Zoological Studies 46(5): 547-560 (2007)
Phylogeography of the Taiwanese Endemic Hillstream Loaches,
Hemimyzon formosanus and H. taitungensis (Cypriniformes:
Balitoridae)
Tzi-Yuan Wang1,2, Te-Yu Liao1,3, and Chyng-Shyan Tzeng1,*
1Department
of Life Science, National Tsing Hua University, 101 Kuang-Fu Road, Sec. 2, Hsinchu 300, Taiwan
Research Center, Academia Sinica, 128 Academia Road, Sec. 2, Nankang, Taipei 115, Taiwan. Tel: 886-2-27898756.
Fax: 886-2-27898757. E-mail: d868210@life.nthu.edu.tw
3Department of Vertebrate Zoology, Swedish Museum of Natural History, SE-104 05 Stockholm, Sweden
2Genomics
(Accepted January 23, 2007)
Tzi-Yuan Wang, Te-Yu Liao, and Chyng-Shyan Tzeng (2007) Phylogeography of the Taiwanese endemic hillstream loaches, Hemimyzon formosanus and H. taitungensis (Cypriniformes: Balitoridae). Zoological Studies
46(5): 547-560. Variations in nucleotide sequences within the mitochondrial control region were used to determine the paleogeography of speciation and diversification of 2 balitorids endemic to Taiwan. Examination of 11
populations of Hemimyzon formosanus and 5 populations of H. taitungensis respectively revealed 23 and 11
haplotypes within the mitochondrial control regions. Utilizing the neighbor-joining method and maximum-parsimony trees, we showed the presence of 3 groups and 2 subgroups in H. formosanus, and 1 group in H. taitungensis. The nested clade analysis, a method with a higher resolution, revealed that the 1 group of H. taitungensis could be further divided into 2 subgroups on the minimum spanning network. The nested clade analysis
predicted the evolutionary divergence of populations in H. formosanus due to past fragmentation; furthermore,
dispersion among populations of H. taitungensis was caused by long-distance colonization. The moderate
gene flow and low genetic divergence within the mitochondrial control region suggest that local range expansion and recent colonization occurred in H. formosanus in west-central Taiwan and in H. taitungensis in eastern
Taiwan. Our study showed that one of the major influences on the speciation of both western H. formosanus
and eastern H. taitungensis was the Central Mountain Range of Taiwan. Moreover, deep genetic divergence
and morphological differences suggest new phylogenetic species exist within H. formosanus.
http://zoolstud.sinica.edu.tw/Journals/46.5/547.pdf
Key words: Balitoridae, Morphology, D-loop, Evolutionary history, Cryptic species.
G
eographical barriers, such as mountains,
may influence the speciation and isolate populations from one another. On the other hand, animals can easily disperse in a region without such
barriers. Determining when dispersal and vicariance have alternately influenced colonization and
subdivision of a species by historical biogeography
is quite complicated, but these events frequently
occur in island systems (Emerson 2002, Wiens
and Donoghue 2004, Tzeng et al. 2006).
Mitochondrial phylogeography can provide important insights into evolutionary patterns of popula-
tion fragmentation and gene flow and offers perspective on speciation, diversification, and colonization of species (Avise 1994, Avise and
Wollenberg 1997).
The Central Mountain Range (CMR) is the
most important natural barrier in Taiwan (Fig. 1),
and it separates many vertebrates and invertebrates such as shrimps, crabs, fishes, frogs, and
lizards into 2 distinct groups or sister species
(Chen 1969, Tzeng 1986, Yang et al. 1994, Chang
and Liu 1997, Chou and Lin 1997, Yeh 1997, Toda
et al. 1998, Lin et al. 2002, Liu 2006, Liu et al.
*To whom correspondence and reprint requests should be addressed. Tel: 886-3-5742765. Fax: 886-3-5742765.
E-mail:cstzeng@life.nthu.edu.tw
547
548
Zoological Studies 46(5): 547-560 (2007)
TAIWAN
N
W1
R2
Linco plateau (LP)
25
R1
R3
W2
CHINA
R4
R5
Miaoli plateau (MP)
R6
R7
Hm
R8
W3
24
R16
R9
Formosa Bank (FB)
Taiwan:
R1. IIan River
R2. Danshui River
R3. Touchien River
R4. Zhonggang River
R5.Houlong River
R6. Taan River
R7. Tachia River
R8. Tadu River
R9. Choshui River
R10. Tzengwen River
R11. Kaoping River
R12. Taimali River
R13. Zhiben River
R14. Peinan River
R15. Hsiukuluan River
R16. Hualien River
E1
A-Li Mountain Shoulder Ridge (ARM)
R15
R10
23
W4
R14
R13
Kaoping Ria Coast
R12
R11
Mainland China:
Hm. Nanpan River
E2
Central Mountaion Range (CMR)
W5
22
120
121
Fig. 1. Sampling locations of Hemimyzon species. The circles represent sampling localities. From figure 3, two rivers are located in
region W1 (R1 and R2) and 3 are in region W2 (R3, R4, and R5). Region W3 consists of 4 rivers (R6, R7, R8, and R9). The Tzengwen
River is located in region W4 (R10), and the Kaoping River is in region W5 (R11). Two (R12 and R13) and 3 rivers (R14, R15, and
R16) are respectively located in regions E2 and E1 of eastern Taiwan. Specimens of H. megalopseos were collected from the Nanpan
River of Yunnan Province, China. More-detailed information is given in table 1.
Wang et al. -- Phylogeography of Hillstream Loaches
2007). Like those species, the geographical distributions of 2 different hillstream loaches are also
divided by the CMR barrier; however, their mitochondrial phylogeography offers a different population history. The benthic, loach-like fish of the
genus, Hemimyzon (Cypriniformes: Balitoridae),
can only survive in highly oxygenated, non-polluted, upper reaches of rivers (Hora 1932, Chen
1980, Tzeng and Shen 1982). The distribution of
Hemimyzon formosanus (Boulenger) is mainly in
rivers of western Taiwan (Tzeng and Shen 1982).
