invasion, genetic variation and species identity of the calanoid copepod sinodiaptomus valkanovi

Invasion, genetic variation and species identity of thecalanoid copepod Sinodiaptomus valkanovi

WATARU MAKINO*, MATTHEW A. KNOX † AND IAN C. DUGGAN†

*Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan†Centre for Biodiversity and Ecology Research, Department of Biological Sciences, University of Waikato, Hamilton, New Zealand

SUMMARY

1. Although Sinodiaptomus valkanovi [sensu Ueda & Ohtsuka (Hydrobiologia, 379, 1998, 159)]

is one of the most common freshwater calanoid copepods in Japan, it was originally

described from specimens collected in Sofia, Bulgaria, as a subspecies of S. sarsi. This

original description raises two issues requiring further investigation. One is whether or not

S. valkanovi should be differentiated from S. sarsi at a species level, and the other is whether

or not records of S. valkanovi from outside of Japan are the result of biological invasions.

2. We examined the gene flow between S. valkanovi and S. sarsi in Japan, using nuclear

DNA (the ribosomal internal transcribed spacers 1 and 2 plus the intervening 5.8S

ribosomal DNA subunit) sequence to clarify if these two taxa are separable at a species

level. We also investigated the population genetic structure of S. valkanovi in Japan, using

the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene, and subsequently of two

recently discovered New Zealand populations, to elucidate the origin and colonisa-

tion ⁄ invasion history of this taxon.

3. In total, 43 and three Japanese populations of S. valkanovi and S. sarsi, respectively, were

analysed. These two taxa did not occur together in any of the localities. Nuclear DNA

analysis did not provide any evidence of gene flow between them, and the inter-taxon

variability of mtCOI was high, corroborating the conclusion of Ueda & Ohtsuka based on

morphological characteristics that S. valkanovi and S. sarsi should be differentiated at the

species level.

4. In Japan S. valkanovi populations are segregated into two areas, around the Seto Inland

Sea (SIS) and northeastern (NE) area. A distinct contrast in the genetic diversity was

observed between the areas; five mtCOI haplotypes were recovered from the NE area

while 38 were observed in the SIS area, despite a similar geographical range and number of

individuals analysed in both areas. Furthermore, S. valkanovi in the SIS area possessed a

mixture of both ancestral and derived haplotypes, while those in the NE area mostly

consisted of derived haplotypes. These results suggest that the NE populations were

founded recently by a limited numbers of individuals from SIS populations.

5. All specimens of S. valkanovi from New Zealand possessed one specific mtCOI

haplotype. In Japan, this haplotype was found only in the NE area, suggesting the origin of

New Zealand S. valkanovi. It is likely that this copepod has on several occasions invaded

areas outside of Japan, one of which was associated with the original description of this

taxon in Bulgaria.

Keywords: biological invasion, copepoda, DNA barcodes, population genetic structure

Correspondence: Wataru Makino, Graduate School of Life Sciences, Tohoku University, 6-3 Aramaki aza aoba, Sendai, Miyagi

980-8578, Japan. E-mail: [email protected]

Freshwater Biology (2010) 55, 375–386 doi:10.1111/j.1365-2427.2009.02287.x

� 2009 Blackwell Publishing Ltd 375

Introduction

A biological invasion is an event in which a popula-

tion is moved beyond its natural range or natural zone

of potential dispersal, usually through human-medi-

ated transport (see Lee, 2002). Biological invasions

appear to be occurring at an increasing rate, and a

number of non-native species have been expanding

their distributions across the world (e.g. Urabe et al.,

2003). It is therefore possible that invaders are

confusing knowledge of biodiversity (e.g. the exis-

tence of cryptogenic species, see Carlton, 1996), and it

can be a serious problem if an invader is described as

a new species in an area to which it has been

introduced. Such risks are rising with the increase in

human movement, especially for small animals that

are easily transported along with plants or fish (as in

spider mites, see Ohashi, Kotsubo & Takafuji, 2003). It

is also generally considered that only a small propor-

tion of invading organisms are able to establish

populations in areas where they are introduced

(Williamson & Fitter, 1996; Lee, 2002). Nevertheless,

unsuccessful invaders might still confuse our under-

standing of biodiversity if they are described as new

species.

The patterns underlying biological invasions may

be revealed using molecular analyses. Invading

organisms generally experience a strong reduction in

genetic variation (see Miura, 2007). An example is the

mud snail Batillaria attramentaria (G. B. Sowerby II,

1855), which was thought to have been introduced to

the west coast of North America in the early 20th

Century with the importation of seed Pacific oyster

(Crassostrea gigas Thunberg, 1793) from Miyagi Pre-

fecture, Japan. In this instance only a few mitochon-

drial gene cytochrome c oxidase subunit 1 (hereafter

mtCOI) haplotypes of B. attramentaria were found

from the west coast of North America, all of which

were consistent with haplotypes found in the Miyagi

Prefecture (Miura et al., 2006). Another example is the

Ponto-Caspican cladoceran Cercopagis pengoi (Ostrou-

mov, 1891) in the Laurentian Great Lakes, where

genetic diversity is much reduced compared with that

in their native range (see Cristescu et al., 2001). Such

phylogeographic studies can therefore be used to

specify the native ranges of organisms (also see Avise,

2000).

Molecular analyses should be particularly useful

in the case of the calanoid copepod Sinodiaptomus

valkanovi Kiefer, 1938 [sensu Ueda & Ohtsuka (1998)

throughout the present study]. This copepod was

originally described from specimens collected in

Sofia, Bulgaria, despite other congeneric species

being distributed through Asia (see Reddy, 1994).

Although the type locality of S. valkanovi is a water

tank in a botanical garden in Sofia (Kiefer 1938, cited

in Ueda & Ohtsuka, 1998), Kiefer (1978, cited in

Ueda & Ohtsuka, 1998) noted that this habitat no

longer existed, and that this species was probably

not native to Bulgaria. It has also been recorded

from California, U.S.A. [as Diaptomus chaffanjoni

Richard, 1897 or Sinodiaptomus sarsi (Rylov, 1923),

where their habitats have since been destroyed; see

Ueda & Ohtsuka, 1998], and recently from New

Zealand (Duggan, Green & Burger, 2006). In New

Zealand S. valkanovi has since been recorded as more

widely established, but is seemingly confined to

artificial water bodies (Banks & Duggan, 2009).

Except for these isolated populations there is no

reliable record of S. valkanovi outside of Japan,

suggesting that S. valkanovi is endemic there (see

Ueda & Ohtsuka, 1998). Populations outside of Japan

are, therefore, assumed to have been introduced,

probably via the transport of aquatic plants from

Japan (Ueda & Ohtsuka, 1998; Duggan et al., 2006).

Although the circumstantial evidence provided

above is highly supportive of this argument, molec-

ular evidence is still necessary to show definitively

(e.g. Xavier et al., 2009) whether S. valkanovi outside

of Japan is indeed an example of a biological

invasion. Another problem with the original descrip-

tion of S. valkanovi is its taxonomic status; S. valkanovi

was originally described as a subspecies of S. sarsi.

While commonly accepted as originally described

(see Reddy, 1994), the most recent revision of this

species has argued for the two taxa to be differen-

tiated at species level (see Ueda & Ohtsuka, 1998).

Both of these taxonomic determinations were based

on morphological characteristics, but require inde-

pendent criteria for further evaluation. Despite the

potential for clarifying the taxonomic status of

S. valkanovi and S. sarsi, no molecular studies have

yet been made on these taxa.

