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ABSTRACT
A new species of Philippine turrid, Gemmula ambara (the amber gem
turrid), is described. The type locality is Balicasag Island, Bohol, Phlippines;
the holotype and several paratypes were collected at the type locality using
tangle nets at depths of 70-120 fathoms. The species occurs offshore across
a number of central Philippines localities; it has also been collected off
Panglao Island using tangle nets and off Aliguay Island using small trawls.
Gemmula ambara had been mistakenly regarded by collectors and dealers
as a small variety of Gemmula diomedea, but a more careful examination
of the shell morphology easily separates the two species. A preliminary analysis
of the phylogeny of Gemmula ambara using standard molecular markers
has revealed that in fact, this species is not closely related to Gemmula
diomedea but instead, is more closely related to Gemmula rosario
and Gemmula lisajoni. It appears that the species of Gemmula
from the Philippines can be divided into two distinct branches, with
Gemmula ambara, Gemmula rosario and Gemmula lisajoni
comprising one branch, and Gemmula diomedea, Gemmula speciosa,
Gemmula kieneri, and Gemmula sogodensis comprising a second
branch.
INTRODUCTION
Venomous marine snails belonging to the superfamily Conoidea are
a significant component of the marine biodiversity of the Philippines and
their venoms are proving to be a promising resource for novel biologically
active compounds. The conoidean snails have traditionally been divided into
three groups: cone snails (family Conidae), auger snails (family Terebridae)
and turrids (family Turridae) — for reviews of these groups see Rockel
et al. 1995; Bratcher and Cernohorsky 1987; Terryn 2007; Powell 1964, 1966.
In numbers of species, the turrids are the largest group of conoideans, but
also the most poorly characterized. Recently, it has become clear that the
family Turridae is not monophyletic (Taylor et al. 1993; Puillandre
et al. 2008); however, a generally accepted taxonomic treatment for the diverse
groups formerly in the family Turridae has not yet been firmly established.
‚ ‚ ‚ ‚ ‚ The type
species of the family Turridae is Turris babylonia (Linnaeus,
1758); this taxon also defines the subfamily Turrinae. Included in the subfamily
Turrinae are several genera, e.g., Turris, Roding 1798,
Gemmula, Weinkauff, 1875, Lophiotoma, Casey, 1904, Polystira,
Woodring, 1928, and Turridrupa, Hedley, 1922. The taxonomy of the
Turridae requires considerable taxonomic attention, with many of
the recently collected Philippine turrid species remaining undescribed. This
is primarily a deep-water group, with most species not accessible to the classical
shallow-water collecting techniques that provided most marine gastropods described
since the work of Linnaeus. In addition, the morphological differences between
many closely related species are subtle, making it easy to confuse closely
related forms.
‚ ‚ ‚ ‚ ‚ In this
work we describe a new species in the Turrinae, in the genus Gemmula,
Weinkauff, 1875 (the “gem turrids”). We define the species based
on its distinctive morphological characteristics, but also establish its phylogenetic
affinities through the use of standard molecular markers. The new species,
the amber gem turrid, Gemmula ambara, is only the first of many undescribed
Philippine turrids that we hope will be characterized in the near future using
a combination of both a shell morphology based taxonomic description, as well
as molecular phylogeny.
METHODS
Specimen collection. The holotype and paratypes
from Balicasag Island were collected by fishermen using tangle nets offshore.
Most of the other specimens examined were purchased through commercial sources
in Mactan, Cebu.
Preparation of genomic DNA. A specimen dissected
by Yuri Kantor on July 1, 2004, (a male, 34 mm in length) in Balicasag Island,
Philippines was used for the molecular analysis. Genomic DNA was prepared
from 5 mg Gemmula ambara proboscis tissue using the Gentra Puregene DNA Isolation
Kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's standard
protocol.
Cloning and sequencing of COI mtDNA. 10 ng of Gemmula
ambara genomic DNA was used as a template for polymerase chain reaction (PCR)
with oligonucleotides corresponding to COI forward primer (5’ GGT CAA
CAA ATC ATA AAG AYA TGY G 3’) and COI reverse primer (5’ TAA ACT
TCA GGG TGA CCA AAR AAY CA 3’). The pcr cycling profiles are as follows:
Initial denaturation (950C, 60s); followed by 40 cycles of denaturation (950C,
20s); annealing (55oC, 20s) and extension (72oC, 30s). The resulting pcr product
was purified by gel electropheresis, recovered from agarose using High Pure
PCR Product Purification Kit (Roche Diagnostics, Indianapolis, IN). The eluted
DNA fragment was annealed to pNEB206A vector using the USER Friendly Cloning
Kit (New England BioLabs, Inc., Beverly, MA) following manufacturer's suggested
protocol and the resulting product transformed into DH5-alpha competent cells.
‚ ‚ ‚ ‚ ‚ The nucleic
acid sequences of the resulting COI-encoding clones were determined according
to the standard protocol for Automated Results.
Molecular phylogeny using 12S mitochondrial ribosomal RNA.
