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European Journal of

PROTISTOLOGY

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European Journal of Protistology 41 (2005) 269–276

www.elsevier.de/ejop

Difflugia mulanensis nov. spec., a freshwater testate amoeba

from Lake Mulan, China

Jun Yanga,b, Ralf Meisterfeldc, Wenjing Zhanga,b, Yunfen Shena,�

aLaboratory of Protozoan Taxonomy and Ecology, Institute of Hydrobiology, Chinese Academy of Sciences,

Wuhan 430072, PR ChinabGraduate School of the Chinese Academy of Sciences, Beijing 100039, PR ChinacInstitut fur Biologie II, RWTH Aachen, Kopernikusstraße 16, 52056 Aachen, Germany

Received 29 April 2005; received in revised form 30 May 2005; accepted 31 May 2005

Abstract

Testate amoebae are an important and abundant component of aquatic ecosystems. Our knowledge of theirdistribution and the species inventory in Asia is still limited. This paper describes a new planktonic Difflugia from LakeMulan, province of Hubei, China. These findings are of taxonomic, as well as biogeographic significance becauseDifflugia mulanensis nov. spec. is a rare and perhaps the only locally distributed member of the well-known urceolatedivision within this most species-rich genus of lobose testate amoebae. We have studied its morphology and biometrywith light and scanning electron microscopical methods. D. mulanensis differs from related species by its very largewide funnel-shaped apertural collar which normally exceeds the width of the test, a relatively long neck, its roundedapical end and the presence of only one nucleus. All shell parameters are normally distributed with low variability.Only the collar height is more variable. This shows that D. mulanensis is a size-monomorphic species.r 2005 Elsevier GmbH. All rights reserved.

Keywords: Testate amoeba; Arcellinida; Difflugia mulanensis nov. spec.; Morphology; Biometry; Plankton; China

Introduction

Difflugia is a morphologically diverse genus of thelobose testate amoebae. The species have been reportedfrom many countries and are widely distributed invarious freshwater habitats. They play an important rolein energy transfer and nutrient cycling in aquatic andterrestrial ecosystems. Over nearly two centuries, morethan 300 species and about 200 sub-species, varieties, orforms of the genus have been described with little regardfor the value of the characters used, the previous

e front matter r 2005 Elsevier GmbH. All rights reserved.

op.2005.05.006

ing author. Tel./fax: +86 27 68780678.

ess: shenyf@ihb.ac.cn (Y. Shen).

literature, or the rules of nomenclature (Meisterfeld2002). Gauthier-Lievre and Thomas (1958), in theirinvestigation of the African Difflugia species, havedivided this genus into ten artificial groups based onthe morphology of the shell, namely lobed, collared,compressed, urceolate, globose, ovoid-globose, elon-gate, acute angled, horned and pyriform. D. urceolata isthe ‘‘name-giving species’’ in the urceolate group whichhas a spherical or ovoid shell furnished with apronounced apertural rim or collar (Gauthier-Lievreand Thomas 1958). Later, Chardez and Gaspar (1982)made a brief review of the diagnostic characters of D.

urceolata and its eight varieties or forms. Notably, D.

urceolata is one of the most widely reported species of

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Figs. 1, 2. Shell outline and position of measured morpho-

metric axes used in this study. 1—total length, 2—body width,

3—collar diameter, 4—neck width, 5—collar height, 6—body

J. Yang et al. / European Journal of Protistology 41 (2005) 269–276270

the genus Difflugia, probably due to its large size anddistinctive shape (Ogden 1983).To permit correct identification to species level, the

description of a new taxon should comprise informationnot only on characters of the shell (if possible by lightand scanning electron microscopy) but also on char-acters of the cell and biometrical data. Although theseare difficult to observe in Difflugia spp., many studiesshow that there are species with similar tests butdifferent cytoplasmic and nuclear structures (Ogdenand Meisterfeld 1989; Ogden 1991).The purpose of this paper is to describe Difflugia

mulanensis nov. spec., a conspicuous freshwater urceo-late species.

length, 7—aperture diameter.

