JOURNAL OF CRUSTACEAN BIOLOGY, 20(3): 522–529, 2000
SYSTEMATICS OF THE EUROPEAN ENDANGERED CRAYFISH SPECIES
AUSTROPOTAMOBIUS PALLIPES (DECAPODA: ASTACIDAE)
Frédéric Grandjean, D. James Harris,
Catherine Souty-Grosset, and Keith A. Crandall
(FG, CSG) Université de Poitiers, Laboratoire de Biologie Animale, UMR 6556, 40 avenue du Recteur
Pineau, F–86022 Poitiers cedex, France (e-mail: [email protected]);
(DJH, KAC) Brigham Young University, Department of Zoology and Monte L. Bean Museum,
574 Widtsoe Building, Provo, Utah 84602–5255, U.S.A.
A B S T R A C T
Mitochondrial DNA sequence variation in the 16S rRNA gene was used to estimate the phylogenetic relationships of Austropotamobius pallipes. The program ModelTest was used to test alternative models of evolution for our data using likelihood ratio tests. Both the minimum evolution tree
with the HKY85 model and the maximum likelihood analysis supports the separation of two major clades (A and B) and three clades within clade A. The two major groups A and B showed genetic differentiation of 4.6% and could be in accordance with the classification on the specific status of A. italicus and A. pallipes. Within clade A, three clades were found corresponding to crayfish sampled in Spain-Italy-France, Austria, and Slovenia. In accordance with morphological data
extracted from recent papers, a new classification based on the presence of three subspecies (italicus, carinthiacus, and carsicus) within A. italicus is proposed.
The white-clawed crayfish Austropotamobius pallipes (Lereboullet, 1858) is included
in the family Astacidae, which contains two
genera in Europe: Austropotamobius with
three species (pallipes, biernhauseri, and torrentium) and Astacus with two species (astacus and leptodactylus), and a single genus
from western North America, Pacifastacus.
Austropotamobius pallipes has a widespread
distribution in Western Europe which extends
from the former Yugoslavia through Italy,
France, Germany, Spain, and into Great
Britain and Ireland, where it reaches the western and northern limits of its range (Holdich,
1996). However, over the past few decades
the number of populations have declined over
this range due to several factors such as habitat alteration, pollution, and disease (Westman, 1985). At present, this species is classified as vulnerable and rare on the red list
of endangered species (Groombridge, 1994).
Several authors have tried to elucidate the
taxonomy by use of morphological criteria
(Bott, 1950, 1972; Karaman, 1963; Albrecht,
1982). Even when geographic-based morphological variation has been detected, uncertainties still exist in the classification of
the different forms, and, consequently, considerable taxonomic confusion in this species
complex persists in the literature. In A. pal-
lipes, Bott (1950) recognized three subspecies
on the basis of morphological criteria: Austropotamobius pallipes pallipes (Lereboullet,
1858) is the most widespread in an area delimited by the Pyrenees and the Alps and
whose northern limit reaches the British Isles
(Ireland and U.K.); A. p. lusitanicus is restricted to the Iberian Peninsula; and the
range of A. p. italicus includes Italy, Slovenia, Austria, and Switzerland. In contrast,
Karaman (1963) named two species, A. pallipes and A. italicus, subdivided the latter into
three subspecies A. i. italicus, A. i. lusitanicus, and A. i. carsicus, with the last one distributed in the former Yugoslavia. More recently, Albrecht (1982) reported one species,
A. pallipes, with several varieties in France,
Spain, and Italy.