On the other hand, H. taitungensis Tzeng and
Shen, the sister species of H. formosanus, is mainly located in eastern Taiwanese rivers. The ability
to adapt to swift currents allows H. taitungensis to
dominate other freshwater fishes in the upstream
regions of eastern rivers (Tzeng and Shen 1982,
Tzeng 1986).
Lin (1957) proposed a number of barriers,
including the CMR, which were thought to have
existed during previous ice ages. For example,
the Miaoli Plateau formed about 300,000 yr ago,
and the Formosa Bank, which is connected to Ali
Mt., are both secondary barriers located in western
Taiwan (Lin 1957, Tzeng 1986). The Formosa
Bank, the land bridge between central Taiwan and
the Nan Mountain range in southern China (Lin
1957), formed during recent ice ages due to lowering of sea levels. The formation of a land bridge
led to the separation of the Northern and Southern
Rivers (Boggs et al. 1979). These barriers and
river systems, like the CMR, have caused various
speciation, diversification, and colonization events
among animal species.
Two modern speciation concepts redefine
“species-level”taxa (Cracraft 1983, Nixon and
Wheeler 1990, Avise 1994, Avise and Wollenberg
1997). The biological species concept (BSC)
states that a species is a reproductively isolated
population, while the phylogenetic species concept
(PSC) defines a species as a group with monophyletically recognizable populations. Evolutionary
history and reproductive ties are 2 related aspects
of both concepts (Avise and Wollenberg 1997).
Thus, by understanding the phylogeography of
species, one can distinguish the relationships
between speciation and population genetics.
According to the species composition and distribution of the freshwater fish fauna of Taiwan,
Tzeng (1986) classified several major biogeographical zones: eastern, northwestern, and southwestern zones and a central intermediate zone
549
(Fig. 1). However, recent phylogeographic patterns have shown some different aspects in comparison to that of Tzeng (Wang et al. 1999, Wang
et al. 2000, Wang et al. 2004). For example,
Varicorhinus barbatulus (Pellegrin) was reported to
be the earliest widespread fish in both western and
eastern Taiwan (Tzeng 1986), but a recent phylogeographic analysis based on molecular data suggests that eastern populations of V. barbatulus
were more recently colonized from southern populations (Wang et al. 2004). The freshwater fish
fauna suggests that most animals may have colonized Taiwan from northern and southern regions
then migrated to central Taiwan (Oshima 1923,
Tzeng 1986). However, mitochondrial (mt)DNA
analysis indicated that the colonization routes of
Acrossocheilus paradoxus (Gunther) were dispersals from central Taiwan to northern and southern
Taiwan; furthermore, the same analysis also indicated that the 3 main regions were isolated during
the last Pleistocene glaciation (Wang et al. 2000).
These phylogeographic patterns point out that the
colonization, emigration, and migration of fish in
Taiwan differ from the currently known routes.
Therefore, in our study, a further phylogeographic
analysis was performed to enhance our understanding of faunal formations of Taiwan.
In contrast to previously studied fishes which
swim well, the hillstream loach is a suitable model
for studying evolutionary history of freshwater fishes because its native populations resulted from
restrictions on hybridization by the various geographical barriers and river courses. In addition,
hillstream loaches are less disturbed by human
activities and have a lower economic value in comparison to other fishes. A phylogeographic analysis of endemic Hemimyzon of Taiwan is therefore
likely to provide a more-accurate scenario of the
influences of the CMR barrier on speciation, subdivision, and diversification of freshwater organisms.
In this study, mitochondrial control region
sequences were used as markers to construct the
phylogeographic patterns of 2 species of
Hemimyzon. A minimum spanning network and
nested clade analysis revealed the spatial patterns
and inferred the historical processes which may
have led to the current distributions. In addition,
genetic and morphological comparisons can further aid our understanding of the geo-historical
influences of diversification at both the inter- and
intraspecific levels.
550
Zoological Studies 46(5): 547-560 (2007)
MATERIAL AND METHODS
Sample collection and morphological measurements
Table 1 and figure 1 indicate the localities and
details of specimens collected from 16 rivers in
Taiwan. In total, 71 extracted DNA samples were
sequenced, including 50 from H. formosanus, 18
from H. taitungensis, and 3 from H. megalopseos
(which was used as the outgroup). The mitochondrial sequences of closely related genera were
obtained from GenBank. Other outgroups utilized
were Jinshaia abbreviate (AY600876), J. sinensis
(DQ105282), and Lepturichthys fimbriata
(DQ105283).
Eighteen morphological measurements and
counts were obtained from each specimen of 59
samples. These samples consisted of 11 individuals of H. taitungensis and 48 individuals of H. formosanus. Measurements made with digital
calipers were rounded up to the nearest 0.1 mm
(Fig. 2).
1
2
3
4
5
10
6
8
9
7
Fig. 2. Measurements taken of Hemimyzon species. 1, standard length, measured from the tip of the head to the posterior
edge of the hypural plate; 2, predorsal length, measured from
the tip of the upper jaw to the anterior edge of the dorsal insertion; 3, prepelvic length, measured from the tip of the upper jaw
to the anterior edge of the pelvic insertion; 4, head length, measured from the tip of the head to the posterior edge of the operculum; 5, snout length, measured from the tip of the head to
the anterior margin of the osseous orbit; 6, head depth, at the
level of the occiput; 7, orbital diameter, the distance between
the horizontal margins of the osseous orbit; 8, body depth, at
the level of the dorsal fin origin; 9, caudal peduncle depth, at
the level of the end of the anal fin base; 10, caudal peduncle
length, measured from the end of the anal fin base to the end
of the hypural plate; the interorbital width is the cross distance
between the upper margins of each osseous orbit; the head
width is the cross distance between the anterior pectoral fin
bases; the body width is the cross distance between the anterior pelvic fin bases; and the mouth width is the cross distance
between the interior corners of the mouth.
