Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Utility of taxon-specific molecular markers for the species identification of herbarium specimens: an example from Desmarestia japonica (Phaeophyceae, Desmarestiales) in Korea

Utility of taxon-specific molecular markers for the species identification of herbarium... Desmarestia japonica (Phaeophyceae, Desmarestiales) was recently established from the Japanese ligulate Desmarestia and is morphologically similar to D. ligulata. This species has been reported only from Japan. However, the taxonomic reports based on additional regional distributions are needed to clarify this taxonomic entity and its species boundaries. Because Desmarestia species have restricted distributions in Korea, we reexamined herbarium specimens of D. ligulata deposited at the National Institute of Biological Resources (South Korea). To improve the amplification efficiency of the polymerase chain reaction and avoid contamination by the DNA of other organisms, we developed taxon-specific molecular markers suitable for DNA barcoding of Desmarestia species. Nuclear ribosomal small subunit RNA (18S rDNA) and mitochondrial cytochrome c oxidase 1 (cox1) regions were selected as target DNA. As a result, both were successfully isolated from herbarium specimens of D. japonica acquired over 10 years. These molecular markers provide useful genetic information for herbarium specimens for which conventional molecular analysis is challenging. Keywords: Nuclear ribosomal small subunit, Mitochondrial cytochrome c oxidase 1, Ligulate Desmarestia, Taxon-specific molecular markers Background Okamura; and D. viridis (O.F.Müller) J.V.Lamouroux Brown algal species of the genus Desmarestia from Korea (Lee and Hwang 2010). Yang et al. (2014) (Desmarestiales) have a worldwide distribution (Guiry revised the taxonomic relationship of Desmarestia and Guiry 2017). Desmarestia species inhabit primarily species and suggested new combinations of the subspe- the cold seawater of higher latitudes of both Northern cies of D. dudresnayi and D. herbacea. On the species and Southern hemispheres but are rarer in warm level, they established D. japonica from Japanese seawater (Graham et al. 2009). The genus includes free Desmarestia species. sulfuric acid-containing species, characterized by many Desmarestia japonica was recently established from branched or foliose macroscopic thalli with pseudo- Japanese Desmarestia species, based on molecular data parenchymatous cell structures (Yang et al. 2014). and morphological characteristics (Yang et al. 2014). Three species of Desmarestia have been reported from This ligulate species had been referred previously to D. Korea, as D. ligulata H. Kawai, T. Hanyuda, D.G.Mülller, ligulata in Japan, and its morphology was described by E.C.Yang, A.F.Peters and F.C.Küpper; D. tabacoides Okamura (1936) and Yoshida (1998)as D. ligulata. Yang et al. (2014) stated that there was no evidence as * Correspondence: eylee1@korea.kr to whether D. japonica occurred in Korea. Thus, there is Microorganism Resources Division, National Institute of Biological Resources, a need to confirm the taxonomic entity and species Incheon 22689, South Korea boundaries. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 2 of 6 In Korea, Desmarestia species show a restricted distri- be included in any DNA analysis. Moreover, fungal and bution in terms of ecological habitats. Thus, the human DNA contamination can occur during conserva- National Institute of Biological Resources (Korea) estab- tion in herbaria. lished the scientific project on the distribution and In this study, we developed taxon-specific molecular genetic diversity of these rare species, and herbarium markers for DNA barcoding of herbarium specimens of specimens of Desmarestia species have been deposited Desmarestia species deposited in the National Institute since 2007. of Biological Resources (Korea). The taxon-specific Herbarium specimens contain valuable information for primer pairs were designed for the amplification of DNA genetic investigations (Nicholls 2009). DNA sequences barcode regions (18S rDNA and cox1). We also report from herbarium specimens can also provide the import- for the first time D. japonica from Korea. ant molecular evidence to solve taxonomic controversies (Goff et al. 1994; Provan et al. 2008; Hughey and Methods Gabrielson 2012; Saunders and McDevit 2012). However, We analyzed herbarium specimens deposited in the most herbarium specimens have been found to not be in National Institute of Biological Resources, Korea (Fig. 1). a suitable condition for molecular biological analyses. The morphological characteristics of 21 specimens of DNA degradation and contamination are still major Korean D. ligulata (Table 1) were measured. Photo- limitations (Taylor and Swann 1994). graphs were taken with a digital camera (C-4040 zoom, Many studies have attempted to overcome the limita- Olympus, Tokyo, Japan) attached to a light microscope tions of herbarium specimens as molecular biological (BX50, Olympus). After the morphological reexamina- materials and to improve molecular tools for DNA tion, we cut a small piece (< 0.5 cm ) to minimize the extraction and amplification of target DNA regions (e.g., damage from herbarium specimens to be used for DNA Taylor and Swann 1994; Meusnier et al. 2008; Prosser et analyses. Images of specimens were obtained with a al. 2016). Next-generation sequencing (NGS) was scanner (Epson, Seiko Epson Corp., Japan, Fig. 1). recently applied to extract genetic information from old For the molecular analyses of the specimens, we used herbarium specimens (e.g., Hughey et al. 2014; Suzuki et the reference sequences of Desmarestia species depos- al. 2016). ited in GenBank (NCBI, National Center for Biotechnol- For the selection of targeted DNA regions, shorter ogy Information). To avoid contamination by fungi and amplicons show higher efficiency