Molecular Phylogeny of Horseshoe Crab
B. Akbar John,
An effort to assess the utility of 650 bp Cytochrome C oxidase subunit I (DNA barcode) gene in delineating the members horseshoe crabs (Family: xiphosura) with closely related sister taxa was made. A total of 33 sequences were extracted from National Center for Biotechnological Information (NCBI) which include horseshoe crabs (Class: Merostomata), beetles (Class: Insecta), common crabs (Class: Melacostraca) and scorpion (Class: Arachnida) sequences. Constructed phylogram through comprehensive dry lab methodology using advanced software predictive tools showed that beetles are closely related with horseshoe crabs than common crabs (Class: Melacostraca). It was interesting to note that terrestrial Scorpion (Class: Arachnida) were distantly related to horseshoe crabs (Class: Merostomata). Phylogram and observed Genetic Distance (GD) data were also revealed that Limulus polyphemus was distantly related to all the other horseshoe crab species. Tachypleus gigas was closely related with Carcinoscorpius rotundicauda than with Tachypleus tridentatus. The observed mean Genetic Distance (GD) value was higher in 3rd codon position in all the selected group of organisms. Among the horseshoe crabs high GC content was observed in L. polyphemus (38.32%) and lowest was observed in T. tridentatus (32.35%). We conclude that COI sequencing (barcoding) could be used in identifying and delineating evolutionary relatedness with closely related species.
Received: October 05, 2010;
Accepted: November 19, 2010;
Published: February 07, 2011
Mitochondrial DNA (mtDNA) analysis has been employed in the evolutionary study
of the animal species for more than 30 years (Brown et
al., 1979; Avise and Walker, 1999). Its higher
mutational rate and lower effective population size than the nuclear DNA make
mtDNA a powerful tool to probe for evolutionary studies. This fact provoked
a proposal to standardize DNA-based species identification by analyzing a uniform
segment of the mitochondrial genome. A library of sequences from taxonomically
verified voucher specimens could be built with this approach which could serve
as DNA identifiers for species, in short, DNA barcodes (Herbert
et al., 2003). For animals, 648 bp segment of the mitochondrial gene
cytochrome C oxidase I (COI), which can be readily recovered from diverse species
with a limited set of primers, was declared as a DNA barcode (Kevin
et al., 2007). For this DNA barcoding approach to be effective, it
must be possible to distinguish between intraspecific and interspecific mtDNA
variation. The simplest test is whether the genetic distance within the species
is lesser than those between species.
There are four extant species of horseshoe crabs, Tachypleus tridentatus,
Tachypleus gigas, Carcinoscorpius rotundicauda and Limulus polyphemus
(Pocock, 1902). The first three species inhabit
the Southeast Asian coast and the last species the East Coast of North America.
These are known as living fossils, have maintained their morphology almost
unchanged for the past 150 million years. The little morphological differentiation
among horseshoe crab lineages has resulted in substantial controversy concerning
the phylogenetic relationship among the extant species of horseshoe crabs, especially
among the three species in the Indo-Pacific region. Earlier studies suggest
that the three species constitute a phylogenetically irresolvable trichotomy
(Xia, 2000). For elucidating their phylogenetic relationships,
two proteins, coagulogen and hemocyanin, have been investigated (Shishikura
et al., 1982; Srimal et al., 1985;
Sugita, 1986). Miyazaki et al.
(1989) first investigated tropomyosin which is one of the major structural
proteins involved in many types of cells, to elucidate prevailing phylogenetic
relationships among horseshoe crabs and his result suggested that L. polyphemus
is phylogenetically differentiated far from the three Asian species. But
the results were not those which were worth studying from the detailed phylogenetic
viewpoint, that is, the patterns of the four species were equally different
from each other. When the proteins were cleaved with chymotrypsin or trypsin,
only smaller differences than those obtained with V8 protease were found (Miyazaki
et al., 1989).
What remains unresolved is the phylogenetic relationship among the three Indo-Pacific
species. T. gigas and T. tridentatus were grouped together on
the basis of morphological traits (Fisher, 1984), but
C. rotundicauda and T. tridentatus appear to be more closely
related on the basis of amino acid sequence divergence of a fibrinopeptide like
protein (Shishikura et al., 1982) and coagulogen
(Srimal et al., 1985) and on interspecific hybridization
studies (Sekiguchi and Sugita, 1980). In a phylogenetic
study of the four species of horseshoe crabs employing two-dimensional electrophoresis
of skeletal and cardiac muscles, the similarity index is the greatest between
T. gigas and C. rotundicauda for cardiac muscles, but between
T. tridentatus and T. gigas for skeletal muscles (Miyazaki
et al., 1987). Phylogenetic analyses to find out the genetic relatedness
among the four horseshoe crab species based on two partial mitochondrial genes,
(16S ribosomal RNA (rRNA) and cytochrome oxidase subunit I (COI)) was also carried
out (Xia, 2000). But comprehensive study on genetic
relatedness among horseshoe crabs and its related sister taxa is scarce.