DNA extraction, polymerase chain reaction
(PCR), and sequencing
A piece of pectoral fin or pelvic fin, of approximately 50 mg, was immersed in 500 µl digestion
buffer (10 mM Tris-HCl (pH 8), 1% SDS, 2 mM
EDTA, 10 mM NaCl, 10 mg/ml DTT, and 0.5 mg/ml
proteinase K; modified from Kocher et al. 1989);
preparations were incubated for 16 h at 50 C in a
dry bath. DNA was isolated and purified by a phenol/chloroform-isoamyl alcohol extraction (Innis et
al. 1989). The control region of mtDNA was amplified and sequenced using the forward primers
(PK2, PK3-1, and U2) and reverse primers (PU3-1
and PU2); the primers were designed in correspondence with the nucleotide positions in the
light-chain of mtDNA of Formosania lacustre
,
(M91245): PK3-1 (L68-L84, D-loop): 5 -TATTTA
,
,
GACCATAAAGC-3 ; U2 (L234-L253, D-loop): 5 ,
AGTAAGAAACCACCAACCAG-3 ; PU3-1 (L912,
,
L898, D-loop): 5 -TTAAGCTACGCTAGC-3 ; PU2
,
(L1046-L1026, 12S-RNA): 5 -GGGCATTCT
,
CACGGGGATGCG-3 ; and PK2 (L164504,
L16530, tRNA Thr): 5 -GTCGACTCTCACCCCTG
,
GCTCCCAAAG-3 .
PCR amplifications were performed in a volume of 50 µl containing 30-100 ng DNA, 200 µM of
each dNTP, 0.3 µM of each primer, and 1 unit of
SuperTaq with the reaction buffer (HT
Biotechnology, Carbridge, UK). The PCR conditions were optimized as follows: a single hot startup cycle of 93 C for 3 min; 35-40 cycles of denaturation at 93 C for 30 s, annealing at 40-55 C for
40 s, and extension at 72 C for 1 min; and a single final extension cycle of 72 C for 10 min. The
amplification procedure was repeated 3 times.
The product mixture was employed as the template for sequencing, which was performed using
the Sequenase PCR Product Sequencing Kit
(United States Biochemical, Illinois, USA) and dyelabeled terminator sequence kits (Applied
Biosystems and Amersham Pharmacia, CA, USA)
on an ABI model 377 automated DNA sequencer.
Each haplotype was submitted to the GenBank
database and assigned the following accession
nos.: AY284892-AY284924 and AY541592AY541597.
°
°
°
°
°
°
Genetic divergence and phylogenetic analysis
Sequences were aligned using ClustalX
(Thompson et al. 1997) and checked visually.
Phylogenetic analyses were performed, first using
the neighbor-joining (NJ) method (Saitou and Nei
551
Wang et al. -- Phylogeography of Hillstream Loaches
1987) with Tamura-Nei (TN) gamma distances
(Tamura and Nei 1993) and 5000 bootstrap replications (Felsenstein 1985), as implemented in
MEGA (vers. 2.1, Kumar et al. 2001). Maximumparsimony (MP) analyses were conducted using a
random addition heuristic search with tree-bisection-reconnection (TBR), and 1000 bootstrap replications in PAUP* (vers. 4.0b10, Swofford 1998).
Genetic diversity was quantified at the inter- and
intra-population levels using DnaSP (vers. 3.99,
Rozas et al. 2003) to calculate the index of haplotype diversity (h) (Nei 1987), estimates of
nucleotide diversity (π) (Nei 1987), and FST for
gene flow (Hudson et al. 1992). A hierarchical
analysis of molecular variance (AMOVA) was performed using Arlequin (vers. 2.0, Schneider et al.
2000) to compare the component of genetic diversity for the variance among subdivisions and
species.
Genetic data were also employed to establish
a minimum spanning network with TCS vers. 1.13
(Clement et al. 2000), a tree that helps reveal
information about distribution patterns (Chiang and
Schaal 1999). The NCA combines genetic and
geographical data to provide inferences about the
recent geographic history of populations at the
intraspecific level (Templeton 1998) and is performed using GeoDis (Posada et al. 2000). A
nested structure derived from the minimum-spanning network, together with information on the geographical distribution of the haplotypes, is used to
estimate 2 geographical measures for each clade:
the clade distance (Dc) and the nested-clade distance (Dn). Dc is a measure of the geographical
extent of a given clade, while Dn measures the
average geographical distance of individuals from
1 clade to another in the next higher-level clade,
within which it is contained. The analysis performed by GeoDis allows inference of a range of
Nested clade analysis (NCA)
Table 1. Sampling localities according to the zoographical zone, major basin, and population. Localities of
the 2 species of Hemimyzon are followed by the number of individuals surveyed (n), the number of unique