in amplification. Thus, other organisms, we developed taxon-specific primer the universal DNA mini-barcode (cox1) with minimal pairs for the amplification of target DNA regions (Fig. 2, length has been adopted for biodiversity analysis (Meus- 18S rDNA and cox1). We selected a putatively conserved nier et al. 2008). However, this short length of the target region among reference DNA sequences of Desmarestia DNA region could not solve the problem of contamin- species. Moreover, the DNA regions conserved with ation. During the preparation and conservation of speci- other organisms were excluded for the primer design as mens, many sources of contamination can be present. much as possible. The universal primer sets for 18S Epiphytic organisms on algal thalli may not be excluded rDNA (A/SSUinR-1 in Lee et al. 2010) and cox1 completely during sample preparation. Many algal speci- (LCO1490/HC02198 in Folmer et al. 1994) were also mens have such epiphytic organisms, and thus, they may tested for comparison. Fig. 1 Herbarium specimens analyzed. These specimens were identified as D. ligulata from morphological examinations. a NIBRAL0000122790. b NIBRAL0000000705. c NIBRAL0000000724 Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 3 of 6 Table 1 Comparison of morphological characteristics of D. japonica Characters Womersley (1987) (as D. ligulata) Yoshida (1998) (as D. ligulata) Yang et al. (2014) Korean specimens (as D. japonica) (in this study) Color Medium brown to greenish brown – Light olive brown Light olive brown Height Usually 20–50 cm tall 60–100 cm tall 0.6–1(− 2) m tall 40–67 cm Width of branches With axis and primary laterals mostly 3–6 mm wide branches 2–6(− 20) mm wide (0.7 −) 1.7–4mm 2–5 mm broad branches wide branches Holdfast Discoid holdfast – Conical or flattened Discoid holdfast Branching type Fronds with frequent opposite Opposite to ca. three Opposite, branches of Opposite, branched branching to 3 orders times branching limited growth branched to to two to three two to three times times Branch morphology More or less linear but tapering Wide linear feather-like Branches stipitate and Branches stipitate apically and basally, dentate, and branches, tapering apically attenuate at base and apex and attenuate at with a faint to conspicuous midrib and basally with midrib base and apex (the axial filament) The DNA extraction, polymerase chain reaction 7 min extension step at 72 °C. Sequencing was con- (PCR), and sequencing adopted the methods described ducted by a commercial service (Genotech, Daejeon, in Lee et al. (2011). We isolated total DNAs from the Korea), and the sequencing chromatograms were assem- subsampled herbarium specimens. We extended the bled with Sequencher 5.4.6 (Gene Codes Corp., Ann incubation time of DNA extraction step (1 h). Moreover, Arbor, MI, USA). The phylogenetic analyses were the incubation times in washing step were also constructed using MEGA version 6 (Tamura et al. 2013). prolonged to improve the quality of DNA eluents. PCR The neighbor-joining method and bootstrap analyses conditions consisted of 3 min at 95 °C, 40 cycles of 30 s (2000 replicates) were used to reconstruct the phylogen- at 94 °C, 30 s at 50 °C, and 1 min at 72 °C, and a final etic tree. Molecular study about Desmarestia herbarium Fig. 2 Primer maps developed. Desmarestia viridis (AJ295828) was used as the reference sequence for the primer-binding positions of 18S rDNA primers (a). Numbers of the cox1 primers represent the position from the cox1 gene start codon (b) Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 4 of 6 specimens had not been carried out in this lab previ- these 18S rDNA sequences alone could not provide suf- ously. All reagents were in a sterile condition and stored ficient genetic information to discriminate interspecific in disposable plastic ware. relationships among Desmarestia species. For the amplification of cox1 sequences (Fig. 2b, one Results forward and two reverse primers), the combinations of Korean Desmarestia species showed a restricted distri- cox1-desm-193F/cox1-desm-504R and cox1-desm-193F/ bution pattern, mainly on the northeastern coast mostly cox1-desm-608R successfully amplified the cox1 region in subtidal habits (Lee and Hwang 2010). Because of Desmarestia species. The primer pair of cox1-desm- Desmarestia species live, herbarium specimens could be 193F/cox1-desm-504R showed high efficiency in amplifi- effective for the molecular investigation. We examined cation (272 bp excluding primer-binding sites). Thus, we herbarium specimens deposited in NIBR collected from used this combination to amplify cox1 from Desmarestia 10 years ago (Fig. 1). First, we selected samples previ- specimens. ously identified as D. ligulata according to morpho- Korean D. japonica samples had the same cox1 logical resemblance (Yang et al. 2014). From the sequence with Japanese D. japonica (HE866773 in Yang et morphological examination, D. ligulata has proliferate al. 2014). A cox1 sequence reported from China as D. viri- pinnate-branched thalli and D. tabacoides typically has dis (KC491233) also had 100% similarity with D. japonica. one or two wide-branched or unbranched foliose thalli Because D. japonica showed below 97.4% similarity with (Table 1). In the case of D. viridis, this species was other Desmarestia species deposited in the GenBank, this distinguished by much longer linear branched thalli. Chinese sample was likely misidentified (Fig. 3). We examined a total of 21 specimens identified as D. ligulata from the Korean coast. The thallus is light olive Discussion brown in color and, when exposed to air, becomes The herbarium specimens examined from the Korean greenish brown. Korean specimens are up to 67 cm in coasts have feather-like features and were smaller than height and have mostly three orders of branching. In the main axes and primary branches, branches were 2 mm wide, but in tall specimens, they were