Bull. Natn. Sci. Mus., Tokyo, Ser. B, 30(2), pp. 55–62, June 21, 2004

Thallus Structure and Reproductive Organs ofNemalionopsis tortuosa (Rhodophyta)
Makoto Yoshizaki
Department of Biology, Toho University.
2–2–1 Miyama, Funabashi, Chiba Pref. 274–8510 Japan.
E-mail: makoto@bio.toho-u.ac.jp

Abstract Detailed morphological studies have been carried out on the vegetative thallus, gametangia and carposporophytes of Nemalionopsis tortuosa Yagi et Yoneda collected from southernJapan. The carpogonium is borne laterally or terminally on a cell of an assimilatory filament. The
gonimoblast initials are produced directly from the fertilized carpogonium, dividing soon afterwards to form several filaments. The gonimoblast filaments produce branches which grow among
the assimilatory filamets, resulting in the formation of a diffuse carposporophyte. On the basis ofthese and other observation, Nemalionopsis is mainteined in the Thoreaceae, Thoreales.
Key words : Nemalionopsis tortuosa, fresh water algae, carposporphyte formation, Batrachospermales, Thoreales, Rhodophyta

Introduction

Materials and MethodsNemalionopsis was estabished by Skuja 1934
with Nemalionopsis shawii Skuja as the typespecies. Since monosporangia are located at thetips of assimilatory filaments.
The genus includes two species at present, andhas been reported from only seven localities
worldwide in Asia and North America (Sheath,Vis and Cole 1993). Nemalionopsis tortuosa wasesatabished by Segi and Yoneda 1949 fromOkichi-izumi, Ehime Prefecture, Japan. The
habitat, thallus structure and monosporangia formation have been described for the species, butgametangia have not been recorded for the genus.
Since the taxonomy of the Florideophyceae isbased on the structure of the carpogonia, the systematic position of the genus has been uncertain.
Recent collections of Nemalionopsis tortuosafrom southern Japan included male and female
plants with carposporophytes, and provided an
opportunity to carry out a detailed study of thegametangial and carposporophyte formation. The
systematic position of the genus is also discussed
on the basis of the results obtained.

Used materials were collected in the irrigation
channel of Khojirogawa River at Kunimi Town,
Nagasaki Prefecture, on March 10, 2001 by myself and May 19, 2001 by Dr. Masafumi Iima,
and were preserved in 10% formalin in water.
After staining with 1% erythrosin, the marerials
were mounted in 50% Karo syrup. Satisfactorythin preparation were eventually obtained byusing a Bright OT/FAS cryostat microtome.
Drawing were made with a camera lucida.
Examind specimens: The type locality, Okichiizumi, Matsuyama, Ehime Prefecture on March19, l942, collecter Y. Okada, and Identified by Y.
Okada, in TNS-Al no. 30809. The irrigation
channel of Khojirogawa River at Kunimi Town,Nagasaki Prefecture, on March 25, 2001, collecter M. Yoshizaki and identified by M. Yoshizaki, in TNS-Al no 156879 and 156880, and onMay 19, 2001, collecter Dr. Masafumi Iima andidentified by M. Yoshizaki.
Thallus morphology
Plants of Nemalionopsis tortuosa Yagi et56

