SYSTEMATIC POSITION AND PHYLOGENETIC

RELATIONSHIPS OF THE CYCLOPHYLLIDEAN CESTODES

An In-silico Study using ITS2 rDNA and Sequence-structure Alignment

Veena Tandon, Devendra Kumar Biswal

Department of Zoology, North-Eastern Hill University,Shillong, Meghalaya, India and Bioinformatics Centre

North-Eastern Hill University, Meghalaya, India

Pramod Kumar Prasad

Department of Zoology, North-Eastern Hill University,Shillong, Meghalaya, India

Chenkual Malsawmtluangi

Department of Zoology, North-Eastern Hill University,Shillong, Meghalaya, India

Keywords: Mesocestoides, Cyclophyllidea Cestodes, ITS2 rDNA, Molecular Morphometrics, Sequence-Structure

alignment.

Abstract: The phylogenetic relationships and systematic position of cyclophyllidean cestodes have always been

controversial and opinions of different authors on the systematic rank and content of this order have varied

greatly. Molecular phylogenetic analysis based on ITS2 rDNA of 16 representatives spanning 6 different

families (Mesocestoididae, Davaineidae, Anoplocephalidae, Taeniidae, Dipylidiidae and Hymenolepididae)

of the Order Cyclophyllidea and one out group from the family Diphyllobothriidae of the Order

Pseudophyllidea confirmed the monophyletic nature of the Order Cyclophyllidea. Further, the results were

validated by bayesian analysis, primary sequence-structure alignment and subsequent molecular

morphometrics analysis. At the major nodes all the trees from various analyses were similar. Mesocestoides

was interestingly accommodated within Cyclophyllidea and served as a sister clade close to the families

Taeniidae, Anoplocephalidae, Hymenolepididae and Dipylidiidae.

1 INTRODUCTION

Cyclophyllidean cestodes are parasites occurring as

adult or larval stage in a wide variety of avian and

mammalian hosts and are cosmopolitan in nature

commonly called tapeworms that live in the

digestive tract of vertebrates as adults and often in

the bodies of various animals as juveniles.

Taxonomists face inconsistent or even contradictory

information when they examine the systematic

relationships between cestodes at higher taxonomic

groupings (Mariaux, 1999). The phylogeny at higher

levels is of little significance as the taxonomy is

primarily based on morphological characters and in

case of cestodes it is often difficult to discern

between secondary loss and convergence of

morphological characters as several authors have

weighed the characters of taxa differently (Khalil et

al., 1994). This applies well to the taxonomic

positions of Mesocestoides, which is quite

complicated by a high degree of non-significant

morphological variation. Cyclophyllidean cestodes

of the Family Mesocestoididae differ from other taxa

in the Order Cyclophyllidea in some important

characteristics. The life cycle of Mesocestoides spp.

requires three hosts and not two; the median ventral

position of the genital atrium and the presence of

bipartite vitelline gland in Mesocestoides spp. appear

to be unique among the Cyclophyllidea (Chandler,

1946).

Although, nowadays, most authors agree that

there are 15 families included in the monophyletic

Order Cyclophyllidea, till date no study has

pinpointed the complete taxonomic linkage of all the

Tandon V., Kumar Biswal D., Kumar Prasad P. and Malsawmtluangi C. (2010).

SYSTEMATIC POSITION AND PHYLOGENETIC RELATIONSHIPS OF THE CYCLOPHYLLIDEAN CESTODES - An In-silico Study using ITS2 rDNA

and Sequence-structure Alignment.

In Proceedings of the First International Conference on Bioinformatics, pages 5-12

DOI: 10.5220/0002690500050012

 SciTePress

15 families persuasively with genetic evidence.

Besides, the taxonomic position of Mesocestoididae

remains unclear as some of the cestodes at the

generic level of the family bear similarity to those of

the Order Pseudophyllidea; one such is the case of

M. lineatus, with a wide range of hosts thus lacking

host specificity, which is atypical of Cyclophyllidea

but resembles the cestodes (viz. Diphyllobothrium

dendriticum) of the Order Pseudophyllidea

(Kamegai et al., 1967).

The phylogeny of cyclophyllidean cestodes has

been reported with aid of 12S rRNA markers of

mitochondrial genomic regions from 21 cestode

species spanning eight families (von Nickisch-

Rosenegk et al., 1999). In the present study, we

address the overall taxonomic resolution of

cyclophyllidean cestodes with a different

phylogenetic marker using a combinatorial approach

of sequence analysis and molecular morphometrics.

The internal transcribed spacer 2 (ITS2), the region

of ribosomal RNA between 5.8S rRNA gene and the

large subunit (28S rRNA) has proven to be

appropriate marker for analysis of microscale

phylogenies of close relatives (Coleman, 2003).

Moreover, the ITS2 sequence data can be subjected

to secondary structure predictions and as the

secondary structure seems to be well conserved, it

can provide clues for higher taxonomic studies

(Schultz et al., 2005). This is quite obvious that

phylogenetic analyses are improvable by inclusion

of molecular morphometrics information in

common sequence analysis (Billoud et al., 2000).

