Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2005? 2005
862
201212
Original Article
DELIMITING SPECIES IN
TETRASTEMMA
M. STRAND and P. SUNDBERG
Biological Journal of the Linnean Society, 2005, 86, 201–212. With 5 figures
Delimiting species in the hoplonemertean genus
Tetrastemma (phylum Nemertea): morphology is not
concordant with phylogeny as evidenced from mtDNA
sequences
MALIN STRAND and PER SUNDBERG*
Göteborg University, Department of Zoology, PO Box 463, SE-405 30 Göteborg, Sweden
Received 2 April 2004; accepted for publication 28 October 2004
The marine genus Tetrastemma contains monostiliferous hoplonemertean (phylum Nemertea) species which mostly
undergo direct development with no free-swimming stages of larvae. The lack of a pelagic phase, and the fact that
many benthic species lay eggs attached to the bottom substrate, are obvious restrictions on dispersal and gene flow.
Nevertheless, some of the species, for example T. candidum and T. melanocephalum, are described as ubiquitous and
are reported from waters all over the world. We studied genetic variation and evolutionary relationships in order to
assess whether they are concordant with external characters in samples of nine morphologically distinct forms formally named as different Tetrastemma species, from different geographical localities. We estimated the phylogeny
and species network based on 539 base pairs of the mitochondrial protein-coding gene cytochrome oxidase-1 (CO1)
for 30 ingroup specimens. From this, we assessed the evolutionary history and phylogenetic relationships between
these forms. We conclude that in most cases there was no correspondence between evolutionary lineage and morphotype. Our results thus indicate that morphological species delimitation in nemerteans may be questionable, and
that this in turn may have a profound effect upon estimates of species diversity within the phylum. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212.
ADDITIONAL KEYWORDS: evolutionary relationships - phylogeography - statistical parsimony.
INTRODUCTION
A central goal in systematic and evolutionary biology
is to find and describe evolutionary lineages, i.e. species, in order to classify and understand evolutionary
processes and mechanisms. Traditionally, newly discovered species have been delimited, diagnosed and
described using a character-based approach: a potentially new species is compared to similar species
already described and considered new if one has
observed some unique, ‘diagnostic’ character states.
Historically, this was a task for the trained and
experienced taxonomist, who was presumed to have a
good ‘feel’ concerning which character differences were
sufficiently significant to give a specimen or group of
*Corresponding author. E-mail: [email protected]
specimens a new name. For obvious reasons, this decision used to be based on morphological characters. It
was also linked to the issue of distinguishing fixed (or
nearly fixed) diagnostic features from those that are
polymorphic within a species. Although perhaps not
explicitly, the taxonomist judged which morphological
differences implied reproductive isolation, and hence
separate species status, even if he or she lacked
knowledge about the genetic basis for these differences. Larger differences were judged to indicate not
just separate species but also different genera, families, or higher taxa. Clearly, these judgements and
decisions reflect traditions and these traditions also
differ between phyla, with the implication that ranks
of higher taxa are not comparable between phyla. The
judgements are also affected by the degree of intraspecific polymorphism and phenotypic plasticity within a
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
201
202
M. STRAND and P. SUNDBERG
taxon - this is particularly evident in many marine
invertebrate groups. This variation is often without
doubt intraspecific, but there are also situations when
the polymorphism in fact instead represents different
species. In other cases it may be the other way around:
species and evolutionary lineages are concealed by a
similar morphology, which will underestimate the true
number of species. This problem has been particularly
evident in small and simply built organisms where
there are few morphological characters available for
study and it is thus difficult to identify evolutionary
lineages based exclusively on morphological information. The number of cryptic species is probably higher
in those taxa with morphological characters that are
either few in number or difficult to observe than, for
example, in birds or gastropods.
The taxonomy of nemerteans (phylum Nemertea)
exemplifies the problem of delimiting species in a
group of invertebrates with few morphological characters. Although there is an increasing number of systematic studies of within-phylum relationships based
on nucleotide sequences (Sundberg & Saur, 1998;
Sundberg, Turbeville & Lindh, 2001; Thollesson &
Norenburg, 2003), the question of species boundaries
is still a matter of how to interpret morphological differences and similarities. External characteristics in
nemerteans are few and mainly confined to eye number, pattern and coloration. Although new species may
be described based on such characters in certain cases,
most of the taxonomy has to rely on anatomical characters accessible through histological sectioning. Most
(all?) animals vary to some degree intraspecifically;
however, nemerteans are so extremely contractile that
the fixation process needed to do the sectioning adds to
this variation by causing contraction artefacts. In the
older literature, this variation is either not investigated, or is interpreted as evidence for several species,
and in some cases even for different genera. Envall &
Sundberg (1993) exemplified this by showing that the
character states that have been used to diagnose the
two genera Oerstediella and Paroerstedia could all be
found in specimens of Oerstedia dorsalis (the type species of genus Oerstedia). Stiasny-Wijnhoff (1930)
described O. dorsalis in meticulous detail, but the
description is clearly based on a contracted specimen
sectioned at an oblique angle and many of the
described character states are obviously artefacts.