As, COI has been proposed as a barcode gene for most of the eukaryotes (Herbert
et al., 2003). To enhance our understanding on the phylogeny of horseshoe
crab and its genetic relatedness with other sister taxa, present study was conducted
to find the phylogenetic cues in mitochondrial COI region and to find out evolutionary
relationship of horseshoe crabs with closely related taxa.
MATERIALS AND METHODS
Sequence features: A total of 33 barcode sequences belong to horseshoe
crab (12), insects including beetle (8), common crab (5) and scorpion (8) were
extracted from National Center for Biotechnological Information (NCBI) via FASTA
format during March 2010. Artemia franciscana (NC_001620.1) was shuffled
among the sequences was clearly occupied separate branch in the phylogram as
an out-group. Pair wise sequence alignment of nucleotide sequences were performed
using ClustalX 2.0.6 (Thomson, 1997). The GC content
of all 33 barcodes was estimated by BioEdit sequence alignment editor (Hall
et al., 1999). MEGA 4.1 Beta3 (Tamura et al.,
2007) was used to construct phylogenetic trees via Neighborhood joining
method using Kimura 2-parameter and to calculate genetic distance of the given
set of sequences in each codon position.
||Total number of organisms used to construct the phylogram
and their respective accession ID with total sequence length (bp) is given.
Percentage of GC content for each organism was calculated using Bioedit
sequence alignment editor V.220.127.116.11 (Hall et al.,
Details of sequences used to generate the phylogram are given in Table
Phylogenetic analysis: A phylogenetic tree was constructed to verify
the efficiency of coxI gene in delineating closely related species of horseshoe
crab and to check its evolutionary relationship with other groups of organisms
which were proved to be closest relatives of xiphosurans. The constructed phylogram
showed two distinct Clads (A and B). Artemia franciscana (NC_001620.1),
used as an out group was clearly branched separately, signified the reliability
of constructed phylogram. Almost all the internal branches showed high bootstrap
Clad A includes horseshoe crabs, insects (beetles) and common crab species and all the scorpion species were distinctly clustered in clad B. As it was expected beetles (Class: Insecta) used in this study was perfectly arranged in clad A showing higher genetic similarity (lesser genetic distance) with horseshoe crab and common crabs were showing higher genetic distance with xiphosurans. Terrestrial scorpion species used in this analysis were clumped together in Clad B indicating their distant genetic relatedness with Horseshoe crab species. The phylogram also showed that L. polyphemus was genetically distinct from 3 other species of horseshoe crabs. Among the three Asian horseshoe crab species T. gigas showed closer genetic relatedness with C. rotundicauda than with T. tridentatus hence they occupied same internal nod in the phylogram.
Among the horseshoe crabs L. plyphemus had comparatively higher GC content than the other species of horseshoe crabs. Average GC content in L. polyphemus was 37.84% followed by C. rotundicauda 35.82%. T. gigas had higher average GC content (33.85%) than T. tridentatus (32.69%) but lower than the other two species.
Mean genetic distance within L. polyphemus at 1st, 2nd and 3rd codon position were 0.01, 0.009 and 0.025, respectively. Likewise for T. tridentatus it was 0.014, 0 and 0.005 at 1st, 2nd and 3rd codon position, respectively. In case of C. rotundicauda the mean genetic distance values were 0.014, 0.014 and 0.05 at 1st, 2nd and 3rd codon position, respectively. Among the Asian horseshoe crab species T. gigas showed closer genetic relatedness (lower GD value) with C. rotundicauda with the value of 0.043, 0.021 and 0.144 at 1st, 2nd and 3rd codon position, respectively than with T. tridentatus with the values of 0.084, 0.028 and 0.242, respectively at 1st, 2nd and 3rd codon position. Calculated genetic distance data showed higher genetic distance in third codon position than its corresponding first and second codon positions (Table 2).