The nucleotide sequences were obtained using PCR and the phylogenetic tree
constructed as described in Heralde et al. (2007).
RESULTS
Morphological
characterization of a new Gemmula species
Range. Collected in the central Philippines, primarily
from Balicasag Island, Bohol, using tangle nets, 70-150 fathoms, and from
Aliguay Island, using small trawls, 30-100 fathoms.
Figure 1. Gemmula
ambara, new species. Shown are three views of the holotype, which is
39.6mm in length.
Remarks.
This medium-sized Gemmula (the holotype is shown in Fig. 1) has the
same general color pattern as Gemmula diomedea, but can be separated
from that species by its distinctively different subsutural region (see Fig.
2). The molecular phylogeny (see next section) suggests that this species
is most closely related to Gemmula rosario and Gemmula lisajoni.
Figure 2. Gemmula
ambara, new species, compared to Gemmula diomedea, Powell, 1964.
The three bottom specimens are Gemmula ambara, and the top specimen
is Gemmula diomedea. Note the difference in the subsutural region.
Description. Shell moderately broad fusiform, length
30-55mm. Overall color of the shell is white, flushed with a distinct violet
or purplish tone in some specimens (particularly when freshly collected),
except for the subsutural area, which is always light amber to dark amber
brown in color. The protoconch has 3-4 translucent yellowish-brown to purplish
brown whorls, axially costate over the last two protoconch whorls. Post-nuclear
whorls 9 - 10.
‚ ‚ ‚ ‚ ‚ The spire
whorls are sculptured with a prominent subsutural fold comprising two strong,
closely-spaced spiral ribs, immediately adjacent to the suture, with the region
in between moderately elevated, such that the two ribs comprise a prominent
amber keel that borders the suture. In earlier spire whorls, there are two
additional ribs in the anterior subsutural region and as the shell increases
in size, the subsutural region can have as many as 6 to 7 spiral ribs, with
the two posterior ones comprising a subsutural keel always the strongest.
There are axial columns between the spiral ribs, quite strong in some specimens.
A diagnostic feature of this species is that the entire subsutural region
up to the peripheral sinus rib is amber in color, and the rest of the shell
(except the colored protoconch) is white.
‚ ‚ ‚ ‚ ‚ In the
first few post-nuclear spire whorls, the most prominent features are the amber
brown subsutural region and the strongly gemmulate sinus rib. By the fourth
post-nuclear whorl, an additional spiral rib anterior to the gemmulate peripheral
sinus rib becomes emergent and in mature specimens, two strong additional
spiral whorls are found anterior to the sinus rib in the larger spire whorls.
‚‚ ‚ ‚ ‚ ‚ In
the body whorl, there are six principal spiral ribs and at the base, numerous
weaker spiral ribs surrounding one major, somewhat crenulate rib. There are
16 spiral ribs with a varying number of weak ribs between the principal raised
spirals along the canal
Comparisons. Gemmula ambara looks superficially
similar to smaller specimens of Gemmula diomedea; it can readily
be distinguished from members of the G. diomedea complex from the
Philippines because the entire subsutural region until the sinus rib at the
periphery is amber brown. In all of the G. diomedea-like specimens
(there are likely to be multiple species in this complex — compare the
specimens tentatively assigned to G. diomedea in Figs. 2 and 3),
the one or two raised subsutural ribs are brown but the remainder of the subsutural
region has the same white background color as the rest of the shell (including
the region between the two brown subsutural spiral ribs — white in G.
diomedea, amber in G. ambara). When preserved, the protoconch
is also diagnostic (see Fig 3): brown in G. ambara, generally white and much
larger in G. diomedea, (although some varieties of G. diomedea,
which may be different species, have a protoconch that is light yellowish).
In addition, most Philippine specimens of Gemmula diomedea have a
higher and narrower spire, and a proportionately shorter canal than Gemmula
ambara. Most specimens of G. diomedea have diameter:length ratio
= 0.30-0.32 while for G. ambara it is 0.33-0.35.
As noted in the molecular phylogeny section that follows, Gemmula ambara
is most closely related to G. rosario and G. lisajoni.
G. rosario is a pure brown shell, uniformly straw brown rather than
amber and white, while G. lisajoni has a distinctive deeply purplish-violet
canal, with a brown body (see Fig. 3). In contrast to G. ambara,
there is no white coloration in either of these species. Other Philippine
forms of Gemmula such as G. speciosa, G. kieneri and
G. sogodensis are both morphologically and genetically even more
distant and would not be mistaken for Gemmula ambara.
Figure 3. A comparison
between Gemmula ambara (A) with Gemmula lisajoni (B), Gemmula
rosario (C) and Gemmula diomedea variety (D). On the bottom
are two of the protoconchs; note the differences in size and color between
the protoconch of Gemmula ambara and Gemmula diomedea. The
specimen of Gemmula ambara shown (A) is Paratype 19; although it
is a dead-collected shell with a bore-hole, it had the best-preserved protoconch
of all the specimens examined. The specimen in D is the form from Aliguay
Island generally assigned to G. diomedea, but similar in shape to
G. congener. The G. diomedea/congener complex needs to be
more carefully analyzed, but all specimens in this group can be reliably separated
from G. ambara.