Intraspecific variation certainly poses a problem for
the taxonomy of nemerteans and it becomes especially
difficult in groups with barely discernible differentiation. In the case of the genus Tetrastemma, differences
in pigmentation patterns or coloration have been
taken as evidence for different species. Sundberg
(1979) showed that the variation within one species of
Tetrastemma encompassed many of the character
states that were reported from other species, and in
those cases treated as diagnostic characters and evidence for separating specimens into different species.
It is clearly a problem to draw up the boundaries of
species in a situation like this where we not only have
few observable characters, but these characters moreover show a high degree of variation. In this study, we
infer the phylogeny based on nucleotide sequences for
a number of Tetrastemma specimens assigned to nine
species on morphological grounds and ask if current
species delimitations are concordant with evolutionary lineages. The idea is that there will be support for
the presence of a distinct species when all the individuals sampled of a putative species appear as each
other’s closest relatives on a gene tree. There are several recent proposals for tree-based methods to test
species boundaries (Wiens & Servedio, 2000;
Templeton, 2001; Sites & Marshall, 2003) based on
genetic data. Unfortunately, most of them cannot be
utilized fully in the case of nemerteans, where it is
often difficult to get the sample sizes needed. Genetic
distances have been used as a measure of species,
genus and family limits (Thorpe & Solé-Cava, 1994)
but we consider the approach flawed and not useful for
species (taxon) delimiting (see also Ferguson, 2002 for
a discussion of the use of genetic divergence as a yardstick for species).
THE
GENUS
TETRASTEMMA
The genus Tetrastemma was erected by Ehrenberg
(1828) and currently contains around 115 species
(Gibson, 1995; Chernyshev, 2003). The morphological
definition of the genus has expanded since its establishment, but there have been no previous attempts to
define it phylogenetically or to specify morphological
synapomorphies for the taxon. Basically, it has been
erected within a tradition of finding combinations of
characters considered to be unique for a group and
hence by implication pointing to natural groups. We
used 18S rDNA to reconstruct the phylogeny for an
extended group of hoplonemerteans (Strand & Sundberg, in press) and found that the included representatives of the genus formed a well-supported
monophyletic group. However, we also found that the
morphology-based diagnosis of the genus does not contain any synapomorphies, and when examined in
detail turns out to be basically the same as for example Amphiporus. To give some examples, when apical
organs are listed as ‘probably present’ in Tetrastemma,
they are ‘present’ in Amphiporus, cerebral organs are
‘generally large’ compared to ‘variably developed
according to species’, cephalic glands are ‘very variable
in size’ in comparison with ‘usually small’, and so on.
Based on this phylogenetic analysis, we concluded
that morphological synapomorphies for the taxon are
‘four eyes; flattened body; well demarcated head’.
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
DELIMITING SPECIES IN TETRASTEMMA
Thus, there is evidence for the monophyletic status
of the taxon Tetrastemma. However, for reasons discussed above, morphological characters are difficult to
use when it comes to the relationships and species
delimitations within the taxon. A careful morphological investigation would require not only the examination of numerous characters, some of which would be
difficult to observe (see e.g. Kirsteuer, 1963), but also
many specimens of each species to cover potential, and
likely, intraspecific variation (Sundberg, 1979; Gibson,
1994). Already in 1893, Riches wrote: ‘As has been
already remarked, this genus is characterized by the
very high degree of variation which its members
exhibit. It is for this reason in many cases extremely
difficult to identify a given species, or to come to anything like a satisfactory conclusion as to the amount
and kind of difference, which justifies specific separation. Such conclusions as are expressed by the recognition of the following species are provisional. They
are, however, based on a consideration of the kind and
degree of variation exhibited in each case’ (Riches,
1893).
Most species in the genus have been described without consideration or documentation of intraspecific
variation and several have been erected exclusively on
external characters, often head pigmentation patterns
or body coloration. However, Chernyshev (1998) and
Manchenko & Kulikova (1996) have documented that
these types of characters also vary within species.
Gibson (1994) concluded that ‘It is more than probable
that several species belong in other genera and/or represent colour varieties of previously established
forms.’ Another often neglected problem is that some
of the characters (e.g. colour pattern, number and
placement of eyes, body shape) seem to develop with
time and differ significantly between juveniles and
adults (we present an instance of this in T. coronatum;
see Results) which means that some of the species
descriptions based on coloration and pigmentation
could very well be of juvenile forms of other species.
In this study we investigate the congruence between
genotype, evolutionary lineages, and morphotypes for
a group of taxonomic species within Tetrastemma that
have been described primarily from their colour patterns. We have identified the included species according to current descriptions and we find the same
colour morphs/species over large geographical scales.
Considering hoplonemertean ecology and reproductive
mode (Norenburg & Stricker, 2002) we believe that
there is reason to doubt the validity of current definitions of a number of these species. We estimated the
phylogeny and haplotype networks between 30 individuals from nine species based on partial sequences
of the CO1 gene, but found little correspondence
between evolutionary relationships and external
morphology.
