# DNA Barcoding for Herbarium Specimens of the Red Alga Meristotheca pilulaora and Molecular Marker Development for Species Identification

## TL;DR

This study used DNA barcoding to correct misidentifications in algal herbarium specimens and developed a molecular marker to distinguish between two red algal species in Korea.

## Contribution

The study developed a cox1 molecular marker for accurate species identification and revealed inter-ordinal misidentifications in herbarium specimens.

## Key findings

- Six out of twelve M. papulosa specimens were misidentified as M. pilulaora or G. textorii.
- A cox1 marker was developed to effectively distinguish between M. pilulaora and G. textorii.
- DNA barcoding provided insights into the geographic distribution of M. pilulaora in Korea.

## Abstract

The red alga Meristotheca pilulaora (Gigartinales, Solieriaceae) has recently been described and newly recorded from Korea based on analyses of a species previously reported as Meristotheca papulosa. We re-examined M. papulosa herbarium specimens deposited in the National Institute of Biological Resources algal herbarium (Korea) to clarify the taxonomic relationship between M. papulosa and M. pilulaora. Using DNA barcoding, we examined M. papulosa specimens and unexpectedly identified Meristotheca pilulaora and Gracilaria textorii (Gracilariales, Gracilariaceae), but not M. papulosa. Thus, we revealed a misidentification at the inter-ordinal level (6/12 specimens). Moreover, we developed a cox1 marker as an effective molecular tool to discriminate between M. pilulaora and G. textorii. The molecular taxonomic results obtained in this study could contribute to gaining useful genetic information for re-examining herbarium specimen identities with similar morphologies and for establishing the geographic distribution of M. pilulaora in Korea.

The genus Meristotheca (Gigartinales, Solieriaceae) comprises edible red algae that are economically important food ingredients in Korea, Japan, and China. In Korea, two species, Meristotheca coacta and Meristotheca papulosa, have been identified, with the latter being predominantly reported. Recently, molecular phylogenetic analysis enabled the identification of Meristotheca pilulaora (Gigartinales; Solieriaceae) on Jeju Island (Korea). In this study, we used a DNA barcoding method to re-examine M. papulosa herbarium specimens deposited at the National Institute of Biological Resources (Incheon, Korea). Specimens were collected from Korean coastal regions between 2009 and 2019. Molecular analyses based on the rbcL and cox1 sequences of the “M. papulosa” herbarium specimens revealed that the specimens were of two other species, M. pilulaora and Gracilaria textorii (Gracilariales; Gracilariaceae). Our work represents a case study for establishing a misidentification at the inter-ordinal level among herbarium specimens without DNA sequence verification. Moreover, we developed a molecular marker for the effective species-level identification of M. pilulaora and G. textorii specimens. The DNA barcoding method provides useful information regarding M. pilulaora distribution and taxonomy.