Molecular Evidence for Genetic Heterogeneity and the Hybrid Origin of Acrorumohra subreflexipinna from Taiwan

2009·8.6 MB·English

by  Ho-Ming Chang

Molecular Evidence
for Genetic Heterogeneity and
the
Hybrid Acrorumohra
Origin of
subreflexipinna
from Taiwan
Ho-MiNG Chang
Department
of Life Science, National Taiwan Normal Ting-Chow
University, No.88, Sec
Rd.,
4,
Division of Botany, Endemic Species Research Ming-Sheng
No.l,
Institute, E. Rd., Chi-Chi,
Nantou Taiwan
552,
Wen-Liang
Chiou
^^'^^^
Taiwan
' '
Wang*
Jenn-Che
Department of Life Science, National Taiwan Normal University No.88, Ting-Chow Sec
Rd.,
4,
Hybridization followed by polyploidization an important mechanism
is
new
driving the formation and
of lineages of ferns other plants (Paun et al,
By means
2007). of diploidization processes, such chromosomal
as rearrange-
ments, intergenome recombination, and gene genomic
silencing, the
constitu-
many
tion of extant taxa might be the outcome of ancient hybridization and
polyploidy (Bowers De
et al, 2003; Bodt et al, 2005; Haufler, 1987; Paun et al,
2007). Hybridization events often begin these cycles and high chromosomal
number
base was
in ferns achieved as the result of repeated cycles of
corresponding
author: e-mail: [email protected]
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polyploidization {Haufler, 1987; Klekowski and Baker, 1966; Nakazato et al,
2006).
Accessing the parentage of hybrids or allopolyploids essential for
is
understanding relationships within taxonomically complex groups. Although
allozyme studies could provide tenable evidence to indicate the possible
origin of hybrid-originated they have been
taxa, rarely utilized distinguish
to
DNA
maternal from However,
lines paternal ones. direct evidence, such as
DNA
nucleotide sequences and fingerprints, can provide more informative
insights into these evolutionary processes than enzymes. In most plants,
genomes
organelle are maternally inherited via female gametes while nuclear
DNA
DNA
Comparing
is biparentally inherited (Sokis et 1992). organellar
al.,
and
hybrid
of taxa their possible parents therefore could reveal the maternal
and
origin (Gastony Yatskievich, 1992; Vogel et al, 1998) while comparing
DNA
show
nuclear of those taxa could both putative parentages (Small
et
al.,
any would
2004). In addition, evolutionary be deposited
trace, theoretically, in
nucleotide sequences and could be detected by DNA-based molecular
DNA
Unique would
technology. be
local variation detectable applicable
if
DNA
markers were chosen (Soltis et al, 1992). markers containing non-coding
shown
regions have been to be the best choice to reconstruct genealogies of
and
hybrid parental populations (Small et 2004; Xiang 2000).
al., al.,
Studies of north temperate ferns have clearly indicated the contribution of
and
hybridization polyploidization to fern evolution (Barrington et 1989;
al,.
Bennert Wagner, Werth Out
et al, 2005; Pinter et al, 2002; 1973; et 1985).
al.,
420 and grow
of the species of lycophytes ferns that in North America, nearly
20%
are of hybrid origin (Flora of North America Editorial Committee, 1993),
and reticulate networks and ploidy levels of most taxonomically complex
groups have been well studied (Barrington, 1986; Stein and Barrington, 1990;
Wagner, 1954, 1962, 1973; Xiang et al, 2000). However, only a few ferns from
other regions have received taxonomic Europe and
attention like those in
North America Takamiya
(e.g., Barrington, 1990; Ebihara et al, 2005; et
al.,
2001; Terada and Takamiya, 2006). Some hybrid ferns have been recorded
from Taiwan (Holttum and Edwards, Kuo, Miyamoto and
1986; 1988, 1990;
Nakamura, now, no
1983), but until direct evidence has been reported
to test
and
verify their parentage.
Acrorumohra
is a small genus with about seven species distributed in
Eastern and Southeastern Asia. This genus has an intermediate morphology
between and
Dryopteris Arachniodes; Acrorumohra were
therefore, species of
once treated in these genera. However, Acrorumohra was treated as an
independent genus Taiwan and
in the Flora of (Shieh 1994) Flora
et
al.,
Reipublicae Popularis Sinicae (Hsieh, 2000) based on the presence of the
zigzag rachis and anadromous pinnules of pinnae. Acrorumohra
subreflex-
ipinna (M. Ogata) H. an endemic species of Taiwan, produces shriveled
Ito,
and abortive spores and has an intermediate morphology between A.