203
MATERIAL AND METHODS
SPECIMENS
AND
DNA
EXTRACTION
Specimens were collected from the littoral as
described by Gibson (1994), or from deeper water by
dredging. External characters were observed under a
dissecting microscope, after which they were placed in
70% or 95% ethanol. The species were identified from
external characters, based on Gibson (1994), Bürger
(1895) and Kirsteuer (1963). (Fig. 1). DNA was
extracted from preserved animals using either a
Chelex (5–10%) solution or QIAamp DNA Mini Kit for
tissue (QIAgen Inc.) following the protocol supplied by
the manufacturer. Sequenced specimens and collection sites are listed in Table 1.
The molecular phylogenetic analyses in both
Sundberg et al. (2001) and Thollesson & Norenburg
(2003) showed unequivocally that Hoplonemertea is a
monophyletic taxon. In the latter analysis, Oerstedia
was in a sister position to Tetrastemma. To our knowledge, this is the closest taxon we could use considering
current phylogenetic knowledge and material available. Since the magnitude of variation within our
ingroup is rather extensive, we preferred an outgroup
within the hoplonemerteans to avoid exceeding the
level of informative variation in CO1. Based on the
analysis of Thollesson & Norenburg (2003) and on
the availability of material and sequences, we chose
four species and specimens from Oerstedia (within
family Tetrastemmatidae) as the outgroup.
AMPLIFICATION,
SEQUENCING AND ALIGNMENT
Amplification of the CO1 gene was carried out by PCR
using a thermal cycler (MJ Research Inc. PTC-100
Programmable Thermal Controller) and eukaryotic
specific end-primers (Folmer et al., 1994): LCO1490
and HCO2198. The gene was amplified in one region of
710 base-pairs. PCRs were performed with up to 10 mL
template in a 50 mL volume with final concentrations
of 10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 0.3 mM of
each primer, 100 mM of each dNTP, 2 units (0.04 U/mL)
of Taq DNA Polymerase (Sigma Product No. D6677).
Thermal cycling was initiated with 1–2 min of denaturation at 94–95 ∞C followed by 35–60 cycles of 30 s
at 94 ∞C, 1 min at 40–50 ∞C, 2 min at 72 ∞C. After
cycling, the reaction was ended with an extension
phase at 72∞ for 7 min. PCR products were purified
with QIAquick PCR Purification Kit (QIAgen Inc.).
Sequencing was carried out either with Cy5-labelled
primers on an ALFexpress automated sequencer
(Pharmacia) following standard procedures, or on a
Beckman Coultier CEQ 2000 (Dye Terminator Cycle
Sequencing) with one exception from standard procedures: primer concentration 3.1 times higher than
standard recommendations.
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
204
M. STRAND and P. SUNDBERG
A
E
B
F
C
G
C(juv)
D
H
Figure 1. The Tetrastemma species included in the analysis. A, T. candidum and T. flavidum. T. candidum lacks distinct
external characters and many records of this species must be of uncertain validity. T. flavidum is similar in external
characters and the distinction between these two species is uncertain. Both are, furthermore, inadequately described and
in need of revision. B, T. laminariae. The species was redescribed by Sundberg (1979); more recently, Chernyshev (2003)
argued, based on observations of the absence of anteriorly directed diverticula of intestinal caecum and more developed
cephalic glands in one syntype specimen, that the species in Sundberg (1979) is not laminariae. However, Sundberg (1979)
showed that these characters vary in the material he examined and we consider the question open as to whether our
specimens from Norway should be classified as laminariae or not. C, T. coronatum. Specimens C and C ( juv) were sampled
at the same time and location. C (juv) is smaller but very similar to C, differing only in pigmentation pattern. The
specimens are genetically practically identical, and we therefore interpret C ( juv) as being a juvenile T. coronatum. D,
T. melanocephalum. E, T. peltatum. F, T. robertianae. G, T. vermiculus H, T. longissimum.
Sequences were edited with Lasergene and aligned
in MegAlign (DNASTAR Inc.). Alignment was
straightforward given the conserved amino-acid
sequence-reading frame.
PHYLOGENETIC
INFERENCE
We used three methods for studying the relationships
between the species/populations: maximum likelihood
(ML), Bayesian analysis, and statistical parsimony
(Templeton et al., 1992).
ML analysis to estimate the haplotype phylogeny
was performed using PAUP 4.0b10 (Swofford, 2000).
MODELTEST v. 3.06 (Posada & Crandall, 1998) was
used to choose the substitution model for our data
based on the Akaike information content. The
selected model was HKY85 + I + G model with
unequal base frequencies (A = 0.2388; C = 0.0696;
G = 0. 1887; T = 0.5029), transitions/transversion ratio
6.78, proportion of invariable sites = 0.521, and
gamma shape parameter = 0.407. Phylogeny was esti-
mated by heuristic search (random addition of
sequences, ten replicates, TBR branch swapping).
Support was estimated from bootstrap analysis (2000
replicates, heuristic search, fast stepwise addition).
Phylogenetic analysis of haplotypes, carried out
using Bayesian inference, was performed with
MrBayes v. 3.06 (Huelsenbeck & Ronquist, 2001)
using the default values of one cold and three heated
Markov chains. Sites were assigned into three partitions depending on position in amino acid triplet (1st,
2nd or 3rd) and allowed to vary independently with
gamma distribution in the GTR model (lset nst = 6).