Recently, several studies have shown that
the application of molecular systematics
could be useful to help taxonomic decisions
especially at specific or intraspecific levels
where taxonomic recognition is based on limited numbers of morphological and ecological traits (Bernatchez, 1995). In A. pallipes,
several attempts have been made to try to resolve its taxonomic status by the use of
molecular markers. The first molecular approach employed to estimate the phylogenetic
relationships among A. pallipes and closely
522
GRANDJEAN ET AL.: SYSTEMATICS OF AUSTROPOTAMOBIUS PALLIPES
related taxa was the use of protein electrophoresis by Albrecht and Von Hagen
(1981). They compared muscle protein in several European species of crayfish but found
no reason to suggest that A. bernhauseri differed from A. pallipes. Furthermore, they considered that there was no valid reason for
classifying A. pallipes and A. torrentium in a
separate genus from Astacus astacus and Astacus leptodactylus. This view was rejected
by Attard and Pasteur (1984), who studied enzyme-coding loci in five species of crayfish
including A. astacus and A. pallipes and
found sufficient diagnostic loci in A. pallipes
to support its status in a separate genus. The
European species were redefined by Brodski
(1983) on the basis of biochemical analysis,
biogeography, and ecology without taking
into account the works of Albrecht and Von
Hagen (1981). In the revised classification,
Astacus astacus and Astacus leptodactylus
were elevated to a new genus called Pontastacus. The genus Austropotamobius was defined as having two subgenera: (1) with one
species: A. torrentium with three races; (2)
with two species: A. pallipes with two subspecies pallipes and bispinosus, and A. italicus with three subspecies italicus, lusitanicus, and carsicus.
Additional studies using allozymes have
been perfomed without clear results (Santucci
et al., 1997; Lörtscher et al., 1997). Recently,
Souty-Grosset et al. (1997) argued that taxonomic problems in A. pallipes are preventing implementation of a suitable management
program and suggested that a review of A.
pallipes is urgently needed.
Mitochondrial DNA (mtDNA) has become
one of the most commonly employed markers for determining genetic relationships
among individuals and species (Avise et al.,
1987; Harrison, 1989). Maternal inheritance
and rapid evolution make the molecule particularly suitable to population genetics and
phylogenetic analysis (Avise, 1989, 1994). In
a survey of four European populations with
regard to restriction site variation in mtDNA,
Grandjean et al. (1997) reported a high genetic variability among white-clawed crayfish
representing populations in Spain, FranceEngland, and Slovenia that could justify the
classification based on three subspecies, A. p.
lusitanicus, A. p. pallipes, and A. p. italicus,
respectively. However, these results were of
limited value due to restrictive sampling that
523
was not fully representative of the range of
this species. In this paper, we used sequence
variation in the mt16S rRNA gene to estimate
the phylogenetic relationships of a broader
sampling of white-clawed crayfish populations across Western Europe.
MATERIALS AND METHODS
Sampling.—We examined 19 crayfish from France (n =
6), Ireland (n = 2), Spain (n = 2), Italy (n = 3), Corsica
(n = 1), Austria (n = 2), Slovenia (n = 2), including one
individual of A. torrentium as an outgroup. Crayfishes
were collected by hand or net and claw, and abdominal
tissue was dissected and frozen at –80°C for DNA isolation. Voucher specimens were preserved in 70% EtOH
and are housed in the collection at the Université de
Poitiers.
DNA Isolation and Amplification.—Total genomic DNA
was isolated from frozen claw or abdominal tissue. Tissue (approximatly 2 g) was ground with plastic pestles
in microcentrifuge tubes that contained 100 mM Tris, 10
mM EDTA, 100 mM NaCl, 0.1% SDS, 50 mM DTT and
10 µg/ml proteinase K (Kocher et al., 1989). Samples
were incubated at 37°C for 4 h, and DNA was extracted
twice with phenol/chloroform/isoamyl alcohol (25:24:1).
The DNA was then precipitated with one volume of isopropanol and 1/10 volume of 3 mM (pH = 5.2) sodium
acetate. The DNA was collected by centrifugation, dried,
and diluted in water to a final concentration of approximately 15 ng/µl.
A 520 base pair (bp) fragment of the mt16S mtDNA
gene was amplified using primers from Crandall and Fitzpatrick (1996). Polymerase chain reaction (PCR) (Saiki
et al., 1988) mixtures contained 1 µl template DNA, 2
µl of each 10 µM primers, 0.5 µl of 25 mM dNTPs, 5 µl
10× reaction buffer, 4 µl of 50 mM Mg2+, 1 unit of Taq
DNA polymerase (PE Applied Biosystems), and 37 µl of
H2O in a programmable thermal cycler (Perkin-Elmer
9600). The DNA was denaturated initially at 95°C for 3
min, then 45 cycles of amplification were carried out under the following conditions: 95°C denaturation for 1 min,
45°C annealing for 1 min, and 72°C extension for 1 min.