The aim of our study was to resolve questions

relating to the taxonomic designation and invasion of

S. valkanovi. To help elucidate whether S. valkanovi

and S. sarsi should be differentiated taxonomically at

the species level, as argued by Ueda & Ohtsuka

376 W. Makino et al.

� 2009 Blackwell Publishing Ltd, Freshwater Biology, 55, 375–386

(1998), we examined gene flow between the two taxa

using the nuclear ribosomal internal transcribed

spacers 1 and 2, plus the intervening 5.8S ribosomal

DNA subunit (hereafter collectively called ncITS). To

help elucidate whether S. valkanovi is truly endemic to

Japan, the genetic variability of S. valkanovi was

analysed using the mtCOI gene. This gene has a

higher mutation rate than ncITS, thus allowing us to

examine genetic variation with finer resolution, and

these results were compared with those from New

Zealand populations.

Methods

Sinodiaptomus valkanovi is, like other calanoid cope-

pods, obligately sexual. Directly upon fertilisation,

eggs pass into an egg-sac that is carried by the adult

female. The eggs hatch as nauplii and pass through

six naupliar instars and five copepodid instars

before becoming mature in the sixth copepodid

stage. Many calanoid copepods produce not only

eggs that hatch immediately but also diapausing

(resting) eggs (see Hutchinson, 1967). Resting eggs

are deposited in the sediment and hatch after

the ‘harsh’ period of the year is over. Due to their

thicker chorion (e.g. Hairston & Old, 1984; Ban &

Minoda, 1991), resting eggs tolerate desiccation and

passage through the gut of predators (Hairston &

Munns, 1984; Hairston & Old, 1984). Thus, resting

eggs are thought to aid passive dispersal (see Zeller,

Reusch & Lampert, 2006). We have confirmed that

S. valkanovi produces resting eggs, at least in two

populations in Japan (localities 10 and 16 in Fig. 1;

also see Table S1). Tomikawa (1971) showed that

S. valkanovi occurred all the year round in a pond

in Hyogo Prefecture (close to our locality 32),

although its abundance was lower in winter than

in summer.

Fig. 1 Localities where Sinodiaptomus valkanovi and S. sarsi were collected in the present study. In Japan S. valkanovi were collected in

the northeast of Japan (i.e. NE area) and around the Seto Inland Sea (SIS area). In panels that magnify the NE and SIS areas, the

localities are provided with a pie chart that represents the frequency of mtCOI groups summarised in Fig. 2 (white, grey, striped

and black bars correspond to haplogroup A, B, C and D respectively) in the population. The number by each pie chart corresponds to a

specific locality code number in Tables S1 & S2. OK and HR in the panel that magnifies the SIS area denote Okayama and Hiroshima

Prefecture respectively. In the North Island, New Zealand, S. valkanovi was collected from an Auckland Domain pond and

Gilmour Lake, Waihi. The pie chart for the New Zealand sites represents haplotype frequencies in the same way as for the Japanese

populations.

Invasion, genetic variation and species identity of Sinodiaptomus valkanovi 377


Sampling and DNA extraction

Sinodiaptomus valkanovi was collected from most of its

range in Japan between 2004 and 2008 (see Table S1).

In New Zealand, S. valkanovi was collected from

Gilmour Lake (Waihi) and a pond in Auckland

Domain (Auckland), both in the North Island

(Fig. 1). In both cases samples were collected with a

conical plankton net and specimens were immediately

preserved using ethanol. Sinodiaptomus sarsi from

Senbaru-ike pond (Okinawa Island) and from the

mainland of Japan was collected to confirm geneti-

cally the latter population as S. sarsi, by comparing

DNA sequences with specimens from the native

Japanese range (i.e. the Ryukyus including Okinawa

Island, see Ueda & Ishida, 1997). Sinodiaptomus spec-

imens were separated morphologically according to

the redescription of S. valkanovi and S. sarsi by Ueda &

Ohtsuka (1998).

For DNA analyses of the Japanese samples, usually

six adult female S. valkanovi or S. sarsi per population

were isolated (see Table S2). Four adult females were

isolated for each New Zealand population. DNA was

extracted individually using the Sigma GenElute

mammalian genomic DNA miniprep kit according

to the manufacturer’s protocol.

Nuclear gene analyses

We amplified ncITS from three individuals of S.

valkanovi ⁄S. sarsi per population (see Table S2) using

the primers ITS1 and ITS4 in Kiesling et al. (2002), in

order to determine if large inter-taxon variations in

this nuclear region (i.e. the lack of gene flow) were

observed. Each 10 lL polymerase chain reaction (PCR)

cocktail contained 1–2 lL of DNA template, 0.1 lL of

EXTaq DNA polymerase (TaKaRa), 1 lL of 10X EX Taq

buffer, 1 lL of each dNTP (2.5 mMM each), and 0.5 lL of

each primer (2.5 lMM). The PCR conditions consisted of

1 min at 95 �C followed by 35 cycles of 30 s at 95 �C,

30 s at 53 �C and 45 s at 72 �C followed by 15 min at

72 �C. PCR products were treated with the ExoSap IT

kit to eliminate unincorporated primers and nucleo-

tide triphosphates, and then sequenced using the

BigDye terminator 3.1 sequencing kit and an ABI 3130

DNA sequencer (Applied Biosystems, Foster City, CA,

U.S.A.). The products were sequenced from one

direction with ITS4. When ambiguous peaks were

observed, another analysis with the ITS1 primer was

executed. The sequences were edited with FINCH TV

and aligned with CLUSTAL X 1.82 (Thompson et al.,

1997). Aligned fragments (673 bp including alignment

gaps) were submitted to the DNA Data Bank of

Japan under Accession numbers AB454171–AB454175

and AB494228–AB494238, and were used for the

inter-taxon comparison.

MtCOI analyses

For S. valkanovi, a c. 1200 bp fragment of the mtCOI

gene was amplified using the primers L1384-COI and

H2612-COI (Machida et al., 2004). Each 10 lL PCR

cocktail contained 1–2 lL of DNA template, 0.1 lL of

EXTaq DNA polymerase (TaKaRa), 1 lL of 10X EX

Taq buffer, 1 lL of each dNTP (2.5 mMM each), and

0.5 lL of each primer (2.5 lMM). The PCR conditions

consisted of 1 min at 95 �C followed by 35 cycles of

30 s at 95 �C, 30 s at 45 �C and 1 min at 72 �C

followed by 15 min at 72 �C. For S. sarsi we initially

tried the primer set of Machida et al. (2004); however,

this primer set did not provide good results. We

therefore used the universal primer set (LCO1490 and

HCO2198) of Folmer et al. (1994). These primers

amplify a shorter region (c. 700 bp, the first half of

the c. 1200 bp fragment) of mtCOI compared with that

used for S. valkanovi. The cocktail for PCR was

identical to that described above except for the

primers. The PCR conditions consisted of 1 min at

95 �C followed by 35 cycles of 30 s at 95 �C, 30 s at

45 �C and 45 s at 72 �C, followed by 15 min at 72 �C.

In both taxa, PCR products were treated with the

ExoSap IT kit and then sequenced using the BigDye

terminator 3.1 sequencing kit and an ABI 3130 DNA

sequencer. The products were sequenced from both

directions with the primers described above for S.

valkanovi, while the products were sequenced only

from one direction with LCO1490 for S. sarsi.