Five separate analyses were run starting from random
trees to assure congruence. In each analysis the Monte
Carlo Markov Chain (MCMC) length was 1000 000
generations with sampling of every 10th generation
chain. Log-likelihood values for sampled trees stabilized after approximately 100 000 generations, burnin was set to 50 000, leaving the last 50 000 sampled
trees for estimating posterior probabilities (or Bayesian support values).
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
DELIMITING SPECIES IN TETRASTEMMA
205
Table 1. The nine Tetrastemma species included in the analysis, together with sampling area and GenBank accession
number. Four Oerstedia species are used as the outgroup. Oerstedia zebra was originally described as Oerstediella zebra,
but that genus was synonomized with Oerstedia by Envall & Sundberg (1998)
Species
Area
Accession no.
Tetrastemma candidum (Müller, 1774)
Tetrastemma coronatum (Quatrefages, 1846)
Tetrastemma flavidum Ehrenberg, 1828
Anglesey, Wales
Faial, Azores
Anglesey, Wales
Càdiz, Spain
Trondheim, Norway
Pico, Azores
Ischia, Italy
Florida, USA
Blanes, Spain
Anglesey, Wales
Tjärnö, Sweden
Ischia, Italy
Tjärnö, Sweden
Càdiz, Spain
Anglesey, Wales
Ischia, Italy
Tjärnö, Sweden
Tjärnö, Sweden
Akkeshi Bay, Japan
Akkeshi Bay, Japan
AY791973
AY791974–76
AY791977
AY791978
AY791979–80
AY791981
AY791987
AY791985
AY791982–84
AY791986
AY791988–89
AY791990–93
AY791994
AY791997
AY791996
AY791995
AY791972
AY791971
AJ436911
AJ436912
Tetrastemma laminariae Ushakov, 1928
Tetrastemma longissimum Bürger, 1895
Tetrastemma melanocephalum (Johnston, 1837)
Tetrastemma peltatum Bürger, 1895
Tetrastemma robertianae McIntosh, 1873
Tetrastemma vermiculus Quatrefages, 1846
Oerstedia
Oerstedia
Oerstedia
Oerstedia
striata Sundberg, 1988
dorsalis Abildgaard, 1806
venusta Iwata, 1954
zebra (Chernyshev, 1993)
Methods for estimating species phylogenies may not
work particularly well when the terminals are population representatives or individuals from the same
population. Our analysis, however, is based on
mtDNA, and we should therefore not have a problem
with recombination and reticulate events. However,
the resulting tree will be a gene tree, not necessarily
representing the phylogeny of the populations/species.
In addition to Bayesian inference and ML, we also
applied a statistical parsimony algorithm (Templeton
et al., 1992) implemented in TCS v. 1.13 (Clement,
Posada & Crandall, 2000) to estimate relationships
between closely related sequences regardless of species category. This program also calculated the significant number of substitutions connecting haplotypes
in the network according to the formulas in Templeton
et al. (1992).
RESULTS
The sequence data for the 34 terminal units included
539 characters, of which 333 were constant, 29 were
parsimony uninformative, and 177 were parsimony
informative. Of the informative sites, 145 substitutions occurred at 3rd position, 2 at 2nd and 30 at 1st.
Within the 34 individual samples there were 29 different haplotypes. To get a baseline for genetic variation within what we a priori consider the same species,
we compared variation in three groups of individuals
with similar external appearance, taken from the
same sampling site and at the same time (see Table 1).
The first group contains five specimens identified as
T. melanocephalum sampled at Tjärnö, Sweden (nos.
15-19 in Table 3). The pairwise difference between the
specimens varies from 0% to 0.2%. A second group consists of three specimens from Faial, Azores identified
as T. coronatum (nos. 6, 32, 33 in Table 3). Two had a
slightly different pigment pattern on the head, but
when considering the size of the animals and general
juvenile appearance we considered these differences
in pigmentation to be a matter of size/age. This is also
supported by the distance values, with differences
ranging from 0.3% to 0.7%. A third group includes
three specimens of T. laminariae from the Trondheim
fjord, Norway (nos. 29, 30, 34 in Table 3) and here the
differences range from 0% to 0.2%.
There are five specimens from two localities around
Ischia, Italy recorded as T. peltatum (nos. 20-24 in
Table 3). Field notes indicate some doubts about identification, and pairwise distances among these five
individuals vary from 0% to 14.6%; the phylogenetic
estimates in Figures 3-5 also indicate that there are
in fact two separate groups. We sequenced
T. melanocephalum from seven sampling sites. There
is little variation among those sampled from Tjärnö
(above), and there are two specimens from the same
site (Blanes, Spain) differing by 1.1%; the remainder
differ by between 11.3 and 15.1%. The species
T. robertianae, originally described by McIntosh
(1873-74) and redescribed by Berg (1973) differs from
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
206
M. STRAND and P. SUNDBERG
Table 2. Interspecific distances within outgroup (%)
Oerstedia
venusta
O. venusta
O. zebra
O. striata
O. dorsalis
O. zebra
O. striata
O. dorsalis
–
10.7
9.0
–
11.5
–
–
0.2
10.7
9.2
1.0
.8
Cumulative fraction
I
.6
II
.4
.2
0
0
5
10
15
20
Nucleotide differences (%)
Figure 2. Plot of the cumulative pairwise differences.
