GENETIC AND MORPHOLOGICAL SYSTEMATIC STUDIES ON THE
CRAYFISH AUSTROPOTAMOBIUS PALLIPES (DECAPODA: ASTACIDAE)
F. Grandjean, N. Gouin, M. Frelon, a n d C. Souty-Grosset
A B S T R A C T
Morphological criteria and mitochondrial D N A (mtDNA) were analyzed to examine the taxonomic and systematic relationships of the endangered crayfish species Austropotamobius pallipes.
Morphological data confirmed the difficulty of differentiating the 3 subspecies: A. p. pallipes, A. p.
italicus, and A. p. lusitanicus. M t D N A analysis by RFLP using 6 endonucleases revealed 5 haplotypes and allowed unequivocal identification of the subspecies. The nucleotide sequence divergence among haplotypes characteristic of each subspecies ranged from 10.93-13.6% and may be
explained by an ancient geographical separation of each subspecies. This distribution of genetic variation agrees with the presumption of 3 refugial regions during the glaciation periods, possibly the
Iberian Peninsula, southern France, and the Balkans. U P G M A analysis from nucleotide distances
placed A. p. italicus and A. p. lusitanicus in a cluster separate from A. p. pallipes, suggesting that
A. p. italicus and A. p. lusitanicus are more closely related.
The Astacidae is the least species-rich of the
three fresh-water crayfish families, containing
less than 2% of the 750 species described in
the world. According to Bott (1950, 1972;
Laurent, 1988; Lowery and Holdich, 1988; Vigneux et al., 1993), this family is widely distributed in Europe and is separated into two
genera, Austropotamobius and A.stacu.s. The
genus Austropotamobius contains two species, A. pallipes Lereboullet and A. torrentium Schrank, both of which are threatened
throughout their ranges and have recently
been classified as vulnerable and rare by the
International Union for the Conservation of
Nature and Natural Resources (LU.C.N.)
(Groombridge, 1994).
Bott (1950, 1972) recognized three subspecies in A. pallipes: pallipes in Great Britain,
Ireland, France, Switzerland, and Corsica;
italicus in Italy, Dalmatia, and Switzerland;
and lusitanicus in the Iberian Peninsula. According to Bott (1950), italicu.s and lusitanicus differ from pallipes by the shape o f the
rostrum and its basal spines, while italicus
and lusitanicus are separated by the presence
of hairs on the upper border of the endopod
of the second male gonopod in lusitanicus.
However, such characters are of limited value
for identifying specimens because they differ considerably from one population to another and the differences between subspecies
are small. Froglia (1978) reported specimens
in northern Piemonte and Liguria that had
characters intermediate between pallipes and
italicus, while A l p a c a (1987) found that no
more than 10% of males of lusitanicus had
hairs on the second gonopod. Females of
italicus and lusitanicus cannot be distinguished by morphological criteria.
Biochemical genetic methods have long
been used to identify species for which morphological characters are unreliable (see
Smith et al., 1994, and references therein).
Albrecht ( 1982) and Albrecht and Von Hagen
( 1981 ) used the electrophoretic patterns of hemocyanins to infer the phylogenetic relationships among European crayfish genera o f the
Astacidae, but the electrophoretic patterns
from animals sampled across Europe showed
no specific variation within and between subspecies. The possibility of misidentifying a
subspecies poses problems for management
schemes, including restocking. In recent
decades, many translocations by humans have
been made, with crayfish coming from Spanish and Italian stocks to restore French stock
decimated by the crayfish plague (Holdich
and Reeve, 1991; Laurent and Suscillon,
1962). These uncontrolled restockings, with
probably different subspecies of A. pallipes,
may alter the genetic structure of French
stock and population mixing should be
avoided. A method for unequivocally identifying these subspecies is clearly desirable.
