Molecular phylogeny and taxonomy of the fern genus Anisocampium (Athyriaceae)

Abstract

We redefine the fern genus Anisocampium based on the results of a phylogenetic analysis using two regions of the chloroplast genome. The redefined genus is separate from Athyrium s. str. and comprises four species, two of which were previously assigned to Athyrium or Kuniwatsukia. Anisocampium paucijugum is reduced to a synonym of A. cumingianum. The four accepted species in Anisocampium are morphologically distinct but the putative hybrid An. ×saitoanum suggests low reproductive isolation in the genus. Anisocampium can be distinguished from related genera by a combination of characters only.

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INTRODUCTION
The classification of the athyrioid ferns remains unset-
tled, even though molecular phylogenetic data have been in-
creasingly used to determine relationships. One ambiguous
genus is Anisocampium C. Presl. (1849), described based on
the imparipinnate leaves and round sori of An. cumingianum
C. Presl. Copeland (1947) added a second species, An. pauci-
jugum (Aderw.) Copel., and stated that Anisocampium shows
an athyrioid affinity.
In 1973, Pichi Sermolli proposed the genus Kuniwatsukia
as a replacement name for Microchlaena Ching (1938), a later
homonym of Microchlaena Wight & Arn. (Sterculiaceae).
Micro chlaena Ching was based on M. yunnanensis (C hrist)
Ch ing (= Microchlaena cuspidata (Bedd.) Ching). Iwatsuki
(1970) considered M. cuspidata to be related to An. cumingia-
num and Athyrium sheareri (Baker) Ching, based on vascular
anatomy of rhizome and stipe, leaf architecture, and chromo-
some number. Hsieh (1985) formally transferred Athyrium
sheareri to Anisocampium.
Recently, Fraser-Jenkins (2006) referred Anisocampium
cumingianum to Athyrium sect . Niponica Ching & Y.T. Hsie h ,
a section established for Athyrium niponicum ( M ett.) H a nce
(Hsieh, 1986). Athyrium niponicum has been recognized as a
member of Athyrium for mo re th an a centu r y afte r its tr ansfer
from Asplenium, but recent molecular analyses have shown
it to have a closer affinity with Anisocampium and Kuniwat-
sukia. In his floristic treatment of Chinese Athyrium, Wan g
& al. (1999) assigned ten species to sect. Niponica, in four
series: ser. Niponica with two species (Ath. niponicum, Ath.
brevisorum (Wall. ex Hook.) Moore) and ser. Biserrulata,
ser. Fallaciosa and ser. Yokoscentia with the remai ning eight
species. Series Niponica was characterized by the creeping
rhizomes, the abruptly narrowed apex of the bipinnate leaf,
the shortened basal pinnules of the stalked, basal pinnae.
Except for Athyrium niponicum, all species differ from the
remaining 160–220 species of Athyrium in leaf dissection
and shape of sori.
A putative hybrid between Athyrium niponicum and Ath.
sheareri (≡ An. sheareri) was reported by Serizawa (1986).
Hybridization suggests close affinities of the two species. The
free venation of Athyrium sheareri is similar to that of Athyrium
and differs from the anastomosing venation of Anisocampium
cumingianum.
Recent molecular phylogenic studies have shown that Athy-
rium s.l. (including Anisocampium) is paraphyletic. Based on
rbcL seq uence s, Sano & al. (2000) foun d Ath. sheareri and Ath.
niponicum to form a clade sister to a clade comprising Cor-
nopteris Nakai and Athyrium that includes Pseudocystoperis
Ching. Based on the trnL-F regions, Wang & al. (2003) found
Kuniwatsukia cuspidata to be cluster ed with Ath. niponicum. In
a molecular analysis using a larger dataset, Adjie & al. (2008)
confirmed that Ath. niponicum, Ath. sheareri and Ath. cuspida-
tum form a monophyletic clade. Anisocampium cumingianum,
the type species of Anisocampium, was lacking in all previous
molecular phylogenetic analyses, however, leaving the relation-
ship between Anisocampium and Athyrium unclear.
In this paper, we describe the molecular phylogeny of the
species considered to be related to Anisocampium. Based on
the results of the analysis and comparative morphology, we
redefine the genus Anisocampium as comprising four species,
the Sino-Japanese An. niponicum and An. sheareri, the Sino-
Himalayan An. cuspidatum, and the tropical and subtropical
Asian An. cumingianum (Fig. 1).