Curve I is based on Table 3 in this study. Curve II depicts
the cumulative differences between the hoplonemertean
species included in Thollesson & Norenburg (2003). These
differences act as reference point for differences between
established species.
the rest of included species by between 9.9% and
19.4%. In the outgroup, distances varied from 0.2% to
11.5% (Table 2). The average difference between the
outgroup and the ingroup species is 15.5 ± 1.48% SD.
Even though genetic differences per se cannot be
used to draw up the boundaries of species, it may still
be illuminating to compare other data sets. The only
study of nemertean systematics based on the mtDNA
CO1 gene so far is by Thollesson & Norenburg (2003).
From a plot (Fig. 2) of the cumulative pairwise distances between the hoplonemertean species in their
study we observe that genetic divergence between
established species starts from 12% and goes up to
around 22%. When plotting the cumulative distances
from our study (only the Tetrastemma-species, curve I
in Fig. 2) we see that around 40% of the distances in
our matrix are of the magnitude found between species in the Thollesson & Norenburg study. However,
we would expect only around 30% of the distances to
be of that magnitude (9/30, number of species/specimens) since that is the proportion of named species
among the distances in our study. Although we stress
that species cannot be defined in this simplistic way, it
is still a clear indication that there are more species in
our sample than indicated by the morphotypes.
ML analysis of the 25 haplotypes in the ingroup,
plus the outgroup species, resulted in four trees with
the same likelihood scores; these trees have the same
topology (Fig. 3) and differ only slightly in branch
lengths. There are morphotypes forming supported
clades (clade B in Fig. 3 for example), and specimens
from the same geographical area forming clades (clade
A in Fig. 3 for example), but the pattern is not consistent; for example, melanocephalum appears not only
in clade B, but also in other (unrelated) places in the
tree. Clade A in Figure 3 thus consists of candidum
and vermiculus sampled from the same area in Wales.
Clade B clusters melanocephalum from Sweden, while
there are several other specimens identified as melanocephalum that are clearly unrelated. Clade C contains specimens identified as melanocephalum and
flavidum from the Mediterranean and Càdiz, just west
of Gibraltar sound. Clade D accommodates two
peltatum haplotypes from Ischia, while other haplotypes collected from Ischia and identified as coming
from the same species are unrelated. Clades E and F
combine geographical area and species identification.
The conclusion from the ML analysis is thus that
there are several cases showing that the same morphotypes do not correspond to evolutionary lineages,
and are thus not the same species.
The Bayesian analysis shows basically the same
pattern (Fig. 4) as the ML analysis, although there is
support for some more inclusive clades in this analysis. Still, the interpretation is the same - the connection between morphotype and evolutionary identity is
blurred, and so is the link between evolutionary lineage and geographical area.
The statistical parsimony analysis calculated the
number of substitutions to connect haplotypes to be
ten at the 95% significance level (Templeton et al.,
1992). Within this limit, there are the following
clusters (Fig. 5): the Swedish melanocephalum, a candidum and a vermiculus from Wales, three melanocephalum from the Mediterranean together with one
flavidum from Càdiz, and another cluster of two of the
four flavidum specimens from Ischia, Italy. Within the
outgroup, two species from Japan are genetically similar and form a group. Remaining specimens/species
are attached to neither other specimens nor groups,
thus reinforcing the ML and Bayesian analyses.