After editing of sequences with FINCH TV, we

aligned the mtCOI sequences from both species with

CLUSTAL X. Aligned fragments of 1162 and 630 bp

were obtained for S. valkanovi and S. sarsi, respec-

tively, and were submitted to the DNA Data Bank of

Japan under Accession numbers AB454127–AB454169

and AB494226–AB494227. The phylogeny of these

mtCOI sequences was investigated using a Bayesian

approach. Hasegawa, Kishino & Yano’s (1985) model

of sequence evolution with site-specific rates for each

codon position (i.e. HKYSS) was selected by the



computer package KAKUSAN (Tanabe, 2007) as the

best-fit model of nucleotide substitution. The Bayesian

analysis was performed using MRRBAYES 3.1.2 (Ron-

quist & Huelsenbeck, 2003). Markov chain Monte

Carlo searches were doubly run with four chains for

3 200 000 generations with sampling every 100 gen-

erations. The number of generations before stationa-

rity of likelihood values (the burn-in) was estimated

graphically as 2 200 000 generations. During these

calculations mtCOI sequence from 631 to 1162 bp of S.

sarsi were treated as missing data.

The phylogenetic data of S. valkanovi were also used

for analysing their population genetic structure in

Japan and New Zealand. Since the distribution of

S. valkanovi in Japan was strongly segregated into two

geographic areas [northeastern (NE) and Seto Inland

Sea (SIS) areas, see Results], the genetic variability of

this species in Japan was analysed with that distribu-

tion pattern considered. Firstly, we applied Tajima’s

(1989) test using ARLEQUIN 2.0 (Schneider, Roessli &

Excoffier, 2000) and found that it did not reject

neutral evolution of the observed sequence

variation in mtCOI for both the NE (D = 3.59,

P(simulated D < observed D) = 1.00) and SIS area (D =

1.32, P(simulated D < observed D) = 0.92). Next, we calcu-

lated haplotype and nucleotide diversities with the

aid of ARLEQUIN and compared the results between

the areas. Difference in these values between the NE

and SIS areas were tested by using t-tests, following

Nei (1987). The population genetic structure was then

investigated by the analysis of molecular variance

(AMOVAAMOVA), also with the aid of ARLEQUIN with 1023

permutations. During this process the fixation index

Fst was also estimated. Finally, a mtCOI haplotype

network that illustrates all connections that have 95%

probability of being the most parsimonious was

drawn by TCS 1.21 (Clement, Posada & Crandall,

2000), so as to visually understand the relationship

among haplotypes recovered from the two geographic

areas. As only one mtCOI haplotype was recovered in

New Zealand (see Results), we attempted no further

analysis for these populations.

Results

Geographic distribution

Sinodiaptomus valkanovi was found from 43 localities in

Japan out of more than 400 lakes ⁄ponds ⁄pools exam-

ined. The 43 populations were geographically sepa-

rated into two major areas: a northeastern part

(hereafter called the NE area) and an area around

the Seto Inland Sea, which is surrounded by Kyushu

and Shikoku Islands and the Chugoku district of

Honshu Island (hereafter the SIS area) (Fig. 1). In both

areas, most individuals of S. valkanovi were collected

from small (median, 0.63 ha) man-made irrigation

ponds in rural ⁄agricultural areas at low altitude

(median, 70 m a.s.l) (see Table S1). In these ponds, S.

valkanovi was found to co-exist with no other calanoid

copepod species except for Neutrodiaptomus formosus

(Kikuchi, 1928), which was distributed from Tokai to

northern Kyushu districts, and was found with S.

valkanovi only in Obaraike and Shimoyamaike ponds

in the SIS area (for locations see Table S1). We found

two S. sarsi populations from Shizuoka Prefecture and

one Okinawa population (Fig. 1, also see Table S1).

Sinodiaptomus sarsi was not found to co-exist with any

other calanoid copepod species.

Genetic differentiation between S. valkanovi and S. sarsi

We found virtually no variation in the ncITS sequence

among nine individuals of S. sarsi from the three

populations. These sequences were not identical to

any of the S. valkanovi ncITS sequences from 126

individuals out of the 43 populations; the aligned

sequence showed three gaps (insertion or deletion)

between S. valkanovi and S. sarsi (Table 1). Thus, the

ncITS data demonstrate that there is no gene flow

between S. valkanovi and S. sarsi populations.

In the mtCOI sequences, the two taxa were found to

have no common haplotypes (see Table S2). Bayesian

phylogenetic analysis clearly revealed that mtCOI

haplotypes recovered from S. sarsi and S. valkanovi

were both monophyletic, showing no evidence of

introgressions between the two taxa (Fig. 2). The

sequence divergence [assessed as Kimura’s (1980)

two-parameter distance; hereafter K2P] between S.

sarsi and S. valkanovi was 23–26%, while intra-taxon

K2P divergences were much smaller (2.4–4.1% and up

to 5.7% in S. sarsi and S. valkanovi, respectively).

Population genetic structure of S. valkanovi in Japan

and New Zealand

For the mtCOI gene of S. valkanovi, 43 haplotypes

were collected from 248 individuals. There was a



distinct contrast in the population genetic composi-

tions between the NE and SIS areas (Table 2). In the

NE area we recovered five haplotypes, among which

haplotypes NE01 and NE05 dominated numerically

(shared by 68% and 28% of individuals in the area,

respectively). NE01 was found in 15 out of 20 NE

populations while NE05 was recovered from eight

populations (see Table S2). In the SIS area, although

its geographic range was smaller and the number of

individuals analysed was similar to that for the NE

area, 38 haplotypes were recovered. Most haplotypes

were found within single populations (i.e. private

haplotypes), while five other haplotypes were shared

with only two-four populations (see Table S2). One

such ‘shared’ haplotype was NE01, which was the

dominant haplotype in the NE. NE01 and SIS05 were

numerically the dominant haplotypes in the SIS area,

but their contributions there were only 12% and 10%,

respectively. As a result, the haplotype diversity in the

SIS area was significantly larger than that in the NE

(t246 = 12.5, P < 0.001) (Table 2). The nucleotide diver-

sity was also larger in the SIS area than in the NE,

although this difference was not statistically signifi-

cant (t246 = 0.28, P > 0.05). A likely reason for the lack

of significance is that haplotypes NE01 and NE05,

which dominated in the NE area, were among the

most genetically distant pairs, with a K2P sequence

difference of 5.2%.

Despite haplotype diversity being apparent be-

tween the two areas, our AMOVAAMOVA indicated that the

contribution of inter-area variation to the total genetic

variation was only 13% (P < 0.01), which was smaller

than that of inter-population variation within the

areas (Table 3). Conducting AMOVAAMOVA in each area

separately, we found that the total genetic variation

was mostly explained by the inter-population varia-

tion (c. 85%) in both cases. The Fst values were

extremely high (0.83–0.85, P < 0.001 in both areas).

Our phylogenetic results reflect the geographical

NE–SIS differentiation of S. valkanovi populations. In

the NE area, dominant haplotypes were NE01 and

NE05, which were genetically distant (see above),

while the remaining haplotypes were their one-base-

pair mutations (Fig. 2). In the SIS area, we also found

the NE01 haplotype, as noted above, and its one-to-

four-base-pair mutations (group A in Fig. 2), which

were derived haplotypes according to the Bayesian

consensus tree. The other haplotypes in the SIS area

fell into the ancestral group D or a group of interme-

diate forms (groups C and B, except NE04 and NE05).