haplotypes, mtDNA lineage, haplotype diversity (h), and nucleotide diversity (π)
Zoographical zone
H. formosanus (Hf)c
Northern zone
Central/intermediate zone
Southern zone
H. taitungensis (Ht)d
Eastern zone
H. megalopseos (Hm)
aThree
Basin
Population n Haplotypes
mtDNA lineage
h ± SD
π ± SD
Ilan River/Taiwan
Danshui River/Taiwan
R1
R2
2
6
2
5
2-2 (W1)
2-2 (W1)
1.000 ± 0.500
0.933 ± 0.122
0.0022 ± 0.0015
0.0032 ± 0.0012
Touchien River/Taiwan
Zhongkong River/Taiwan
Houlong River/Taiwan
Taan River/Taiwan
Tachia River/Taiwan
Tadu River/Taiwan
Choshui River/Taiwan
R3
R4
R5
R6
R7
R8
R9
5
2
3
6
3
5
6
2
1a
2a
4a
2
1
1
2-1 (W2)
2-1 (W2)
2-1 (W2)
2-1 (W2,W3)
2-1 (W3)
2-1 (W3)
2-1 (W3)
0.400 ± 0.237
0.000 ± 0.000
0.667 ± 0.314
0.800 ± 0.172
0.667 ± 0.314
0.000 ± 0.000
0.000 ± 0.000
0.0009 ± 0.0006
0.0000 ± 0.0000
0.0007 ± 0.0008
0.0029 ± 0.0011
0.0023 ± 0.0013
0.0000 ± 0.0000
0.0000 ± 0.0000
Tzengwen River/Taiwan
R10
7
3
3-2 (W4)
0.524 ± 0.209
0.0012 ± 0.0007
Kaoping River/Taiwan
R11
5
2
3-3 (W5)
0.400 ± 0.237
0.0005 ± 0.0005
Taimali River/Taiwan
Zhiben River/Taiwan
Peinan River/Taiwan
Hsiukuluan River/Taiwan
Hualien River/Taiwan
R12
R13
R14
R15
R16
2
2
4
2
8
1
1
4
2b
4b
2-4 (E2)
2-4 (E2)
2-3 (E1)
2-3 (E1)
2-3 (E1)
0.000 ± 0.000
0.000 ± 0.000
1.000 ± 0.177
1.000 ± 0.500
0.643 ± 0.184
0.0000 ± 0.0000
0.0000 ± 0.0000
0.0047 ± 0.0016
0.0011 ± 0.0011
0.0014 ± 0.0006
Nanpan River/China
Hm
3
2
-
0.667 ± 0.314
0.0082 ± 0.0023
populations share a common Hf8 haplotype. bTwo populations share a common Ht8 haplotype. cThe average haplotype
diversity and nucleotide diversity of all pooled samples within the species were 0.495 ± 0.013 and 0.0149 ± 0.0022, respectively. dThe
average haplotype diversity and nucleotide diversity of all pooled samples within the species were 0.889 ± 0.064 and 0.0049 ± 0.0010,
respectively.
552
Zoological Studies 46(5): 547-560 (2007)
phylogeographic processes, including range
expansion (either contiguously or by long-distance
colonization), isolation by distance due to restricted gene flow, fragmentation of populations (including extinction of intermediate populations), and
various combinations of these possibilities. There
are 4 steps in the NCA: constructing a haplotype
network, nesting clades on the network, testing for
geographic associations, and determining inferences about the processes that have generated
the pattern. An inference key was provided by
Posada and Templeton (2005), and updates (the
most recent of which was used here) are available
on the GeoDis website.
RESULTS
Mitochondrial DNA variations
In both H. formosanus and H. taitungensis,
the length of the D-loop regions within mitochondria ranged from 893 to 899 base pairs (bp). In
addition, the average nucleotide compositions in
the D-loop region were 31.8% T, 18.3% C,
36.0% A, and 13.9% G. Furthermore, the
nucleotide compositions indicated that this region
within both species was AT-rich, and similar observations were found in many other vertebrates
(Brown el al. 1986, Tzeng et al. 1992, Lee et al.
1995, Perdices and Doadrio 2001).
In H. formosanus, 23 haplotypes were identi-
(a) NJ tree
(b) MP tree
Hf5(R2)
58
Hf22(R1)
0.01
Hf23(R1)
Hf4(R2, n = 2) W1
4 bp deletion
Hf1(R2)
Hf2(R2)
Hf3(R2)
95
Hf9(R5)
Hf8(R4,R5,R6, n = 5)
Hf6(R3, n = 4) W2 group
Hf7(R3)
H. formosanus
Hf11(R6)
Hf12(R6, n = 3)
100
Hf13(R7, n = 2)
63
Hf10(R6)
W3
Hf14(R7)
Hf15(R8,
n
=
5)
93
Hf16(R9, n = 6)
100 Hf18(R10)
Hf17(R10, n = 5)W4
group
Hf19(R10,)
99
Hf20(R11, n = 3) W5
group
Hf21(R11)
Ht6(R14)
80
Ht4(R14)
Ht7(R15)
Ht3(R14)
Ht8(R15,R16, n = 6)
E H. taitungensis
Ht9(R16)
Ht5(R14)
Ht10(R16)
Ht11(R16)
Ht1(R12, n = 2)
Ht2(R13, n = 2)
100
Hm1
100
80
Hm2(n = 2)
J. abbreviata
J. sinensis
90
L. fimbriata
H. megalopseos
84
51
60
I
100
79
90
55
99
II
60
III
100
87
81
99
Hf3
Hf2
Hf1
Hf4
Hf22
Hf23
Hf5
Hf6
Hf7
Hf9
Hf8
Hf11
Hf12
Hf13
Hf10
Hf16
Hf14
Hf15
Hf17
Hf18
Hf19
Hf20
Hf21
Ht7
Ht3
Ht10
Ht8
Ht5
Ht6
Ht4
Ht9
Ht11
Ht1
Ht2
W1
W2
W3
W4
W5
E
Hm1
Hm2
J. abbreviata
J. sinensis
L. fimbriata
Fig. 3. Phylogenetic tree of the 2 balitorid fishes. (a) Neighbor-joining (NJ) phylogram constructed from Tamura-Nei (gamma) distances in the D-loop region with 3 major groups in Hemimyzon formosanus. (b) Three groups within the maximum-parsimony (MP)
cladogram. The numbered nodes indicate NJ/MP bootstrap values (%). Values for each subgroup are shown. Abbreviations are
explained in table 1.