up to 4 mm wide. Gross morphology, with feather-like pinnate branching, was similar to that of Japanese ligulate Desmarestia spe- cies. Representative specimens showing morphological differences were also analyzed using molecular methods. The universal primer set for 18S rDNA (Lee et al. 2010) produced fungal 18S rDNA sequences from the total genomic DNA extracts of herbarium specimens. The 18S rDNA sequenced showed high similarity with Agaricus bisporus var. bisporus (CP015465, 520/ 527(99%) from D. ligulata). However, we successfully isolated 18S rDNA (MF363011) and cox1 (MF363010) sequences of D. japonica from three specimens, using our taxon-specific primer pairs: NIBRAL0000000724 (Gangneung March 7, 2006), NIBRAL0000122790 (Gangneung May 8, 2009), and NIBRAL0000000705 (Goseong July 23, 2005). Using forward primer A (Medlin et al. 1988; Lee et al. 2010), two reverse primers (Fig. 2a) produced PCR bands from DNA extracts of D. ligulata. The combin- ation A/18S-desm-233R produced 213 bp, and A/18S- desm-670R amplified 650 bp of 18S rDNA without primer-binding sites. The three 18S rDNA sequences had the same sequence and 100% similarity with D. japonica (HE866912-HE866915, Yang et al. 2014). However, these 18S rDNA regions also had identical Fig. 3 Phylogenetic relationship among Desmarestia species. A sequences with D. aculeata (HE866893-4), D. distans neighbor-joining tree was analyzed with 2000 replicates of the boot- (HE866923), D. latifrons (HE866916), D. ligulata strap values. Himantothallus grandifolius was used as an outgroup (HE866917-22), and D. muelleri (HE866924-5). Thus, Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 5 of 6 the taller samples described by Yoshida (1998)as D. The overall morphologies of D. japonica specimens ligulata and Yang et al. (2014)as D. japonica (Table 1). were variable in branching and branch width (Fig. 1, However, they were similar in the color, branching pat- Table 1). Moreover, their morphologies were similar to tern, and height of Japanese plants as well as Australian those of D. ligulata. plants (Womersley 1987). Lamouroux’s(1813) illustra- In this study, we found D. japonica from herbarium tion of D. ligulata showed that some primary laterals of specimens in NIBR using the taxon-specific primer pair the frond were dichotomous and some secondary (Fig. 2). These specimens were collected over 10 years laterals did not branch oppositely. However, we did not ago and were first identified as D. ligulata based on find such dichotomous branches within our Korean morphological characteristics (Fig. 1). A Chinese cox1 specimens whereas we did mostly observe opposite sequence (KC491233) of D. viridis also showed 100% branching in secondary laterals. similarity in the cox1 region with Japanese D. japonica. Molecular phylogenetic studies of Desmarestia species These results indicate an extended distribution in Korea were conducted to establish new species and to recon- and China for D. japonica (Fig. 3). Consequently, a mo- struct the phylogenetic relationships (Tan and Druehl lecular taxonomic reexamination of the morphological 1996; Yang et al. 2014). As a result, the key reference resemblance among D. japonica, D. ligulata, and D. sequences of the 18S rDNA and cox1 region are viridis is needed in future studies. available in GenBank. Thus, we selected these DNA sequences as the target regions for taxon-specific Conclusions molecular markers of Desmarestia species. We developed taxon-specific primer sets to amplify the DNA degradation in dried algal specimens and 18S rDNA and cox1 regions without contaminants (e.g., contamination are the major reasons for the failure of fungi and epiphytic organisms) and successfully isolated DNA analyses. The universal cox1 primer pair could not DNA regions from herbarium specimens over 10 years amplify the cox1 region from herbarium specimens of old. From these results, we confirmed the presence of D. Desmarestia. In the case of 18S rDNA, fungal DNAs japonica from Korea and China. We believe that the were amplified. Thus, a primer pair is required having new molecular markers we have developed also provide high specificity and efficiency in amplifying the target useful information for DNA barcoding species of the DNA region from herbarium samples. In this study, we economic seaweed Desmarestia. developed new primer pairs having short fragments of Abbreviations PCR to enhance the efficiency of amplification cox1: Mitochondrial cytochrome c oxidase 1; NCBI: National Center for (Meusnier et al. 2008) and the specificity for the target Biotechnology Information; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; rDNA: Ribosomal DNA plant samples (Fig. 2). The primer pairs developed could successfully Acknowledgements amplify 18S rDNA and cox1 regions from specimens of Not applicable Desmarestia species. When the universal primers were Funding used in the analyses, the samples showed no PCR band This study was supported by the National Institute of Biological Resources (cox1) or amplified fungal 18S rDNAs. The 18S rDNA (NIBR201701103), Ministry of Environment of South Korea. and cox1 region could provide robust results for the Availability of data and materials finding of taxonomic entities of D. japonica.This re- All datasets analyzed during the current study are available from the port of D. japonica is the first regarding the distribution corresponding author on reasonable request. of D. japonica after the establishment of this species Authors’ contributions based on Japanese specimens (Yang et al. 2014). SRL conducted the research, analyzed the molecular result, and prepare the The isolated 18S rDNA sequence of Desmarestia draft. EYL designed the research, analyzed the herbarium specimens, and prepared the draft in part. Both authors read and approved the final species could not provide a taxonomic resolution at the manuscript. interspecific level and was not a suitable marker to analyze the taxonomic entities of Korean samples. The Ethics approval and consent to participate Not applicable cox1 region has been selected frequently as a standard marker for algal DNA barcode use (Lane et al. 2007). In Consent for publication this study, the cox1 region provided suitable genetic Not applicable information to examine the taxonomic entity of D. Competing interests japonica from Korea. Yang et al. (2014) also found The authors declare that they have no competing interests. effective taxonomic resolution of the cox1 region, reflecting species delimitations among Desmarestia Publisher’sNote species and proposed the cox1 region as a potential Springer Nature remains neutral with regard to jurisdictional claims in barcode marker for the genus Desmarestia. published maps and institutional affiliations. Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 6 of 6 Author details Womersley HBS. The marine benthic flora of Southern Australia, Part II / HBS Marine Research Institute, Pusan National University, Busan 46241, South Womersley. Adelaide: South Australian Government Printing Division; 1987. Korea. Microorganism Resources Division, National Institute of Biological Yang EC, Peters AF, Kawai H, Stern R, Hanyuda T, Bárbara I, Müller DG, Strittmatter Resources, Incheon 22689, South Korea. M, Prud’Homme van Reine WF, Küpper FC. Ligulate Desmarestia (Desmarestiales, Phaeophyceae) revisited: D. japonica sp. nov. and D. Received: 9 November 2017 Accepted: 26 January 2018 dudresnayi differ from D. ligulata. J Phycol. 2014;50:149–66. Yoshida T. Marine algae of Japan. Tokyo: Uchida Rokakuho Publishing; 1998. References Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3:294–9. Goff LJ, Moon DA, Coleman AW. Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales). J Phycol. 1994;30:521–37. Graham LE, Graham JM, Willcox LW. Algae. San Francisco: Benjamin Cummings; Guiry MD, Guiry GM. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. 2017. http://www.algaebase.org, searched on 05 June 2017. Hughey JR, Gabrielson PW. Comment on “acquiring DNA sequence data from dried archival red algae (Florideophyceae) for the purpose of applying available names to contemporary genetic species: a critical assessment”. Botany. 2012;90:1191–4. Hughey JR, Gabrielson PW, Rohmer L, Tortolani J, Silva M, Miller KA, Young JD, Martell C, Ruediger E. Minimally destructive sampling of type specimens of Pyropia (Bangiales, Rhodophyta) recovers complete plastid and mitochondrial genomes. 2014. Sci Rep. 2014;4:5113. Lamouroux JVF. Essai sur les genres de la famille des thalassiophytes non articulées. Annales du Muséum d'Histoire Naturelle. 1813;20:43–5. pl 8 Lane CE, Lindstrom SC, Saunders GW. A molecular assessment of northeast Pacific Alaria species (Laminariales, Phaeophyceae) with reference to the utility of DNA barcoding. Mol Phylogenet Evol. 2007;44:634–48. Lee JW, Hwang IK. Dictyotales, Desmarestiales. In: Anon, editor. Algal flora of Korea. Volume 2, number 2. Heterokontophyta: Phaeophyceae: Ishigeales, Dictyotales, Desmarestiales, Sphacelariales, Cutleriales, Ralfisales, Laminariales. Incheon: National Institute of Biological Resources; 2010. p. 19–69. Lee S-R, Oak JH, Chung IK, Lee JA. Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR, PCR-RFLP, and sequencing. J Appl Phycol. 2010;22:699–707. Lee S-R, Oak JH, Keum YS, Lee JA, Chung IK. Utility of rbcSgene asanovel target DNA region for brown algal molecular systematics. Phycol Res. 2011;59:34–41. Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA coding regions. Gene. 1988;71:491–9. Meusnier I, Singer GA, Landry JF, Hickey DA, Hebert PD, Hajibabaei M. A universal DNA mini-barcode for biodiversity analysis. BMC Genomics. 2008;9:214. Nicholls H. Time to sequence the ‘red and the dead’. Nature. 2009;458:812. Okamura K. Nippon kaisô shi [Descriptions of Japanese algae]. Tokyo: Uchida Rokakuho; 1936. Prosser SW, de Waard JR, Miller SE, Hebert PD. DNA barcodes from century-old type specimens using next-generation sequencing. Mol Ecol Resour. 2016;16: 487–97. Provan J, Booth D, Todd NP, Beatty GE, Maggs CA. Tracking biological invasions in space and time: elucidating the invasive history of the green alga Codium fragile using old DNA. Divers Distrib. 2008;14:343–54. Saunders GW, McDevit DC. Acquiring DNA sequence data from dried archival red Submit your next manuscript to BioMed Central algae (Florideophyceae) for the purpose of applying available names to contemporary genetic species: a critical assessment. Botany. 2012;90:191–203. and we will help you at every step: Suzuki M, Segawa T, Mori H, Akiyoshi A, Ootsuki R, Kurihara A, Sakayama H, • We accept pre-submission inquiries Kitayama T, Abe T, Kogame K, Kawai H, Nozaki H. Next-generation sequencing of an 88-year-old specimen of the poorly known species Liagora � Our selector tool helps you to find the most relevant journal japonica (Nemaliales, Rhodophyta) supports the recognition of Otohimella � We provide round the clock customer support gen. nov. PLoS One. 2016;11:e0158944. � Convenient online submission Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9. � Thorough peer review Tan IH, Druehl LD. A ribosomal DNA phylogeny supports the close evolutionary � Inclusion in PubMed and all major indexing services relationships among the Sporochnales, Desmarestiales, and Laminariales � Maximum visibility for your research (Phaeophyceae). J Phycol. 1996;32:112–8. Taylor JW, Swann EC. DNA from herbarium specimens. In: In Ancient DNA. New Submit your manuscript at York: Springer; 1994. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Utility of taxon-specific molecular markers for the species identification of herbarium specimens: an example from Desmarestia japonica (Phaeophyceae, Desmarestiales) in Korea

Lee, Sang-Rae; Lee, Eun-Young
Fisheries and Aquatic Sciences , Volume 21 (1) – Mar 26, 2018
Download PDF
6 pages

Loading next page...
