil attempt to estimate the divergence time of Mankyua, calibration points to estimate the divergence time of specifically for its split from the closest lineage and the crown Ophioglossaceae. We incorporated four fossil constraints from age. Therefore, in the present study, we explored the a reassessment of the fern fossil record and the root of the phylogenetic position of Mankyua within Ophioglossaceae and resulting tree was used as a calibration point based on the estimated the divergence time of M. chejuense. concurrent appearance of fossils belonging to each of these lineages in the Middle Devonian. We employed a relaxed Materials and Methods molecular clock model (Drummond et al., 2006) relying on uncorrelated rates drawn from a log-normal distribution, and DNA sequence and phylogenetic analyses a Yule tree prior for speciation. Two independent Markov All DNA sequences were retrieved from GenBank chain Monte Carlo runs were performed with 5,000,000 (Appendix 1). The rbcL and matK sequences were aligned generations each, with every 500 generation sampled. A burn- using CLUSTAL W (Larkin et al., 2007), implemented in in of 10% per run was discarded after assessing convergence Geneious version 7.1.7 (Kearse et al., 2012). The alignment with Tracer version 1.4.1. To obtain an estimate of the was further examined and slightly edited manually as phylogenetic tree with mean divergence time and 95% highest necessary. For the combined cpDNA datasets, we selected six posterior density (HPD) intervals, the program TreeAnnotator taxa of two genera, Huperzia and Isoetes, as outgroups (Pryer v.1.7.5 was used as it summarizes the post burn-in trees and et al., 2004). A total of 42 accessions for 37 taxa were used their parameters. for phylogenetic analysis. The combined data set was analyzed by maximum parsimony (MP), maximum likelihood (ML), and Results Bayesian inference (BI) method. The MP phylogenetic analyses was performed using PAUP* version 4.0b10 Phylogenetic position of Mankyua (Swofford, 2002). Characters were treated as unordered and The combined cpDNA data set included a total of 42 all character transformations were weighted equally. The MP accessions. A total 2,032 aligned sites for concatenated cpDNA analyses were conducted using heuristic search options with characters were used for MP, ML, and BI analyses. Of the simple stepwise addition of taxa, tree-bisection-reconnection 2,032 characters, 836 were constant, 123 were variable but branch swapping, and saving multiple trees. Bootstrap values parsimony-uninformative, and 1,073 were parsimony- (Felsenstein, 1985) were calculated from 1,000 replicate informative characters. The MP analysis found one most Korean Journal of Plant Taxonomy Vol.