hasseltii
(Blume) Ching and A. (Baker) H. Given morphological
diffracta
Ito. its
characteristics, A. subreflexipinna has been suspected as a hybrid of these two
species (Moore, 2000). Moreover, the fact that A. subreflexipinna always grows
CHANG
ET HYBRID OF ACRORUMOHRA
ORIGIN SUBREFLEXIPINNA
AL.:
sympatrically with two
the later species reinforces the reasonable hypothesis
of its hybrid origin. The narrowly defined genus Acrorumohra' was followed
'
name
and
the scientific subreflexipinna' used throughout
'A. the study,
is
although palynological and unpublished breeding
data indicates Fl
a sterile
it
DNA
hybrid. In this study, chloroplast, mitochondria and nucleus markers
were used
to identify the parentage of this suspected hybrid. Furthermore, the
hypothesis that hybrid populations in Taiwan each originated independently
was
tested. In addition to haplotype comparison, genetic variation in different
was
populations determined
to clarify lineage relationships.
Materials and Methods
Acrorumohra
Plants of subreflexipinna were sampled from
three sites in
Taiwan: Mt. Howeishan, Lake Chunglingchih and Kentuerhshan
Mt.
(Fig.
1).
Leaf tissue of four to 11 individuals per population was collected molecular
for
Ten
analyses. individuals of the two putative parent species, A. hasseltii and
were sampled Two
A. diffracta, also in each sympatric site (Table plants of
1].
Dryopteris polita Rosenst. were also sampled to detect any possible parental
relationship because based on phylogenetic
analysis of a chloroplast trnS-rps4
and
data D. polita A. and
set, hasseltii are sister species (Li Lu, 2006).
Two
chloroplast intergenic spacers [trnL-trnF and trnS-rps4 IGS) and one
mitochondrial intron [nadS intron which have been frequently used
2], for
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phylogenetic analysis lower taxonomic were employed
at levels, to reveal
maternal history, introns of the single-copy nuclear gene pgiC (including
vi^hile
introns 14 and 15, and exon 15) w^ere used to observe bi-parental inheritance.
These sequences chosen because
w^ere of their significant phylogenetic
information relative to other fragments and the availability of usable primers
(Ishikawa et al, 2002; Nadot et al, 1995; Smith and Cranfill, 2002; Vangerow
et al, 1999).
Dry or fresh tissues of young leaves were homogenized with liquid nitrogen.
DNA
mg
Genomic was
extracted from ca. 100 of leaf tissue by using a Plant
DNA
Genomic Mini USA). The PGR
Kit (Viogene, amplification of segments
all
was performed in an ABI thermocycler (9700). Primers for trnL-trnF IGS,
GCG CAA GTT GCG GTA GAA
CTG
trnLF-11 5'- CGA-3' and 5'-CTG
trnLF-12
TAG GGA GTG AG
GTA-3', were modifications of those utilized by Taberlet
et
AAG
GGA AGT GAT
The primers 5'-GGG GTT GA-37
al. (1991). tsr4-f/tsr4-r
AGG GTT GGA ATG GGT GGT ATA TGG
5'-GGG nadh2-f/nadh2-r 5'-GGG
C-3',
GGA GAG GTG GAA GTT
TGG-375'-GGG
TGG-3', and pgiG-14fA/pgiC-16rA 5'-
GTG GTT GTG GGT GTT TTG GTG GAT TAG TTG GAG
AG-375'-GTT
GT-3'
were developed study Smith and Vangerow
for this referring to Granfill
(2002),
and PGR
Ishikawa were
et al. (1999) et al. (2002), respectively. reactions
DNA,
carried out in 20 reactions containing 2 unstandardized template
^iL |iL
mmol/L
0.2 of each dNTP, 0.8 units of Taq polymerase (ABgene, USA) and
6.25 pmol each of the forward and reverse primers, and programmed min
for 5
min min and min
at 95^C, 35 cycles of 1 at 95°G, 1 at annealing temperature 2
at 72X, followed by a 8 min extension 72°G. The anneahng temperature was
at
59°G in amplifying the chloroplast trnL-trnF IGS and the mitochondrial nad5
G When
and
intron 52 in amplifying the chloroplast trnS-rps4 fragment.
2,
amplifying nuclear pgiC intron 14-15 segment, annealing was performed
at
57°G for the first 3 cycles, at 55=C for the next 3 and at 54=G for the final 29.
PGR
products were directly sequenced, using one amplification primer, on an
ABI 373A automated sequencer (Applied Biosystems, USA) with the Taq Dye
Dideoxy Terminator Gycle Sequencing Kit (Applied Biosystems). For the
electrophoresed bands with lengths greater than 500 bp, sequences were
determined both pgiC 14-15 segments
in directions. Additionally, intron of
all
A. subreflexipinna samples and 3-5 samples each population
in of A.