MATERIALS AND METHODS
Taxon sampling and source of sequences. —
A tot al of 20
athyrioid species of ferns, including eight species of Athyrium,
three of Cornopteris, two of Diplazium, three of Deparia, and
all four species of Anisocampium were sam pled . The species of
Molecular phylogeny and taxonomy of the fern genus Anisocampium
(Athyriaceae)
Yea-Chen Liu,1,2 Wen-Liang Chiou1 & Masahiro Kato3
1 Division of Botanical Garden, Taiwan Forestry Research Institute, 53 Nan-Hai Road, Taipei, Taiwan
2 Current address: Depart ment of Biological Resources, National Chiayi Universit y, Chiayi, Taiwan
3 Department of Botany, National Museum of Nature and Science, Tsukuba 305-0005, Japan
Authors for correspondence: Wen-Liang Chiou, chiou@tfri.gov.tw; Masahiro Kato, sorang@kahaku.go.jp
Abstract
We redefine the fern genus Anisocampium based on the results of a phylogenetic analysis using two regions of the
chloroplast genome. The redef ined genus is separate f rom Athyrium s.str. and comprises four species, two of which were
previously assigned to Athyrium or Kuniwatsukia. Anisocampium paucijugum is reduced to a synonym of A. cumingianum.
The four accepted species in Anisocampium are morphologically distinct but the putative hybrid An. ×saitoanum suggests low
reproductive isolation in the genus. Anisocampium can be disti ngu is he d fro m re la te d ge ner a by a combi na tion of ch ara ct ers onl y.
Keywords
Anisocampium; Athyrium; Kuniwatsukia; rbcL; trnL-F

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Athyrium represent all five sections recognized by Ching and
Hsieh (H si eh , 1986), or six of the 14 sec tions pro posed by Wang
(1997). Three species of sect. Niponica, i.e., Ath. niponicum,
Ath. biserrulatum, and Ath. yokoscense, are the type species
of ser. Niponica, ser. Biserrulata, and ser. Yokoscentia, re spe c-
tively. The species of Diplazium and Deparia were selected as
outgroups, because they are the closest relatives of Athyrium
and Anisocampium (Sa no & al., 200 0; Wang & al., 20 03; Adjie
& al., 2008). In addition to the rbcL and trnL-F sequence s avail-
able in GenBank (Appendix), we obtained new sequences of
Anisocampium cumingianum (rbcL, HM156338) and Athyrium
biserrulatum (rbcL, HM156336; trnL-F, HM156337). Voucher
specimens are deposited in the Herbarium of Taiwan Forestry
Research Institute (TAIF).
DNA extraction, PCR amplification and sequencing. —
Total genomic DNA of Anisocampium cumingianum and Athy-
rium biserrulatum was extracted from silica-gel-dried leaves
using the modified cetyl trimethyl ammonium bromide (CTAB)
procedure of Doyle & Doyle (1987). Two plastid genome regions
(rbcL, trnL-trnF) were amplified separately using the stan-
dard polymerase chain reaction (PCR) and published primer
sets (primer ESRBCL1F, ESRBCL1361R from Schuettpelz
& Pryer, 2007 for the rbcL gene; primer f from Taberlet &
al., 1991; Fern-1 from Trewick & al., 2002 for the trnL-trnF
region including the trnL intron and the trnL-trnF IGS). The
resulting PCR products were purified using GFX PCR DNA
and Gel Band Purification kits according to the manufacturer’s
instructions, then directly sequenced. Sequencing reactions
were conducted using the DYEnamic ET Dye Terminator Cycle
Sequencing Kit. Sequences were analyzed using MegaBACE
100 0 DNA Analysis Syst ems (Amersham Biosciences, Taipei,
Tai wan) following the manufacturer’s protocol.
Sequence alignment and phylogenetic analyses. —
Align-
ments of all sequences were generated using CLUSTAL X
(Thompson & al., 1997) and subsequently edited manually in
Bio Edit (Hall, 1999) for both intron and IGS of the trnL-F region.
Ambiguously aligned regions were excluded from all subse-
quent analyses. Phylogenetic analyses were conducted using
both maximum likelihood and Bayesian inference of phylogeny.
Maximum likelihood analyses were conducted using PHYML
v.2.4.3 (Guindon & Gascuel, 2003) under the optimal model of
sequence evolution. For each dataset, an Akaike information
criterion as implemented in jModeltest v.0.1.1 (Posada, 2008)
was us ed to sele ct the model of nucleotide subs tit ution , incl ud i ng
gamma shape and proportion of invariant sites. Maximum like-
lihood bootstrap support (MLBS) values were estimated from
1000 replicates in PHYML. Bayesian inference was performed
with MrBayes v.3.0b 4 (Ronqu ist & Huel senbe ck , 2003). Ma rkov
chains were run for 10,000,000 generations and sampled every
1000 generations, and the first 1000 trees were the “burn-in” of
the chain. An allcompat consensus tree was calculated to obtain
topology with average branch lengths as well as posterior prob-
abilities (PP) for all resolved nodes.