553
Wang et al. -- Phylogeography of Hillstream Loaches
(a) H. formosanus
3-1
Hf3
Hf1
Hf2
Hf4
Hf23
Hf5
Hf22
W1
Hf17
9
Hf19
12
1-3
Hf6
Hf7
15
Hf18
Hf8
3-2
Hf15
W4
Hf9
Hf10
Hf14
8
Hf16
Hf11
1-2
Hf20
Hf12
Hf21
3-3
2-1
Hf13
W5
W2+W3
1-1
28
E
1-5
2-3
Hf3
Hf7
1-4
E2
1-8
Hf9
Hf1
Hf8
Hf10
2-4
Hf11
Hf2
1-9
Hf4
Hf5
Hf6
1-6
E1
1-7
(b) H. taitungensis
Fig. 4. Minimum spanning haplotype network. (a) In Hemimyzon formosanus; (b) in H. taitungensis. Unfilled circles indicate unsampled intermediate haplotypes with a single mutation from the neighboring haplotype. Numbers next to the dotted and dashed lines indicate the numbers of mutational events which occurred.
554
Zoological Studies 46(5): 547-560 (2007)
fied, most of which were not shared among different populations (Table 1). However, 1 haplotype
(Hf8) was shared in populations from 3 rivers: the
Zhongkong (R4), Houlong (R5), and Taan (R6)
Rivers. On the other hand, in H. taitungensis, 11
haplotypes were discovered, and 1 shared haplotype (Ht8) existed in populations from the
Hsiukuluan (R15) and Hualien (R16) Rivers.
,
A 4-bp indel located at the 5 -end of the control region in mtDNA was identified in populations
of both species. The indel was observed in all
haplotypes of the Kaoping River (R11) in H. formosanus (Fig. 3a), and was present in all haplotypes of H. taitungensis.
Among H. formosanus, the average haplotype
diversity (h) of samples from all populations was
0.495 ± 0.013, and the average nucleotide diversity (π) of samples from all populations was 0.0149
± 0.0022 (Table 1). On the other hand, in H.
taitungensis, the average haplotype diversity of
samples from all population was 0.889 ± 0.064,
and the average nucleotide diversity of samples
from all populations was 0.0049 ± 0.0010. By
comparison, the average haplotype diversity was
about 2 times higher in H. taitungensis than in H.
formosanus. The average nucleotide diversity was
3 times higher in H. formosanus than in H. taitungensis.
Phylogenetic patterns
Populations of H. formosanus were divided
into 3 major groups: group I consisted of populations W1, W2, and W3; group II contained population W4, and group III consisted of population W5
(Fig. 3a). The phylogenetic relationships between
each group were supported by high bootstrap values and by high confidence from the interior
branch test (Nei et al. 1985, Nei and Kumar 2000)
(data not shown). In addition, the 3 major groups
diverged at different genetic distances. A similar
pattern of evolutionary relationships was also
observed in the MP tree (Fig. 3b). Although the
W1 group might have appeared paraphyletic in the
MP tree, it appeared monophyletic in the NJ tree
(with a bootstrap value of 95) and in the haplotype
network (with 3 mutational events). Therefore, W1
could be classified as a subgroup of group I.
Populations from central Taiwan (W2 and W3)
were shown to be a hybrid group, which could be
separated into 2 subgroups with intermediate bootstrap values (of 63 and 79) in both the NJ and MP
trees. The slightly lower bootstrap values were
because the haplotypes from the Taan River were
classified into both groups W2 and W3. Group W2
included populations from the Touchien (R3),
Zhongkong (R4) and Houlong (R5) Rivers, and 1
individual from the Taan River (R6). On the other
hand, group W3 consisted of populations from the
Tachia (R7), Tadu (R8), and Choshui (R9) Rivers,
and 4 individuals from the Taan River (R6).
Although AMOVA indicated genetic structural differences within the subgroups (Table 2), populations from W2 and W3 were indiscernible and were
classified into a single group.
From both the NJ and MP trees of H. taitungensis, a monophyletic group (E) with high bootstrap values (100 and 99) was comprised of all
haplotypes with a short branch length, and all following subdivisions revealed low bootstrap values,
implying no further subdivisions, unlike the deep
genetic divergence among groups I, II, and III of H.
formosanus.
Haplotype networks and the NCA
The haplotype networks were generally congruent with the NJ and MP trees (Fig. 4). Eleven
populations of H. formosanus were divided into 3
major groups (clades 3-1, 3-2, and 3-3) in the haplotype network. Greater mutational events were
discovered among clades 3-1 (groups
W1+W2+W3), 3-2 (group W4), and 3-3 (group
W5). Furthermore, clade 3-1 contained clades 2-1
and 2-2; clade 2-1 consisted of groups W2 (clades
1-2 and 1-3) and W3 (clade 1-1), and clade 2-2
included group W1. Although W3 formed a subgroup, groups W2 and W3 merged in the haplotype network due to the shared haplotypes from
the Taan River (Hf8, Hf10, Hf11, and Hf12). Both
the phylogenetic analysis and haplotype network
classified the 11 populations of H. formosanus into
3 major groups and 2 subgroups.
However, in H. taitungensis, results from the
constructed NJ and MP trees were inconsistent
with the results from the haplotype network
because 2 subgroups (clades 2-3 and 2-4) were
identified in the latter method. Clade 2-3 (E1) represented populations from 3 east-central rivers,
while clade 2-4 (E2) represented 2 southeastern
populations (from the Taimali and Zhiben Rivers).
This inconsistency could be explained by the
enhanced sensitivity of the haplotype network
(Templeton 1998); moreover, in the“Discussion”
section, the haplotype network results were given
greater emphasis over the NJ and MP tree results.
555
Wang et al. -- Phylogeography of Hillstream Loaches
The most recent NCA key (Posada and
Templeton 2005) was used to infer the most likely
geographic patterns and their associations in the
evolutionary history (Table 3). The information
provided by the NCA key indicated that the 3 major
divisions were caused by past fragmentation in H.
formosanus, and long-distance colonization/range
expansion occurred in H. taitungensis.
(E2) of H. taitungensis. This genetic distance was
almost equal to the value between the Kaoping
population and the remaining populations of H. formosanus (3.91% ± 0.66% to 4.09% ± 0.65%;
mean value, 4.03% ± 0.64%), but was 1/3 lower
than the mean distance between the 2 species
(mean value, 5.87% ± 0.81%).