PGR
were The
hasseltiiand A. cloned. products of the nuclear
diffracta
TAB
IX
segment were purified by electrophoresis using buffer on a 1.2%
agarose gel. Electrophoresed bands were cut and eluted using the Gel-M gel
DNA
yT&A
extraction system (Viogene). Purified nuclear was cloned with the
cloning kit (Yeastern Biotech, Taiwan) following the manufacturer's protocol.
PGR
Five to eight colonies were chosen to perform colony using TA-F forward
DNA
TA-R
and reverse primers (Yeastern Biotech). Purified nuclear was
Ml
sequenced with 3 universal and reverse primers which are located on the
DH5a When
vector termination any haplotype was
different detected,
site.
PGR
repeated reactions using a different Taq polymerase (Genomics, Taiwan)
DNA
or using from another three colonies were chosen to check whether was
it
a real variant or not. All sequences were deposited in the GenBank nucleotide
CHANG
ET HYBRID OF ACRORUMOHRA
ORIGIN SUBREFLEXIPINNA
AL.:
DNA
sequence database, and accession numbers and corresponding
their
regions Table
are listed in
1.
The
sequences were aligned by BioEdit and manual and
7.0 correction,
compared
with nucleotide sequences available through GenBank determine
to
their boundaries of coding region. Haplotypes were named the
after first letter
and
of the specific epithet, followed by lowercase and number
a
letter to
minor
designate different, haplotypes (those differing from correspond-
their
ing major haplotype only one base) of A. Genetic
at hasseltii. diversity
at
DNA
population and was
species levels estimated with the software package
Sequence Polymorphism (DnaSP Rozas The
4.20.2, et al, 2003). haplotype
and
diversity [h] nucleotide diversity of these three populations were
(ti)
and
calculated separately Genetic
totally. differentiation Nei, 1982)
(yst.
among
these three populations and between pairs of populations was
also
calculated by this package, but not Fst or Nst, was used because the three
y^t,
sampled populations were the only ones of interest (Lynch and Crease, 1990).
Because no was
variation detected in the nuclear sequences of A.
diffracta,
only the A. hasseltii haplotypes cloned from A. subreflexipinna were used
when
analyzing genetic and among
diversity differentiation the populations
of
A. subreflexipinna. Haplotypes of A. hasseltii and A. subreflexipinna were
identified and coded by direct sequence comparison, and unrooted haplotype
networks were constructed with program TCS
the 1.21 (Clement
et 2000).
al.,
GC
and
Total aligned length content of the sequences of nuclear pgiC intron
14-15, chloroplast trnL-trnF IGS and trnS-rps4 IGS, and mitochondrial nadS
intron 2 were 725 bp/37.8%, 268 bp/34.9%, 374 bp/36.5%, and 728 bp/
Low GC
52.6%,
respectively. content of chloroplast segments agreed with
the AT-rich property of most non-coding spacers (Graur and
Li, 2000).
and
In the chloroplast mitochondria segments, 50 individuals of A.
all
subreflexipinna and A. hasseltii had the same nucleotide sequences but were
different from those of A. diffracta and D. polita (Tables 2-4). In the nuclear
pgiC 14-15
intron sequences, A. subreflexipinna possessed both the A.
and
hasseltii the A. diffracta haplotypes (Table but not that of D. polita (data
5)
not shown). pgiC intron 14-15 sequences of A. diffracta from the three
populations were same
all the (haplotype 'D'), but those of A. hasseltii and A.
subreflexipinna in each population had two to three haplotypes (Tables and
5
There were two major (Ha and Hb) and another three minor haplotypes
6).
Hbl
and Hb2) found
(Hal, in A. hasseltii (Table These minor haplotypes
5).
from
differ their corresponding major haplotypes only one and were
base,
at
found
in the three populations respectively. In total, five and four haplotypes
were found in A. hasseltii and A. subreflexipinna, respectively.
When
calculating haplotype diversity and nucleotide
[h] diversity
[n]
(Table 6), the haplotype "D" was, a priori, removed from the genetic pool of ^.
subreflexipinna to avoid interference in comparison with that of A.
hasseltii.
among
A. haplotype
In hasseltii, diversity these three populations ranged
[h]
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1
from and was
0.533 to 0.689, 0.724 at the species level. In A. subreflexipinna,
it
among
haplotype diversity populations ranged from 0.400 and was
to 0.667,
it
among
0.674 at the species level. Nucleotide diversity the three populations
[n]
of A. hasseltii ranged from 0.00074 to 0.00446, and was 0.00485 the
at
it
among
species level. In A. subreflexipinna, nucleotide diversity populations
ranged from 0.00055 to 0.00553, and was 0.00466 the species
at level.
it
For the nuclear pgiC segment of A. hasseltii and A. subreflexipinna, the Ha
Hb
haplotype could be clearly distinguished from by with
six sites different
base and two The Ha
pairs indel (Table haplotype has minor
sites Fig. a
5; 2).
type (Hal) with a single base difference. This minor haplotype found only
in
is
the Mt. Kentuerhshan population and
of A. hasseltii A. subreflexipinna
On
Hb
(Fig. the other hand, the haplotype has two single base change
2).
minors (Hbl and Hb2) occurring respectively in Lake Chunglingchih and Mt.