Material and methods

Samples

A population of D. mulanensis nov. spec. was sampledin Lake Mulan, Hubei Province, China in July 2003 andJuly 2004. Detailed information on the lake has beengiven by Yang et al. (2004a) and a map with the locationof the lake by Yang and Shen (2005). The samples weretaken from the upper layer of the water by horizontalhauls with a plankton net (64 mm mesh size). The watertemperature was 32–34 1C and the pH 6.0.

Light and scanning electron microscopy

Specimens for light microscopy (Zeiss Axioplan 2imaging System, Germany) and scanning electronmicroscopy (X-650 HITACHI, Japan) observationswere prepared following the procedures of Yang et al.(2004a).

Measurements

The seven morphometric characters are defined inFigs. 1, 2. All measurements were taken directly fromfresh material at a magnification of 320� using anocular micrometer. The statistical analysis was per-formed using the computer program STATISTICA,version 6.0.

Results and discussion

Classification summary

Class Lobosea Carpenter, 1861; Order ArcellinidaKent, 1880; Family Difflugiidae Wallich, 1864; GenusDifflugia Leclerc, 1815.

Type locality

Plankton, Lake Mulan, Hubei Province, China(311060N; 1141280E).

Type specimen

The holotype specimen (HB2003046) has been depos-ited on a permanent microscope slide in the collection ofthe Laboratory of Protozoan Taxonomy and Ecology,Institute of Hydrobiology, Chinese Academy ofSciences, Wuhan, PR China.

Etymology

The specific epithet (mulanensis) is named after LakeMulan, where the first living specimens of this specieswere found.

Morphology of the test

The shell is yellowish to brown and semitransparent.It looks like a spherical to elliptical vase with a largeapertural collar. The test is 1.16–1.61 times longer thanthe diameter of the collar, symmetrical in longitudinaland apertural view (Figs. 3–5, 14). It consists of a largecircular collar and a tall shell belly with a distinct neck.The aperture is circular (Fig. 4). The collar is dish-like orlow-funnel-shaped, forming an angle of 1151–1601; theapertural funnel has a height of 11–24% of the totallength, and its basal (neck) diameter is 0.39–0.74 timesthe collar diameter; its margin is ragged (Figs. 3, 4, 7).The belly (or main body) is circular in cross section, thenarrowest cross section (neck) lies just below the collar,gradually expanding (71–171); the maximal width isnormally located in the posterior 22–31% of the shell,with its greatest diameter 0.73–1.06 times the collardiameter (Figs. 3, 14). The aboral end is always

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Figs. 3–13. LM photographs of Difflugia mulanensis. 3: lateral view. 4: apertural view showing the circular aperture which is

surrounded by a conspicuous large collar. 5: bottom view showing the round aboral end. 6: the ovular nucleus. 7: lateral-oblique

view. 8–13: specimens with extended pseudopodia, Figs. 10, 11 and 13 with gas bubbles, Fig. 12 with epipodes attached to the

internal wall. Scale bars 20mm (6); 50mm (3–5, 7–13).

J. Yang et al. / European Journal of Protistology 41 (2005) 269–276 271

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Figs. 14–17. SEM photographs of Difflugia mulanensis. 14: lateral view. 15: detail of the shell surface, note the tight packing of small

angular and polymorphic particles and medium flattish pieces of quartz to give a smooth surface. 16, 17: surface of the shell showing

incorporated broken diatom frustules. Scale bars 3mm (16, 17); 6mm (15); 30mm (14).

J. Yang et al. / European Journal of Protistology 41 (2005) 269–276272

rounded, never with a protuberance, horn or spine(Figs. 3, 5). The shell wall is composed of small angularparticles and medium flattish pieces of quartz or otherforms of silica (xenosomes) (Figs. 15–17), includingpieces which appear to be parts of the frustules of centricdiatoms (Figs. 16, 17), fitted together to give a smoothsurface and a regular outline (Figs. 3, 14). The collar ismade of smaller flattish particles (Fig. 4). No obvious

cement structures are recognizable in the scanningelectron microscope.