Further, we found that the distribution of haplotypes

in group D were restricted to a part of Okayama and

Table 1 Base position of polymorphic sites in aligned ncITS sequence of Sinodiaptomus valkanovi and S. sarsi

Taxa

Recovered

sequences

Base position

1 1 1 1 2 2 2 2 2 3 3 3 4 5 5 5 5 6 6 6 6

4 4 5 6 6 9 1 2 5 8 1 3 3 3 9 0 0 0 9 0 3 5 8 4 5 6 6

0 2 0 0 2 7 5 1 6 6 1 0 3 5 1 1 4 7 4 2 1 5 3 4 4 0 3

S. valkanovi SV1 A G – T T A T T A A A C C C T T A T C – C C T T G A C

SV2 A G – T T A T T A A A T C C T T A C C – C G T T G A C

SV3 A G – T T A T T A A A C C C T T A C C – C C T T G A C

SV4 A G – T T A T T A A A T C C T T A C C – C C T T G A C

SV5 A A – T T A T T A A A C C C T T A C C – C C T T A A C

SV6 A G – T T A T T A A A C T T T T A C T – C C T T G A C

SV7 A G – T T A T T A A A C C C T T A C C – C G T T G A C

SV8 A G – T T A T T A A A Y C C T T A C C – C C T T G A C

SV9 A G – T T A T T A A A Y Y Y T T A C Y – C C T T G A C

SV10 A G – T T A T T A A A Y C C T T A Y C – C S T T G A C

SV11 A G – T T A T T A A A C C C T T A Y C – C S T T G A C

SV12 A G – T T A T T A A A Y C C T T A C C – C S T T G A C

SV13 A G – T T A T T A A A T C C T T A C C – C S T T G A C

SV14 A R – T T A T T A A A Y C C T T A C C – C S T T R A C

S. sarsi SS1 G G A C C G C C G C R C C C – Y M C C C T C A C G G Y

SS2 G G A C C G C C G C G C C C – C C C C C T C A C G G C

Analyses were made for three animals per locality (see Table S2 for relationships between localities and haplotypes).

–, an alignment gap.



Hiroshima Prefectures in Honshu Island, and it

appears that this haplogroup was surrounded by the

derived haplogroups (see Fig. 1). These results indi-

cate that S. valkanovi populations in the SIS area (as a

whole, not in a specific population there) are the

mixture of ancestral and divergent haplotypes of

different ‘ages’, while those in the NE area consist of

mostly ‘younger’ haplotypes. The star-like clade of

1.00

0.99

0.68

0.90

1.00

1.00

1.00

0.56

Sinodiaptomus valkanovi

Sinodiaptomus sarsi

A

B

D

SIS02SIS03SIS05SIS25SIS01

NE03SIS27

SIS04NE01SIS11SIS34SIS19SIS36NE02

SIS26SIS13SIS15SIS14


NE04NE05

SIS37SIS21SIS22SIS24SIS23SIS20


SIS12SIS16

SIS28SIS29SIS18SIS10SIS08SIS07

SAR02SAR03SAR01

Neocalanus plumchrus (AB093143)Heterocope appendiculata

0.1

0.99

1.00

0.91

1.00

1.00

1.00

0.56

SIS22

SIS24SIS21

SIS23

SIS20

D

SIS07

SIS18

SIS10

SIS08SIS29

SIS28SIS12

SIS16

SIS06

SIS35

SIS09SIS17

15 steps

S. valkanovi haplotype network

SIS32SIS31

SIS33 SIS30

NE04

NE05

B

SIS37

A

SIS01

SIS02

SIS03

SIS04

SIS05

SIS25

SIS27

SIS26

SIS13

SIS14

SIS15

SIS19SIS34

SIS11

NE03

NE02

16 steps

NE01

SIS36

C

C

Fig. 2 Phylogenetic relationships in the mtCOI haplotypes of Sinodiaptomus valkanovi and S. sarsi, with the sequences of Neocalanus

plumchrus (AB093143, Machida et al., 2004) and Heterocope appendiculata (AB454170, this study) as outgroups. The numbers by the

nodes represent Bayesian posterior probabilities. For S. valkanovi, names of haplotypes that were found in the NE area are labelled in

red. Also, mtCOI haplotype networks drawn by TCS software were redrawn with a few simplifications. Each haplotype is indicated by

a circle with its assigned name. Red and blue circles represent haplotypes that were recovered from the NE and SIS area, respectively,

and the area of the circle roughly corresponds to the number of individuals that possessed the haplotype. Small, white small circles

represent missing intermediate haplotypes. All connections between haplotypes indicated by solid lines are parsimonious at a 95%

level, while the level was 90% for dashed lines that connect two haplotypes with the designated number of missing haplotypes. It was

not possible to connect all haplotypes obtained in the present study at more than 90% parsimonious level. The right hand vertical bars

represent groups A, B, C and D in the present study.

Table 2 Populations of Sinodiaptomus valkanovi in the present study

Area

No. of populations

sampled

No. of animals

sequenced

No. of haplotypes

recovered

No. of

polymorphic sites H p

NE 20 119 5 60 0.463 (0.039) 0.0206 (0.0101)

SIS 23 129 38 113 0.954 (0.006) 0.0250 (0.0122)

H denote haplotype diversity as the probability that two randomly chosen haplotypes are different, while p denote nuclerotide

diversity as the probability that two randomly chosen nucleotide sites are different (sample variation in parentheses).



group A implies a recent population expansion from a

limited numbers of founders (see Avise, 2000).

Two populations were analysed from New Zealand,

as recently identified by Banks & Duggan (2009). The

first was from a pond at the Auckland Domain, while

the second was from Gilmour Lake, Waihi, c. 110 km

from the Auckland pond. All individuals collected

from the two New Zealand populations possessed the

haplotype NE05, the second most dominant haplotype

in the NE area of Japan, which was not present in

the SIS area (see Table S2 and Fig. 2).

Discussion

Taxonomic status of S. valkanovi and S. sarsi

Our ncITS analysis directly showed the lack of gene

flow between S. valkanovi and S. sarsi. Therefore, our

study genetically corroborates the conclusion of Ueda

& Ohtsuka (1998), which was based on morphological

characteristics, that S. valkanovi and S. sarsi are

different species.

For the sequence divergence of ncITS in other

members of family Diaptomidae, which includes

Sinodiaptomus, Thum & Harrison’s (2009) data showed

that the average inter-specific ncITS variation (as K2P

model, including alignment gaps) among six Skisto-

diaptomus species ranges from 1.53% to 9.84% (acces-

sion numbers EU582651–EU582685, 680 bp, including

alignment gaps). The K2P differentiations in ncITS

between S. valkanovi and S. sarsi, calculated from data

shown in Table 1, were 1.8–2.6% (average, 2.2%),

falling into lower end of that between Skistodiaptomus

species. As for the mtCOI in copepods, sequence

divergences among morphologically similar, but rec-

ognisably distinct congeneric species (such as

S. valkanovi and S. sarsi in the present study),

commonly go beyond 15–20% (e.g. Bucklin et al.,

1999; Machida et al., 2006; Thum & Harrison, 2009).

Our results (23–26% as K2P differentiation) corrobo-

rate the results of previous studies. Our study,

therefore, has explicitly demonstrated that DNA

sequence divergences between S. valkanovi and S.

sarsi are indeed of a magnitude that differentiates

these taxa at the species level, using two genes that are

inherited in a different manner. This also indicates

that there is no signature of introgressive hybridisa-

tion (see Lee & Frost, 2002; Taylor, Sprenger & Ishida,

2005) or shared ancestory between the two species.

