Morphological differentiation, mitochondrial and nuclear DNA variability between geographically distant populations of Daphnia galeata and Daphnia cucullata (Anomopoda, Daphniidae) Текст научной статьи по специальности «Биологические науки»

Journal of Siberian Federal University. Biology 4 (2010 3) 434-453

УДК 575 + 591.151/.158:595.324

Morphological Differentiation, Mitochondrial

and Nuclear DNA Variability

Between Geographically Distant Populations

of Daphnia galeata and Daphnia cucullata (Anomopoda, Daphniidae)

Elena I. Zuykovaa*, Nickolai A. Bochkareva, Anna S. Semenovab and Alexey V. Katokhinc

a Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences, 11 Frunze, Novosibirsk, 630091 Russia b Atlantic Research Institute of Marine Fisheries

and Oceanography, 5 Dm. Donskoy, Kaliningrad, 236022 Russia c Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentyev, Novosibirsk, 630090 Russia 1

Received 3.12.2010, received in revised form 10.12.2010, accepted 17.12.2010

Although members of genus Daphnia (Anomopoda, Daphniidae) are the most common water invertebrates and are considered as model organisms for many taxonomic, ecological and evolutionary studies their systematics remains unresolved. Here, morphological differentiation and genetic polymorphism between the geographically distant populations of the sister species Daphnia galeata Sars, 1864 and Daphnia cucullata Sars, 1862 in the Curonian Lagoon, a large shallow freshwater lagoon of the Baltic Sea (Russia, Kaliningrad Oblast) and Novosibirsk Reservoir (Russia, Novosibirsk Oblast) are presented. The divergence between species and their populations was analyzed based on traditional morphological traits and a large set of morphometric traits describing the body shape. The traits describing the shape of head and helmet, and spine were the most variable morphological characters. Phylogenetic relationships between species andpopulations were constructed based on variation in mitochondrial 16S and 12S rRNA genes and nuclear ITS2 rDNA sequences. The mitochondrial DNA divergence between D. galeata and D. cucullata species was significant and reflected their monophyletic origin, whereas intraspecific genetic distances are estimated as insignificant.

Keywords: Daphnia galeata, Daphnia cucullata, morphological variation, mitochondrial DNA, nuclear DNA, genetic divergence

* Corresponding author E-mail address: ih@eco.nsc.ru

1 © Siberian Federal University. All rights reserved

Introduction

Cladoceran of genus Daphnia (Anomopoda, Daphniidae) are the most common invertebrates in water ecosystems. Many species of this genus are used as model organisms in the different field of biology including toxicology, biogeography, and evolutionary ecology. The most reliable taxonomic keys of some Daphnia species were developed by S.M. Glagolev (1986). However, the systematics of many Daphnia species complexes remains unresolved and morphological distinction between some species is often lacking. The main cause of taxonomic confusion consists in remarkable morphological plasticity in response to ecological and genetic factors. The body shape, helmet and tail spine sizes were shown to depend on water temperature, turbulence, quantity of available food, and presence of invertebrate and vertebrate predators (Hebert, Grewe, 1985; Mort, 1989; Sorensen, Sterner, 1992; Burns, 2000; Lass, Spaak, 2003; Laforsh, Tollrian, 2004). Both considerable morphological variability and similarity may be due to interspecific hybridization and introgression as it was shown for species of Daphnia longispina complex based on genetic studies (Taylor, Hebert, 1992; Colbourne, Hebert, 1996; Schwenk et al., 1998; Gießler et al., 1999; Schwenk et al., 2000; Hobsk et al., 2004; Gießler, Englbrecht, 2009). At present time both mitochondrial and nuclear genetic markers have a wide use for delineation of Daphnia species and phylogenetic relations assignment between them (Taylor et al., 1996; Schwenk et al., 1998; Gießler, 2001; Duffy et al., 2004; Petrusek et al., 2008). These studies deal with both geographically limited and distant Daphnia populations inhabiting different waterbodies of Western Europe and North America. Meanwhile, the study of genetic diversity of Daphnia populations from Russian water bodies is extremely shallow (Bychek, Müller, 2003; Kotov et al., 2006; Ishida, Taylor, 2007). Besides, often genetic studies

of daphniids are not confirmed by analysis of the taxonomic traits, therethrough generate obvious mistakes in species identification. Different statistical methods on quantitative and qualitative morphological data sets were successfully applied to reveal traits useful for species delineation (Dodson, 1981; Schwartz et al., 1985; Benzie, 1988; Giepler, 2001; Duffy et al., 2004).