RES U LT S
The phylogenetic relationships of Anisocampium we re de-
duced from the combined rbcL and trnL-F sequences (in total
2003 base pairs) (Fig. 2). Anisocampium was sister, with high
support (PP = 1.0, MLBS = 98%), to a clade comprising Athy-
rium and Cornopteris which showed high support (PP = 1.0,
MLBS = 99%). Monophyly of eac h of the th ree genera was also
robu st with hig h sup por t for each (PP = 1.0, MLBS = 100%). In
Anisocampium, a clade of An. sheareri and An. cumingianum
was sister to An. cuspidatum (≡ Ath. cuspidatum), and together
sister to An. niponicum (≡ Ath. niponicum), although support
for the first two clades was not very high.
P
60°E 90°E 120°E 150°E
Anisocampium niponicum
Anisocampium ×saitoanum
Anisocampium cumingianum
P
90°E 120°E 150°E
0°
30°N
60°N
Anisocampium sheareri
Anisocampium cuspidatum
Fig. 1.
Distribution of Anisocampium.

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DISCUSSION
The Anisocampium clade is distinct from the Athyrium
clade, and similar to the topology of previous analyses (Sano
& al., 2000; Wang & al., 2003; Schuettpelz & Pryer, 2007;
Adjie & al., 2008). In the taxonomic definition below, we re-
define the genus Anisocampium to include An. cuspidatum
(≡ Kuniwatsukia cuspidata) and An. niponicum (≡ Athyrium
niponicum), and separate the genus from Athyrium and Cor-
nopteris. The results are not consistent with current taxo-
nomic treatments based on morphological comparisons (Kato,
1977; Ching, 1978; Kato & Kramer, 1990; Wang, 1997; Wang
& al., 1999). Kuniwatsukia and Anisocampium have been
treated as synonyms of Athyrium (Kato, 1977; Fraser-Jenkins,
2006), while Cornopteris has long been presumed to be re-
lated to Diplazium (Ching, 1945; Wang, 1997) or Athyrium
(Tagawa, 1959; Kato, 1979). Based on morphological similar-
ity, Fraser-Jenkins (2006) placed Ath. cumingianum (≡ An.
cumingianum), along with Ath. niponicum, in Athyrium sect.
Niponica, while recognizing Cornopteris. He also noted the
similarity of Ath. cumingianum with Ath. cuspidatum and Ath.
sheareri.
An alternative classification to ours is to recognize Athy-
rium s.l. as including Anisocampium and Cornopteris. Cor-
nopteris is characterized by exindusiate sori and fleshy pro-
jections (corni) at the base of costae and costules, by which it
0.01
Athyrium vidalii
Athyrium nakanoi
Athyrium strigillosum
Athyrium otophorum
Athyrium biserrulatum
Athyrium filix-femina
Athyrium rupestre
Athyrium yokoscense
Anisocampium sheareri
Anisocampium cumingianum
Anisocampium cuspidatum
Anisocampium niponicum
Diplazium nipponicum
Diplazium hachijoense
Deparia lobatocrenata
Deparia petersenii
Deparia conilii
Cornopteris decurrentialata
Cornopteris crenulatoserrulata
Cornopteris christenseniana
0.76
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.68
0.95
0.92
85
92
85
81
100
99
100
100
98
86
77
100
100
100
69
Fig. 2.
Phylogenetic tree of
Anisocampium and allied gen-
era based on rbcL and trnL-F
region sequences. Tree topology
is deduced from Bayesian infer-
ence analysis; numbers indicate
values of posterior probabilities
(above branches) and bootst rap
(below branches); numbers less
than 0.5 or 50 are not shown.
Three species of Deparia and
two of Diplazium are outgroups.

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is distinguished from Athyrium. Kato (1977), Kato & Kramer
(1990) and Fraser-Jenkins (2006) treated Anisocampium and
Kuniwatsukia as synonyms of Athyrium, but separated Cornop-
teris. Our phylogenetic analysis indicates that such a circum-
scription results in the paraphyly of Athyrium.
Like Adjie & al. (2008), we did not find any unique diag-
nostic characters to define Anisocampium. The genus can only be
defined and distinguished from the closely related Athyrium and
Cornopteris by a combination of features of the stipe base, projec-
tions, spines and sori (Table 1). The base of the stipe is as broad
as the upper part of the stipe in Anisocampium, while the stipe is
broader near the base with lateral pneumatophores and narrowed
to the very base in Athyrium, Cornopteris and Deparia within
the athyrioid ferns (Liew, 1972; Kato, 1977, 1979, 1984). The
stipe base is persistent after the leaves wither. They are arranged
tightly around the short, erect rhizomes in most species of these
two genera, or are loosely arranged on the creeping rhizomes in,
e.g., Athyrium atkinsonii Bedd. and Cornopteris decurrentialata
Nakai. The persistent stipe base, called a trophopod (Johnson,
1986), is possibly a derived, adaptive trait in plants in the temper-
ate zone. The persistent stipe base is absent in Anisocampium and
Diplazium, even in species with erect rhizomes.