Mitochondrial DNA (mtDNA), by virtue of
its simple structure, maternal inheritance, and
relatively rapid evolutionary rates, has been
widely used in recent years to study population structure, taxonomy, and phylogeny of
animal species (Avise et al., 1979 a, b, 1987;
S a u n d e r s et al., 1986; H a r r i s o n , 1989). H o w ever, little w o r k has b e e n d o n e o n the p h y l o genetic relationships between closely related
species o f crustaceans f r o m total m t D N A an-
where m and m�are the numbers of restriction fragments
in DNA sequences x and y, respectively, and in, is the
number of fragments shared by the two sequences. The
number of nucleotide substitutions per site d can be estimated by
a l y z e d b y e n d o n u c l e a s e s , b e c a u s e it is d i f f i cult to e x t r a c t m t D N A ( K o m m et al.,
B r a s h e r e t al.,
1982;
1992a, b; G r a n d j e a n et al.,
1993). In crayfish, a f e w phylogenetic studies that h a v e b e e n r e p o r t e d h a v e b e e n b a s e d
o n a n u c l e o t i d e s e q u e n c e d a t a set f r o m the 16
S r e g i o n o f the m i t o c h o n d r i a l g e n o m e in the
where r is the number of bases per restriction site (Nei
and Li, 1979). When different kinds o f enzymes with different r values are used, the mean number of nucleotide
substitutions can be estimated by the formula given by
Nei and Tajima (1981):
family C a m b a r i d a e (Crandall, 1993; Crandall
and Fitzpatrick,
1996). Recently, G r a n d j e a n
and Souty-Grosset (1996) described a method
for extracting total m t D N A f r o m crayfish.
This m e t h o d has n o w b e e n u s e d to e v a l u a t e
where In, = m ,lA, + 2 m ii" and k refers to the kth class of
t h e t a x o n o m i c s t a t u s o f A. p a l l i p e s b y a n a -
restriction enzymes.
l y z i n g g e n e t i c d a t a in the c o n t e x t o f m o r phological classifications according to the cri-
teria provided by Bott (1950, 1972).
MATERIALS AND M E T H O D S
Sixty specimens of A. pallipe.s were collected, 15 from
each of 4 locations across the range of the subspecies in
Europe: Magnerolle and Gatineau Rivers (Department o f
Deux-S�vres, France), Rizana River (southern Slovenia),
and a pond located in the province of Leon (Spain). Crayfish were transported alive to the University of Poitiers
for analysis.
Morphological C h a r a c t e r s . - T h r e e characters were used
for classification: (1) the number of spines behind the cervical groove; (2) the apex to rostrum length ratio (A/R),
with measurements made to the nearest 0.1 mm using an
eyepiece micrometer; and (3) the presence or absence of
hairs on the upper border of the endopod of the second
male gonopods.
Genetic A n a l y s i s . - T h e heart, green glands, ovaries, and
testes were removed and the m t D N A extracted as d e scribed by Grandjean and Souty-Grosset (1996).
M t D N A samples were digested with 6 restriction endonucleases, four 6-base cutters (Bgl II, P.st I, Hind III,
Xlw I) and two 4-base cutters (Acc II, H a e III), according to the manufacturer's instructions (Gibco BRL, Life
Technologies S.A.R.L., Cergy Pontoise Cedex, France).
The resulting restriction fragments were separated in 1.2%
agarose gels in Tris-EDTA buffer (30 mM; 60 mM) for
15 h at 30 volts. Gels were stained with SYBR™ Green
I (FMC Bioproducts, Tebu S.A., 78610 Le Perray en Yvelines, France) and visualized with an UV light transluminator. The restriction fragment patterns from each of
the 6 endonucleases were identified by a letter, each individual being characterized by a composite haplotype
of 6 letters.
The total proportion of shared fragments (S-value) between two individuals was calculated from the following equation (Nei and Li, 1979).
RESULTS
Morphological Data
The number of cervical grooves ranged
from 1-5 for the French samples (mean = 4
± 0.84 and 3.73 ± 0.96 for Magnerolle and
Gatineau, respectively), whereas it ranged between 1 and 2 for the Spanish and Slovenian
samples (mean = 1.4 ± 0.51 and 1.6 ± 0.63,
respectively). No significant differences were
found between French samples (t-test =
0.311) or between Spanish and Slovenian
samples (t-test = 0.316). The means were significantly different between French and the
other two populations (P � 0.01 ). The A/R ratios were 0.227 ± 0.026 and 0.23 ± 0.027 for
the French (Magnerolle and Gatineau, respectively), 0.293 ± 0.016 for the Spanish,
and 0.316 ± 0.046 for the Slovenian populations. No significant differences were found
between the two French samples (t-test =
0.806) or between Spanish and Slovenian
samples (t-test = 0.955). The means were significantly different between French and the
other two populations (P � 0.01).
The specimens coming from France and
Slovenia had no hairs on the upper border of
the endopod of the second male gonopod.
Only 1 of 12 males in the Spanish sample had
hairs on this area.