The purpose of this study is to perform comparative morphological analysis of the body shape variability using multivariate statistical method and to evaluate the variability of the 16S and 12S mitochondrial DNA and the ITS2 nuclear DNA markers in geographically distant populations of sister species D. galeata Sars, 1864 and D. cucullata Sars, 1862 (D. longispina complex) from Novosibirsk Reservoir of West Siberia and the Curonian Lagoon of the Baltic Sea.

Materials and Methods

Study areas

Novosibirsk Reservoir (54°28'N, 82°23'E) is a large artificial water body in the Ob River's valley located in two regions: Novosibirsk Oblast and Altai Territory. Some reservoirs characteristics are given in Table 1. In winter this water body is covered by ice in the whole. According to literature data zooplankton community was originated from zooplankton of drowned flood-plane water bodies belonging to the river channel. The reservoir is used for recreation and fishing. In different periods of the reservoir's formation three species D. longispina, D. cucullata, and D. hyalina among genus Daphnia were identified (Solonevskaya, 1961; Bityukov, 1964; Pomerantseva, 1976; Kotikova, 1985). At present D. cucullata and D. longispina inhabit in the lacustrine part of the reservoir and D. cucullata has being dominated since 1995 (Ermolaeva, 2007).

Table 1. Some characteristics of the waterbodies investigated, specimens number in morphological and genetic data sets

__

?

2 £

a e a e a

m 13 <u

M a 13

c3 jm a e r < •f ~ na 1 1

Waterbody

13

=1

<

D. galeata

y

M

ft a o S oa

2

K M

6 2 H

1 1

D. cucullata

y

M

ft a o S oa

2

M M M

6 2 H

1 1

Novosibirsk Reservoir

Curonian Lagoon

113 1082 8.8

;.3 25.0

1584 6.2 3.8

5.8

75

71

17

19

10

31

The Curonian lagoon (55°18'N, 20°55'E) is a large shallow freshwater lagoon of the Baltic Sea is subjected to strong anthropogenic impact. Some characteristics of the lagoon are provided in Table 1. The continuing eutrophication of the lagoon is accompanied by water "hyperbloom" under the mass development of blue-green algae (Alexandrov, Dmitrieva, 2006). Their biomass significantly exceeds the level conditioning the secondary pollution of the water body in some year. According to hydrochemical data and the structural and functional characteristics of zooplankton, the Curonian Lagoon belongs to eutrophic water bodies with a transition to a hypereutrophic stage (Alexandrov et al., 2006; Semenova, Alexandrov, 2009). This water body is covered by ice for a short winter period. According to literature data several Daphnia species were registered in the Curonian Lagoon, namely D. longispina, D. hyalina, D. cucullata, D. cristata, and D. pulex (Szidat, 1926; Schmidt-Ries, 1940; Kiselite, 1957; Naumenko, 1994; Pliuraite, 2003). At present, D. galeata is dominant species and D. cucullata is subdominant one.

Sampling

For studies of morphological and genetic variability of Daphnia specimens in Novosibirsk Reservoir the zooplankton samples were taken in August, 2008 with the Apstein net (mesh

size 250 ^m). For studies of morphological variability of Daphnia in the Curonian Lagoon we used the samples collected from April to September, 2008. For study of their genetic polymorphism the samples were collected in May-June and September, 2009. In the Curonian Lagoon the samples were taken with a Van-Dorn bathometer.

The samples were preserved in 5 % (or 4 %) formalin solution with sucrose (Haney, Hall, 1973) for morphological and morphometric analyses. For genetic analysis of Daphnia species zooplankton samples were stored directly in ethanol (90-95 %) until DNA was extracted.

Morphological analysis

Daphnia species were identified according to the keys presented in the recent literature (Glagolev, 1986; FloPner, Kraus, 1989). Females of D. galeata h D. cucullata in the forth or fifth age-size groups were photographed for digital morphological analysis in lateral view under AxioScan microscope (Carl Zeiss, Germany) (*50 or xi00 magnitude) (for sample size see Table 1) To analyze a body shape 23 morphological measurements were made using the digital images with the AxioVision software. The morphometric characters were taken according to the set given in Zuykova, Bochkarev (2010). Three characters were additionally used, namely,

7

6

7

4

3

4

4

0

3

3

3

the distance from center of the eye to the point of tail spine attachment (O.l.t.sp), the distance from the antennulae tip to the rostrum tip (a.r) and the helmet angle (helmet angle).