After the last cycle, a final extension was carried out at
72°C for 5 min. Successful PCR bands were purified using a GeneClean II kit (Bio 101). These products were
then sequenced using an ABI 377 automated sequencer
and the ABI Big-dye Ready-Reaction kit (PE Applied
Biosystems) but with a quarter of the recommended reaction size.
Sequence Analysis.—The resulting sequences were
aligned using Clustal X (Thompson et al., 1997). The
alignment contained few gaps, which were treated as
missing in subsequent analyses. In phylogeny estimation,
it is necessary to justify a model of evolution used to
model character-state changes (Huelsenbeck and Crandall, 1997). We used the program ModelTest (Posada and
Crandall, 1998) to test alternative models of evolution for
our data using likelihood ratio tests. This program tests
a number of null hypotheses, including: (1) equal base
frequencies, (2) equal transition/transversion rates, (3)
equal rates within transition and transversion classes, (4)
equal rates of substitution, and (5) no invariable sites. We
tested all these hypotheses in order to optimize a model
of evolution. This model was then used in a minimum
524
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 20, NO. 3, 2000
Table 1. Tests of hypotheses relating to the model of evolution appropriate for phylogeny reconstruction (Huelsenbeck and
Crandall, 1997), (JC (Jukes and Cantor, 1969), F81 (Felsenstein, 1981), HKY (Hasegawa et al., 1985), and GTR (Rodriguez
et al., 1990). ti = transition nucleotide substitution, tv = transversion nucleotide substitution. P-values were obtained using
the computer program ModelTest (Posada and Crandall, 1998). Note: Due to the performance of multiple tests, the
significance level of rejection of the null hypothesis should be adjusted via the Bonferroni correction to 0.01.
Null hypothesis
Equal base frequencies
Equal ti/tv rates
Equal ti and equal tv rates
Equal rates among sites
Proportion of invariable sites
Models compared
–lnL0
–lnL1
–2lnl
d.f.
P
H0: JC69
H1: F81
H0: F81
H1: HKY85
H0: HKY85
H1: GTR
H0: HKY85
H1: HKY85+G
H0: HKY85
H1:HKY85+invar
1,350
1,304
1,304
1,276
1,276
1,274
1,276
1,275
1,276
1,275
46
–92
3
< 0.000001
28
–56
1
< 0.000001
2
–4
3
0.5533
1
–2
1
0.1070
1
–2
1
0.1564
evolution (Rzhetsky and Nei, 1992a, b) and a maximum
likelihood (Felsenstein, 1981) search. The likelihood
search was performed using random sequence addition
and setting parameters to values indicated in the model
optimization. Both searches were heuristic, with TBR
branch swapping using PAUP (4.0 version D64, Swofford, 1998). Confidence in the nodes of the resulting tree
was assessed using the bootstrap procedure (Felsenstein,
1985).
RESULTS
Mitochondrial DNA (16S) sequences ~520
bp in length were obtained from 19 individuals, one from the outgroup A. torrentium and
18 from A. pallipes. The sequences have been
deposited in GenBank under accession numbers AF237590–AF237610. With these data,
we rejected the null hypothesis of equal base
frequencies (Table 1). There appears to be an
AT bias in these data; A = 0.3349, C = 0.1122,
G = 0.2097, T = 0.3432. Likewise, we rejected the null hypothesis of equal rates of
transitions and transversions, with an estimated ti/tv ratio of 2.049. We failed to reject
the rate homogeneity hypothesis, equal rates
among classes of transitions and transversions, and invariable sites (Table 1). Thus, the
model justified by our data is the HKY85
model (Hasegawa et al., 1985). With this
model, we estimated the minimum evolution
tree (Fig. 1). The minimum evolution search
resulted in just a single tree supporting two
major clades (labelled A, including individuals from Austria, France, Italy, Slovenia, and
Spain; and B, including individuals from
France, Corsica, Italy, and Ireland) and three
distinct clades within clade A (labelled 1, with
Italian, Spanish, and French specimens; 2,
with Austrian ones; and 3, with Slovenian and
French ones). Similarly, we used this model
to estimate the maximum likelihood tree (Fig.