 
/lp/springer-journals/utility-of-taxon-specific-molecular-markers-for-the-species-c9bddK0nM0

References (27)

Publisher
Springer Journals
Copyright
Copyright © 2018 by The Author(s)
Subject
Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
eISSN
2234-1757
DOI
10.1186/s41240-018-0085-0
Publisher site
See Article on Publisher Site

Abstract

Desmarestia japonica (Phaeophyceae, Desmarestiales) was recently established from the Japanese ligulate Desmarestia and is morphologically similar to D. ligulata. This species has been reported only from Japan. However, the taxonomic reports based on additional regional distributions are needed to clarify this taxonomic entity and its species boundaries. Because Desmarestia species have restricted distributions in Korea, we reexamined herbarium specimens of D. ligulata deposited at the National Institute of Biological Resources (South Korea). To improve the amplification efficiency of the polymerase chain reaction and avoid contamination by the DNA of other organisms, we developed taxon-specific molecular markers suitable for DNA barcoding of Desmarestia species. Nuclear ribosomal small subunit RNA (18S rDNA) and mitochondrial cytochrome c oxidase 1 (cox1) regions were selected as target DNA. As a result, both were successfully isolated from herbarium specimens of D. japonica acquired over 10 years. These molecular markers provide useful genetic information for herbarium specimens for which conventional molecular analysis is challenging. Keywords: Nuclear ribosomal small subunit, Mitochondrial cytochrome c oxidase 1, Ligulate Desmarestia, Taxon-specific molecular markers Background Okamura; and D. viridis (O.F.Müller) J.V.Lamouroux Brown algal species of the genus Desmarestia from Korea (Lee and Hwang 2010). Yang et al. (2014) (Desmarestiales) have a worldwide distribution (Guiry revised the taxonomic relationship of Desmarestia and Guiry 2017). Desmarestia species inhabit primarily species and suggested new combinations of the subspe- the cold seawater of higher latitudes of both Northern cies of D. dudresnayi and D. herbacea. On the species and Southern hemispheres but are rarer in warm level, they established D. japonica from Japanese seawater (Graham et al. 2009). The genus includes free Desmarestia species. sulfuric acid-containing species, characterized by many Desmarestia japonica was recently established from branched or foliose macroscopic thalli with pseudo- Japanese Desmarestia species, based on molecular data parenchymatous cell structures (Yang et al. 2014). and morphological characteristics (Yang et al. 2014). Three species of Desmarestia have been reported from This ligulate species had been referred previously to D. Korea, as D. ligulata H. Kawai, T. Hanyuda, D.G.Mülller, ligulata in Japan, and its morphology was described by E.C.Yang, A.F.Peters and F.C.Küpper; D. tabacoides Okamura (1936) and Yoshida (1998)as D. ligulata. Yang et al. (2014) stated that there was no evidence as * Correspondence: eylee1@korea.kr to whether D. japonica occurred in Korea. Thus, there is Microorganism Resources Division, National Institute of Biological Resources, a need to confirm the taxonomic entity and species Incheon 22689, South Korea boundaries. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 2 of 6 In Korea, Desmarestia species show a restricted distri- be included in any DNA analysis. Moreover, fungal and bution in terms of ecological habitats. Thus, the human DNA contamination can occur during conserva- National Institute of Biological Resources (Korea) estab- tion in herbaria. lished the scientific project on the distribution and In this study, we developed taxon-specific molecular genetic diversity of these rare species, and herbarium markers for DNA barcoding of herbarium specimens of specimens of Desmarestia species have been deposited Desmarestia species deposited in the National Institute since 2007. of Biological Resources (Korea). The taxon-specific Herbarium specimens contain valuable information for primer pairs were designed for the amplification of DNA genetic investigations (Nicholls 2009). DNA sequences barcode regions (18S rDNA and cox1). We also report from herbarium specimens can also provide the import- for the first time D. japonica from Korea. ant molecular evidence to solve taxonomic controversies (Goff et al. 1994; Provan et al. 2008; Hughey and Methods Gabrielson 2012; Saunders and McDevit 2012). However, We analyzed herbarium specimens deposited in the most herbarium specimens have been found to not be in National Institute of Biological Resources, Korea (Fig. 1). a suitable condition for molecular biological analyses. The morphological characteristics of 21 specimens of DNA degradation and contamination are still major Korean D. ligulata (Table 1) were measured. Photo- limitations (Taylor and Swann 1994). graphs were taken with a digital camera (C-4040 zoom, Many studies have attempted to overcome the limita- Olympus, Tokyo, Japan) attached to a light microscope tions of herbarium specimens as molecular biological (BX50, Olympus). After the morphological reexamina- materials and to improve molecular tools for DNA tion, we cut a small piece (< 0.5 cm ) to minimize the extraction and amplification of target DNA regions (e.g., damage from herbarium specimens to be used for DNA Taylor and Swann 1994; Meusnier et al. 2008; Prosser et analyses. Images of specimens were obtained with a al. 2016). Next-generation sequencing (NGS) was scanner (Epson, Seiko Epson Corp., Japan, Fig. 1). recently applied to extract genetic information from old For the molecular analyses of the specimens, we used herbarium specimens (e.g., Hughey et al. 2014; Suzuki et the reference sequences of Desmarestia species depos- al. 2016). ited in GenBank (NCBI, National Center for Biotechnol- For the selection of targeted DNA regions, shorter ogy Information). To