Genetic Data
The composite restriction patterns of all six
enzymes for each individual (N = 60) are
given in Table 1. Among the six restriction
enzymes used, only Xho I produced a mono-
morphic pattern. All the other enzymes gave
two or more restriction patterns (Table 1). Bgl
II gave two patterns with pattern A common
to French and Slovenian individuals. Three
patterns were produced by Acc II and by Hind
III. Ace II gave profiles specific to each subspecies (Fig. 1). Profile A was found in
French animals, B in Slovenian animals, and
C in Spanish animals. Hind III gave patterns
A and B in French individuals, but the difference between them was the gain or loss
o f a single restriction site of the major band.
Pattern C was shared by Spanish and Slovenian individuals. Pst I did not cut the mtDNA
from Spanish or Slovenian animals. Among
the four profiles obtained by Hae III, two (B
and C) were found in Slovenian animals; profile A was found in French animals and D in
Spanish individuals. Thus, five different composite haplotypes were detected among the 60
crayfish representing the four populations
(Table 2). The mitochondrial DNA nucleon
diversity values ranged from 1.16-13.6%
(Table 2) and the three subspecies were genetically well differentiated. The low variation of mtDNA size, estimated from the restriction profiles for an endonuclease given,
is probably due to the small fragments unrevealed by SYBRTM Green I coloration. This
may produce a small bias in the calculation
o f the actual number of restriction sites and
shared bands. However, the nucleotide divergence percentage estimated between haplotypes should not change considerably.
U P G M A revealed two major clusters, one
containing haplotypes 1 and 2 representing
the A. p. pallipes subspecies, the second containing haplotypes 3 (A. p. italicus) and 4 (A.
p. lusitanicus) (Fig. 2).
DISCUSSION
The morphological results are in agreement
with the morphological characters proposed
by Bott (1950) for discriminating between
the three subspecies of the white-clawed crayfish. Austropotamobius p. pallipes had an
A/R ratio of approximately 0.22, while the
other subspecies, A. p. lusitanicus and A. p.
italicu.s, had ratios greater than 0.29. This
agrees with Laurent and Suscillon (1962) who
Fig. 1. The three restriction patterns produced by the
endonuclease Acc / / discriminate the three subspecies of
A ustropotamobius pallipes. Lane 1: restriction pattern (A)
produced from A. p. /�allipes; Lane 2: profile B characterizing A. p. italicus� Lane 3: profile C for A. p. lusi-
tanicus. On the right side are given the fragment size
markers in base pairs (bp) of Lambda-phage DNA digested by Hind III.
Table 2. Values (%) above the diagonal are pairwise
estimates of nucleotide divergence, p, as calculated in Nei
and Li (1979). Values below the diagonal are calculations
of total proportion of shared fragments, F, of six restriction enzymes.
reported A/R ratios of approximately 0.20 in
five French populations and around 0.32 in
one Italian population. This ratio, however,
does not discriminate between the subspecies
A. p. lusitanicus and A. p. italicus. Similarly,
the number of spines behind the cervical
groove allowed the discrimination of A. p.
pallipes from the other two. In A. p. pallipes
the number of spines was significantly larger
than in A. p. italicus or lusitanicus. This is
in accordance with the data of Laurent and
Suscillon (1962), who found a mean of 2.8
spines on A. pallipes sampled in five French
populations, while A. p. italicus had only one
spine. Similar observations have been made
on crayfish from Ireland, France, Italy, and
Slovenia (Albrecht, 1982). With respect to the
morphological differentiation between A. p.
italicus and A. p. lusitanicus, only 1 Spanish
Fig. 2. U P G M A phenogram of affinities based on genetic distances from five composite haplotypes of subspecies of Austropotamobius pa(lipes. The letter designations refer to single enzyme genotypes in the order presented in Table 1 (uncut mtDNA: profile 0).
male revealed the presence of hairs on the
second gonopod. These results are in agreement with the findings of Almaça (1987) who
showed that the presence of hairs on the second male gonopods was not widespread in A.
p. lusitanicus (lower than 10% of males).
These results show that there is no substantive basis for recognizing A. p. italicus and A.
p. lusitanicus. Generally, geographical location
was used to infer identity (Almaqa, 1987).