A principal component analysis (PCA) was performed to estimate morphological variation just as it has been done for other Daphnia species (Schwartz et al., 1985; Benzie, 1988). This analysis calculates new variables (principal component) which are linear combinations of the original characters and allows distinguishing the most significant characters. Obtained variables were normalized and centered. The components were estimated as new traits, and then an average loading value, an error in mean, and a standard deviation were calculated for each sample. To estimate the significance of morphological divergence between all Daphnia samples based on the average loading values the Student t-test was applied (Efimov, Kovaleva, 2005). As the first principal component accounts for the most variation and explains the size variability, hence the body shape parameters between the Daphnia samples were analyzed in the space of the second and third PCA axes. The PCA variables were used as input in UPGMA analysis to estimate the divergence among the samples. All statistical analyses were performed using STATISTICA version 6.0 (StatSoft Inc., USA), SNEDECOR version 5.0 (ODS Soft, Novosibirsk, Russia), and PAST version 2.05 (http://palaeo-electronica.org) softwares.

DNA analysis

Ethanol-preserved animals were used for analysis of nucleotide polymorphism. Total DNA was extracted from a single individual (female or male) or an ephippium using a 5 % suspension of Chelex 100 resin (BioRad). Before use in PCR the extracted DNA was stored under -20°C. The polymerase chain reaction was used to amplify the 16S and 12S mitochondrial genes and the

ITS2 region of nuclear DNA including part of flanking 5.8S and 28S ribosomal RNA genes. The primers and conditions for PCRs in a 20 ^l reaction volume were as following: 2-5 ^l DNA homogenate, 0.2 pM dNTPs, 2 ^l 10* PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mMKCl), 2.5 mM MgCl2, 0.5 ^M of each primer and 1 unit of Thermus aquaticus DNA polymerase (Taq-pol).

The 16S gene was amplified using the originally designed primers:

16Sin-F 5 '-TTTGTAAATGGCCGCAGTA-3'

and

16Sin-R 5' -CGGTTTGAACTCAGATCAT-GTA-3'.

A thermocycler (BIS-N, Novosibirsk, Russia) was run for 2 min at 94 °C (1 cycle), followed by 30 s at 94 °C, 30 s at 56 °C, 1 min 45 s at 72 °C (35 cycles) and extension for 2 min at 72 °C.

The 12S gene was amplified using the primers:

12S-F 5'-ATGCACTTTCCAGTACATCTAC-3'

and

12S-R 5'-AAATCGTGCCAGCCGTCGC-3' (Colbourne, Hebert, 1996). A thermocycler was run for 2 min at 94 °C (1 cycle), followed by 1 min 30 s at 94 °C, 45 s at 58 °C, 1 min 30 s at 72 °C (35 cycles) and extension for 6 min at 72 °C.

The ITS2 region was amplified using the specially designed forward primer 5.8Fr 5'-CCCTGAACGGTGGATCACTA -3' and a reverse primer according to Taylor et al. (2005) 28SD2BR 5'-TTAGAAGGAGTTTACCTCCCGCTTAGG -3'. A thermocycler was run at 2 min at 94 °C (1 cycle), followed by 1 min at 94 °C, 45 s at 53 °C, 1 min at 72 °C (35 cycles) and extension for 6 min at 72 °C.

The PCR products were separated on 1 % agarose 1* TAE gel (Low EEO Standart agarose, BIOZYM, Russia) in the presence of ethidium bromide and photographed under UV light. A 1-2 kb DNA ladder (MEDIGEN, Novosibirsk, Russia) was used for the estimation of the amplicon length.

The amplified products were purified using a kit from BIOSILICA (Novosibirsk, Russia) and both stands were sequenced on an automated sequencer ABI PrISM 3100 Avant Genetic Analyzer (Applied Biosystems, USA) using Big Dye terminator sequencing kit (Applied Biosystems, USA) at the Center of DNA Sequencing of Siberian Branch of the Russian Academy of Science (Novosibirsk, Russia, http://sequest.niboch.nsc.ru). The DNA sequences were first automatically aligned using the CLUSTALW algorithm and then manually edited. The nucleotide sequences of the newly analyzed specimens were deposited in GenBank (see Table 2 for accession numbers).