Overall dispersal capacity of S. valkanovi inferred

from mtCOI data

In Japan, S. valkanovi populations were segregated

geographically between the NE and SIS areas. The

mtCOI haplotype compositions also contrasted be-

tween these areas. In both areas, however, the Fst was

extremely high (c. 0.85), demonstrating that gene flow

among populations is extremely small even over small

spatial scales. As S. valkanovi possesses diapausing

eggs, this copepod could be expected to disperse

easily by passive means, as observed in other zoo-

plankton taxa that have diapausing eggs, such as the

cyclical parthenogenetic cladocerans and rotifers (e.g.

Caceres & Soluk, 2002; Green, Figuerola & Sanchez,

2002; Cohen & Shurin, 2003). Our data do not indicate

this to be the case, however. Our data do confirm

(qualitatively) what has been found in cladocerans

and rotifers, that there are strong inter-population

genetic differentiations at a local scale, although some

specific haplotypes may occur over broad geographic

areas (e.g. Gomez, 2005; Ishida & Taylor, 2007).

Table 3 Results of A M O V AA M O V A for Sinodiaptomus valkanovi populations in Japan

Design Source of variation Degree of freedom Sum of squares Percentage of variation

Within Japan Among areas 1 357 13.4

Among populations within areas 41 3058 73.4

Within populations 212 465 13.2

Total 254 3880

Within the NE area Among populations within area 19 1293 83.6


Total 119 1508

Within the SIS area Among populations within area 22 1765 85.7


Total 134 2014



Such a ‘provincialism’ again contradicts the general

assumption that zooplankton with diapausing eggs

have a high potential for passive dispersal. One

hypothesis that explains this discrepancy is that rapid

population growth after a historical colonisation event

from a few founding individuals, which has been

repeatedly observed, prevents allele frequency

changes due to gene flow (the persistent founder

effects hypothesis, sensu Boileau & Hebert, 1991; also

see Boileau, Hebert & Schwartz, 1992). Such ‘persis-

tent effects’ of the historical founders would be

further enhanced not only by the presence of a large

diapausing egg bank but also by the rapid adaptation

of resident populations to local conditions (the

monopolisation hypothesis, De Meester et al., 2002).

These mechanisms may also be applicable to

S. valkanovi populations in Japan, since gene flow

among populations is extremely small, despite

S. valkanovi producing diapausing eggs. If this is the

case we can therefore consider that the successful

spread of S. valkanovi probably occurs via the dispersal

of small numbers of individuals to a new ‘frontier.’

It is noteworthy that most of the habitats for

S. valkanovi in Japan are currently man-made (irriga-

tion) ponds. Although we cannot be sure exactly when

each pond was built, the oldest irrigation ponds in

Japan date back to c. 391 AD (Nogyodobokure-

kishikenkyukai, 1988). Sixteen hundred years, as an

extreme scenario, is obviously not adequate to allow

the mtCOI gene to diversify to the extent found in the

present study (up to 5.7%), if one applies conventional

mtDNA molecular clocks that are used for calanoid

copepods elsewhere (1.4–2.3% per million years,

Dooh, Adamowicz & Hebert, 2006). We therefore do

not consider that the haplotype composition of

S. valkanovi in such ‘young’ irrigation ponds reflects

only what happened in each pond; rather, it should

reflect the history of a ‘unit of local populations,’

which might persist for longer time than any single

population.

MtCOI data reveal the invasion corridor of

S. valkanovi

Differences in mtCOI haplotype diversity among the

study areas suggest that the invasion corridor of

S. valkanovi started from the SIS area in Japan and

‘jumped’ twice, firstly to the NE area in Japan and

later to New Zealand.

In Japan, S. valkanovi populations were segregated

geographically between the NE and SIS areas. It could

be argued that past vicariance, the splitting of a once

contiguous distribution, is the most important factor

explaining the segregated distribution of S. valkanovi

in Japan. This idea, however, would not be likely for

the following reasons. Firstly, the Hokuriku-Tokai

area that separates the two groups of S. valkanovi

populations has not had a history of submergence

since its uplift over 6 million years ago (see Yonekura

et al., 2001), implying that there has been no geo-

graphical event that could split a contiguous distri-

bution into the current NE and SIS areas. Secondly,

with a vicariance scenario we would expect that the

genetic differentiation between the current NE and

SIS areas would be like the phylogeographic Category

I of Avise (2000), in which there are prominent genetic

gaps distinguishing deep allopatric lineages in a gene

tree. Our mtCOI data do not match this pattern, as

most of the S. valkanovi individuals in the NE area

possess the mtCOI haplotype NE01, which is also

distributed in the SIS area.

A reduced haplotype diversity in the NE area, on

the other hand, is consistent with the characteristics of

invading populations (Cristescu et al., 2001; Miura

et al., 2006), implying that dispersal from the SIS area

to the NE area may have played a significant role. This

possibility is also supported by the number of mtCOI

haplotypes derived from the haplotype NE01, being

larger in the SIS area than the NE, suggesting the SIS

area is the origin of the haplotype NE01 (and more

broadly the origin of group A also). The importance of

dispersal from the SIS to the NE area is also supported

by the diversity of ncITS genotypes (see Table S2),

being larger in the SIS area than the NE, although our

ncITS analysis was primarily for examining the gene

flow between S. valkanovi and S. sarsi. The number of

ncITS genotypes recovered, however, was 11 in the

SIS area and five in the NE area, and two major

genotypes there (SV3 and 6, see Table 1) were also

found in the SIS area. The ncITS gene diversity, which

is the expected heterozygosity calculated by ARLEQUIN,

was larger in the SIS area (0.850 ± 0.021) than in

the NE area (0.762 ± 0.021), and this difference is

statistically significant [t(64) = 2.97, P < 0.01]. From

these results we argue that the SIS area is the original

locality of S. valkanovi and that long-distance dispersal

from the SIS area therefore provides the best expla-

nation for the formation of current NE populations.



It should be noted that a clear phylogeographic

structure, which probably reflects the result of natural

colonisation, was found only in the SIS area despite

the limited dispersal capacity of S. valkanovi. In the NE

area the mtCOI haplotypes consisted of numerically

major ones (NE01 and NE05) that are genetically

relatively distant. The haplotype NE01 was broadly

distributed in the NE area and shared between the

two areas. Interestingly, there may be a possibility

that S. valkanovi was absent in the NE area until

recently (see Ueda & Ohtsuka, 1998). Indeed Tomik-

awa’s (1971) attempt to collect S. valkanovi in 1969–70

in the Hokuriku and Tohoku districts (where S.

valkanovi is not uncommon) was unsuccessful. These

results imply that the dispersal from the SIS to NE

area was rapid (which is why a phylogeographic

structure was unclear), and may have been achieved

by human activities (in view of the apparently limited

natural dispersal capacity of S. valkanovi). The mtCOI

haplotype NE05 may seem to be an exceptional case

for this scenario, however, because it was recovered

only from the NE. Neither did we recover any

‘closely’ related haplotypes to NE05 in the SIS area.

However, we speculate that increasing sampling

efforts may reveal the presence of this and ⁄or closely

related haplotypes in the SIS area. If so, that would

mean that S. valkanovi has invaded the NE area at least

twice independently.