Morphology of the cell

The cell body possesses long, sometimes branched,finger-like, and fine granular pseudopodia with rounded

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ends (Figs. 8, 9, 11). The number of pseudopodiagenerally varies from one to half a dozen, and theirmeasurement ranges in length generally from 10 to120 mm and ranges in width from 3 to 8 mm (usuallyabout 5 mm) (Figs. 8–11, 13). Living specimens usuallymove slowly over a glass slide in an upright positionwith the aperture facing the slide by a generally steadyflow of cytoplasm (Fig. 9). In moving specimens the cellbody commonly occupies the greater part of the anteriorshell cavity around the aperture, leaving the posteriorportion of the shell empty (Fig. 13). Notably, in somespecimens, one or two pseudopodia usually extend fromthe aperture to the back of the shell. In fully extendedcondition, the pseudopodia may exceed the aboral endalong the shell (Figs. 10, 13). Epipodes attached to theinternal walls have been seen (Fig. 12). A single sphericalnucleus is generally located in the posterior part of thecell. It is of ovular type (see Meisterfeld 2002) with manynucleoli and has a diameter range of 22–25 mm (Fig. 6).A gas vacuole was often clearly visible in the anteriorbody part of floating specimens (Figs. 10, 11, 13).

Table 1. Morphometric characteristics of Difflugia mulanensis nov

Charactersa x M SD

Total length (1) 108.4 109 7.32

Body width (2) 69.9 69 3.84

Collar diameter (3) 78.4 78 6.19

Neck width (4) 44.8 45 3.55

Collar height (5) 19.3 19 3.84

Body length (6) 88.8 90 5.36

Aperture diameter (7) 35.4 36 3.36

aNumbers 1–7 in parentheses designate features as shown in Figs. 1 and 2

CV, coefficient of variation in %; M, median; Max, maximum; Min, minimu

standard error of arithmetic mean; x, arithmetic mean.

Figs. 18,19. Frequency plots of body dimensions in Difflugia mulan

(character 1 as shown in Fig. 1). 19: histogram showing the size fre

Biometry

Table 1 shows the detailed morphometric character-ization of D. mulanensis nov. spec. The new species ischaracterized by a remarkable uniformity of size. Withthe exception of the collar height which is highlyvariable (CV 19.90), total length, body width, collardiameter, neck width, body length and aperturediameter are relatively constant with a low variability(CV between 5.49 and 9.49) (Table 1). All variables arenormally distributed, which indicates that D. mulanensis

is a size-monomorphic species (Figs. 18, 19). Frequencyanalyses of the other morphometric characters (collardiameter, neck width, collar height, body length andaperture diameter) show almost the same results.Morphometry provides additional criteria for differen-tiation between D. mulanensis and related species.Table 2 illustrates that the shell dimensions in

D. mulanensis show positive correlation at po0:0520:001, especially total length is well correlated withcollar height (r ¼ 0:7125; n ¼ 140) and body length

. spec. from Lake Mulan, China

SE CV Min Max n

0.62 6.75 90 128 140

0.32 5.49 61 81 140

0.52 7.90 65 95 140

0.30 7.92 36 59 140

0.32 19.90 11 29 140

0.45 6.04 73 103 140

0.28 9.49 27 47 140

. Data based on random selection of specimens. Measurements in mm.m; n, number of individuals investigated; SD, standard deviation; SE,

ensis. 18: histogram showing the size frequency of total length

quency of body width (character 2 as shown in Fig. 1).

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Table 2. Correlation coefficients between morphometric characteristics in Difflugia mulanensis nov. spec. from Lake Mulan, China

(n ¼ 140)

Characters TL (1) BW (2) CD (3) NW (4) CH (5) BL (6) AD (7)

TL (1) —

BW (2) 0.4090*** —

CD (3) 0.6138*** 0.5391*** —

NW (4) 0.2739** 0.4479*** 0.4399*** —

CH (5) 0.7125*** 0.1487 0.4717*** 0.1614 —

BL (6) 0.8631*** 0.4477*** 0.5067*** 0.2562** 0.2692** —

AD (7) 0.3122*** 0.4145*** 0.4597*** 0.9591*** 0.1965* 0.2825*** —

Significant relationship �po0:05, ��po0:01, ���po0:001. TL, total length; BW, body width; CD, collar diameter; NW, neck width; CH, collar height;BL, body length; AD, aperture diameter (see Figs. 1 and 2).