An estimation of the divergence between sequences and the construction of a neighbor-joining (NJ) phylogram based on Kimura 2-parameters (with pairwise deletion of the gaps and missing sites) was conducted in Molecular Evolutionary Genetics Analysis software version 4.0 (MEGA 4) (Saitou, Nei, 1987; Tamura et al., 2007). One thousand bootstrap replicates were run to assess the statistical support in the tree nodes. Additionally, we analyzed the phylogenetic relationships among individuals using minimum evolution (ME) and maximum parsimony (MP) methods. For comparative analysis the sequences of respective fragments for Daphnia species from GenBank database were included into analyses.

Results

Morphological variability

Morphological analysis of the Daphnia populations based on the main qualitative characters traditionally used in taxonomic keys (Glagolev, 1986) allowed identification of D. galeata and D. cucullata species in the Curonian Lagoon and Novosibirsk Reservoir. These characters included the shape of the antennulae mound, insertion and length of aesthetasks, presence of ocellus, the crest in frontal view, rostrum shape and length, head shape near the eye

and the ventral margin of the head (Fig. 1, 2). In addition we use some traits of males (Fig. 1 K - P, U, V, Fig. 2 J). Subsequently, analysis of the body shape was carried out based on the morphometric traits describing body shape only.

The body shape of D. galeata from the Curonian lagoon was found to be remarkably changeable. At first, this can be explained by seasonal variability, because the morphological analysis was carried out with the samples taken during the whole growing season. The most significant morphological differences concerned helmet size and form. So, D. galeata specimens collected in April were characterized by a rounded head or had a very small helmet (Fig. 1 A, F). The individuals collected in May had both a large and medium-scale helmet; in September the individuals with a large helmet were registered only. Thus, the D. galeata specimens from the Curonian lagoon were divided into three groups with respect to their helmet size and shape. D. cucullata specimens presented the separate forth group (Fig. 1 N - P, V). The sample of D. galeata in Novosibirsk Reservoir was more homogeneous. The only significant difference among individuals was related to the helmet size (Fig. 2 A - F). The second group in Novosibirsk Reservoir was presented by D. cucullata specimens (Fig. 2 G, H).

Figure 3a displays the morphological divergence between all groups and samples generated by principal component analysis at the space of the first two axes. The first PCA axis was formed by approximately equal positive loadings of all characters and this axis reflects a dimensional variability (69.01 %) in the common Daphnia samples (Table 3). The most remarkable differences were registered between all samples of D. galeata and D. cucullata and between the populations of these species (Table 4). The most significant divergence among all D. galeata samples was found between the rounded head

Table 2. List of specimens' designations and corresponding GenBank accession numbers of Daphnia species genetically examined in this study