Makoto Yoshizaki

Yoneda (Fig. 1) grow on rocks or stone walls attaching by means of a small discoidal holdfast,
2–3 mm in diam. Usually one, or sometimes twoor three erect fronds are producing from a holdfast. They are terete, soft, gelatinous in texture,
grow to a height of 2.3 m, and are up to 2 mm indiam, with two to 10 times iregularly dichotomously branches. Sometimes older plants produce several short buranches at the lower basal
parts of the thallus. Thallus structure is multiaxial, consisting of medullary filaments and assimilatory cortical filaments (Fig. 2a). The medullaryfilaments are 7–10 m m in diam. and interlace
with one another, forming a core. The assimilatory filaments usually branch dichotomously one tothree times, but only in the upper parts. Thelower parts of assimilatory filaments are terete,
4–8 m m in diam, and the basal parts of the assimilatory filaments produce rhizoidal filaments
which contribute the structure of the medulla(Fig. 2f). The upper parts of the assimilatory filaments are composed of barrel-shaped cells.
Sometimes the cells of the assimilatory filaments
are barrel-shaped continuously form base to thetip (Fig. 2f).
Reproduction
Plants of Nemalionopsis tortuosa are unisexual. Spermatangia (spn) are borne terminally or intwos or threes in short small clusters. (Fig. 2b, c).
The spermatangia are ovoid 8–10 m m long and
6–9 m m in diam. Liberated spermatia are spherical, measuring 10 m in diam.
Carpogonia (cp) are formed in abundance inthe younger parts of the thallus. The carpogonium is borne laterally or terminally on a cell of anassimilatory filament. They are one-celled (Fig.
2e, d), urceolate in shape, 12–15 m m long and
6–7 m m wide at the base, each having a strightand cylindrical trichogyne (tr) that is up to1200 m m long and 3–4 m m in diam.
More than one spermatium (sp) may attach toa trichogyne (Fig. 2e). After fertilization, the trichogyne withere or cut off at the base. The fertilized carpogonium produces a protuberance on

the upper side (Fig. 2f) from which a gonimoblast initials is cut off by a cell division (Fig.
2g). Two to five gonimoblast initials are produced
from the same zygote (Figs. 3, 4) which divide toform gonimoblast filaments that develope in oneof two ways (Fig. 4). One develops upperwardly
parallel to the assimilatory filaments. The otherdevelops downwardly toward the medulla, butdoes not mix with the medullary filaments. Instind they run out parallel to the surface of thethallus, extending among the assimilatory filaments and give rise to a diffuse carposporophyte.
The tips of gonimoblast filaments produce many
short, erect branches (Fig. 4). The short erectbranches branch again resultng in another orderof branches grow among the assimilatory filaments. The carposporangia are formed on thetips of these branches of the gonimoblast filaments (Fig. 4). The mature carposporangia are
ovoid 12-14 m m long and 8–10 m m in diam. Only
the terminal cells produce carposporangia, andthe gonimoblast filaments remain after carpospore have been released.
Discussion
Pueschel et Cole (1982) have proposed the establishment of the order Batrachospermales on
the basis of thallus construction, pattern of life
cycle, chloroplast structure and their presence infresh water, and including three families: Batrachospermaceae, Lemaneaceae and Thoreaceae.
Vis, Saunders, Sheath, Dunse and Entwisleet(1998) circumscribed the Batrachospermales as ahaving heterotricous life history; absence oftetraspore formation; a two-layered pit plug, theouter layer of which is dome-shaped; and astrictly fresh water habitat, the include three families: Batrachospermaceae, Lemaneaceae andPsilosiphonaceae. On their studies, the family
Thoreaceae does not appear to be a naturalgrouping within the Batrachospermales.
Multiaxial thallus construction in found in theNemaliales, including the Helminthocladiaceae,
Dermonemataceae and Galaxauraceae among
marine families, and the Thoreaceae among

Thallus Structure and Reproductive Organs of Nemalionopsis tortuosa (Rhodophyta)Fig. 1. Specimen of Nemalionopsis tortuosa Yagi et Yoneda.
The irrigation channel of Khojirogawa River at Kunimi Town, Nagasaki Prefecture, on March 25, 2001, TNSAl no 156879.

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Fig. 2. The vegetative structure of thallus, and male and female sexual organs and gonimoblast initiations ofNemalionopsis tortuosa. a. Cross section of the younger part of thallus, Showing cortical assimilatory filaments and filamentous medulla; b, c. Spermatangia are borne terminally or in twos or threes in short small
clusters. d. two carpogonia (cp) are produced terminally on the assimilatory filaments; e. Spermatia (sp) attached on the tip of trichogyne. f. Postfertilization stage, showing the gonimoblast initial (gi) developingfrom the upper part of the zygote (zyg); g. Gonimoblast initials (gi) developing, one of then is already cut off
from the zygote, and other one is arising from other side.