In New Zealand, S. valkanovi populations possessed

only a single mtCOI haplotype, which was consistent

with the NE05 haplotype in Japan. We can therefore

argue that S. valkanovi in New Zealand has a Japanese

origin, and most likely from the NE area of the present

study. While the occurrence of exotic zooplankters

such as Daphnia pulicaria Forbes, 1893 (Urabe et al.,

2003) has been genetically confirmed in Japan, this is

apparently the first study to confirm genetically a

Japanese freshwater zooplankter that has invaded

another country. In many regions, there are com-

monly species that cannot be confidently classified as

native or introduced (e.g. Carlton, 1996). The present

study provides a good example of how to resolve the

cryptogenic status of species by genetic analyses.

Interestingly, the habitat types of S. valkanovi are

similar between Japan and the non-indigenous pop-

ulations, with recently constructed ponds (i.e. a

frontier) being the only known sites of establishment

in New Zealand, Bulgaria and California (see Banks &

Duggan, 2009). As only a single haplotype was

observed in the New Zealand populations, despite a

geographical separation of >100 km between the

invaded ponds, introduction into that country prob-

ably comprised a single event, with subsequent

spread occurring from one site to another. Unfortu-

nately, populations recorded in Bulgaria and Califor-

nia no longer exist, so we cannot elucidate whether all

non-indigenous populations of this species are

derived from a single emigration event from Japan,

or whether this species has been dispersed many

times from single or multiple sources in Japan.

However, we believe other undiscovered populations

probably already exist elsewhere, which may be used

to test this hypothesis.

Acknowledgments

We wish to thank the Urabe laboratory, Kawata

laboratory and Chiba laboratory, Tohoku University,

for providing both laboratory facilities and meaning-

ful suggestions during this project. Akifumi S. Tanabe

provided logistic support for the phylogenetic analy-

ses, and Hajime Yoshino collected S. sarsi from

Senbaruike, Okinawa, and provided constructive

comments on earlier versions of the manuscript, for

which we are grateful. The Japanese component of

this research was supported by grants from the Japan

Society for the Promotion of Science (Nos. 16770011

and 19770010) and in part by the Water Resources

Environment Technology Center (No. 2008–06). The

NZ component of this research was funded in part by

FRST grants UOWX0501, UOWX0505, and a Univer-

sity of Waikato Research grant. We thank Professor A.

Hildrew and the anonymous referees, whose com-

ments significantly improved our manuscript.

References

Avise J.C. (2000) Phylogeography: The History and Forma-

tion of Species. Harvard University Press, Cambridge,

447 pp.

Ban S. & Minoda T. (1991) The effect of temperature on the

development and hatching of diapause and suitaneous

eggs in Eurytemora affinis (Copepoda: Calanoida) in the

sediment of Lake Ohnuma, Hokkaido, Japan. Bulletin of

the Plankton Society of Japan, Special Volume, 299–308.

Banks C.M. & Duggan I.C. (2009) Lake construction has

facilitated calanoid copepod invasions in New Zea-

land. Diversity and Distributions, 15, 80–87.



Boileau M.G. & Hebert P.D.N. (1991) Genetic conse-

quences of passive dispersal in pond-dwelling cope-

pods. Evolution, 45, 393–400.

Boileau M.G., Hebert P.D.N. & Schwartz S.S. (1992) Non-

equilibrium gene frequency divergence: persistent

founder effects in natural populations. Journal of

Evolutionary Biology, 5, 25–39.

Bucklin A., Guarnieri M., Hill R.S., Bentley A.M. &

Kaartvedt S. (1999) Taxonomic and systematic assess-

ment of planktonic copepods using mitochondrial COI

sequence variation and competitive, species-specific

PCR. Hydrobiologia, 401, 239–254.

Caceres C.E. & Soluk D.A. (2002) Blowing in the wind: a

field test of overland dispersal and colonization by

aquatic invertebrates. Oecologia, 131, 402–408.

Carlton J.T. (1996) Biological invasions and cryptogenic

species. Ecology, 77, 1653–1655.

Clement M., Posada D. & Crandall K.A. (2000) TCS: a

computer program to estimate gene genealogies.

Molecular Ecology, 9, 1657–1659.

Cohen G.M. & Shurin J.B. (2003) Scale-dependence and

mechanisms of dispersal in freshwater zooplankton.

Oikos, 103, 603–617.

Cristescu M.E.A., Hebert P.D.N., Witt J.D.S., MacIsaac

H.J. & Grigorovich I.A. (2001) An invasion history

for Cercopagis pengoi based on mitochondrial gene

sequences. Limnology and Oceanography, 46, 224–

229.

De Meester L., Gomez A., Okamura B. & Schwenk K.

(2002) The monopolization hypothesis and the dis-

persal-gene flow paradox in aquatic organisms. Acta

Oecologica, 23, 121–135.

Dooh R.T., Adamowicz S.J. & Hebert P.D.N. (2006)

Comparative phylogeography of two North American

‘glacial relict’ crustaceans. Molecular Ecology, 15, 4459–

4475.

Duggan I.C., Green J.D. & Burger D.F. (2006) First New

Zealand records of three non-indigenous zooplankton

species: Skistodiaptomus pallidus, Sinodiaptomus valkano-

vi, and Daphnia dentifera. New Zealand Journal of Marine

and Freshwater Research, 40, 561–569.

Folmer O., Black M., Hoeh W., Lutz R. & Vrijenhoek R.

(1994) DNA primers for amplification of mitochondrial

cytochrome c oxidase subunit I from diverse metazoan

invertebrates. Molecular Marine Biology and Biotechnol-

ogy, 3, 294–299.

Gomez A. (2005) Molecular ecology of rotifers: from

population differentiation to speciation. Hydrobiologia,

546, 83–99.

Green A.J., Figuerola J. & Sanchez M.I. (2002) Implica-

tions of waterbird ecology for the dispersal of aquatic

organisms. Acta Oecologica, 23, 177–189.

Hairston N.E. Jr, Munns W.R. Jr (1984) The timing of

copepod diapause as an evolutionarily stable strategy.

American Naturalist, 123, 733–751.

Hairston N.E. Jr & Old E.J. (1984) Population differences

in the timing of diapause: adaptation in a spatially

heterogeneous environment. Oecologia, 61, 42–48.

Hasegawa M., Kishino H. & Yano T. (1985) Dating of the

human-ape splitting by a molecular clock of mito-

chondrial DNA. Journal of Molecular Evolution, 22, 160–

174.

Hutchinson G.E. (1967) A Treatise on Limnology, Vol. 2.

Introduction to Lake Biology and the Limnoplankton. J.

Wiley and Sons, New York.

Ishida S. & Taylor D.J. (2007) Mature habitats associated

with genetic divergence despite strong dispersal

ability in an arthropod. BMC Evolutionary Biology, 7,

52.

Kiesling T.L., Wilkinson E., Rabalais J., Ortner P.B.,

McCabe M.M. & Fell J.W. (2002) Rapid identification of

adult and naupliar stages of copepods using DNA

hybridization methodology. Marine Biotechnology, 4,

30–39.

Kimura M. (1980) A simple method for estimating

evolutionary rates of base substitutions through com-

parative studies of nucleotide sequences. Journal of

Molecular Ecology, 16, 111–120.

Lee C.E. (2002) Evolutionary genetics of invasive species.

Trends in Ecology and Evolution, 17, 386–391.

Lee C.E. & Frost B.W. (2002) Morphological stasis in the

Eurytemora affinis species complex (Copepoda: Temor-

idae). Hydrobiologia, 480, 111–128.