The high FST values indicated the occurrence
of low gene flow between the groups (except for H.
taitungensis and group I of H. formosanus; Table
4). A moderate FST value was calculated between
groups E1 and E2, and slightly higher FST values
were calculated between groups W1 and W2 and
between groups W1 and W3. In addition, a higher
frequency of gene flow appeared between groups
W2 and W3 in west-central Taiwan.
Genetic differentiation
Deep genetic divergence was observed
between the Kaoping population (W5) and the
remaining populations of H. formosanus. The
genetic distance within group W5 (0.05% ± 0.05%)
was the lowest, while group W1 had the highest
within-group distance (0.35% ± 0.12%; Table 4).
The genetic distance between groups was the
highest between groups W3 and W5 (4.09% ±
0.65%), and was the lowest between groups W2
and W3 (0.41% ± 0.13%).
In H. taitungensis, the genetic distance within
the southern group E2 (0.77% ± 0.25%) was 3
times higher than in the central group E1 (0.24% ±
0.07%). In addition, the genetic distance between
these 2 groups was 0.91% ± 0.25%, which is
slightly higher than the genetic distance within
group E2, and it was much higher than the distance within group E1 (Table 4).
The interspecific genetic distance was 3.99%
± 0.65% between the Kaoping population (W5) of
H. formosanus and the 2 southeastern populations
Morphological comparisons
Due to the deep genetic divergence (4.03%)
of the southern population of H. formosanus, it is
possible to classify the Kaoping population as a
new biological or phylogenetic species or subspecies. Morphological comparisons can help
explain this variance in genetic diversity. Fortyeight specimens of H. formosanus and 11 H.
taitungensis specimens were morphometrically
analyzed. In total, 18 measurements and counts
were made (Table 5). According to the biogeographic analysis described by Tzeng (1986), we
separated H. formosanus ranges into 3 main
zones (northern, southern, and central/intermedi-
Table 2. Hierarchical analysis based on the genetic distance among species,
subdivisions and distinct zones of Hemimyzon formosanus
Source of variation
d.f.
Sum of
squares
Variance
components
Percent of
variation
Among species
Among subdivisions within species
Within subdivisions
Total
1
5
60
66
589.632
299.877
67.302
956.811
19.26579
6.83732
1.12169
27.22480
70.77*
25.11**
4.12**
Among subdivisions
Among populations within subdivisions
Within populations
Total
4
5
33
42
275.881
19.020
16.310
311.211
7.72604
0.90929
0.49424
9.12956
84.63**
9.96**
5.41**
Among distinct zones
Among subdivisions within distinct zones
Within subdivisions
Total
2
2
44
48
179.709
105.851
43.240
328.801
3.17979
5.32995
0.98273
9.49248
33.50
56.15**
10.35**
* p < 0.05; ** p < 0.01.
556
Zoological Studies 46(5): 547-560 (2007)
ate zones). The northern zone was comprised of
group W1; the central zone included groups W2
and W3; while groups W4 and W5 belonged to the
southern zone. Unfortunately, none of these measurements could be utilized to establish group W5
as a new species due to large morphological variations within the central group. However, after the
specimens were excluded from the central/intermediate zone, 2 measurements were determined
that can help discern specimens between the
northern and southern zones. The 2 measurements are the values of the lateral line scales and
the ratios of the standard length to the caudal
peduncle depth. According to the ANOVA test,
some groups exhibited a significant difference in
these 2 measurements.
DISCUSSION
The phylogeographical and morphological
analyses of H. formosanus and H. taitungensis
offer a new scenario for the speciation of aquatic
organisms in Taiwan. Our data suggest that the
population of H. formosanus in western Taiwan
can be divided into 3 groups and 2 subgroups.
This division pattern is partially congruent with the
patterns of other freshwater fishes. In addition,
mean genetic distances revealed that the Kaoping
population of H. formosanus (group W5) is more
divergent from the other H. formosanus populations than from H. taitungensis. These results indicate the presence of cryptic species in the Kaoping
River and an unusual evolutionary history for these
2 balitorids in Taiwan.
Phylogeographical implications
The phylogenetic tree and NCA inferences
present a scenario of the evolutionary history of H.
formosanus in Taiwan: the spatial patterns are the
results of 3 major fragmentations followed by subsequent local range expansion and colonization.
The 3 major groups (W1+W2+W3, W4, and W5) of
H. formosanus are potentially consequences of
past fragmentations (Table 3). The genetic differ-
Table 3. Chains of inference from the nested clade analysis. Haplotype and clade designations are given in
figure 4
Clade
Inference chain
Inferred pattern
Hemimyzon formosanus
1-1
1-2-3-5-6-13-14-15 NO
2-1
1-2-11-12-13-14 NO
3-1
1-2-11-12-13 YES
Total 1-2-11-12-13-14 NO
H. taitungensis
2-3
1-2-3-4 NO
Total 1-2-11-12-13 YES
Past fragmentation (PF) or long-distance colonization (LDC)
Colonization event is inferred, perhaps associated with recent fragmentation (CRF)
Past fragmentation followed by range expansion (PF-RE)
Past fragmentation (PF)
Restricted gene flow with isolation by distance (RGF)
Long-distance colonization possibly coupled with subsequent fragmentation (LDC-SF) or past
fragmentation followed by range expansion (PF-RE)
Table 4. Genetic divergence and standard deviation (%) within (bold) and between (lower left matrix) different subdivisions; the upper right matrix indicates the FST value. High FST values (0.8-1.0) reveal low gene
flow between subdivisions
W1
W2
W3
W4
W5
E2
E1
aModerate
W1
W2
W3
W4
W5
E2
E1
0.35 ± 0.12
0.81 ± 0.25
0.94 ± 0.25
2.32 ± 0.49
3.91 ± 0.66
5.71 ± 0.85
5.77 ± 0.87
0.650a
0.18 ± 0.09
0.41 ± 0.13
2.61 ± 0.54
4.01 ± 0.67
5.87 ± 0.86
5.94 ± 0.88
0.618a
0.455a
0.26 ± 0.09
2.65 ± 0.54
4.09 ± 0.65
6.06 ± 0.87
6.12 ± 0.90
0.878
0.925
0.883
0.12 ± 0.07
4.03 ± 0.67
6.21 ± 0.92
6.36 ± 0.96
0.956
0.977
0.950
0.971
0.05 ± 0.05
3.99 ± 0.65
4.47 ± 0.70
0.939
0.957
0.936
0.955
0.961
0.77 ± 0.25
0.91 ± 0.25
0.908
0.926
0.907
0.925
0.918
0.512a
0.24 ± 0.07
FST values (0.3-0.7); low FST values (0-0.2).