Fleshy projections (corni) in the groove on the adaxial sur-
face at the base of the costae and costules are characteristic of
Cornopteris, while they are absent from Anisocampium and
Athyrium. There are spines in many species of Athyrium and
Cornopteris, but none in Anisocampium. The spines are continu-
ations of the interrupted ends of ridges along the groove of the
costae and costules on the adaxial surface of the leaf. The sori
are rounded-reniform, shortly U- or J-shaped and indusiate in
most species of Anisocampium, but they are also oblong-linear
in An. niponicum. In Cornopteris, however, they are rounded
to linear and exindusiate and in Athyrium usually linear or
J-shaped and indusiate.
Diagnostic characters separating Anisocampium and related
genera are shown in Table 1. A key for their identification and
a brief taxonomic treatment of the revised genus Anisocampium
are given below.
KEy TO AnisocAmpium AND RELATED
gENERA
1. Groove of costa interrupted at base and not confluent with
groove of rachis; multicellular hairs present on stipe, rachis
and costae, rarely few or absent ................... Deparia
1. Groove of costa continuous with groove of rachis; multicel-
lular hairs absent or sometimes present ................... 2
2. Sori exindusiate; fleshy projections present at base of costa
....................................................Cornopteris
2. Sori indusiate; fleshy projections absent .................. 3
3. Sori linear or oblong, not J- or U-shaped; groove of ra-
chis and costa U-shaped with flat base in cross section
..................................................... Diplazium
3. Sori various in shape, linear, oblong, J- or U-shaped, or
round-reniform; groove of rachis and costa V-shaped in
cross section ................................................. 4
4.
Rhizome ascending or shortly creeping; stipe base usually
broadened, with pneumatophores on sides, narrowed down-
ward; spines at base of costules and midvein present or
absent adaxially; lamina 2- or 3-pinnate, sometimes pinnate
or bipinnatifid, usually gradually narrowed to apex
......
...................................................... Athyrium
4. Rhizome creeping or ascending; stipe base not broadened,
devoid of pneumatophores; spines absent; lamina pinnate
or bipinnatifid, in A. niponicum bipinnate to tripinnatifid,
abruptly narrowed to apex or terminated by pinna conform
to lateral pinnae .............................Anisocampium
Anisocampium C. Presl, Epimel. Bot.: 52. 1849; Tardieu &
Christensen in Lecomte, Fl. Gén. l’Indo-Chine 7(7): 281.
1940; Copeland, Gen. Fil.: 151. 1947; Ching in Acta Phy-
totax. Sin. 16(3): 13. 1978 – Type: Anisocampium cum-
ingianum C. Presl.
= Kuniwatsukia Pic. Serm. in Webbia 28: 455. 1973; Ching in
Acta Phytotax. Sin. 16(3): 13. 1978 ≡ Microchlaena Ching
in Bull. Fan Mem. Inst. Biol. 8: 322. 1938, non Micro-
chlaena Wall. ex Wight & Arn. 1834; Iwatsuki in Acta
Phytotax. Geobot. 24: 188. 1970 – Type: Kuniwatsukia
cuspidata (Bedd.) Pic. Serm., syn. nov.
= Athyrium Roth sect. Niponica Ching & Y.T. Hsieh in Bull.
Bot. Res., Harbin 6(4): 131. 1986, pro parte – Type: Athy-
rium niponicum (Mett.) Hance, syn. nov.
Rhizome creeping or ascending to shortly erect (in A. cus-
pidatum), radially symmetrical or dorsiventral (in A. cumingia-
num), apex scaly. Leaves more or less distant from each other,
stipe base scaly, not swollen, with 1 (in A. niponicum) or 2 leaf
traces in rhizome, deltoid-ovate or ovate-oblong, imparipinnate
to 3-pinnatifid, abruptly reduced to pinnatifid apex or bearing
terminal pinna conforming with lateral pinnae; pinnae lanceo-
late or narrowly elliptic, base of lower pinnae inequilateral;
veins free or anastomosing (in A. cumingianum); spines absent
on adaxial surface at base of costae, costules and midvein. Sori
oblong, J-shaped, horseshoe-shaped, round-reniform, indusiate;
indusia conforming with sori, margin ciliate, or erose, often fu-
gacious. Spores bilateral, with perispore. Chromosome number
x = 40. Four species.
Habitat. – Mesic forests from near sea level to 1900 m alt.