Machida R.J., Miya M.U., Nishida M. & Nishida S. (2004)

Large-scale gene rearrangements in the mitochondrial

genomes of two calanoid copepod Eucalanus bungii and

Neocalanus cristatus (Crustacea), with notes on new

versatile primers for the srRNA and COI genes. Gene,

332, 71–78.

Machida R.J., Miya M.U., Nishida M. & Nishida S. (2006)

Molecular phylogeny and evolution of the pelagic

copepod genus Neocalanus (Crustacea: Copepoda).

Marine Biology, 148, 1071–1079.

Miura O. (2007) Molecular genetic approaches to eluci-

date the ecological and evolutionary issues associated

with biological invasions. Ecological Research, 22, 876–

883.

Miura O., Torchin M.E., Kuris A.M., Hechinger R.F. &

Chiba S. (2006) Introduced cryptic parasites exhibit

different invasion pathways. Proceedings of the National

Academy of Sciences of the United States of America, 103,

10818–19823.

Nei M. (1987) Molecular Evolutionary Genetics. Columbia

University Press, New York.



Nogyodobokurekishikenkyukai (1988) Daichi eno kokuin,

konoshimaguni wa ikanishite warewareno seizonkiban ni

nattaka. Nogyodobokurekishikenkyukai, Tokyo, 187 pp

(in Japanese).

Ohashi K., Kotsubo Y. & Takafuji A. (2003) Distribution

and overwintering ecology of Tetranychus takajujii

(Acari: Tetranychidae), a species found from Kinki

district, Japan. Journal of the Acarological Society of Japan,

12, 107–113. (in Japanese with English abstract).

Reddy Y.R. (1994) Copepoda: Calanoida: Diaptomidae. Guide

to the Identification of the Microinvertebrates of the

Continental Waters of the World 5. SPB Academic

Publishing, Hague.

Ronquist F. & Huelsenbeck J.P. (2003) MrBayes 3:

Bayesian phylogenetic inference under mixed models.

Bioinformatics, 19, 1572–1574.

Schneider S., Roessli D. & Excoffier L. (2000) ARLEQUIN,

A Software Program for Population Genetic Analysis,

Version 2.00. Genetics and Biometry Laboratory, Uni-

versity of Geneva, Switzerland.

Tajima F. (1989) Statistical methods for testing the neutral

mutation hypothesis by DNA polymorphism. Genetics,

123, 585–595.

Tanabe A.S. (2007) KAKUSAN: a computer program to

automate the selection of a nucleotide substitution

model and the configuration of a mixed model on

multilocus data. Molecular Ecology Notes, 7, 962–964.

Taylor D.J., Sprenger H.L. & Ishida S. (2005) Geographic

and phyogenetic evidence for dispersed nuclear intro-

gression in a daphniid with sexual propagules. Molec-

ular Ecology, 14, 525–537.

Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F.

& Higgins D.G. (1997) The ClustalX windows inter-

face: flexible strategies for multiple sequence align-

ment aided by quality analysis tools. Nucleic Acids

Research, 24, 4876–4882.

Thum R.A. & Harrison R.G. (2009) Deep genetic diver-

gences among morphologically similar and parapatric

Skistodiaptomus (Copepoda: Calanoida: Diaptomidae)

challenge the hypothesis of Pleistocene speciation.

Biological Journal of the Linnean Society, 96, 150–165.

Tomikawa T. (1971) Ecological studies on a freshwater

copepod, Sinodiaptomus valkanovi KIEFER. II. Meta-

morphosis, growth, seasonal and geographical distri-

bution. Japanese Journal of Limnology, 32, 32–39.

Ueda H. & Ishida T. (1997) Species composition and

description of limnoplanktonic copepods from Oki-

nawa. Plankton Biology and Ecology, 44, 41–54.

Ueda H. & Ohtsuka S. (1998) Redescription and taxo-

nomic status of Sinodiaptomus valkanovi, a common

limnoplanktonic calanoid copepod in Japan, with

special comparison to the closely related S. sarsi.

Hydrobiologia, 379, 159–168.

Urabe J., Ishida S., Nishimoto M. & Weider L.J. (2003)

Daphnia pulicaria, a zooplankton species that suddenly

appeared in 1999 in the offshore zone of Lake Biwa.

Limnology, 4, 35–41.

Williamson M. & Fitter A. (1996) The varying success of

invaders. Ecology, 77, 1661–1666.

Xavier R., Santos A.M., Lima F.P. & Branco M. (2009)

Invasion or invisibility: using genetic and distribution

data to investigate the alien or indigenous status of

the Atlantic populations of the peracarid isopod,

Stenosoma nadejda (Rezig 1989). Molecular Ecology, 18,

3283–3290.

Yonekura N., Kaizuka S., Nogami M. & Chinzei K. (Eds)

(2001) Regional Geomorphology of the Japanese Islands. 1.

Introduction to Japanese Geomorphology. University of

Tokyo Press, Tokyo, 349 pp. (in Japanese).

Zeller M., Reusch T.B.H. & Lampert W. (2006) A

comparative population genetic study on calanoid

freshwater copepods: investigation of isolation-by-

distance in two Eudiaptomus species with a different

potential for dispersal. Limnology and Oceanography, 51,

117–124.

Supporting Information

Additional Supporting Information may be found in

the online version of this article:

Table S1. Information on sampling localities in the

present study.

Table S2. Information on molecular analyses in the

present study.

As a service to our authors and readers, this journal

provides supporting information supplied by the

authors. Such materials are peer-reviewed and may

be re-organized for online delivery, but are not copy-

edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.

(Manuscript accepted 4 July 2009)



Makino, Knox, Duggan. Freshwater Biology. Electronic supplement 1. Information on sampling localities in the present study. For

locality types M and N denote a man-made irrigation pond and a natural pond, respectively.