557
Wang et al. -- Phylogeography of Hillstream Loaches
entiation within the species of H. formosanus is 2-3
times greater than those seen in A. paradoxus and
V. barbatulus. The different degrees of divergence, along with the regional separations
deduced by NCA inferences, imply the occurrence
of early fragmentations. Furthermore, the molecular clock calculations suggest that fragmentations
appeared during the early Pleistocene (Wang et al.
2007). Therefore, this deep divergence may have
been due to earlier subdivisions related to isolated
refugia, lower gene flow, or specific habits separating populations of H. formosanus from north-cen-
tral and southern Taiwan.
On the other hand, recent fragmentations and
local range expansion took place in central Taiwan.
Herein, the inference of nested clade 3-1 suggests
that the separation of the northern and central
groups likely resulted from recent fragmentation
followed by range expansion. In addition, clade 21 (W2+W3) was inferred to have resulted from a
colonization event followed by recent fragmentation in central Taiwan. During the middle or late
Pleistocene, a decrease in the sea level or flooding
in central Taiwan resulted in changes in the river
Table 5. Morphological comparison of Hemimyzon formosanus and H. taitungensis (units: mm).
Unbranched fin-rays are represented as Roman numerals and branched rays are represented as Arabic
numerals. n is the number of individuals sampled; p is the number of populations within each area. Bold
and underlined letters indicate differences between the 2 species and 2 subdivisions, respectively
H. formosanus
Distinct zone
Northern zone
Subdivision
n (p)
W1
4 (1)
Dorsal fin rays
iii-8
Anal fin rays
ii-5
Pectoral fin rays
x-xi/10-11
Ventral fin rays
iv/9
Lateral line scales
68-71 (70)**
Standard length
32.0-59.4 (43.0)
Standard length /
6.67-7.96 (7.43)
body depth
Standard length /
4.18-4.83 (4.54)
body width
Standard length /
4.95-5.33 (5.17)
head length
Standard length /
6.15-7.66 (6.95)
caudal peduncle length
Standard length /
8.29-8.89 (8.61)**
caudal peduncle depth
Head length /
1.62-1.74 (1.68)
head depth
Head length /
0.87-0.97 (0.92)
head width
Head length /
2.13-2.17 (2.15)
snout length
Head length /
4.62-5.23 (4.93)
orbital diameter
Head length /
1.88-2.21 (2.04)
interorbital width
Caudal peduncle
1.13-1.44 (1.25)
length / caudal
peduncle depth
Head width / mouth width 2.74-3.00 (2.88)
H. taitungensis
Central/intermediate zone
W2
6 (2)
W3
25 (4)
Southern zone
Eastern zone
W4
8 (1)
W5
5 (2)
E1+E2
11 (3)
iii-8
iii-8
ii-5
ii-5
x-xii/9-11
x-xii/8-11
iv/7-10
iii-v/8-11
70-76 (74)
68-75 (71)**
58.6-74.0 (65.0) 37.6-73.5 (52.07)
5.95-7.89 (6.91) 5.48-9.09(7.02)
iii-8
ii-5
ix-xii/10-11
iii-iv/8-9
72-76 (74)**
45.1-60.0 (52.9)
7.27-8.34 (7.65)
iii-8
ii-5
xii/10-11
iv-v/9-10
70-73 (72)
34.7-49.1 (51.7)
7.17-8.45 (7.92)
iii-8
ii-5
xii-xv/10-13
v-vii/10-11
78-81 (79)
49.9-73.0 (61.0)
6.27-8.80 (7.50)
3.81-4.56 (4.29)
3.57-5.40(4.15)
4.22-4.97 (4.72)
3.99-4.75 (4.47)
4.54-5.29 (4.89)
4.61-5.83 (5.04)
4.33-5.69(4.86)
4.74-5.61 (5.11)
4.47-5.27 (4.86)
4.70-5.37 (5.01)
6.81-8.06 (7.46)
6.22-9.41(7.41)
6.99-7.87 (7.58)
6.33-8.00 (7.22)
6.16-7.23 (6.57)
9.11-10.57 (9.85) 8.05-11.79(10.1)** 9.40-10.76 (10.2) 10.52-12.93 (11.5)** 9.16-10.85 (9.76)
1.74-1.94 (1.86)
1.63-2.01(1.83)
1.69-1.93 (1.79)
1.73-2.31 (2.02)
1.77-2.26 (1.97)
0.91-1.05 (0.97)
0.81-1.05(0.93)
0.85-1.04 (0.93)
0.89-1.05 (0.98)
0.94-1.13 (1.00)
1.90-2.30 (2.06)
1.84-2.23(2.04)
1.88-2.25 (2.05)
1.97-2.07 (2.02)
1.93-2.51 (2.18)
5.08-6.00 (5.56)
4.11-6.45(5.49)
5.00-6.33 (5.63)
4.72-5.47 (5.00)
5.19-7.76 (6.02)
1.76-2.31 (1.98)
1.58-2.15(1.85)
1.67-2.18 (1.86)
1.81-2.07 (1.97)
2.11-2.41 (2.22)
1.14-1.54 (1.33)
1.05-1.85(1.38)
1.21-1.51 (1.35)
1.51-1.71 (1.59)
1.34-1.76 (1.49)
2.51-2.93 (2.73)
2.42-3.28(2.87)
2.50-2.84 (2.66)
2.52-2.88 (2.70)
2.41-3.20 (2.85)
** p < 0.01 by one-way ANOVA with each other
558
Zoological Studies 46(5): 547-560 (2007)
systems (Lin 1957, Emery et al. 1971); thus the
hillstream loach populations may have dispersed
and interacted with each other in central Taiwan.