Distribution. – Japan, Korea, China, Taiwan, Vietnam,
Laos, Thailand, Myanmar, India, Bhutan, Nepal, Sri Lanka,
Philippines, Indonesia (Fig. 1).
KEy TO THE SpECIES Of AnisocAmpium
1. Lamina pinnate, apex of lamina pinnatifid, not conforming
with lateral pinnae .......................................... 2
1. Lamina terminated by a pinna conforming or subconform-
ing to lateral pinnae ......................................... 4
2. Lamina pinnate throughout; pinnae usually shallowly
lobed; sori round ..............................1. A. sheareri
2. Lamina bipinnatifid or bipinnate; pinnae lobed more than
2/3 to costa; sori oblong, J-shaped, or round ............. 3
3. Lamina bipinnate; pinnules free, lobed ...2. A. niponicum

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3. Lamina bipinnatifid; pinnules not free, at least adnate to
costa by broad base .....................2a. A. ×saitoanum
4. Pinnae to 7 pairs, lanceolate, base cuneate or round; veins
anastomosing ........................... 3. A. cumingianum
4. Pinnae 10 pairs or more, linear, base truncate; veins free
..............................................4. A. cuspidatum
1. Anisocampium sheareri (Baker) Ching in Acta Bot. Yunnan.
7(3): 314. 1985; Wang & al., Fl. Reipubl. Pop. Sin. 3(2):
75, pl. 15, f. 1–6. 1999 ≡ Nephrodium sheareri Baker in
J. Bot. 13: 200. 1875 ≡ Dryopteris sheareri (Baker) C. Chr.,
Index Filic.: 292. 1905 ≡ Athyrium sheareri (Baker) Ching
in Christensen, Index Filic., Suppl. 3: 44. 1934; Iwatsuki,
Ferns Fern Allies Jap.: 232, photo 151(1, 2). 1992; Kato in
Iwatsuki & al., Fl. Jap. 1: 203. 1995 – Type: China, Shearer
s.n. (K, photo!).
= Nephrodium polypodiforme Makino in Bot. Mag. (Tokyo) 13:
58. 1899 ≡ Dryopteris polypodiformis (Makino) C. Chr.,
Index Filic.: 285. 1905 – Syntypes: Japan, Z. Matsumura
s.n., Jul. 17, 1882 (TI), Y. Yoshinaga s.n., 1887, M. Makino
s.n., Aug. 1887, 1889, Nov. 1892 (K, photo!).
= Aspidium otarioides Christ in Bull. Acad. Int. Géogr. Bot. 11:
247. 1902 ≡ Dryopteris otarioides (Christ) C. Chr., Index
Filic.: 282. 1905 – Type: China, E. Bodinier 2050 (P, photo!).
= Nephrodium isolatum Baker in Gard. Chron., n.s., 14: 494.
1880 – Type: China, Maries s.n. (K, photo!).
Distribution. – Japan, South Korea (Cheju Is.), China
(North Central, Southeast, South Central), Taiwan (new record,
Hualien Co., Y.Y. Huang 620; deposited in HAST).
2. Anisocampium niponicum (Mett.) Y.C. Liu, W.L. Chiou
& M. Kato, comb. nov. ≡ Asplenium niponicum Mett. in
Ann. Mus. Bot. Lugduno-Batavi 2: 240. 1866 ≡ Athy-
rium niponicum (Mett.) Hance in J. Linn. Soc., Bot. 13:
92. 1872; Tardieu & Christensen in Lecomte, Fl. Gén.
l’Indo-Chine 7(7): 273. 1940; Dickason in Ohio J. Sci. 46:
123. 1946; Iwatsuki, Ferns Fern Allies Jap.: 239, photo
160(1, 2). 1992; Shieh & al. in Editorial Committee of
the Flora of Taiwan, Fl. Taiwan, ed. 2, 1: 424. 1994; Kato
in Iwatsuki & al., Fl. Jap. 1: 210. 1995; Wang & al., Fl.
Reipubl. Pop. Sin. 3(2): 132, pl. 25, f. 1–8. 1999; Liu &
al., Fern Fl. Taiwan, Athyrium: 34, fig. 14, map 14, photo
14. 2009 – Type: Japan, Keiske (K. Ito), Siebold, Buerger
s.n. (L, photo!).
= Asplenium uropteron Miq. in Ann. Mus. Bot. Lugduno-Batavi
3: 174. 1867 ≡ Athyrium uropteron (Miq.) C. Chr., Index
Filic.: 147. 1905 – Type: Japan, Keiske (K. Ito) s.n. (L,
photo!).
Table 1.
Comparison of Anisocampium and related genera.