Thallus Structure and Reproductive Organs of Nemalionopsis tortuosa (Rhodophyta)Fig. 3. Carposporophyte development of Nemalionopsis tortuosa. Post fertilization stages, showing variousstages in the develoed stages of gonimoblast filaments.

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Fig. 4. Mature carposporophyte of Nemalionopsis tortuosa. Well developed carposporophyts, showing twostages of diffused gonimoblast filaments and forming clusterts of carposporangia (cspn).
Abbreviations used in the figures: af-assimilatory filament, cp-carpogonium, cspn-carposporangium, gigonimoblast initial, gf-gonimoblast filament, rhf-rhizoidal filament, sp-spermatium, spn-spermatangium, trtrichogyne, wtr-withered trichogyne.

Thallus Structure and Reproductive Organs of Nemalionopsis tortuosa (Rhodophyta)

freshwater families. According to Migita (1986),
released spores develop into a heterotichous habit
composed of prostrate and erect filaments. Haraand Chihara (1974) observed in N. tortuosa thatmany discoid chloroplasts without pyrenoid arepresent in each cell. Based on these features, itappeares to place the Thoreaceae in the Batrachospermales. In additional feature of the Batarachospermales, pit plugs have two cap layers oneither side of the pit plug core and also show adome-shaped elaboration of the outer layer of theplug cap (Pueschel et Cole 1982). Acording toMueller, Sherwood, Pueschel, Gutell and Sheath(2002), the pit plugas of the gametophytic andchantransia stages of the Thoreaceae, contein twocap layers, the outer one of which is typicallyplate-like, though occasionally inflated ones havebeen seen. No pit plug cap membrane has beeenobserved.
Sheath and Mueller (1999) sequenced the rbcL
and 18S ribosomal DNA on the members offresh water rhodophytan Balbiania and its relatives. Vis, Saunders, Sheath, Dunse and Entwisle(1998) sequenced the rbcL and 18S ribosomal
DNA on the menbers of Batrachospermales, inall trees, Thorea violacea was not closely related
to the other taxa of the Batrachospermales.
Based these data, they considered again that theThoreaceae does not appear to be a naturalgrouping within the Batrachospermales. These
proposal were supported by Hanyuda, Kumano,Arai, Suzawa, Iima, Ueda (2001) on the sequenced the rbcL and 18S ribosomal DNA on theJapanese menbers of Batrachospermales, especially on the members of Thoreaceae. Both papers of Sheath, Mueller and Sherwood (2000)
and Mueller, Sherwood, Pueschel, Gutell andSheath (2002), proposed the Thoreales to include
two genera Nemalionopsis and Thorea.
Because of their multiaxial thallus construction, pit plugs that lack outer cap layers that are
not universally dome-shaped. The phylogenetic
relationships of the genera were investigatedusing a combination of DNA sequence analysis(rbcL and 18S rRNA genes) and transmission
electron microscopy. In addition, analysis of the61

secondary structure of the 18S rRNA gene in Nemaliopsis and Thorea reveals an additional helix,
which is not present in any of the other taxa within the Rhodophyta. The carpogonial branches inThorea consist of only a single cell. The carpogonial morphology of Nemalionopsis tortuosa issimilar to that of Thorea okadai which is borneon a basal cell of an asimilatory filament and isalmost cylindrical in morphology (Yoshizaki1986). Nemalionopsis is distingushed fromThorea by the position of the sporangia in thethallus. The monosporangia of Nemalionopsis
are situated at the apex of the assimilatory filaments, whereas the monosporangia of Thorea arelocated at the base of assimilatory filaments.
Among freshwater genera, diffuse gonimoblasts
are observed only in Thorea and Sirodotia.
Sirodotia is classified in the Batrachospermaceae
because of its uniaxial thallus construction.
Multiaxial thallus construction, one-celled carpogonia and diffuse gonimoblasts are significant
features that can now be employed to characterize the family Thoreaceae in the order Thoreales.
Acknowledgements
I wish to express my sincere thanks to Professer Max H. Hommersand, who has been partly
reeding English. Also, I woul like to express
thanks to Dr. Masafumi Iima, who provide thefresh materials.
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