However, such characters are of limited
value for identifying individuals, because
they differ considerably between populations
and the differences between subspecies are
small. Many subspecies of A. pallipes erected
on the basis of morphological data are now
considered to be varieties. Only a few studies have examined the phylogenetic relationships between subspecies of A. pallipes using
molecular markers. In a taxonomic examination of A. pallipes from different parts of Europe, Albrecht ( 1982) and Albrecht and Von
Hagen (1981) revealed no difference in the
electrophoretic patterns of general proteins
between populations, despite the differences
in a number o f morphological characters.
More recently, Attard and Pasteur (1984) and
Attard and Vianet (1985) found a low level
of heterozygosity ranging from 0 . 0 1 3 - 0 . 0 2 6
between French and Irish populations of A. p.
pallipes. Agerberg (1990), and Fevolden and
Hessen (1989) found a lack of discrimination between four populations of another species of Astacidae, Astacus astacus Linne,
sampled on a large scale in Sweden and Norway, respectively. In a more recent study,
Fevolden et al. (1994) found only minor genetic variation from 12 Norwegian populations. These results suggest that allozyme
analysis may not be suitable to discriminate
phylogenetic relationships among genera of
the Astacidae and other genera of crayfish
(Nemeth and Tracey, 1979; Brown, 1980; Busack, 1988, 1989).
Our results show considerable genetic differences between the three subspecies, confirming the suitability of mtDNA for determining phylogenetic relationships among
closely related species. There were clear differences in the mtDNA patterns of the three
subspecies, indicating that RFLP analysis
could be a fairly simple diagnostic technique
for distinguishing between subspecies o f A.
pallipes. All restriction enzymes tested (Ace
II, Hind III, Pst I, B�//, and Hae III), except
Xho I, allowed us to discriminate among two
or three subspecies (Table 1). Each of these
enzymes except for Pst I (profile 0 found in
Spanish and Slovenian individuals), cut the
mtDNA fragment of each subspecies at least
two times and thereby provide an internal assay for enzyme activity. Since the three subspecies of white-clawed crayfish shared many
common morphological features, the use of
mtDNA RFLP provided an unequivocal identification of each subspecies.
U P G M A analysis, based on nucleotide distance between haplotypes, placed A. p. italicus and A. p. lusitanicus in a cluster separate
from A. p. pallipes. This agrees with the morphological data, which suggest that A. p. italicus and A. p. lusitanicus are more closely related. However, this result does not support
the classification proposed by Karaman
(1962), who cast doubt on the taxonomic position of A. pallipes. He considered pallipes
and italicus to be species and lusitanicus to
be a subspecies of italicus. The high level of
nucleotide divergence found between A. p.
lusitanicus and A. p. italicus supports a classification based on three subspecies. However, there is no a priori level of genetic divergence associated with taxonomic rank.
The identification of intraspecific taxa such
as subspecies would require further examination of the nature of geographical variation over the species range, and the interpretation of variation in haplotype frequencies in
different sites.
The practical implication of this study is
that the great nucleotide divergence between
haplotypes characterizing the three subspecies
suggests that mixing of populations should be
avoided in restocking. This is most important for the establishment o f restocking operations in France. Laurent and Suscillon
(1962) argued that attempts to restock waterways decimated by the crayfish plague
from animals coming from Spanish and Italian stocks were made to reconstitute French
stocks. According to these authors, the three
subspecies may be present in the French hydrographic basin system. Thus, restocking,
performed without genetic characterization of
animals, may alter the genetic structure of
populations in a hydrographic basin.
Biogeographical Implication
The three subspecies sampled from French,
Spanish, and Slovenian populations were well
differentiated from one another, confirming
an ancient geographical separation of each
subspecies in these countries. This genetic
variation agrees with the presumption of three
refugial regions during the glaciation periods,
possibly the Iberian Peninsula, southern
France, and the Balkans. The present range
of these subspecies may be explained by repopulation events by specimens from these
refuges which survived and diverged in the
southern refuges during several ice ages. This
shows the importance of the geographical
barriers formed by the Pyrenees and Alps to
the lack o f gene flow allowing divergence to
accumulate. Our preliminary data support this
statement, but data on mtDNA haplotype frequencies over a wide range is required for
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RECEIVED: 30 July 1997.
ACCEPTED: 26 January 1998.
Address: Laboratoire de Biologie Animale, U M R
C N R S n° 6556, Universitd de Poitiers, 40 avenue du
Recteur Pineau, 86022 Poitiers cedex, France. (e-mail:
[email protected])