2). This search resulted in six equally likely
trees. Again, the maximum likelihood analysis supports the separation of the same two
major clades and three clades within clade
A. All but clade 3 are supported by high bootstrap values (more than 80). A parsimony
search weighting transversions : transitions,
2:1, resulted in 44,733 equally parsimonious
trees. All the clades labeled in Figs. 1 and 2
were present in the strict consensus tree of the
parsimony search except clade 3.
The average of genetic divergence within
clade A and clade B were 1.6 ± 1% and 1.4
± 1%, respectively. The average between the
two clades was 4.6 ± 0.9%.
DISCUSSION
One of most important questions in the
taxonomy of the white-clawed crayfish A.
pallipes is to know what is the level of taxonomic rank between populations located in
France, Ireland, and England and those located
in Spain, Italy, Austria, and former Yugoslavia. This question is particularly important
to design suitable management programs for
this endangered species (Grandjean et al.,
1998). On the basis of several morphological criteria such as the shape of the rostrum
and the number of spines, Karaman (1962)
classified them in two species A. pallipes and
A. italicus respectively. This assumption differs from Bott (1950), who defined three subspecies. Even if there is no a priori level of
genetic divergence associated with taxonomic
rank, Avise (1994) reported that the magnitude of nucleotide sequence divergence could
be an effective gauge of taxonomic standing.
GRANDJEAN ET AL.: SYSTEMATICS OF AUSTROPOTAMOBIUS PALLIPES
525
Fig. 1. Single tree derived from a minimum evolution analysis based on mt16S rDNA characters using HKY 85
model (Hasegawa et al., 1985) from PAUP (4.0 version D64: Swofford, 1998) rooted using A. torrentium. Numbers
at nodes indicate bootstrap support (500 replicates). Labels A and B indicate major clades of the crayfish Austropotamobius pallipes based on individuals sampled from western Europe; clade A = A. italicus and clade B = A.
pallipes. The subclades labelled A1, A2, A3 indicate subspecies of A. italicus: A. i. italicus, A. i. carinthiacus, and
A. i. carsicus, repectively.
Our results clearly define two major groups,
one containing crayfish sampled in France,
Ireland, and northern Italy (Figs. 1, 2, clade
B) and the other one including animals sampled in France, Italy, Spain, Slovenia, and
Austria (Figs. 1, 2, clade A). If we refer to the
geographical range of the white-clawed crayfish described by several authors (Laurent,
1988), then clade A could represent A. italicus and clade B could represent A. pallipes.
526
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 20, NO. 3, 2000
Fig. 2. Strict consensus of 6 trees derived from a maximum likelihood analysis based on mt16S rDNA characters
using the HKY85 model (Hasegawa et al., 1985) from PAUP (4.0 version D64: Swofford, 1998) rooted using A. torrentium. Numbers at nodes indicate bootstrap support (500 replicates). Labels A and B indicate major clades of the
crayfish Austropotamobius pallipes based on individuals sampled from western Europe; clade A = A. italicus and clade
B = A. pallipes. The subclades labelled A1, A2, A3 indicate subspecies of A. italicus: A. i. italicus, A. i. carinthiacus, and A. i. carsicus, respectively.
The separation of two Italian populations in
each of the two clades is in accordance with
the work of Santucci et al. (1997), who reported a range separation of A. pallipes and
A. italicus between northern and southern
Italy, respectively. Concerning the French
populations, it is well known that crayfish
from other countries have been widely introduced into France by anthropological activities (Laurent and Suscillon, 1962). Our results
seem to confirm this, because some of the individuals sampled in France are more closely
related to clades from Slovenia (Fig. 1, clade
A3) and Spain (Fig. 1, clade A1) than to others sampled from France (Fig. 1, clade B).
The degree of genetic differentiation between haplotypes from the main clades (Fig.