Here, we combine sequence with structural

information and apart from the biological problem,

address the different in-silico practices in vogue for

phylogeny studies using ITS2 r-DNA.

2 MATERIALS AND METHODS

2.1 Sequence Alignment and Molecular

Phylogenetic Analysis

The Cyclophyllidean Cestoda sequences from

several geographical locations spanning six different

families and one from the Pseudophyllidea order

(taken as out group) were retrieved from the NCBI

GenBank databases in the present study. Nucleotide

sequences were aligned and edited using ClustalW

(Thompson et al., 1994). A phylogenetic tree was

constructed using the Neighbor-Joining as well as

Maximum Parsimony methods in MEGA 4.0

(Tamura et al., 2007)). Branch support was given

using 1000 bootstrap replicates. Maximum

parsimony was accomplished with gaps treated as

missing data and all characters coded as “unordered”

and equally weighted.

2.2 Bayesian Phylogenetic Analysis

A Bayesian analysis using MrBayes V 3.12

(Ronquist and Huelsenbeck, 2003) was carried out

for tree construction using a general time reversible

substitution model (GTR) with substitution rates

estimated by MrBayes. Metropolis-Coupled Markov

Chain Monte Carlo (MCMCMC) sampling was

performed with two incrementally heated chains that

were combinatorially run for 20,000 generations.

The convergence of MCMCMC was then monitored

by examining the value of the marginal likelihood

through generations. Coalescence of substitution rate

and rate model parameters were also examined.

Average standard deviation of split frequencies was

checked and the generations were kept on adding

until the standard deviation value was below 0.01.

The values slightly differed because of stochastic

effects. The sample of substitution model parameters

and samples of trees and branch lengths were

summarized by the “sump burnin” and “sumt

burnin” commands, respectively. The values in the

following commands were adjusted as per the 25%

of our samples. A cladogram with the posterior

probabilities for each split and a phylogram with

mean branch lengths were generated and

subsequently read by the tree drawing program Tree

view V1.6.6 (Page, 1996).

2.3 ITS2 Secondary Structure

Prediction and Sequence Structure

Alignment

ITS2 secondary structures of the cestodes were

folded with the help of MFold (Zuker, 2003) by

screening for thermodynamically optimal and

suboptimal secondary structures (default settings,

with T=25

C). The secondary structures in Vienna

(dot-bracket-dot) format was used as an input for

MARNA (Siebert and Backofen, 2005) to calculate

sequence-structure multiple alignment. However,

there was a limitation with the online server that the

maximum length of one RNA sequence is restricted

to 500 bases; hence some of the ITS sequences

whose exact boundary information was available

from GenBank graphics view was trimmed for

facilitating MARNA to run. Some more cestode

sequences were also taken whose 5.8S, 28S and

ITS2 regions were clearly defined so as to include in

the sequence-structure multiple alignment dataset. A

phylogenetic tree was created using ProfDistS

(Wolf. et al., 2008) that takes the multiple aligned

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sequence-structure as an input and a consensus tree

was built using RNA/DNA structure profile

neighbor-joining method with 100 bootstraps.

Besides, the GC content of the ITS 2 regions was

calculated using Oligo Calculator available at

http://www.pitt.edu/~rsup/OligoCalc.html.

3 RESULTS

3.1 Neighbour-Joining (NJ) and

Maximum Parsimony (MP) Trees

GenBank accession numbers of ITS2 sequences for

the cestodes spanning 6 families of the Order

Cyclophyllidea and one from the Order

Pseudophyllidea (as out group) are given in Table 1.

The evolutionary history was inferred using the NJ

method (Saitou and Nei, 1987) and the bootstrap

consensus tree (Fig. 1), inferred from 1000

replicates, depicted an overall robust topology of the

cyclophyllidean cestodes’ phylogeny. Branches

corresponding to partitions reproduced in less than

50% bootstrap replicates are collapsed. The

evolutionary distances were computed using the

Maximum Composite Likelihood method (Tamura

et al., 2004) and are in the units of the number of

base substitutions per site. All positions containing

gaps and missing data were eliminated from the

dataset (complete deletion option). There was a total

of 168 positions in the final dataset.

With MP method (Dayhoff et al., 1965) the most

parsimonious tree drawn had the length=727. The

percentage of replicate trees in which the associated

taxa clustered together in the bootstrap test (1000

replicates) are shown next to the branches (Fig. 2)

Figure 1: Neighbor-Joining Tree.

and the MP tree was obtained using the Close-

Neighbor-Interchange algorithm (Nei and Kumar,

2000) with search level 3 in which initial trees were

obtained with the random addition of sequences.

After deletion of the positions containing the gaps,

there were a total of 168 positions in the final dataset

out of which 139 were parsimony informative.

The phylogenetic analysis using the distance and

character state methods showed very good bootstrap

values (Figs. 1 & 2) and all the six cyclophyllidean

families depicted reliable monophyletic groupings.

Bootstrap values for the six monophyletic groups

ranged from 70-100%.

Figure 2: Maximum Parsimony Tree.