DISCUSSION
The study involves littoral nemerteans of the two genera Oerstedia and Tetrastemma that probably have a
limited rate of dispersal since they are, as far as we
know, direct developers. We can therefore expect lim-
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
5
6
15.0
14.6
14.6
16.9
14.0
10.1
11.1
-
4
1 - 10.7 10.7 11.5 16.5 15.7 14.6
2
0.2
9.2 16.3 15.9 15.0
3
9.0 16.1 15.7 14.8
4
16.7 15.7 16.5
5
14.2 13.8
6
8.7
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
3
8
2
7
1
14.4
14.4
14.4
17.1
14.2
10.5
10.5
1.5
-
9
14.6
14.6
14.6
16.9
14.4
10.1
10.7
1.5
0.7
-
10
15.9
15.0
15.0
17.3
15.3
10.9
11.1
11.7
11.5
11.9
-
11
14.8
14.2
14.4
17.5
16.5
13.4
13.0
15.3
15.1
15.1
14.6
-
12
15.7
16.7
16.9
18.2
14.4
7.5
8.8
11.1
11.3
11.3
10.9
13.0
-
13
14.4
14.2
14.2
16.7
13.6
10.5
10.3
1.9
1.5
1.1
11.7
15.0
11.3
-
14
14.2
14.4
14.6
14.2
12.8
11.3
10.9
9.8
9.4
10.0
11.7
14.2
11.5
9.4
-
15
14.6
14.8
15.0
14.6
12.6
11.5
11.1
10.0
9.6
10.1
12.1
14.4
11.7
9.6
0.1
-
16
14.4
14.6
14.8
14.4
12.8
11.3
10.9
9.8
9.4
10.0
11.9
14.2
11.5
9.4
0.0
0.2
-
17
14.4
14.6
14.8
14.4
12.8
11.3
10.9
9.8
9.4
10.0
11.9
14.2
11.5
9.4
0.0
0.2
0.0
-
18
14.4
14.6
14.8
14.4
12.8
11.3
10.9
9.8
9.4
10.0
11.9
14.2
11.5
9.4
0.0
0.2
0.0
0.0
-
19
18.0
16.7
16.5
17.8
18.2
13.4
13.4
15.3
15.5
15.9
14.2
16.7
14.4
15.1
15.5
15.7
15.5
15.5
15.5
-
20
18.2
16.7
16.5
17.6
18.0
13.4
13.4
15.3
15.5
15.9
14.0
17.1
14.8
15.1
15.7
15.9
15.7
15.7
15.7
1.7
-
21
16.7
15.9
15.7
17.1
15.5
10.3
11.7
12.8
12.6
12.8
10.3
14.6
11.7
12.6
11.3
11.5
11.3
11.3
11.3
14.4
14.6
-
22
16.7
15.9
15.7
17.1
15.5
10.3
11.7
12.8
12.6
12.8
10.3
14.6
11.7
12.6
11.3
11.5
11.3
11.3
11.3
14.4
14.6
0.0
-
23
15.3
13.8
14.0
16.7
16.1
10.5
11.1
11.7
11.7
11.9
11.5
13.8
10.0
11.7
11.7
11.9
11.7
11.7
11.7
12.6
12.8
9.4
9.4
-
24
17.1
16.7
16.5
18.4
15.1
10.0
11.5
10.5
10.5
10.3
11.1
15.9
11.1
10.1
13.2
13.6
13.4
13.4
13.4
14.2
13.8
10.7
10.7
10.5
-
25
16.3
16.1
15.9
17.6
15.7
11.1
11.3
13.0
12.5
12.6
12.5
14.6
12.5
12.8
13.2
13.4
13.2
13.2
13.2
15.1
14.6
12.1
12.1
11.3
11.1
-
26
17.3
16.9
16.7
17.3
1.7
14.8
14.0
14.6
14.4
14.6
15.1
16.7
15.0
14.2
13.4
13.2
13.4
13.4
13.4
18.2
17.6
16.3
16.3
16.3
15.7
16.7
-
27
15.9
16.3
16.3
18.2
15.5
13.4
13.2
14.6
14.2
14.2
14.2
14.0
14.0
13.4
13.4
13.6
13.4
13.4
13.4
18.8
18.8
15.0
15.0
15.0
13.4
15.3
16.1
-
28
14.8
14.2
14.4
16.9
13.4
9.2
10.9
9.8
10.0
10.1
10.7
14.8
9.6
9.4
10.9
11.1
10.9
10.9
10.9
13.8
13.6
11.3
11.3
10.0
9.0
11.1
14.4
13.8
-
29
14.6
14.0
14.2
16.9
13.2
9.0
10.7
9.6
9.8
10.0
10.9
14.8
9.4
9.2
10.7
10.9
10.7
10.7
10.7
13.6
13.4
11.1
11.1
9.8
8.8
10.9
14.2
13.6
0.2
-
30
15.7
15.5
15.3
15.7
14.2
10.1
11.1
11.5
11.5
11.5
11.9
13.4
12.3
11.1
9.8
10.1
10.0
10.0
10.0
15.0
15.1
11.7
11.7
11.1
10.9
12.1
14.6
15.1
10.1
10.1
-
31
16.1
16.3
16.1
16.1
14.8
0.7
8.4
10.5
10.9
10.5
11.3
13.8
7.8
10.9
11.7
11.9
11.7
11.7
11.7
13.8
13.8
10.7
10.7
10.9
10.3
11.5
15.3
13.8
9.6
9.4
10.5
-
32
15.7
15.5
15.3
15.7
14.4
0.3
8.0
9.8
10.1
9.8
10.5
13.4
7.1
10.1
11.3
11.5
11.3
11.3
11.3
13.0
13.0
10.3
10.3
10.1
10.0
11.1
15.0
13.4
8.8
8.6
10.5
0.7
-
33
14.6
14.0
14.2
16.9
13.2
9.0
10.7
9.6
9.8
10.0
10.9
14.8
9.4
9.2
10.7
10.9
10.7
10.7
10.7
13.6
13.4
11.1
11.1
9.8
8.8
10.9
14.2
13.6
0.1
0.0
10.1
9.4
8.6
-
34
Table 3. Pairwise percentage nucleotide differences between ingroup (Tetrastemma) specimens/populations, together with outgroup (Oerstedia species). Tetrastemma species have been identified from external characteristics. Oerstedia venusta and O. zebra data are from Thollesson & Norenburg (2003). Oerstedia: 1.
striata; 2. venusta; 3. zebra; 4. dorsalis. Tetrastemma: 5. candidum (Wales, UK); 6, 32, 33. coronatum (Faial, Azores); 7. flavidum (Wales); 8. flavidum (Càdiz,
Spain); 9-11. melanocephalum (Blanes, Spain); 12. melanocephalum (Florida, USA); 13. melanocephalum (Wales, UK); 14. melanocephalum (Ischia, Italy); 15-19.