1, clades A and B) is relatively high, 4.6 ±
0.9% (Table 2). This value is comparable with
the level of differentiation observed between
species in several crustaceans, particularly
crayfish. Based on sequences for the same region of the mt16S rRNA gene used in this
study, Crandall (1996) reported a mean nu-
0.081
0.083
0.077
0.079
0.085
0.083
0.071
0.081
0.075
0.067
0.069
0.071
0.088
0.083
0.087
0.089
–
0.059
0.057
0.057
0.057
0.065
0.059
0.051
0.063
0.058
0.022
0.020
0.026
0.031
0.000
0.002
–
0.057
0.055
0.055
0.055
0.063
0.057
0.049
0.061
0.056
0.020
0.018
0.024
0.031
0.000
–
0.046
0.043
0.034
0.043
0.048
0.043
0.034
0.049
0.043
0.003
0.003
0.006
0.012
–
0.053
0.053
0.051
0.053
0.060
0.055
0.046
0.053
0.051
0.015
0.015
0.015
–
0.039
0.042
0.037
0.037
0.041
0.043
0.033
0.043
0.037
0.004
0.006
–
0.039
0.037
0.037
0.037
0.043
0.039
0.031
0.043
0.037
0.002
–
0.036
0.039
0.035
0.035
0.041
0.041
0.030
0.041
0.035
–
0.006
0.010
0.023
0.006
0.012
0.010
0.019
0.002
–
0.012
0.016
0.030
0.012
0.018
0.018
0.026
–
0.025
0.028
0.012
0.023
0.029
0.027
–
0.008
0.006
0.033
0.006
0.012
–
0.008
0.010
0.027
0.006
–
0.002
0.004
0.027
–
0.031
0.033
–
0.002
–
–
A*1-2 Bologne IT
A*3- Las Lilas FR
A*4-5 Rizana SLO
A*6- Garrel FR
A*7- Tafalla SP
A*8- San Esteban SP
A*9- Las Lilas FR
A*10- Plansee AUS
A*11- Plansee AUS
B*12- Fertagh IR
B*13- Val Renard FR
B*14- Genes IT
B*15- Martinière FR
B*16- Corsica
B*17- Blessington IR
B* 18- Gace FR
19- A. torrentium
19
18
17
16
15
14
13
12
11
10
9
8
7
6
4–5
3
1–2
Table 2. Pairwise mt16 S sequence divergence (adjusted for missing data) for 19 crayfish calculated using REAP 4.0 (1–18 represent individuals of Austropotamobius pallipes collected
from various western European populations; 19 is the outgroup representing an individual of A. torrentium collected in Slovenia). IT = Italy, FR = France, SLO = Slovenia, SP = Spain, AUS
= Austria, IR = Ireland.
GRANDJEAN ET AL.: SYSTEMATICS OF AUSTROPOTAMOBIUS PALLIPES
527
cleotide sequence divergence between Orconectes luteus and O. medius of 4% and between O. macrus and O. nana of 7%. In lobsters, Sarver et al. (1998) found a mean
mt16S rDNA nucleotide sequence divergence
of 7.3% among three recognized species,
Panulirus longipes, P. cygnus, and P. marginatus. Therefore, the sequence divergence
revealed in this study between the two clades
appears to be in accordance with the classification on the specific status of A. pallipes
and A. italicus. However, according to Avise
(1994), taxonomic implications based only on
mtDNA data are not recommended, and additional information such as morphological
data or nuclear genes are necessary because
discrepancies can exist between gene trees
and species trees. Morphological differences
between A. pallipes and A. italicus are well
documented. According to several authors
(Bott, 1950; Karaman, 1962; Albrecht, 1982),
A. italicus differ from A. pallipes by the shape
of the rostrum and its basal spines. Additional
studies have confirmed the taxonomic suitability of these morphological criteria to discriminate A. pallipes from A. italicus. Grandjean et al. (1998) reported an A/R ratio
(length of apex (A) to size of rostrum (R))
of approximately 0.22 for A. pallipes and 0.29
for A. italicus. Similar results have been proposed by Laurent and Suscillon (1962) who
have revealed (A/R) ratios of approximately
0.20 in five French populations (putatively A.
pallipes) and around 0.32 in one Italian population (putatively A. italicus). Similarly, A.
pallipes has a larger number of spines behind
the cervical groove than A. italicus, with a
mean of 2.8 spines on A. pallipes and only
one spine for A. italicus (Bott, 1950; Laurent
and Suscillon, 1962). In agreement with the
mtDNA results reported here, a high level of
genetic variation based on allozyme analysis
was reported by Santucci et al. (1997) for
populations sampled in France (A. pallipes)
and Italy (A. italicus). Thus, genetic (from nuclear and mitochondrial genomes), morphological, and geographical data are concordant
in confirming the specific status A. pallipes
and A. italicus proposed by Karaman (1963).