Character Anisocampium Cornopteris AthyriumaDiplaziumbDepariac
Rhizome Creeping or erect Creeping or erect Erect or ascending,
few creeping
Creeping, ascending
or erect
Creeping, ascending
or erect
Stipe base Not persistent Persistent Persistent, rarely not Not persistent Persistent or not
Scale margin Entire Entire Entire Toothed, rarely entire Entire
Multicellular hairs Absent Present or absent Absent, rarely present Absent, rarely present Present, rarely absent
Unicellular hairs Present Present or absent Present or absent Absent Absent
Pinna- or pinnule base Inequilateral Equilateral Inequilateral Equilateral Equilateral
SpinedAbsent Present Present or absent Absent Absent
Continuation of grooveeConfluent Confluent Confluent Confluent Not confluent
Shape of groovefV-shaped V-shaped V-shaped Widely U-shaped,
rarely V-shaped
Shallowly V-shaped
ProjectionsgAbsent Present Absent Absent Absent
Veins Free or anastomosing Free Free Free or anastomosing Free or anastomosing
Sori Round-reniform, U-
or J-shaped, or oblong
Round or oblong Round-reniform,
U- or J-shaped, linear,
or oblong
Linear or oblong Round-reniform,
U- or J-shaped, linear,
or oblong
Indusium Present Absent Present, rarely absent Present Present
Indusium margin Ciliate, lacerate, erose
or subentire
—Entire, erose, rarely
ciliate
Entire or erose Entire, erose, lacerate
or ciliate
Chromosome number 40 40 or 41 40 41 40 or 41
a Including Pseudocystopteris.
b Including Allantodia.
c Including Athyriopsis, Dictyodroma, Dryoathyrium, Lunathyrium, and Triblemma.
d Spines are continuations from ends of interrupted ridges embanking grooves at insertions of costae, costules and midveins on adaxial surface of lamina.
e Grooves on adaxial surface of costae and costules are confluent with grooves on costules and midveins or not confluent because of a separating ridge.
f Grooves are V-shaped or U-shaped with flat bottom in cross section.
g Fleshy projections (corni) in grooves at base of pinnae and/or pinnules differ from spines, which are continuous with ridges.

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= Athyrium biondii Christ in Nuovo Giorn. Bot. Ital., n.s., 4: 91.
1897 – Type: China, G. Giraldi s.n. (B, photo!).
= Athyrium yunnanense Christ in Bull. Geogr. Bot. Mans. 17:
134. 1907 – Type: China, Cavalerie 3864 (K, photo!).
= Athyrium fissum Christ in Notul. Syst. (Paris) 1: 47. 1909 –
Type: China, Henr y 1310 7 (P, photo!).
Distribution. – Japan, Korea, China (Northeast, North
Central, Southeast, South Central), Taiwan, southern Vietnam,
Myanmar (central), northeast India (Arunachal Pradesh). An-
isocampium niponicum is also in north east India (Man ipur and
Tripura) (C.R. Fraser-Jenkins, pers. comm.).
Note. – Ogura (1921) and Kato (1977) found single-leaf
trace vasculature in An. niponicum, Ath. yokoscense (Franch.
& Sav.) Christ, and Ath. vidalii (Franch. & Sav.) Nakai, which
is contrasted with the bina r y leaf trace in most athy rioid ferns.
The leaf trace is divided into two and united to a single U-
shaped bundle in cross section in the upper part of the stipe.
2a. Anisocampium ×saitoanum (Sugim.) M. Kato, comb. nov.
≡ Athyrium sheareri f. saitoanum Sugim., Keys Herb. Pl.
Jap. 3: 404. 1966 ≡ Athyrium ×saitoanum (Sugim.) Seriz.
in J. Phytogeogr. Taxon. 34: 68, f. 1–3. 1986 – Type: Japan,
Y. Saito s.n. (Herb. Sugimoto, photo!).
Distribution. – Western Japan; the distribution of Aniso-
campium ×saitoanum ext end s over most of the range of overlap
of A. niponicum and A. sheareri.
Note. – Anisocampium ×saitoanum is a putat ive hybr id de-
rived from crosses between An. niponicum and An. sheareri. It
has been recorded from 18 prefectures in western Japan (Fig. 1;
Serizawa 1986; Kurata & Nakaike, 1997). The parental spe-
cies are phylogenetically the farthest from each other in the
genus in Japan. Anisocampium ×saitoanum can reproduce to
a certain degree in culture, indicating incomplete reproductive
isolation between the species (T. Oka, pers. comm.). The hybrid
may therefore be reproducing in nature or may be the result
of multiple origins. Either case suggests a close affinity of the
two parental species.
3. Anisocampium cumingianum C. Presl, Epimel. Bot.: 59.
1849; Tardieu & Christensen in Lecomte, Fl. Gén. l’Indo-
Chine 7(7): 281. 1940; Copeland, Fern Fl. Philipp. 3: 418.