melanocephalum (Tjärnö, Sweden); 20-24. peltatum (Ischia, Italy); 25. robertianum (Tjärnö, Sweden); 27. vermiculus (Wales, UK); 28. vermiculus (Càdiz, Spain);
29, 30, 34. laminariae (Trondheim, Norway); 31. longissimum (Pico, Azores)
DELIMITING SPECIES IN TETRASTEMMA
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
207
208
M. STRAND and P. SUNDBERG
100
100
O. striata
O. venusta
O. zebra
O. dorsalis
T. candidum W
T. vermiculus W
100
T. melanocephalum SW
T. melanocephalum SW
T. longissimum PI
T. flavidum CAD
100
T. melanocephalum BL
T. melanocephalum BL
100
T. melanocephalum ISC
100
T. peltatum ISC
T. peltatum ISC
100
T. laminariae N
E
T. laminariae N
T. vermiculus ISC
T. coronatum FAI
F
100
T. coronatum FAI
T. coronatum FAI
T. melanocephalum W
T. robertianae SW
T. melanocephalum BL
T. peltatum ISC
T. flavidum W
T. peltatum ISC
T. melanocephalum FL
T. vermiculus CAD
100
100
A
B
C
D
Figure 3. The haplotype phylogeny estimated by maximum likelihood analysis (see Material and methods) of the 25
haplotypes of nine morphotypes/assumed species of Tetrastemma, together with four outgroup species ( Oerstedia striata,
O. venusta, O. zebra and O. dorsalis). There of the four equally likely phylogenies all agreed in topologies but differed
slightly in branch lengths. Bootstrap values (100%) are indicated above the branches for supported clades (based on 2000
replicates). Abbreviations: W = Wales, UK; FAI = Faial, Azores; PI = Pico, Azores; CAD = Càdiz area, Spain; BL = Blanes
area, Spain; ISC = Ischia, Italy; FL = Florida, USA; SW = west coast, Sweden; N = Trondheim area, Norway. Clades A–F
are discussed in the text.
ited gene flow between distant populations and the
validity of many nemertean species with wide geographical distributions is doubtful. We have shown
that individuals of a species-level taxon fail to cluster
together and there are several possible explanations.
It could be a case of incomplete lineage sorting of
ancestral polymorphisms, i.e. suggesting that the
putative species is valid but has very recently
diverged. However, we consider this explanation
unlikely in the light of the high degree of genetic
divergence. Another possibility is that the estimated
phylogeny fails to match the population/species histories. Our analyses are based on mitochondrial DNA
genotypes that provide information of haplotypes/
genetic lineages (Palumbi, 1996) and what we estimate is the gene tree. Once again, the high degree of
genetic divergence is a strong indication that the morphotypes do not correspond to evolutionary lineages.
Another reason behind the lack of correspondence
between morphology and evolutionary lineages is that
we are dealing with the presence of multiple, unrelated, species hidden by previous taxonomy. In this
case, this is the explanation we favour when considering the high degree of genetic divergence in combination with the previously mentioned (see Introduction)
difficulties and deficiencies of existing nemertean
taxonomy.
INTRASPECIFIC
VARIATION IN MORPHOLOGICAL
CHARACTERS
There are few external characters in nemerteans,
although they serve to identify many species. Current
systematic decisions rest on anatomical characters.
These are observed from histological sections, which
may be a problem when characters are of the kind
‘extension of oesophagus’, ‘shape of cerebral organ’,
etc., since these animals are extremely contractile and
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
DELIMITING SPECIES IN TETRASTEMMA
209
T. melanocephalum FL
72
T. vermiculus CAD
T. candidum W
100
A
T. vermiculus W
T. flavidum CAD
100
75
T. melanocephalum BL
54
C
T. melanocephalum BL
T. melanocephalum ISC
100
100
86
T. melanocephalum SW
T. melanocephalum SW
B
T. longissimum PI
T. coronatum FAI
100
64
T. coronatum FAI
F
T. coronatum FAI
93
T. melanocephalum W
57
T. robertianae SW
T. flavidum W
T. melanocephalum BL
T. peltatum ISC
T. peltatum ISC
100
T. laminariae N
100
T. laminariae N
D
E
T. peltatum ISC
T. peltatum ISC
T. vermiculus ISC
O. striata
O. dorsalis
100
100
O. venusta
O. zebra
Figure 4. The haplotype phylogeny estimated by Bayesian analysis (see Material and methods), of the 25 haplotypes of
nine morphotypes/assumed species of Tetrastemma, together with four outgroup species (Oerstedia striata, O. venusta,
O. zebra and O. dorsalis). The numbers above branches represent probability values. Abbreviations as per Fig. 3.
hence prone to fixation artefacts. Although there have
been a number of studies exposing the high degree of
variation (Berg, 1972; Sundberg, 1979, 1984), it
appears not to have been taken into account by many
authors. Sundberg (1979) studied the variation within
a set of specimens considered to be conspecific since
they had the same general appearance, were collected
at the same time, and at the same sampling spot.