Species Label Country Location Developmental stage Sex GenBank accession numbers

16S 12S ITS2

1 2 3 4 5 6 7 8 9

D. cucullata NRCul Russia Novosibirsk Reservoir adult female HM067407 HM100080 HM161684

D. cucullata NRCu2 Russia Novosibirsk Reservoir adult female HM067408 HM100081 HM161685

D. cucullata NRCu3 Russia Novosibirsk Reservoir adult female HM067409 HM100082 HM161686

D. cucullata NRCu4 Russia Novosibirsk Reservoir adult female - HM100083 HM161687

D. galeata NRG1 Russia Novosibirsk Reservoir adult female HM067430 HM100087 HM161698

D. galeata NRG2 Russia Novosibirsk Reservoir adult female HM067431 HM100088 HM161699

D. galeata NRG3 Russia Novosibirsk Reservoir adult female HM067432 HM100089 HM161700

D. galeata NRG4 Russia Novosibirsk Reservoir adult female HM067433 HM100090 HM161701

D. galeata NRG5 Russia Novosibirsk Reservoir adult female HM067434 HM100091 HM161702

D. galeata NRG6 Russia Novosibirsk Reservoir ephippium HM067435 HM100092 HM161703

D. galeata NRG7 Russia Novosibirsk Reservoir ephippium HM067436 - HM161704

D. cucullata CoLCul5 Russia Curonian Lagoon adult female HM067410 HM100084 HM161681

D. cucullata CoLCuló Russia Curonian Lagoon adult female HM067411 HM100085 HM161682

D. cucullata CoLCul7 Russia Curonian Lagoon adult female HM067412 HM100086 HM161683

D. galeata CoLGl Russia Curonian Lagoon juv female HM067413 HM161705 HM161688

D. galeata CoLG2 Russia Curonian Lagoon juv female HM067414 HM161706 HM161689

D. galeata CoLG3 Russia Curonian Lagoon adult female HM067415 HM161707 HM161690

D. galeata CoLG4 Russia Curonian Lagoon adult female HM067416 HM161708 HM161691

D. galeata CoLG5 Russia Curonian Lagoon adult female HM067417 HM161709 HM161692

D. galeata CoLG7 Russia Curonian Lagoon adult female HM067418 HM100093 HM161693

D. galeata C0LG8 Russia Curonian Lagoon juv female HM067419 HM100102 -

D. galeata CoLG9 Russia Curonian Lagoon adult female - HM100094 -

D. galeata CoLGlO Russia Curonian Lagoon juv female HM067420 HM100095 -

D. galeata CoLGll Russia Curonian Lagoon adult female HM067421 HM100096 -

D. galeata CoLG14 Russia Curonian Lagoon juv male HM067422 HM100103 -

D. galeata C0LGI8 Russia Curonian Lagoon adult female HM067423 HM100097 -

D. galeata CoLG19 Russia Curonian Lagoon juv male HM067424 HM100104 -

D. galeata CoLG20 Russia Curonian Lagoon adult female HM067425 HM100098 HM161694

D. galeata CoLG21 Russia Curonian Lagoon adult female HM067426 HM100099 -

D. galeata CoLG22 Russia Curonian Lagoon juv male HM067427 HM100105 HM161695

D. galeata CoLG23 Russia Curonian Lagoon adult female HM067428 HM100100 HM161696

D. galeata CoLG24 Russia Curonian Lagoon juv male HM067429 HM100101 HM161697

Continued table 2

D. galeata Canada Guelph Lake AF064187*

D. galeata Russia Volga River AY115492*

D. cucullata Germany Thalersee FJ943783*

D. cucullata Germany Klostersee FJ943782*

D. cucullata Netherlands Tjeukemeer FJ943784*

D. cucullata Germany Hattstein Weiher FJ178307*

D. cucullata Czech Repub. Medlov Pond AF277270*

D. cucullata Slovenia Lake Bled AF277269*

D. cucullata Russia Lake Glubokoe AF277272*

D. galeata Canada AY730382*

D. galeata USA AY730396*

D. galeata USA AY730397*

D. galeata Japan AY730398*

D. galeata England AY730401*

D. cucullata Germany AY730402*

Fig. 1. Daphnia morphology from the Curonian Lagoon. D. galeata A-P: A-G. female, lateral view; H, I. Head, female, lateral view; J. Postabdomen, female, lateral view; K, L. male, lateral view; M. Head, male, lateral view; N. Antenna I, male; O. Postabdomen, male; P. Limb I, male; D. cucullata Q-V: Q-S. female, lateral view; T. Head, female, lateral view; U. male, lateral view; V. Head, male, lateral view. Scale bars 200 ¡im for A-I, K, L, R, S; 100 ¡m for J, M-P, T-V

Fig. 2. Daphnia morphology from Novosibirsk Reservoir. D. galeata A-J: A-F. female, lateral view; G. Head, female, lateral view; H. Postabdomen, female, lateral view; I. Postabdominal claw, female; J. male, lateral view; D. cucullata K,L: K. female, lateral view; L. Head, female, lateral view. Scale bars 200 ^m for A-G, K; 100 ^m for H, J, L; 50 ^m for I

form and an intermediate one inhabiting the Curonian Lagoon.

With respect to the second and third PCA axes the morphological divergence between all D. galeata samples was smaller, except the rounded head form from the Curonian Lagoon (Fig. 3 b). The second PCA axis (12.44 %) loaded primarily on the head characters (l.cap., m.v.cap.), the eye position (O.m.v.), helmet size and form (l.helm, m.v.helm., helmet angle), and length tail spine (l.t.sp). The third PCA axis (4.30 %) was formed by the loadings of the characters of the eye (O, O.m.v.cap), helmet form (m.v.helm, helmet angle), rostrum form and length (r.m.v., a.r) and the carapace characters (w.br., r.W.v., w.cap.d) (Table 3). Almost all samples and forms significantly differed with respect to the loadings

into the third PCA axis, except the D. cucullata samples (Table 4).