1960; Sledge in Bull. Brit. Mus. (Nat. Hist.) Bot. 2(11):
281. 1962; Tagawa & Iwatsuki, Fl. Thailand 3(3): 444,
f. 47(5–7). 1988; Manickam & Irudayaraj, Pterid. Fl. W.
Ghats: 231, pl. 178. 1991; Wang & al., Fl. Reipubl. Pop.
Sin. 3(2): 77, pl. 15, f. 7–8. 1999 ≡ Athyrium cumingia-
num (C. Presl) Ching in Christensen, Index Filic., Suppl.
3: 40. 1934; Fraser-Jenkins in Shaffer-Fehre, Rev. Handb.
Fl. Ceylon 15(B): 548. 2006 ≡ Aspidium otaria Kunze ex
Mett., Abh. Sencken berg. Naturf. Ges. 2: 318. 1858 ≡ Neph-
rodium otaria (Kun ze ex Met t.) Baker in Hoo ke r & Baker,
Syn. Fil.: 288. 1867 – Type: Philippines, Cuming 239 (K!).
= Phegopteris paucijuga Al der w. in Bu ll. Jard. Bo t. Buit enzorg ,
se r. 2, 16: 26. 1914 ≡ Anisocampium paucijugum (Alderw.)
Copel., Gen. Fil.: 151. 1947 – Type: Java, C.A. Backer 8290
(BO, photo!), syn. nov.
Distribution. – China (southern Yunnan), Taiwan (Liu &
al., 2000), Laos, Thailand, southern Myanmar, southern and
northeast India, Philippines (Luzon), Indonesia (Bali, East
Java, newly recorded to South Sulawesi [Bantimurung], T. Oka
100608011, TNS), Sri Lanka. Anisocampium cumingianum also
occurs in the Lesser Sunda Islands (Sumbawa, Timor).
Note. – The lamina of An. paucijugum, described from
eastern Java, is relatively small, i.e., 12 cm long and has two or
three pinnae on each side of the rachis. The size of the leaf and
the number of pinnae are within the range of variation of An.
cumingianum. The other cha r acter ist ics, e.g., the impa r ipinnate
leaf, anastomosing veins, and round sori, are shared with An.
paucijugum and An. cumingianum.
Anisocampium cumingianum is the most de rived spe cies in
the genus in its dorsiventral rhizome (vs. radially symmetrical),
the imparipinnate lamina (vs. paripinnate lamina with gradu-
ally less dissected apical part), and the anastomosing veins
(vs. free veins).
4. Anisocampium cuspidatum (Bed d.) Y.C. Liu, W.L. Chiou &
M. Kato, comb. nov. ≡ Lastrea cuspidata Be dd., Fer ns Br it .
India: t. 118. 1870 ≡ Aspidium cuspidatum Mett. in Abh.
Senckenberg. Naturf. Ges. 2: 376. 1858, non Desv. 1827 ≡
Nephrodium cuspidatum Baker in Hooker & Baker, Syn.
Fil.: 260. 1867, non C. Presl 1825 ≡ Dryopteris cuspidata
(Bedd.) Christ in Philipp. J. Sci., C 2: 205. 1907 ≡ Micro-
chlaena cuspidata (Bedd.) Ching in Acta Phy totax. Sin. 9:
99. 1964; Iwatsuki in Acta Phytotax. Geobot. 24: 188. 1970
≡ Kuniwatsukia cuspidata (Bedd.) Pic. Serm. in Webbia 28:
455. 1973; Tagawa & Iwatsuki, Fl. Thailand 3(3): 442, f.
47. 1988; Wang & al., Fl. Reipubl. Pop. Sin. 3(2): 78, pl. 16.
1999 ≡ Athyrium cuspidatum (Bedd .) M. Kato in Bot. Mag.
(Tokyo) 90: 27. 1977 ≡ Dryopteris khasiana C. Ch r., Index
Filic.: 272. 1905 ≡ Polypodium elongatum Wall. ex Hook.,
Sp. Fil. 4: 234. 1862, non Aiton 1789 ≡ Phegopteris elongata
J. Sm., Hist. Fil.: 233. 1875, non E. Fourn. 1872 ≡ Lastrea
elongata Bedd . ex Cla rke, Tra ns. Lin n. Soc. Lon don, Bot. 1:
513. 1880, non C. Presl 1836 ≡ Dryopteris elongata (Bedd.
ex Clarke) Kuntze, Revis. Gen. Pl. 2: 811. 1891 – Type:
Nepal, Wallich Cat. No. 309 (NY, photo!).
= Aspidium yunnanense Christ in Bull. Herb. Boissier 6: 965.