avoid contamination by fungi and amplicons show higher efficiency in amplification. Thus, other organisms, we developed taxon-specific primer the universal DNA mini-barcode (cox1) with minimal pairs for the amplification of target DNA regions (Fig. 2, length has been adopted for biodiversity analysis (Meus- 18S rDNA and cox1). We selected a putatively conserved nier et al. 2008). However, this short length of the target region among reference DNA sequences of Desmarestia DNA region could not solve the problem of contamin- species. Moreover, the DNA regions conserved with ation. During the preparation and conservation of speci- other organisms were excluded for the primer design as mens, many sources of contamination can be present. much as possible. The universal primer sets for 18S Epiphytic organisms on algal thalli may not be excluded rDNA (A/SSUinR-1 in Lee et al. 2010) and cox1 completely during sample preparation. Many algal speci- (LCO1490/HC02198 in Folmer et al. 1994) were also mens have such epiphytic organisms, and thus, they may tested for comparison. Fig. 1 Herbarium specimens analyzed. These specimens were identified as D. ligulata from morphological examinations. a NIBRAL0000122790. b NIBRAL0000000705. c NIBRAL0000000724 Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 3 of 6 Table 1 Comparison of morphological characteristics of D. japonica Characters Womersley (1987) (as D. ligulata) Yoshida (1998) (as D. ligulata) Yang et al. (2014) Korean specimens (as D. japonica) (in this study) Color Medium brown to greenish brown – Light olive brown Light olive brown Height Usually 20–50 cm tall 60–100 cm tall 0.6–1(− 2) m tall 40–67 cm Width of branches With axis and primary laterals mostly 3–6 mm wide branches 2–6(− 20) mm wide (0.7 −) 1.7–4mm 2–5 mm broad branches wide branches Holdfast Discoid holdfast – Conical or flattened Discoid holdfast Branching type Fronds with frequent opposite Opposite to ca. three Opposite, branches of Opposite, branched branching to 3 orders times branching limited growth branched to to two to three two to three times times Branch morphology More or less linear but tapering Wide linear feather-like Branches stipitate and Branches stipitate apically and basally, dentate, and branches, tapering apically attenuate at base and apex and attenuate at with a faint to conspicuous midrib and basally with midrib base and apex (the axial filament) The DNA extraction, polymerase chain reaction 7 min extension step at 72 °C. Sequencing was con- (PCR), and sequencing adopted the methods described ducted by a commercial service (Genotech, Daejeon, in Lee et al. (2011). We isolated total DNAs from the Korea), and the sequencing chromatograms were assem- subsampled herbarium specimens. We extended the bled with Sequencher 5.4.6 (Gene Codes Corp., Ann incubation time of DNA extraction step (1 h). Moreover, Arbor, MI, USA). The phylogenetic analyses were the incubation times in washing step were also constructed using MEGA version 6 (Tamura et al. 2013). prolonged to improve the quality of DNA eluents. PCR The neighbor-joining method and bootstrap analyses conditions consisted of 3 min at 95 °C, 40 cycles of 30 s (2000 replicates) were used to reconstruct the phylogen- at 94 °C, 30 s at 50 °C, and 1 min at 72 °C, and a final etic tree. Molecular study about Desmarestia herbarium Fig. 2 Primer maps developed. Desmarestia viridis (AJ295828) was used as the reference sequence for the primer-binding positions of 18S rDNA primers (a). Numbers of the cox1 primers represent the position from the cox1 gene start codon (b) Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 4 of 6 specimens had not been carried out in this lab previ- these 18S rDNA sequences alone could not provide suf- ously. All reagents were in a sterile condition and stored ficient genetic information to discriminate interspecific in disposable plastic ware. relationships among Desmarestia species. For the amplification of cox1 sequences (Fig. 2b, one Results forward and two reverse primers), the combinations of Korean Desmarestia species showed a restricted distri- cox1-desm-193F/cox1-desm-504R and cox1-desm-193F/ bution pattern, mainly on the northeastern coast mostly cox1-desm-608R successfully amplified the cox1 region in subtidal habits (Lee and Hwang 2010). Because of Desmarestia species. The primer pair of cox1-desm- Desmarestia species live, herbarium specimens could be 193F/cox1-desm-504R showed high efficiency in amplifi- effective for the molecular investigation. We examined cation (272 bp excluding primer-binding sites). Thus, we herbarium specimens deposited in NIBR collected from used this combination to amplify cox1 from Desmarestia 10 years ago (Fig. 1). First, we selected samples previ- specimens. ously identified as D. ligulata according to morpho- Korean D. japonica samples had the same cox1 logical resemblance (Yang et al. 2014). From the sequence with Japanese D. japonica (HE866773 in Yang et morphological examination, D. ligulata has proliferate al. 2014). A cox1 sequence reported from China as D. viri- pinnate-branched thalli and D. tabacoides typically has dis (KC491233) also had 100% similarity with D. japonica. one or two wide-branched or unbranched foliose thalli Because D. japonica showed below 97.4% similarity with (Table 1). In the case of D. viridis, this species was other Desmarestia species deposited in the GenBank, this distinguished by much longer linear branched thalli. Chinese sample was likely misidentified (Fig. 3). We examined a total of 21 specimens identified as D. ligulata from the Korean coast. The thallus is light olive Discussion brown in color and, when exposed to air, becomes The herbarium specimens examined from the Korean greenish brown. Korean specimens are up to 67 cm in coasts have feather-like features and were smaller than height and have mostly three orders