Within the A. italicus group (clade A, Figs.
1, 2), three branches have a high boostrap
value (more than 80), corresponding to animals sampled in Italy-Spain-France (clade
A1), Austria (clade A2), and Slovenia-France
(clade A3). These three clusters are not in ac-
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 20, NO. 3, 2000
cordance with the taxonomic proposals of
previous authors (Bott, 1950; Karaman, 1963;
Brodski, 1983) for several reasons. Concerning the presence of two subspecies in Spain
(A. i. lusitanicus) and Italy (A. i. italicus) described by all authors, our results indicate
closely related haplotypes from animals sampled in Spain and Italy (clade A1, Figs. 1, 2).
The genetically close relationship between
samples from Spain and Italy has also been
reported by Santucci et al. (1997) from allozyme analysis. Molecular data reported here
are concordant with morphological studies
that show that A. i. italicus and A. i. lusitanicus are separated only by the presence of
hairs on the upper border of the endopod of
the second male gonopod in A. i. lusitanicus
(Bott, 1950). However, recent works have
shown that this criterion has a limited value
for separating specimens because a high percentage (around 90%) of male specimens
from Spain have no hairs on the gonopod (Almaça, 1987; Grandjean et al., 1998). These
molecular results do not justify the classification based on the existence of two subspecies in each of these two countries and
help confirm the assumption proposed by
Laurent (1988) concerning the anthropological origin of Spanish crayfish stock.
The position of Austrian crayfish (clade A2,
Figs. 1, 2) in a cluster separate from Italian/Spanish/French ones is not really surprising considering the work of Albrecht (1982),
Machino and Fuereder (1996), and Machino
(1997a). Albrecht (1982) reported that crayfish
from Austria represent an independent variety
A. p. carinthiacus that differs from those from
Italy by the chocolate brown color on the upper side of the chelae and high numbers of
spines on the merus of the third maxilliped and
behind the cervical groove. In a recent study,
Machino and Fuereder (1996) supported Albrecht’s (1982) assumption in spite of a minor difference they observed in the number of
spines behind the cervical groove. Our results
are in accordance with these morphological
criteria that validate a subspecies level of the
Austrian crayfish as A. i. carinthiacus.
The third cluster in the A. italicus complex
(clade A3, Figs. 1, 2) is constituted by crayfish sampled in Slovenia and France. This result is in accordance with those found from
allozyme analysis by Santucci et al. (1997).
They reported 12% divergence between populations sampled in Italy and Slovenia. Moreover, Laurent and Suscillon (1962) and
Machino (1997b) also showed some differences in the A/R ratio between Slovenian and
Italian populations. These results could support the subspecies status A. i. carsicus given
by Brodsky (1983).
CONCLUSIONS
Results based on sequences from the mt16S
rRNA gene give support to morphological
and allozyme data and allow us to propose a
new classification for the white-clawed crayfish Austropotamobius based on four species:
A. torrentium, A. biernhauseri (distributed in
Switzerland and not sampled in this study),
A. pallipes, and A. italicus. The last species
includes three subspecies A. i. italicus, A. i.
carinthiacus, and A. i. carsicus.
It is clear that some specimens of A. italicus italicus and A. italicus carsicus have been
introduced into France. Further work is
needed to examine the geographical extent
to which these populations have spread and
their effect on indigenous populations.
ACKNOWLEDGEMENTS
We thank the many people who assisted in obtaining
samples for this study: Y. Machino (Austria), N. Budihna (Slovenia), J. Dieguez-Uribeondo (Spain), J. R.
Reynolds (Ireland), and all members of the Conseil Superieur de la Pêche (C. S. P.). This work was supported
by C. S. P. of France (to FG), and NSF IBN–97–02338
and the Alfred P. Sloan Foundation (to KAC).
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RECEIVED: 20 January 1999.
ACCEPTED: 1 March 2000.