1898 ≡ Microchlaena yunnanensis (Christ) Ching in Bull.
Fan Mem. Inst. Biol. Bot. 8: 325, t. 6. f. 1. 1938; Dickason
in Ohio J. Sci. 46: 120. 1946 ≡ Dryopteris yunnanensis
(Christ) Copel., Gen. Fil.: 122. 1947 ≡ Aspidium stenolepis
Christ, Bull. Herb. Boissier 7: 22. 1899 – Type: China,
A. Henry 9038A (US, NY, photo!).
Distribution. – China (South Central, Southwest Central),
northeast Myanmar, Northern Thailand, Bhutan, northeast
India, central and east Nepal. According to C.R. Fraser-Jenkins
(pers. comm.), Anisocampium cuspidatum also occurs in the
western Himalayas (Pithoragash, India). Hooker (1862: 234)
thought that the type specimen of Polypodium elongatum, col-
lected in Kew Gardens, originated in Sri Lanka, but Clarke
(1880: 512) doubted it (Fraser-Jenkins, 2006).
Note. – Anisocampium cuspidatum is distinct in having
shortly creeping rhizomes with an ascending apex and radially
arranged leaves with linear pinnae.

830
TAX ON
60 (3) • June 2011: 824–830Liu & al. •
Phylogeny and taxonomy of Anisocampium
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ACKNOWLEDgMENTS
We th an k C.R. Fra ser-Jen kin s for pr ovidin g di st r ib ut ion da ta for
this group of ferns, T. Oka for useful infor mation on A. cumingianum
and A. ×saitoanum, Titien Ng Pr aptos uw ir yo for pr ovidi ng a phot o of
the type specimen of A. paucijugum, X. Cheng for providing infor-
mation on the distribution of A. cuspidatum, and D.E. Boufford for
correcting the English of the manuscript. We thank the curators of
BO, HAST, K, KYO, PE, PNH, PYU, SYSU, and TAIF for allowing
us to examine specimens in their herbar ia. T his study was suppor ted
by a Grant-in-Aid for Scientific Study from the Japan Society of the
Promotion of Science to M.K. and from the Taiwan Nat ional Science
Council ( NSC-97-2313-B-054-005-MY3) to W.L.C.
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Appendix.
Species, sources and GenBan k accession s (rbcL, trnL-F) used in this st udy.
Anisocampium cumingianum C. Presl, J.M. Chen s.n., Taiwan (TAIF), HM156338, FJ807659. Anisocampium cuspidatum (Bedd.) Y.C. Liu, W.L. Chiou &
M. Kato, EU329029, EU329073 (Adjie & al., 2008). Anisocampium niponicum (Mett.) Y.C. Liu, W.L. Chiou & M. Kato, EU329042, EU329085 (Adjie & al.,
200 8). Anisocampium sheareri (Bake r) Ching in Y.T. Hsieh, EU329047, EU32909 0 (Adjie & al., 20 08). Athyrium biserrulatum H. Christ, YCLiu 9370, China
(TAIF), HM156337, HM156336. Athyrium filix-femina (L.) Roth, EU329032, EU32 90 76 (Adji e & al ., 2008). Athyrium nakanoi Mak ino, EU329039, EU32 90 82
(Adjie & al., 2008). Athyrium otophorum (Miq.) Koid z., EU329 04 4, EU329087 (Adjie & al., 2008). Athyrium rupestre Kodama, EU 3290 45, EU329088 (Adjie
& al., 2008). Athyrium strigillosum (Moore ex Lowe) Moore ex Salom, EU329049, EU329092 (Adjie & al., 2008). Athyrium vidalii (Franch. & Sav.) Nakai,
EU329052, EU329095 (Adjie & al., 2008). Athyrium yokoscense (Franch. & Sav.) H. Christ, EU329055, EU329098 (Adjie & al., 2008). Cornopteris christense-
niana (Koidz.) Tagawa, EU329061, EU329104 (Adjie & al., 2008). Cornopteris crenulatoserrulata Nakai, EU329062, EU329105 (Adjie & al., 2008). Cornopteris
decurrentialata ( Ho ok.) Naka i, EU329063, EU329106 (Adji e & al ., 200 8). Deparia conilii (Fr an ch . & Sav.) M. Ka to , EU 329 065, EU 32910 8 (Ad jie & al. , 20 08) .
Deparia lobatocrenata (Tagawa) M. Kato, EU 329 06 6, EU329109 (Adjie & al., 20 08). Deparia petersenii (Ku nze) M. Kato, EU3290 64 , EU 32910 7 (Adjie & al.,
200 8). Diplazium hachijoense Nakai, EU329068, EU329111 (Adjie & al., 2008). Diplazium nipponicum Tagawa, EU329067, EU329110 (Adjie & al., 2008).