A dendrogram constructed using average values of the first three principal components suggested that there are three main distinct clusters (Fig. 4). The first cluster consisted of the D. galeata specimens from both water bodies. The D. cucullata populations comprised the second cluster. Finally, the rounded head form of D. galeata from the Curonian Lagoon was separated into a distinct group, mainly due to head shape near the eye and the ventral margin of the head.

Mitochondrial DNA variability

16S mtDNA. For thirty Daphnia individuals 481 bp of the 16S gene were sequenced. Additional

Fig. 3. Plot of clouds distributions and centroids of the common samples of D. galeata and D. cucullata from the Curonian Lagoon (CoL) and Novosibirsk Reservoir (NR) according to the morphological variables in the space of the first and second (A) and second and third (B) PCA axes; ± standard deviation. Open cirles - D. cucullata (CoL), black circles - D. cucullata (NR); grey circles - rounded form of D. galeata (CoL), open diamonds -helmeted form of D. galeata (CoL), grey squares - intermediate form of D. galeata (CoL), grey triangles - D. galeata (NR)

2 sequences for D. galeata were obtained from GenBank database (Table 2). The pairwise distances for the 16S fragment within D. galeata and D. cucullata species were 0.002 and 0.004, respectively. The divergence between these species was 0.022. There were 7 conservative sites through multiple alignment 522 nucleotides of length. The overall transition/transversion bias was R = 3.862.

A neighbour-joining analysis (the 16S sequence for Eubosmina coregoni was used as outgroup, GenBank #EU650747) produced a tree with the high bootstrap support for two branches corresponding to D. galeata and D. cucullata species, 89 and 88 %, respectively (Fig. 5). The topology indicated monophyletic origin of these groups. However, two D. cucullata specimens (NRCu2 and NRCu3) from Novosibirsk Reservoir formed a separate group with high bootstrap support, 85 %. Minimum evolution and maximum parsimony analyses (trees not presented) resulted in identical topologies with slightly less bootstrap support for the branches.

12S mtDNA. For the 12S gene 7 sequences of 610 bp for D. cucullata and 24 sequences of 608 bp for D. galeata were obtained. Additional 17 sequences for both species from GenBank database were included into analysis. The sequence for E. coregoni was chosen as outgroup (GenBank #AF494467). The within-specific pairwise distances for the 12S fragment were 0.002 for D. galeata and 0.003 for D. cucullata. The divergence between species was 0.083. If the sequences obtained from GenBank database were eliminated from the analysis the genetic distances within and between species were 0.001 and 0.075, respectively. There were 8 conservative sites through 12S multiple alignment 743 nucleotides of length. The overall transition/transversion bias was R = 2.356.

NJ-tree agreed in topology with NJ-tree based on 12S sequences (Fig. 6). There were two

Table 3. Component loadings of the morphological characters of the common Daphnia samples into the

first three PCA axes. Major loadings are asterisked.

Character Loadings

1 PCA 2 PCA 3 PCA

L 0.24* 0.00 0.10

o.t.sp 0.23* -0.08 0.05

W 0.23* -0.08 0.10

w.br. 0.21* -0.07 0.35*

w.cap. 0.24* 0.02 -0.09

l.cap. 0.21* 0.24* 0.01

l.helm. -0.15 0.38* -0.14

O 0.19 -0.12 0.35*

lr 0.21* 0.14 -0.15

O.m.v. 0.16 0.31* 0.15

r.m.v. 0.22* 0.04 -0.22*

m.v.cap. 0.19 0.30* -0.03

m.v.helm. -0.05 0.53* 0.22*

Or 0.24* 0.06 -0.10

O.w.cap. 0.22* 0.15 -0.22*

cap.d. 0.21* 0.19 -0.01

r.W.v. 0.21* -0.16 0.29*

w.cap.d. 0.22* -0.14 0.30*

l.t.sp. 0.14 0.30* -0.06

d.l.t.sp. 0.23* -0.06 0.05

v.l.t.sp. 0.23* -0.05 -0.07

helmet angle 0.19* -0.22* -0.36*

a.r. 0.19* -0.17 -0.42*

l.cl. 0.18* -0.06 -0.14

Cumulative % 69.01 12.44 4.30

Table 4. P-values between average values of the first three eigenvectors for pairwise comparison of the common samples of Daphnia (f-test)

Eigenvectors P-value

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

1 - - 0.001 0.001 - 0.001 0.001 0.001 - 0.001 0.001 - 0.001 0.001 0.001

2 0.001 0.001 - 0.001 - 0.001 0.001 0.001 0.001 0.001 - 0.001 0.001 - 0.001

3 0.001 0.001 0.001 0.001 0.001 0.001 - 0.001 - 0.01 0.001 0.001 0.001 - 0.001

Note: number samples see Fig. 4; differences assuming CD-test are marked by bold type.

