Taxonomic reexamination of 17 species of Nitella subgenus Tieffallenia (Charales, Charophyceae) based on internal morphology of the oospore wall and multiple DNA marker sequences

In an attempt to reconstruct the natural taxonomic system for Nitella, 17 species of Nitella subgenus Tieffallenia were reexamined using SEM observations of the internal morphology of the oospore wall (IMOW) and phylogenetic analyses of 4553 base pairs from multiple DNA markers (atpB, rbcL, psaB, and ITS-5.8S rRNA genes). Our SEM observations identified three types of IMOW: homogeneous (HG), weakly spongy (W-SG), and strongly spongy (S-SG) types. Based on differences in the IMOW, species with reticulate or tuberculate oospore wall ornamentation in the external morphology of the oospore wall (EMOW) were subdivided into two distinct groups (characterized by the HG or S-SG types of IMOW, respectively), which were robustly separated from each other in our molecular phylogenetic analyses. In our molecular phylogeny, the subgenus Tieffallenia consisted of four robust monophyletic groups-three clades of the HG type and a spongy (S-SG and W-SG) type clade-that were characterized by differences in the IMOW and EMOW. In addition, our SEM observations and sequence data verified the distinct status of five species (N. japonica Allen, N. oligospira A. Braun, N. vieillardii stat. nov., N. imperialis stat. nov., and N. morongii Allen) that R. D. Wood had assigned as infraspecific taxa. Moreover, our SEM observations of the IMOW also suggested that N. megaspora (J. Groves) Sakayama originally identified by LM includes at least two distinct species, characterized by W-SG and S-SG types of IMOW, respectively.