J. Yang et al. / European Journal of Protistology 41 (2005) 269–276274

(r ¼ 0:8631; n ¼ 140) at po0:001, while neck width ishighly correlated with aperture diameter at po0:001(r ¼ 0:9591; n ¼ 140). The only exception is collarheight, which is correlated with neither body- norneck-width.

Ecology

At present, D. mulanensis has only been found in theoligotrophic, slightly acid Lake Mulan. It has not beendiscovered in our study of eutrophic lakes in the sameregion, like Lake Donghu and Lake Nanhu in theChangjiang valley. Perhaps, like D. biwae (Yang andShen 2005), D. mulanensis has a restricted geographicaldistribution with endemic areas located in East Asia,because it was not found in Europe and North Americawhere numerous investigations of testate amoebae faunahave been made. In addition, D. mulanensis shows adistinct annual distribution, with highest populationdensities in summer and autumn. During the rest of theyear one can hardly find any individuals in the plankton.In summer and autumn the water temperatures arearound 30 1C. Whether this indicates an alternationbetween a benthic phase in winter and a planktonicstage in summer, as in other planktonic Difflugia species(D. hydrostatica/limnetica), is not clear so far.

Comparison with similar species

According to the sub-divisions of Gauthier-Lievreand Thomas (1958), D. mulanensis nov. spec. belongs tothe urceolate group of the genus Difflugia. Chardez andGaspar (1982) have made a brief review of thediagnostic features of D. urceolata and its eight varietiesor forms. Species within the group are normallydistinguished by their shell size, morphology and thecomposition of the shell. A very important characteristicwhich distinguishes D. mulanensis from all other speciesof the urceolate Difflugia is the diameter of the funnel-shaped collar that usually exceeds the maximum width

of the shell. Considering its overall appearance, D.

mulanensis should be compared with eight other similarDifflugia species, sub-species or forms, namely D.

amphoralis Hopkinson, 1909, D. amphoralis cornuta

Gauthier-Lievre and Thomas, 1958, D. amphoralis

globosa Gauthier-Lievre and Thomas, 1958, D. urceola-

ta Carter, 1864, D. urceolata lageniformis Gauthier-Lievre and Thomas, 1958, D. urceolata lageniformis

minor Gauthier-Lievre and Thomas, 1958, D. urceolata

minor Deflandre, 1926 and D. urceolata sphaerica

Playfair, 1917 (Table 3).Difflugia amphoralis Hopkinson has a similar size

(115 mm); however, it can be clearly distinguished fromD. mulanensis by having a fusiform (spindle-shaped) andnot an elongate ovoid shell, an aboral end pointed like aGothic arch (not rounded) and a much smaller collar. D.

amphoralis cornuta Gauthier-Lievre and Thomas and D.

amphoralis globosa Gauthier-Lievre and Thomas differfrom D. mulanensis in having a larger size (175–183 mmand 180–228 mm) and in having a relatively short neckthat widens abruptly to the belly of the shell. Besides, D.

amphoralis cornuta has two or three aboral horns.Although smaller specimens (104–152 mm) of D. am-

phoralis globosa have been reported from a puddle withmany filamentous green algae (Spirogyra) near theShudu River in Zhongdian County, Yunnan Province,China by Yang et al. (2004b), they can be easilydistinguished from D. mulanensis by the shell shape.Morphologically, D. urceolata Carter appears to be

closely related to D. mulanensis; it differs from D.

mulanensis in having a larger size (204–398 vs.90–128 mm), a pronounced but shorter and slightlyreflexed apertural collar and in having numerous nuclei.Another two similar forms, D. urceolata lageniformis

Gauthier-Lievre and Thomas and D. urceolata lagen-

iformis minor Gauthier-Lievre and Thomas, can beclearly differentiated from D. mulanensis by having alarger size (240–380 and 150 vs. 90–128 mm) and aslightly bent backward collar. D. urceolata minor

Deflandre has a similar size (124–170 mm) and lowfunnel-shaped collar, but it can be distinguished by a