Fig. 4. UPGMA-dendrogram based on morphometric data for six samples of D. galeata and D. cucullata from the Curonian Lagoon (CoL) and Novosibirsk Reservoir (NR) (Euclidean distance between the average loading values into the second and third PCA axes). 1 - helmeted form ofD. galeata (CoL), 2 - rounded form ofD. galeata (CoL), 3 - intermediate form of D. galeata (CoL), 4 - D. cucullata (CoL), 5 - D. galeata (NR), 6 - D. cucullata (NR)

clusters with bootstrap support of the branches for D. galeata 99 % and D. cucullata 100 %. The topology of the 12S NJ-tree also indicated the monophyletic origin of these species.

ITS2 nuclear DNA. Between 1075 and 1087 bp of the ITS2 region were sequenced for 7 specimens of D. cucullata and for 17 specimens of D. galeata. Additional 6 ITS2 sequences were obtained from GenBank database and D. longispina ITS2 sequence (Poland, GenBank #AY730404) was used as the outgroup. Pairwise distances within D. galeata and D. cucullata species ranged from 0.002 to 0.05, respectively, with divergence between these species 0.013. There were 11 conservative sites in the ITS2 region through multiple alignment 1131 nucleotides of length. The overall transition/transversion bias was R = 1.652.

The phylogenetic relationships between D. galeata and D. cucullata species identified using the ITS2 region (tree is not presented) were generally consistent with the branching

topology of trees based on mitochondrial DNA. The ITS2 sequences were also subjected to NJ and ME analyses. All methods produced a nearly identical topology with respect to species. But the support for a branch that resolves the position both species was lost. One D. cucullata specimen (NRCu3) from Novosibirsk Reservoir clustered together with D. galeata.

Discussion

The use of traditionally taxonomic keys has allowed identification of D. galeata and D. cucullata species in the Curonian Lagoon and Novosibirsk Reservoir. We suppose that enormous morphological variability, nomenclatural problems and the use of the inappropriate key for the identification of species within Daphnia longispina complex by previous studies could result in delineation of D. longispina and D. hyalina species in the investigated water bodies. The remarkable fact was that D. galeata was not recognized in the species composition of

Fig. 5. A phylogenetic tree constructed using the neighbor-joining method (NJ) based on mitochondrial 16S rDNA sequences for D. galeata and D. cucullata. The NJ was rooted with Eubosmina coregoni. The number above the branches represents the bootstrap confidence limit (1000 replicates)

zooplankton community in Novosibirsk Reservoir until recently (Ermolaeva, 2007). Based on the morphometric analysis we have shown that the geographically distant populations of D. galeata differed between each other based on head length, shape of the ventral margin of the head, helmet length, slope and shape, the position and diameter of the eye, rostrum shape, some characters of the carapace and length tail spine. D. cucullata

was characterized by less interpopulation morphological variability compared with D. galeata. However, despite the marked differences the geographically distant populations of these Daphnia species clustered together confirming their species identity.

Interpopulation variability of the 16S and 12S mitochondrial genes for the studied species is negligible and the consistency in the topology of the

Fig. 6. A phylogenetic tree constructed using the neighbor-joining method (NJ) based on mitochondrial 12S rDNA sequences for D. galeata and D. cucullata. The NJ was rooted with Eubosmina coregoni. The number of the branches represents the bootstrap confidence limit (1000 replicates)

NJ-trees for both markers was found. Additional analyses of the phylogenetic relationships between closely related species D. galeata and D. cucullata using the minimum evolution and maximum parsimony methods also showed a concordant topology. Meanwhile, the intraspecific genetic distances for the 16S gene were higher than those for the 12S gene but the interspecific genetic distances were lower. An additional point is that the genetic divergence within D. cucullata was more significant as compared with D. galeata, whereas the morphology of the first species was less variable. The deletion from the analysis of the sequences obtained from GenBank database for the 12S gene resulted in reduction of the differences between the D. cucullata specimens inhabiting Novosibirsk Reservoir and the Curonian Lagoon. Our data are consistent with data on the phylogenetic relationships of D. cucullata and D. galeata populations in the water bodies of Western Europe, which also marked monophyletic and sister relationships (Schwenk et al., 2000; Petrusek et al., 2008). We did not find divergence between European and Siberian D. galeata populations using both mitochondrial markers, as it was shown earlier for European and North American populations (Taylor et al., 1996). However, we found that 16S gene was scarce conservative sites in comparison with the 12S gene, whereas for the North American D. laevis complex has been shown opposite (Taylor et al., 1998).