However, owing to the increase in the sea level or
the appearance of geographic boundaries, the
populations may have been re-isolated from one to
another. Alternate dispersal and vicariance events
may have been the driving forces for interactions
of groups W2 and W3; therefore, slightly diversified subgroups still exist as supported by the NJ
tree. This explanation is supported by the identification of a shared haplotype (Hf8) and moderate
gene flow (FST = 0.455) between the 2 populations
in the central region. Similar inferences were discovered in the analyses of A. paradoxus and V.
barbatulus in central Taiwan as well.
The haplotype network divided the populations of H. taitungensis into 2 nested clades (Fig.
4). The causes of the separation of clades 2-3
(group E1) and 2-4 (group E2) remain uncertain.
Two possible causes are proposed: the separation
could have resulted from long-distance colonization coupled with subsequent fragmentation, or
from recent fragmentation followed by 1 or more
range expansions. Group E1, which includes most
of the haplotypes and was constructed as a lowerlevel clade, was inferred to have restricted gene
flow due to isolation by distance (Table 3). As suggested by the center of the haplotype network
(clade 1-4), the Hualien River (R16) could be the
expansion center for H. taitungensis in eastern
Taiwan. In our study, a long geographical distance
between the expansion center and group E2 was
observed. In addition, the shared haplotype (Ht8)
and moderate FST value (0.512) between groups
E1 and E2, evidence for the occurrence of gene
flow, imply that at least 1dispersal event took place
in eastern Taiwan. Therefore, long-distance colonization coupled with subsequent fragmentation
appears to be a better explanation for the results
of the haplotype network. A low genetic distance
(< 1%) and moderate gene flow indicate at least 1
recent colonization event in eastern Taiwan, which
is similar to the pattern observed for V. barbatulus
during the last Pleistocene glaciation.
The molecular clock estimates of the
cytochrome b gene revealed that the speciation
and sequential subdivisions of Hemimyzon species
occurred approximately 2-4 million yr ago (Wang
et al. 2007). The speciation time of the 2 species
is in the vicinity of the formation time of the CMR
barrier, which implies that the CMR barrier may
have participated in separating the common
ancestor of H. formosanus and H. taitungensis into
their present distributions in western and eastern
Taiwan. Sequential subdivisions in H. formosanus
may have played crucial roles in the genetic variations in northern, central, and southern populations, the strength of which is correlated to different divergence times and degrees of isolation in
the west.
Genetic and morphological implications
The deep genetic divergence implies that
cryptic species might have arisen from the southern population of H. formosanus, which is unique
in differing from the shallow genetic divergence
known for other freshwater fishes in Taiwan. The
AMOVA indicated significant spatial patterns of
genetic structure among each group within the distinct zones (Table 2). Both the phylogenetic tree
and NCA supported the divergence patterns of
each group. Furthermore, the results indicated
slight morphological differences between the
northern and southern groups (Table 5). A recent
study (Chen and Chang 2005) also described several morphological differences between the southern population of H. formosanus and other populations, which supports the southern population
being a cryptic species. Both measurements could
be utilized to determine the possibility of the cryptic
species being classified as a distinct species from
H. formosanus.
From our study, a 4-bp indel was identified in
1 cryptic species from the Kaoping population
(W5) and all populations of H. taitungensis.
However, this indel was absent from the northern
population (W1) and most of the central populations (W2 and W3) of H. formosanus, except for 1
haplotype in the Taan River. Thus the cryptic
species exhibited a higher degree of similarity to
H. taitungensis than to H. formosanus. Moreover,
the genetic distance (3.99% ± 0.65%) between the
Kaoping population (W5) of H. formosanus and
southeastern population (E2) of H. taitungensis
was equal to the mean distance (4.03% ± 0.64%)
between the W5 group and other populations of H.
formosanus. The deep genetic distance between
the Kaoping population and other populations of H.
formosanus implies a new phylogenetic species in
southern Taiwan. The phylogeographic analysis,
the variance in the morphology/mitochondrial
sequence length, the deep genetic distance, and
Wang et al. -- Phylogeography of Hillstream Loaches
the isolated habitats all suggest that the Kaoping
population of H. formosanus may have resulted
from early fragmentation, and it may correspond to
a new cryptic species.
In conclusion, the phylogeography reveals
that during the Pleistocene, the main factor separating populations of H. formosanus was sequential fragmentations, and the main cause for the
emergence of H. taitungensis was long-distance
colonization. Genetic and morphological variations
imply that the Kaoping population of H. formosanus represents a phylogenetic species or
subspecies. Future studies should focus on
detailed analyses of anatomical comparisons and
reproductive barriers to confirm the identity of the
cryptic species.
Acknowledgments: We are grateful to Yong-Zhou
Chang (Tzu Chi University, Hualien, Taiwan),
Chun-Huo Chiu (National Chiao Tong University,
Hsinchu, Taiwan), and Hung-Du Lin (National
Cheng Kung University, Tainan, Taiwan) for kindly
providing the samples. We also thank Tiffany
Chang, Pei-Fang Chuang, James Khoo, Jonathan
Ready, and anonymous reviewers for their valuable comments on the manuscript. This research
was financially supported by the National Science
Council of Taiwan (NSC86-2311-B-002-028-B17
and NSC87-2311-B-002-015-B17) and sample collecting permission was obtained from the Council
of Agriculture, Executive Yuan (Taipei, Taiwan).
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