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PRES

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Table 3. Comparison of Difflugia mulanensis nov. spec.with eight related urceolate species or sub-species. All measurements in mm. (?—data not available)

Characters Difflugia

mulanensis

Difflugia

amphoralis

Difflugia

amphoralis

cornuta

Difflugia

amphoralis

globosa

Difflugia

urceolata

Difflugia

urceolata

lageniformis

Difflugia

urceolata

lageniformis

minor

Difflugia

urceolata minor

Difflugia

urceolata

sphaerica

Total length 90–128 115 175–183 180–228 204–398 240–380 150 124–170 250–300

Body width (BW) 61–81 70 100–108 105–120 193–426 250–285 110 116–118 250–300

Collar diam. (CD) 65–95 ? ? ? ? 125–180 ? ? 200

Aperture diameter 27–47 ? 50–55 50–75 87–198 125–200 40 72–80 70–120

Aboral end Rounded Like an aboral

Gothic arch

2 to 3 aboral

horns

Rounded Rounded or

with one or

more

protuberances

Rounded Rounded Rounded Rounded

Shell outline Elongate ovoid

like a vase, with

a distinct long

neck, large,

circular, flat or

funnel-like

collar, rim

never bent

backward

Fusiform, with

an aboral

extremity like a

Gothic arch

and a slightly

widened but not

upturned

apertural rim

Longish

elliptical,

distinct

apertural rim or

collar that is

bent backward

Longish

elliptical, and a

apertural rim or

collar that is

bent backward

Ovoid or

circular in

lateral view,

sometimes with

irregular, blunt,

aboral

protruberances,

and slightly

bent backward

apertural rim or

collar

Longish ovoid,

distinct

apertural rim or

collar, often

bent backward

Longish ovoid,

large apertural

collar, often

bent backward

Longish ovoid,

large apertural

collar, often

bent backward

Spherical,

apertural rim or

collar that is

usually bent

backward

Shell composition Small angular

particles and

medium flattish

pieces of quartz

Chitinous

material,

surfaced with

amorphous

scales, but few

sand-grains

Small to

medium

amorphous

particles and

flattish quartz

Small to

medium

amorphous

particles and

flattish quartz

Small to

medium

polymorphic

particles and

flattish quartz

Small to

medium

polymorphic

particles and

flattish quartz

Small to

medium

polymorphic

particles and

flattish quartz

Small to

medium

polymorphic

particles

incorporates the

Porifera

spicules

Small to

medium

polymorphic

mineral

particles

CD4BW Yes No No No No No No No No

Neck Long Short Short Long Short Short Short Short Short

Nucleus Single, ovular Single ? ? Multinucleate ? ? ? ?

Biometry Monomorphic ? ? ? Polymorphic ? ? ? ?

Description based

on material from

China Great Britain Africa Africa Great Britain Africa Sudan Venezuela and

Congo

Africa

Data source Original Cash and

Hopkinson

1909

Gauthier-Lievre

and Thomas

1958

Gauthier-Lievre

and Thomas

1958

Cash and

Hopkinson

1909; Ogden

1983

Gauthier-Lievre

and Thomas

1958

Gauthier-Lievre

and Thomas

1958

Deflandre 1926;

Gauthier-Lievre

and Thomas

1958

Playfair 1917;

Gauthier-Lievre

and Thomas

1958

J.Yangetal./EuropeanJournalofProtisto

logy41(2005)269–276

275

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smaller apertural collar that does not reach themaximum width of the shell. D. urceolata sphaerica

Playfair differs from D. mulanensis by its larger size(250–300 mm), its spherical shell and by its smaller,slightly backward apertural collar.The differences from the existing species or sub-

species of the urceolata- or amphoralis-like group ofDifflugia are so obvious that a description as a newspecies is justified.

Acknowledgements

This work was supported by the National NaturalScience Foundation of China (Grant No. 30270185), theKnowledge Innovation Program of the Chinese Acad-emy of Sciences (Grant No. KSCX 2-2-06) and theFoundation of Donghu Experimental Station of LakeEcosystems, the Chinese Academy of Sciences (GrantNo. 200202).

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