of branching. In the main axes and primary branches, branches were 2 mm wide, but in tall specimens, they were up to 4 mm wide. Gross morphology, with feather-like pinnate branching, was similar to that of Japanese ligulate Desmarestia spe- cies. Representative specimens showing morphological differences were also analyzed using molecular methods. The universal primer set for 18S rDNA (Lee et al. 2010) produced fungal 18S rDNA sequences from the total genomic DNA extracts of herbarium specimens. The 18S rDNA sequenced showed high similarity with Agaricus bisporus var. bisporus (CP015465, 520/ 527(99%) from D. ligulata). However, we successfully isolated 18S rDNA (MF363011) and cox1 (MF363010) sequences of D. japonica from three specimens, using our taxon-specific primer pairs: NIBRAL0000000724 (Gangneung March 7, 2006), NIBRAL0000122790 (Gangneung May 8, 2009), and NIBRAL0000000705 (Goseong July 23, 2005). Using forward primer A (Medlin et al. 1988; Lee et al. 2010), two reverse primers (Fig. 2a) produced PCR bands from DNA extracts of D. ligulata. The combin- ation A/18S-desm-233R produced 213 bp, and A/18S- desm-670R amplified 650 bp of 18S rDNA without primer-binding sites. The three 18S rDNA sequences had the same sequence and 100% similarity with D. japonica (HE866912-HE866915, Yang et al. 2014). However, these 18S rDNA regions also had identical Fig. 3 Phylogenetic relationship among Desmarestia species. A sequences with D. aculeata (HE866893-4), D. distans neighbor-joining tree was analyzed with 2000 replicates of the boot- (HE866923), D. latifrons (HE866916), D. ligulata strap values. Himantothallus grandifolius was used as an outgroup (HE866917-22), and D. muelleri (HE866924-5). Thus, Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 5 of 6 the taller samples described by Yoshida (1998)as D. The overall morphologies of D. japonica specimens ligulata and Yang et al. (2014)as D. japonica (Table 1). were variable in branching and branch width (Fig. 1, However, they were similar in the color, branching pat- Table 1). Moreover, their morphologies were similar to tern, and height of Japanese plants as well as Australian those of D. ligulata. plants (Womersley 1987). Lamouroux’s(1813) illustra- In this study, we found D. japonica from herbarium tion of D. ligulata showed that some primary laterals of specimens in NIBR using the taxon-specific primer pair the frond were dichotomous and some secondary (Fig. 2). These specimens were collected over 10 years laterals did not branch oppositely. However, we did not ago and were first identified as D. ligulata based on find such dichotomous branches within our Korean morphological characteristics (Fig. 1). A Chinese cox1 specimens whereas we did mostly observe opposite sequence (KC491233) of D. viridis also showed 100% branching in secondary laterals. similarity in the cox1 region with Japanese D. japonica. Molecular phylogenetic studies of Desmarestia species These results indicate an extended distribution in Korea were conducted to establish new species and to recon- and China for D. japonica (Fig. 3). Consequently, a mo- struct the phylogenetic relationships (Tan and Druehl lecular taxonomic reexamination of the morphological 1996; Yang et al. 2014). As a result, the key reference resemblance among D. japonica, D. ligulata, and D. sequences of the 18S rDNA and cox1 region are viridis is needed in future studies. available in GenBank. Thus, we selected these DNA sequences as the target regions for taxon-specific Conclusions molecular markers of Desmarestia species. We developed taxon-specific primer sets to amplify the DNA degradation in dried algal specimens and 18S rDNA and cox1 regions without contaminants (e.g., contamination are the major reasons for the failure of fungi and epiphytic organisms) and successfully isolated DNA analyses. The universal cox1 primer pair could not DNA regions from herbarium specimens over 10 years amplify the cox1 region from herbarium specimens of old. From these results, we confirmed the presence of D. Desmarestia. In the case of 18S rDNA, fungal DNAs japonica from Korea and China. We believe that the were amplified. Thus, a primer pair is required having new molecular markers we have developed also provide high specificity and efficiency in amplifying the target useful information for DNA barcoding species of the DNA region from herbarium samples. In this study, we economic seaweed Desmarestia. developed new primer pairs having short fragments of Abbreviations PCR to enhance the efficiency of amplification cox1: Mitochondrial cytochrome c oxidase 1; NCBI: National Center for (Meusnier et al. 2008) and the specificity for the target Biotechnology Information; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; rDNA: Ribosomal DNA plant samples (Fig. 2). The primer pairs developed could successfully Acknowledgements amplify 18S rDNA and cox1 regions from specimens of Not applicable Desmarestia species. When the universal primers were Funding used in the analyses, the samples showed no PCR band This study was supported by the National Institute of Biological Resources (cox1) or amplified fungal 18S rDNAs. The 18S rDNA (NIBR201701103), Ministry of Environment of South Korea. and cox1 region could provide robust results for the Availability of data and materials finding of taxonomic entities of D. japonica.This re- All datasets analyzed during the current study are available from the port of D. japonica is the first regarding the distribution corresponding author on reasonable request. of D. japonica after the establishment of this species Authors’ contributions based on Japanese specimens (Yang et al. 2014). SRL conducted the research, analyzed the molecular result, and prepare the The isolated 18S rDNA sequence of Desmarestia draft. EYL designed the research, analyzed the herbarium specimens, and prepared the draft