DOI: 10.6084/m9.figshare.27143343
title: Assessment of nine markers for phylogeny, species and haplotype identification of <i>Kappaphycus</i> species and <i>Eucheuma denticulatum</i> (Solieriaceae, Rhodophyta)

description: Molecular studies have contributed to the taxonomy of carrageenan-producing <i>Kappaphycus</i> spp. and <i>Eucheuma denticulatum</i>. However, unresolved species complexes and the lack of standardization in the use of genetic markers impede the identification of specimens and the delineation of a robust taxonomic framework. Here, nine molecular markers (<i>cox</i>1, <i>cox</i>2–3 spacer, <i>cox</i>2, <i>cox</i>3, COB, ITS, <i>psb</i>A, UPA and <i>rbc</i>L) were used to generate a multilocus phylogeny for 113 fresh eucheumatoid samples and four herbarium specimens. Analyses of species delineation and genetic distances confirmed the monophyly of currently accepted taxa. These analyses suggest that clades previously reported as <i>K. striatus</i> KS1 and KS2 are conspecific, and that <i>E. denticulatum</i> EDA ‘spinosum’ and EDB ‘endong/cacing’ are also conspecific. The results also unveiled possible new taxa from Hawaii and Indonesia. Each molecular marker and combinations thereof were assessed with regard to species identification, ease of amplification and sequencing, and haplotype characterization. All genetic markers recorded at least 94% success in the amplification and sequencing of fresh specimens, with <i>cox</i>1 being the most phylogenetically informative. Automatic partitioning, phylogenetic and tree-based assessments showed <i>cox</i>1, <i>cox</i>2–3 spacer, <i>cox</i>2 and <i>rbc</i>L were able to correctly identify species while <i>cox</i>1+ <i>rbc</i>L, COB+<i>rbc</i>L, <i>cox</i>2+ <i>rbc</i>L or <i>cox</i>1+ COB+<i>rbc</i>L trees best represented the phylogeny with consistently high nodal support. Among individual markers, <i>cox</i>1 identified the greatest number of haplotypes, while UPA, partial <i>rbc</i>L (750 bp), ITS, <i>cox</i>3 and <i>cox</i>2–3 spacer were able to retrieve information from herbarium specimens of 12–16 years of age. These molecular results provide a basis for a database essential for the taxonomic framework, cultivar development and germplasm conservation of eucheumatoids. Mitochondria <i>cox</i>1, <i>cox</i>2–3 spacer, <i>cox</i>2 and plastid <i>rbc</i>L can be used for species identification and <i>cox</i>1 for haplotype detection of eucheumatoids.<i>cox</i>1+<i>rbc</i>L, COB+<i>rbc</i>L, <i>cox</i>2+<i>rbc</i>L or <i>cox</i>1+COB+<i>rbc</i>L are the most cost-effective molecular markers for phylogenetic inference.The most comprehensive up to date multilocus phylogeny of <i>Kappaphycus</i> spp. and <i>Eucheuma denticulatum</i> is presented. Mitochondria <i>cox</i>1, <i>cox</i>2–3 spacer, <i>cox</i>2 and plastid <i>rbc</i>L can be used for species identification and <i>cox</i>1 for haplotype detection of eucheumatoids. <i>cox</i>1+<i>rbc</i>L, COB+<i>rbc</i>L, <i>cox</i>2+<i>rbc</i>L or <i>cox</i>1+COB+<i>rbc</i>L are the most cost-effective molecular markers for phylogenetic inference. The most comprehensive up to date multilocus phylogeny of <i>Kappaphycus</i> spp. and <i>Eucheuma denticulatum</i> is presented.