Etymology. The amber colored subsutural region,
characteristic of this species, is the origin of the name ambara.
Types. A summary of type specimens is given in Table
1. The holotype will be deposited in the Philippine National Museum. Paratypes
will be deposited in the Museum National d'Histoire Naturelle in Paris; the
Academy of Natural Sciences in Philadelphia, Pennsylvania; the Harvard Museum
of Comparative Zoology, in Cambridge, Massachusetts, the Natural History Museum,
Geneva, Switzerland. All of these museums will also be provided with a printed
version of this manuscript.
Table 1:
Gemmula ambara, Types examined.
Molecular phylogeny of Gemmula ambara
A molecular phylogeny of Gemmula ambara was carried out; the results are shown
in Figure 4 and Table 2. Phylogenetic trees based on 12S ribosomal mitochondrial
DNA sequences were constructed using several standard methods (see Methods);
the data reveal that of the species of Gemmula analyzed, Gemmula
ambara is more closely related to Gemmula rosario and Gemmula
lisajoni than to the other Gemmula species for which sequences
were available (Gemmula sogodensis, Gemmula speciosa, Gemmula
diomedea and Gemmula kieneri). This result was rather surprising,
since shells of Gemmula ambara have generally been confused with
smaller specimens of Gemmula diomedea, as indicated above, since
these forms share a white and brown color pattern.
‚The COI barcode sequence for Gemmula
ambara was obtained and compared to other COI Gemmula sequences
previously determined. The results for the barcode of all Philippine Gemmula
species analyzed so far are in Table 2. As would be expected for distinct
species, the barcode sequences diverge significantly from each other within
the homologous COI interval.
Table 2. Barcode
sequences (CO1)
Figure 4. Phylogenetic analysis using 12S mitochondrial DNA sequences. Phylogenetic tree based on a small number of 12S rRNA sequences for the genus Gemmula showing strong support for placement of Gemmula ambara in a clade with G. lisajoni and G.rosario to the exclusion of other Gemmula species and the outgroup Turris spectabilis. Identical trees with similar support values were generated using Bayesian methods with posterior probabilities (indicated by the numbers) and maximum likelihood analysis with bootstrap analysis.
DISCUSSION
In this paper, a new turrine species from the Philippines, the amber
gem turrid, Gemmula ambara, is characterized. Most specimens examined
have come from the Balicasag/Aliguay region of the Central Philippines where
specimens have either been caught by tangle nets or using a small trawl at
depths >30 fathoms. At these localities, it appears that the species is
not uncommon, but has apparently been confused with similarly colored forms
in the genus Gemmula that belong to the Gemmula diomedea complex.
As is described above, there are a number of distinctive morphological features
that reliably differentiate the shells of Gemmula ambara from the
smaller Gemmula diomedea specimens of the same size as G. ambara.
‚ ‚ ‚ ‚ ‚ The molecular
phylogeny reveals that contrary to expectations from the similarities in color
and shell morphology of Gemmula ambara to G. diomedea, it
is most closely related to Gemmula lisajoni and Gemmula rosario,
although the color pattern of G. ambara is distinct and easily separable
from those species. With a more careful examination of morphological characters
of these species, certain more subtle shell morphological characteristics
appear to be better correlated with the true relationships than is color pattern.
For example, the sculpture of the subsutural region and the size of the protoconch
may prove to be a better indicator of genetic affinities, with color pattern
potentially more sensitive to environmental factors.
‚ ‚ ‚ ‚ ‚ Of the
species for which molecular data are available in the Philippines that are
conventionally assigned to the genus Gemmula, there now appear to
be two very distinctive subgroups. One includes Gemmula speciosa,
Gemmula sogodensis, Gemmula kieneri and Gemmula diomedea.
The other includes Gemmula rosario, Gemmula lisajoni and
the species we characterized in this work, Gemmula ambara. Other
forms in Gemmula need to be analyzed and added to the phylogenetic
tree. Recent evidence to be described elsewhere (M. Astilla and G. Concepcion,
unpublished), suggests that some Gemmula species prey on terebellid
polychaetes. Whether this prey preference is shared by G. ambara remains
to be established.
‚ ‚ ‚ ‚ ‚ Clearly,
the first step to obtain a comprehensive framework for understanding the gem
turrids is to elucidate the taxonomy and phylogenetic relationships within
the group. A number of new species of Gemmula have been described
from the Philippines (Olivera 1999; Olivera 2004). Recently, a molecular characterization
of venom components of some of the Turrinae has been initiated (Heralde et
al. 2008; Watkins et al. 2006) and therefore, it is now possible to systematically
investigate pharmacologically-active compounds in this group of species, including
Gemmula ambara. However, the taxonomic and phylogenetic definition
of the undescribed turrine species from the Philippines clearly needs to be
addressed in a timely manner, to keep pace with the molecular characterization
of their venom components.