in part. Both authors read and approved the final species could not provide a taxonomic resolution at the manuscript. interspecific level and was not a suitable marker to analyze the taxonomic entities of Korean samples. The Ethics approval and consent to participate Not applicable cox1 region has been selected frequently as a standard marker for algal DNA barcode use (Lane et al. 2007). In Consent for publication this study, the cox1 region provided suitable genetic Not applicable information to examine the taxonomic entity of D. Competing interests japonica from Korea. Yang et al. (2014) also found The authors declare that they have no competing interests. effective taxonomic resolution of the cox1 region, reflecting species delimitations among Desmarestia Publisher’sNote species and proposed the cox1 region as a potential Springer Nature remains neutral with regard to jurisdictional claims in barcode marker for the genus Desmarestia. published maps and institutional affiliations. Lee and Lee Fisheries and Aquatic Sciences (2018) 21:8 Page 6 of 6 Author details Womersley HBS. The marine benthic flora of Southern Australia, Part II / HBS Marine Research Institute, Pusan National University, Busan 46241, South Womersley. Adelaide: South Australian Government Printing Division; 1987. Korea. Microorganism Resources Division, National Institute of Biological Yang EC, Peters AF, Kawai H, Stern R, Hanyuda T, Bárbara I, Müller DG, Strittmatter Resources, Incheon 22689, South Korea. M, Prud’Homme van Reine WF, Küpper FC. Ligulate Desmarestia (Desmarestiales, Phaeophyceae) revisited: D. japonica sp. nov. and D. Received: 9 November 2017 Accepted: 26 January 2018 dudresnayi differ from D. ligulata. J Phycol. 2014;50:149–66. Yoshida T. Marine algae of Japan. Tokyo: Uchida Rokakuho Publishing; 1998. References Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3:294–9. Goff LJ, Moon DA, Coleman AW. Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales). J Phycol. 1994;30:521–37. Graham LE, Graham JM, Willcox LW. Algae. San Francisco: Benjamin Cummings; Guiry MD, Guiry GM. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. 2017. http://www.algaebase.org, searched on 05 June 2017. Hughey JR, Gabrielson PW. Comment on “acquiring DNA sequence data from dried archival red algae (Florideophyceae) for the purpose of applying available names to contemporary genetic species: a critical assessment”. Botany. 2012;90:1191–4. Hughey JR, Gabrielson PW, Rohmer L, Tortolani J, Silva M, Miller KA, Young JD, Martell C, Ruediger E. Minimally destructive sampling of type specimens of Pyropia (Bangiales, Rhodophyta) recovers complete plastid and mitochondrial genomes. 2014. Sci Rep. 2014;4:5113. Lamouroux JVF. Essai sur les genres de la famille des thalassiophytes non articulées. Annales du Muséum d'Histoire Naturelle. 1813;20:43–5. pl 8 Lane CE, Lindstrom SC, Saunders GW. A molecular assessment of northeast Pacific Alaria species (Laminariales, Phaeophyceae) with reference to the utility of DNA barcoding. Mol Phylogenet Evol. 2007;44:634–48. Lee JW, Hwang IK. Dictyotales, Desmarestiales. In: Anon, editor. Algal flora of Korea. Volume 2, number 2. Heterokontophyta: Phaeophyceae: Ishigeales, Dictyotales, Desmarestiales, Sphacelariales, Cutleriales, Ralfisales, Laminariales. Incheon: National Institute of Biological Resources; 2010. p. 19–69. Lee S-R, Oak JH, Chung IK, Lee JA. Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR, PCR-RFLP, and sequencing. J Appl Phycol. 2010;22:699–707. Lee S-R, Oak JH, Keum YS, Lee JA, Chung IK. Utility of rbcSgene asanovel target DNA region for brown algal molecular systematics. Phycol Res. 2011;59:34–41. Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA coding regions. Gene. 1988;71:491–9. Meusnier I, Singer GA, Landry JF, Hickey DA, Hebert PD, Hajibabaei M. A universal DNA mini-barcode for biodiversity analysis. BMC Genomics. 2008;9:214. Nicholls H. Time to sequence the ‘red and the dead’. Nature. 2009;458:812. Okamura K. Nippon kaisô shi [Descriptions of Japanese algae]. Tokyo: Uchida Rokakuho; 1936. Prosser SW, de Waard JR, Miller SE, Hebert PD. DNA barcodes from century-old type specimens using next-generation sequencing. Mol Ecol Resour. 2016;16: 487–97. Provan J, Booth D, Todd NP, Beatty GE, Maggs CA. Tracking biological invasions in space and time: elucidating the invasive history of the green alga Codium fragile using old DNA. Divers Distrib. 2008;14:343–54. Saunders GW, McDevit DC. Acquiring DNA sequence data from dried archival red Submit your next manuscript to BioMed Central algae (Florideophyceae) for the purpose of applying available names to contemporary genetic species: a critical assessment. Botany. 2012;90:191–203. and we will help you at every step: Suzuki M, Segawa T, Mori H, Akiyoshi A, Ootsuki R, Kurihara A, Sakayama H, • We accept pre-submission inquiries Kitayama T, Abe T, Kogame K, Kawai H, Nozaki H. Next-generation sequencing of an 88-year-old specimen of the poorly known species Liagora � Our selector tool helps you to find the most relevant journal japonica (Nemaliales, Rhodophyta) supports the recognition of Otohimella � We provide round the clock customer support gen. nov. PLoS One. 2016;11:e0158944. � Convenient online submission Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9. � Thorough peer review Tan IH, Druehl LD. A ribosomal DNA phylogeny supports the close evolutionary � Inclusion in PubMed and all major indexing services relationships among the Sporochnales, Desmarestiales, and Laminariales � Maximum visibility for your research (Phaeophyceae). J Phycol. 1996;32:112–8. Taylor JW, Swann EC. DNA from herbarium specimens. In: In Ancient DNA. New Submit your manuscript at York: Springer; 1994. www.biomedcentral.com/submit

Journal

Fisheries and Aquatic Sciences – Springer Journals

Published: Mar 26, 2018

There are no references for this article.