Howeishan
populations of A. hassehii (Fig. In A. subreflexipinna, genetic
2(i]).
among
variation different individuals and/or populations directly came from
different haplotypes of A. hasseltii. For example, in A. subreflexipinna of Mt.
Kentuerhshan, except haplotype was
for the that identical to A. diffracta, there
were two haplotypes (Ha and Hal] were found
that also in A. hasseltii of the
sympatric site. Nuclear haplotypes of A. hasseltii in Mt. Howeishan and Lake
Chunglingchih were identical except the two minors (Hbl and Hb2).
for
However, only one major haplotype (Ha) was found in A. hasseltii and A.
The Hb
subreflexipinna of Mt. Kentuerhshan. and derivatively minor
haplotypes were found
neither in A. hasseltii nor A. subreflexipinna of Mt.
Kentuerhshan.
CHANG
ET HYBRID ORIGIN OF ACRORUMOHRA
AL.: SUBREFLEXIPJNNA
D.polita A G
The
among
level of divergence the three populations could not be revealed
by
the organellar fragments because only one haplotype was
detected in each
species (Tables 2-4). For nuclear pgiC 14-15
intron sequences, however,
DnaSP
analysis revealed high among
levels of genetic
differentiation three
= =
populations of A. hasseltii and A. subreflexipinna 0.44377 and
(ysx Yst
0.26399; Table
Additionally, higher levels of genetic were
7). differentiation
also detected between northern and
southeastern populations (A-B and A-C;
Table two
of these species while was
found between
7) differentiation
little
those northern two (B-C; Table For same on
this fragment, the other hand,
7).
had
A. only one haplotype and no
diffracta indicated
pattern of population
and
Hybridization parentage.—
Similar to the traditional circumscription
of
species, hybrid species and hybrid parentage are usually postulated
initially
based on
morphological and
characters degree of spore/pollen abortion
Acrorumohra
(Barrington, 1989, 1990). subreflexipinna suspected
as a
is
natural hybrid between A. hasseltii and A. diffracta (Moore, 2000) because A.
subreflexipinna has abortive spores and intermediate morphology between
A.
hasseltii and A. and occurs sympatrically with two
diffracta, these
species. In
addition, A. subreflexipinna' s spores show no germination, but those of A.
and
hasseltii A. germinate more 80%
diffracta at a rate of than (unpublished
Therefore, A.
data). subreflexipinna appears to be a sterile Fl hybrid.
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D.
polita
Acrorumohra
subreflexipinna with perennial however, could occupy
habit,
its
an original habitat for a long time despite of spores being Repeated
all sterile.
where
hybridization the putative parents sympatrically might
exist also
replenish the stock of hybrid.
this
genomes
Organelle are generally maternally inherited in monilophytes
(Gastony and Yatskievich, 1992; Vogel et ah, 1998). The assumption that
chloroplast and mitochondria are maternally inherited adopted through this
is
was
study. All organellar sequence data indicated that A. hasseltii the maternal
parent of A. subreflexipinna. Nuclear pgiC sequences indicated that A.
was genome
donor
diffracta the other of this hybrid.
Acrorumohra
In addition to the three taxa of discussed here, another
was
species, A. yoroii (Seriz.) Shieh, reported in the second edition of Flora of
Taiwan (Shieh Taiwan, grows high montane and
et al, 1994). In in regions
it
never sympatrically with other three taxa of Acrorumohra. Samples of that
species were also collected from Taiwan and sequenced. has organellar and
It
nuclear sequences different from those of A. subreflexipinna, and phylogenetic
analysis indicates a distant relationship between them (data not shown).
There are three other species of this narrowly defined genus. Acrorumohra
dissecta Ching ex Hsieh distributed in a few locations of southwestern
is
China, and A. obtusissima (Mett. ex Kuhn) Ching and A. undulata (Bedd.)
we
Ching Though
are distributed throughout Sri Lanka. cannot reject the
hypothesis, the possibility of these species contributing to the formation of this
extremely low because and
hyljrid of their restricted habitats disjunct
is
from
distribution hybrid.
this