Locality

Spcecies and Code Prefecture or Latitude Longitude Altitude Area

country No. Domain Name (deg min sec) (deg min sec) (m a.s.l.) (ha) Type

Sinodiaptomus 1 Hokkaido a pond in Nakajimakoen 43 02 44 N 141 21 09 E 30 2.19 M

valkanovi 2 Aomori Kumazawa tameike 40 43 07 N 140 32 58 E 20 6.27 M

Japan 3 Santa tameike 40 43 52 N 140 35 47 E 35 6.57 M

NE area 4 Akita Tazawa onuma 39 38 13 N 140 36 29 E 90 10.43 N

5 Iwate a pond in Morioka 39 37 58 N 141 05 52 E 175 0.08 M

6 Miyagi Araitsutsumi tameike 38 06 56 N 140 51 06 E 15 1.36 M

7 Neko tameike 38 07 08 N 140 48 57 E 35 0.14 M

8 Yamagata a pond in Oishida 38 32 35 N 140 22 06 E 75 4.48 M

9 a pond in Mogamikanayama 38 53 07 N 140 18 58 E 160 0.91 M

10 Mikokubonuma 38 14 54 N 140 12 05 E 540 1.26 N

11 Fukushima a pond in Koriyama 37 20 50 N 140 15 05 E 305 0.29 M

12 a pond in Ten'ei 37 16 04 N 140 09 25 E 420 0.13 M

13 Niigata a pond in Satohonjyo 38 11 45 N 139 30 03 E 20 0.52 M

14 a pond in Tochiomizusawa 37 30 22 N 138 59 55 E 50 0.56 M

15 Suimentei tameike 37 31 02 N 138 59 23 E 35 1.49 M

16 a pond in Kariha 37 27 10 N 138 37 38 E 15 0.39 M

17 Sawada tameike 37 46 07 N 139 15 25 E 25 0.85 M

18 Imoriike 36 51 58 N 138 10 28 E 735 0.99 N

19 Gunma a pond in Ota 36 22 17 N 139 19 19 E 139 0.11 M

20 Saitama Higashishimonuma tameike 36 05 56 N 139 22 59 E 46 0.09 M

Sinodiaptomus 21 Oita Shichimataike 33 28 54 N 131 42 21 E 55 0.71 M

valkanovi 22 Kawakubo tameike 33 12 44 N 131 39 24 E 65 0.47 M

Japan 23 a pond in Moeda 32 59 36 N 131 34 30 E 115 2.20 M

SIS area 24 a pond in Usuki 33 00 57 N 131 41 10 E 118 0.13 M

25 Ehime Miyanotaniike 33 54 48 N 133 16 00 E 100 0.36 M

26 a pond in Iyokomatsu 33 53 22 N 133 06 35 E 40 0.18 M

27 Tokushima Kawabataike 34 09 02 N 134 28 12 E 25 0.11 M

28 Keida tameike 34 10 51 N 134 26 57 E 114 0.26 M

29 Hinoike 33 59 42 N 134 31 30 E 20 0.60 M

30 a pond in Mukaiyama 34 00 55 N 134 31 35 E 26 0.29 M

31 Gamodaike 33 50 06 N 134 44 50 E 4 1.19 N

32 Hyogo a pond in Ono 34 50 25 N 134 54 19 E 50 0.28 M

33 Okayama Furuato tameike 35 02 41 N 134 09 20 E 125 2.39 M

34 Obaraike 34 58 05 N 133 58 22 E 225 3.21 M

35 a pond in Mimasaka 35 01 40 N 134 09 08 E 115 0.01 M

36 a pond in Maniwa 35 04 00 N 133 48 42 E 210 1.84 M

37 Hiroshima Suganoike 34 24 26 N 133 19 28 E 45 1.54 M

38 Hamaike 34 28 21 N 133 15 47 E 50 1.76 M

39 Ohginoike 34 22 55 N 132 44 13 E 190 0.66 M

40 a pond in Hirasako 34 24 32 N 133 18 06 E 20 0.40 M

41 a pond in Shobara 34 49 24 N 132 55 29 E 296 0.70 M

42 a pond by Ueda Shogakko 34 41 45 N 132 54 45 E 466 0.04 M

43 Shimoyamaike 34 29 24 N 132 54 59 E 356 1.55 M

Sinodiaptomus - Auckland Auckland Domain pond 36 51 90 S 174 46 25 E 80 0.18 M

valkanovi - Wahili Gilmour Lake 37 24 40 S 175 50 75 E 100 0.94 M

New Zealand

Sinodiaptomus 44 Shizuoka a pond in Kakegawa 34 44 02 N 138 02 19 E 45 0.51 M

sarsi 45 a pond in Omaezaki 34 41 29 N 138 07 33 E 50 0.89 M

Japan 46 Okinawa Senbaruike 26 14 56 N 127 45 49 E 100 1.02 N

Makino, Knox, Duggan. Freshwater Biology. Electronic supplement 2. Information on molecular analyses in the present study.

Molecular analyses

mtCOI ncITS

Species and Locality No. of inds Recovered haplotypes No. of inds Recovered genotypes

country No. analysed (No. of inds) analysed (No. of inds)

Sinodiaptomus 1 6 NE01 (5), NE05 (1) 3 SV3 (2), SV8 (1)

valkanovi 2 6 NE01 (6) 3 SV3 (3)

Japan 3 6 NE01 (6) 3 SV3 (2), SV8 (1)

NE area 4 5 NE05 (5) 3 SV3 (2), SV8 (1)

5 6 NE01 (6) 3 SV4 (2), SV8 (1)

6 6 NE01 (3), NE02 (3) 3 SV8 (2), SV4 (1)

7 6 NE05 (5), NE04 (1) 3 SV3 (2), SV8 (1)

8 6 NE01 (5), NE03 (1) 3 SV9 (2), SV4 (1)

9 6 NE01 (6) 3 SV6 (1), SV8 (1), SV9 (1)

10 6 NE01 (6) 3 SV6 (1), SV8 (1), SV9 (1)

11 6 NE05 (6) 3 SV4 (2), SV8 (1)

12 6 NE01 (4), NE05 (2) 3 SV8 (2), SV9 (1)

13 6 NE01 (6) 3 SV3 (2), SV8 (1)

14 6 NE01 (4), NE05 (2) 3 SV4 (2), SV8 (1)

15 6 NE01 (6) 3 SV9 (2), SV3 (1)

16 6 NE01 (6) 3 SV4 (1), SV8 (1), SV9 (1)

17 6 NE01 (6) 3 SV4 (2), SV8 (1)

18 6 NE01 (6) 3 SV4 (2), SV8 (1)

19 6 NE05 (6) 3 SV3 (3)

20 6 NE05 (6) 3 SV3 (2), SV9 (1)

Sinodiaptomus 21 6 NE01 (6) 3 SV3 (3)

valkanovi 22 5 NE01 (5) 3 SV3 (3)

Japan 23 6 SIS03 (3), SIS05 (2), SIS04 (1) 3 SV3 (2), SV12 (1)

SIS area 24 6 SIS03 (5), NE01 (1) 3 SV3 (2), SV12 (1)

25 6 SIS01 (6) 3 SV2 (3)

26 6 SIS01 (3), SIS19 (3) 3 SV2 (1), SV3 (1), SV12 (1)

27 6 SIS02 (6) 3 SV1 (3)

28 5 SIS26 (4), SIS27 (1) 3 SV10 (3)

29 6 SIS05 (6) 2 SV2 (1), SV10 (1)

30 6 SIS05 (5), SIS25 (1) 3 SV2 (3)

31 6 SIS21 (2), SIS20 (1), SIS22 (1), SIS23 (1), SIS24 (1) 3 SV1 (3)

32 6 SIS36 (4), SIS37 (2) 3 SV6 (2), SV2 (1)

33 6 SIS06 (6) 3 SV1 (3)

34 6 SIS16 (2), SIS14 (2), SIS13 (1), SIS15 (1) 2 SV1 (2)

35 6 SIS06 (5), SIS35 (1) 3 SV1 (3)

36 6 SIS29 (5), SIS28 (1) 3 SV1 (3)

37 6 SIS07 (3), SIS08 (1), SIS09 (1), SIS10 (1) 3 SV5 (2), SV10 (1)

38 6 SIS17 (5), SIS18 (1) 3 SV5 (3)

39 6 SIS12 (4), SIS11 (1), NE01 (1) 3 SV2 (1), SV3 (1), SV13 (1)

40 5 SIS07 (5) 3 SV2 (1), SV10 (1), SV14 (1)

41 6 SIS30 (3), SIS31 (2), SIS23 (1) 3 SV7 (2), SV11 (1)

42 6 SIS33 (6) 3 SV7 (3)

43 6 SIS17 (2), SIS34 (2), NE01 (2) 2 SV3 (2)

Sinodiaptomus - 4 NE05 (4) 0

valkanovi - 4 NE05 (4) 0

New Zealand

Sinodiaptomus 44 5 SAR2 (2), SAR3 (3) 3 SS1 (2), SS2 (1)

sarsi 45 6 SAR3 (6) 3 SS1 (3)

Japan 46 6 SAR1 (5), SAR2 (1) ! 3 SS2 (3)

invasion, genetic variation and species identity of the calanoid copepod sinodiaptomus valkanovi

Documents