A clear resolution between the ITS2 sequences of nuclear DNA for D. galeata and D. cucullata species from both Novosibirsk Reservoir and the Curonian Lagoon was not found. The genetic divergence was lower than it was calculated for mitochondrial DNA. The possibility of interspecific hybridization is suggested by the lack of divergence among the ITS2 sequences of specimens from the studied populations. This finding, in turn, indicates also

that both species are insufficiently isolated from each other and demonstrate sister relationship. The existence of hybridization between different populations of these species has been previously shown using other DNA markers (Schwenk et al., 1998; Gießler et al., 1999; Schwenk et al., 2001; Taylor et al., 2005; Ishida, Taylor, 2007; Petrusek et al., 2008; Gießler, Englbrecht, 2009).

Conclusion

The most important finding of our study is the absence of any significant morphological and genetic divergence between the geographically distant D. cucullata and D. galeata populations. The existence of separate phylogenetic lineage of D. cucullata in Novosibirsk Reservoir may be a result from flooding from various water bodies during the process of its formation. Mitochondrial and nuclear DNA significant variation among different morphotypes of D. galeata from the Curonian Lagoon was absent too. Such low level of the divergence within these morphs may be due to either their conspecific or hybrid origin of the intermediate morphs with inheritance of maternal mitogenome of D. galeata. On the other hand, the rounded head morph of D. galeata from the Curonian Lagoon observed at the beginning of spring enormously distinguished it from both other morphs and D. cucullata based on morphometric analysis. However, its specific delineation remains in abeyance.

In general, we have demonstrated significant morphological and genetic similarity among the geographically distant D. galeata and D. cucullata populations from two large water bodies in Russia.

Acknowledgments

We thank N.M. Korovchinsky, A.A. Kotov, and A.Yu. Sinev for advice on Daphnia morphology and for help with preparing of the Daphnia drawings.

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Морфологическая изменчивость и генетический полиморфизм географически удаленных популяций Баркта Са1ва1а и Баркта Сысыима (Anomopoda, Daphniidae)

Е.И. Зуйкова3, Н.А. Бочкарев а, А.С. Семенова ь, А.В. Катохин с

а Институт систематики и экологии животных СО РАН, Россия 630091, Новосибирск, ул. Фрунзе, 11 ьАтлантический научно-исследовательский институт рыбного хозяйства и океанографии, Россия 236022, Калининград, Дм. Донского, 5 с Институт цитологии и генетики СО РАН, Россия 630090, Новосибирск, Лаврентьева, 10

Несмотря на то, что представители р. Daphnia (Anomopoda, Daphniidae) являются одними из наиболее распространенных водных беспозвоночных и используются в качестве модельных организмов в таксономических, экологических и эволюционных исследованиях, их систематика остается весьма запутанной. Настоящее исследование посвящено изучению морфологической дифференциации и генетической изменчивости географически удаленных популяций сестринских видов Daphnia galeata Sars, 1864 и Daphnia сисЫШа Sars, 1862 (Anomopoda, Daphniidae) из пресноводной части Балтийского моря - Куршского залива (Россия, Калининградская область) и Новосибирского водохранилища (Россия, Новосибирская область). Морфологическая дивергенция между видами и их популяциями оценивалась по диагностическим признакам и на основании анализа изменчивости формы тела по набору морфометрических признаков. Самыми изменчивыми были признаки, характеризующие форму головы, шлема и хвостовой иглы. Реконструкция филогенетических отношений между видами выполнена на основе изменчивости 16S и 12S генов митохондриальной ДНК и фрагмента ITS2 ядерной ДНК. Дивергенция между видами D. galeata и D. сисЫШа на основе генов митохондриальной ДНК была значительной и свидетельствует об их монофилетическом происхождении, тогда как внутривидовые генетические дистанции оцениваются как незначительные.

Ключевые слова: Daphnia galeata, Daphnia сисЫШа, морфологическая изменчивость, митохондриальная ДНК, ядерная ДНК, генетическая дивергенция.