Around 100 specimens were cross-sectioned and the
best sections (those sectioned at a perpendicular
angle) were used to numerically describe the variation
in a number of characters normally used when
describing hoplonemertean species. He was able to
show that many key characters varied within the sample to such a degree that most states used to erect species were covered. We suspect that several described
Tetrastemma species have been described without taking into account intraspecific variation. On the basis of
results presented in this study, we are also confident
that many reports of a particular species in fact represent observations of different species.
OUTGROUP
RELATIONSHIPS
There are four alleged Oerstedia species in the outgroup. The phylogenetic analyses places them in a
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
210
M. STRAND and P. SUNDBERG
robertianae SW
laminariae N
melanocephalum SW
laminariae N
melanocephalum SW
melanocephalum W
melanocephalum SW
flavidum W
candidum W
vermiculus W
melanocephalum BL
melanocephalum ISC
peltatum I
melanocephalum BL
peltatum ISC
melanocephalum BL
peltatum ISC
vermiculus ISC
vermiculus CAD
flavidum CAD
peltatum I
coronatum FA I
coronatum FA I
Outgroup
coronatum FA I
longissimum PI
dorsalis
striata
zebra
melanocephalum FL
venusta
Figure 5. Statistical parsimony analysis of the 34 sequences. Each bar indicates one substitution, and only genotypes
connected by equal or less than the significant number of ten substitutions are connected. Dotted frames around a name
indicate two identical genotypes. Dots plus dashes indicate three identical genotypes. Abbreviations as per Fig. 3.
monophyletic clade separate from Tetrastemma and
we have no reason to question this result. The genetic
differences between outgroup species (Table 2) vary
from 11.5% to 0.2%; the latter figure is the percentage
difference between O. venusta and O. zebra, which differ by only one substitution. Oerstedia venusta was
described by Iwata (1954); the short description does
not list any characters distinguishing it from O. dorsalis. We interpret Iwata’s comment that ‘the outer
feature of this specimen differs mainly from species
known in the genus’ to mean that the species was
erected on external characters. Gibson (1995) considered it a nomen dubium. It is not possible from the
schematic figure in Iwata (1954) to confirm whether
the colour and pigmentation pattern resemble what
we have observed in O. dorsalis specimens. Still, it is
genetically separated from dorsalis and when the geographical range is also taken into account, we conclude that it is a separate species. However, it is
almost genetically identical to O. zebra, which
Chernyshev (1993) described from the Sea of Japan.
When comparing the external characters between
these two species, they are within the range of variation we see in O. dorsalis. Since the two species are,
furthermore, geographically rather close we conclude
that O. zebra is a junior synonym of O. venusta.
Oerstedia striata Sundberg, 1988 was established by
Sundberg & Jansson (1988) as a separate species from
O. dorsalis based on allozyme data, and it is highly
variable when it comes to external characters; specimens differ in both coloration and pigmentation patterns (Gibson, 1994).
© 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212
DELIMITING SPECIES IN TETRASTEMMA
CONCLUSIONS
We found little agreement between morphotypes and
evolutionary lineages in the sampled Tetrastemma
specimens. Other comparable genetic studies on nemerteans are limited. Envall & Sundberg (1998) showed
in a study of the 16S gene a similar ambiguity between
morphological and genetic data in the hoplonemertean
genus Ototyphlonemertes. There is also evidence of
intraspecific gene differentiation over a small geographical scale between populations of the heteronemertean Parborlasia corrugatus in an allozyme study
conducted by Rogers et al. (1998), although the results
are difficult to compare with divergence in nucleotide
sequences. There are studies of other marine invertebrates showing large morphological differences
between populations, but at the same time little
sequence divergence in CO1 (e.g. vestimentiferan tube
worms; Black et al., 1998). On the other hand, there are
also studies showing opposite results, with high morphological similarity and large sequence divergence in
CO1 (e.g. Cirripedia; Wares, 2001), which would be
interpreted as limited gene flow and a possible speciation process not apparently reflected in morphology.
The conclusion from our analyses is that external
characters do not correspond to evolutionary lineages,
and hence not to what we would interpret as species,
however defined. Our interpretation is that characters
used to describe species in Tetrastemma are inadequate for identifying evolutionary lineages, and that
we are probably dealing with several cryptic species.
Likewise, however, there are also cases of conspecific
species with different external characters. This will of
course have a profound effect on the true number of
nemertean species and estimates of their diversity. We
doubt that a solution can be found in further anatomical study, unless one went into very fine detail (for
example electron microscopy); this seems to be prohibitive considering time and cost. It may be that
nemertean taxonomy will depend on nemerteans being
identified by genotyping, as has been recently suggested for other taxa (Blaxter, 2003; Tautz et al., 2003).
ACKNOWLEDGEMENTS
We thank Inger Holmquist for technical assistance,
and Svetlana Maslakova who provided us with watercolours of Oerstedia venusta and O. zebra. This study
was financially supported by the Swedish Research
Council (to PS), Helge Ax:son Johnsons stiftelse,
Kungliga Fysiografiska Sällskapet and Rådman och
Fru Ernst Collianders stiftelse.
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