Mean GD value within the four species of horseshoe crabs was 0.095, 0.033 and 0.301 at 1st, 2nd and 3rd codon position respectively. The GD value between Insects (beetle) and horseshoe crabs were 0.201, 0.094 and 0.468 at 1st, 2nd and 3rd codon position respectively proved that horseshoe crabs are genetically more closer to insects followed by common crabs (GD values: 1st = 0.256; 2nd = 0.108 and 3rd = 0.527). Terrestrial scorpion species were showing higher mean GD value with horseshoe crabs (1st = 0.445; 2nd = 0.174 and 3rd = 1.016) clearly demarcated their distant genetic relatedness (Table 3). Figure 1 shows the results.
||Average genetic distance (GD) between four available species
of horseshoe crabs observed in different codon positions
||The evolutionary history was inferred using the Neighbor-Joining
method (Saitou and Nei, 1987). The optimal tree with
the sum of branch length = 1.67404499 is shown in figure. The percentage
of replicate trees in which the associated taxa clustered together in the
bootstrap test (500 replicates) is shown next to the branches (Felsenstein,
1985). The tree is drawn to scale, with branch lengths in the same units
as those of the evolutionary distances used to infer the phylogenetic tree.
The evolutionary distances were computed using the Kimura 2-parameter method
(Kimura, 1980) and are in the units of the number
of base substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding.
All positions containing gaps and missing data were eliminated from the
dataset (Complete deletion option). There were a total of 434 positions
in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura
et al., 2007)
||Mean genetic distance (GD) values of different groups of organisms
with reference to horseshoe crab at all the possible codon position indicating
3rd codon position shows higher GD value
The constructed phylogram clearly indicated that horseshoe crabs are distantly
related with scorpions at DNA level. Among the horseshoe crabs L. polyphemus
showed distant genetic relatedness with other 3 species of Asian horseshoe crabs.
This result was also well corresponded with previous finding by Miyazaki
et al. (1987) where in two dimensional electrophoresis techniques
clearly demarcated L. polyphemus from the three other species. T.
gigas showed closer genetic relationship with C. rotundicauda than
with T. tridentatus. This observation was also well coincide with the
previous findings (Xia, 2000) where partial mitochondrial
DNA sequence analysis revealed grater similarity index between T. gigas
and C. rotundicauda than with T. tridentatus.
Insects were showing higher genetic similarity (lesser genetic distance) with
xiphosurans (Horseshoe crabs) than the common crabs indicating that horseshoe
crab might probably evolved from the ancient aquatic insects. Eurypterids (e.g.,
sea scorpions) have traditionally been regarded as close relatives of horseshoe
Crabs; together forming a group called Merostomata. Subsequent studies placed
eurypterids closer to the arachnids (e.g., spiders, terrestrial scorpions, mites
and ticks) in a group called Metastomata (Pavlicek et
al., 2008). There has also been a prevailing idea that eurypterids are
closely related to terrestrial scorpions (Raz et al.,
2009). The most recent study of relationships between arachnids and their
relatives recognized Eurypterida, Xiphosura and Arachnida as three major groups,
but was not able to resolve details between them (Shultz,
2007). Same result was reflected in the present study by separating scorpion
species in a clad B indicating that horseshoe crabs have lesser genetic relatedness
with terrestrial scorpions (Fig. 1).
Another interesting observation made from the calculated genetic distance data
was higher genetic distance was observed in third codon position than its corresponding
first and second codon positions. Similar observation was made by Ward
et al. (2005) while barcoding of fishes from Australian waters. Simmons
et al. (2006) also observed that greater phylogenetic signal is often
found in parsimony-based analyses of third codon positions of protein-coding
genes relative to their corresponding first and second codon positions, even
for early-derived basal clades (Khan et al., 2010;
Siemion and Przemyslaw, 1994). Average genetic distance
among the different groups of test organisms used in this study showed higher
GD value at 3rd codon position indicating that detailed study on
3rd codon position might reveal possible evolutionary information
among the closely related groups of organisms.
The COI sequence in the phylogram constructed clearly clustered the selected
species in individual group, proving the efficacy of COI gene in delineating
the members of evolutionarily cryptic groups of organisms. Constructed phylogram
and genetic distance data clearly proved that horseshoe crabs are more genetically
related to insects (Class: Insecta) than with common crabs and are distantly
related with terrestrial scorpions. But further studies need to be conducted
to prove this concept by analyzing total mitochondrial DNA sequence. It is also
evident from the present study that the greater phylogenetic signal is often
found in third codon position relative to their corresponding first and second
codon positions as reported by Ward et al. (2005)
and Simmons et al. (2006).
This research work was funded by IIUM internal endowment fund (Ref No. EDW B100960435). We wish to express our sincere gratitude to International Islamic University Malaysia (IIUM) and Institute of Oceanography and Maritime Study (INOCEM) for providing infrastructural facilities. One of us extent his thanks to Dr. Shahriza, University Malaysia Terengganu (UMT) for his valuable comments on this present work.
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