3.2 Bayesian Analysis

Bayesian analysis of the alignment retained the same

topology and supported the branches with good

bootstrap values (Fig. 3), though there were slight

variations in the placing of some species of the

Family Taeniidae (Taenia saginata from China and

Taenia taeniaeformis from India) that were grouped

in another node from the rest of the Taenia species.

Diphyllobothrium latum of the Order

Pseudophyllidea was rooted as an out group.

Figure 3: Bayesian Analysis Phylogeny.

SYSTEMATIC POSITION AND PHYLOGENETIC RELATIONSHIPS OF THE CYCLOPHYLLIDEAN CESTODES -

An In-silico Study using ITS2 rDNA and Sequence-structure Alignment

Table 1: Cyclophyllidean Cestode species used in this study with the respective GenBank accession numbers for the

corresponding ITS 2 sequences. (*) Designated as outgroup.

Species GenBank Accession No. Order & Family

ymenolepis diminuta India_Meghalaya FJ939132.1 Cyclophyllidea: Hymenolepididae

ymenolepis diminuta Japan AB494475.1 Cyclophyllidea: Hymenolepididae

ymenolepis microstoma Japan AB494478.1 Cyclophyllidea: Hymenolepididae

ymenolepis nana Japan AB494477.1 Cyclophyllidea: Hymenolepididae

ipylidium caninum China AM491339.1 Cyclophyllidea: Dipylidiidae

Taenia saginata China AY825540.1 Cyclophyllidea: Taeniidae

chinococcus granulosus China AY389985.1 Cyclophyllidea: Taeniidae

Taenia serialis Canada DQ099571.1 Cyclophyllidea: Taeniidae

Taenia crassiceps California DQ099564.1 Cyclophyllidea: Taeniidae

Taenia taeniaeformis India_Andhra Pradesh EU051352.1 Cyclophyllidea: Taeniidae

Taenia taeniaeformis India_Meghalaya FJ939133.1 Cyclophyllidea: Taeniidae

oniezia expansa Japan AB367793.1 Cyclophyllidea: Anoplocephalidae

noplocephala perfoliata Germany AJ578153.1 Cyclophyllidea: Anoplocephalidae

esocestoides spp. USA AF119697.1 Cyclophyllidea: Mesocestoididae

aillietina beveridgei Australia AY382318.1 Cyclophyllidea: Davaineidae

aillietina dromaius Australia AY382320.1 Cyclophyllidea: Davaineidae

*Diphyllobothrium latum Japan AB302387.1 Pseudophyllidea:Diphyllobothriidae

3.3 Secondary Structure Analysis and

GC Content

3.3.1 ITS2 Secondary Structures

The ITS 2 secondary structures (Figs. 4, 5A-I) were

analyzed for conserved stem and loop. The

Hymenolepis species showed characteristic hallmark

of ITS 2 secondary structure, i.e., four helices were

clearly visible in secondary structures with third one

as the longest. However, the third helix contains a

side branch (Fig. 4). Species of Mesocestoides and

Raillietina also maintained common secondary ITS2

core structure. Taenia and Echinococcus species

showed a lot of variation in the secondary structure

with many extra helices, loops and side branches.

UGGU motif (Fig. 4) in the secondary structure was

present in almost all the species of Hymenolepis

genus and the U-U mismatch motif was completely

absent in them. Secondary structures of species,

belonging to the same genus showed high overall

structural similarity except Taenia species in which

considerable differences were noticed. The grouping

of the families Taeniidae, Mesocestoididae and

Hymenolepididae together in phylogenetic trees

(Figs. 1, 2 & 3) forming a monophyletic group was

supported by ITS2 secondary structure similarity.

3.3.2 GC Content

The GC content in the ITS2 region was calculated

(Table. 2) and it was found that for Taenia species

the GC content varied from 54.6% to 62.6%. For

species of Hymenolepis, it ranged from 43.7% to

54%. Taenia species showed a higher GC content

compared to others. The GC content also somewhat

reflected grouping pattern of the organisms in the

phylogenetic tree. Among many other factors, GC

content is one of the factors related with stability of

the secondary structure.

Table 2: Percentage of GC content in the ITS2 region of

various cyclophyllidean cestodes.

Organism name GC Content

Taenia saginata 62.4%

Taenia serialis 62.6%

Taenia crassiceps 57%

Taenia taeniaeformis 61.6%

Taenia pisiformis 54.6%

Hymenolepis nana 50.5%

Hymenolepis diminuta 43.7%

Raillietina beveridgei 50.5%

Raillietina australis 49.4%

Mesocestoides spp. 58.8%

Anoplocephala perfoliata 46%

Echinococcus granulosus 59%

Figure 4: ITS2 Secondary Structure showing UGGU

motif: Hymenolepis nana.

UGGU

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5A 5B 5C

5D 5E 5F

5G 5H

5I 5J

Figure 5A-J: ITS2 Secondary structures of Cyclophyllideab cestodes.

SYSTEMATIC POSITION AND PHYLOGENETIC RELATIONSHIPS OF THE CYCLOPHYLLIDEAN CESTODES -

An In-silico Study using ITS2 rDNA and Sequence-structure Alignment

3.3.3 Primary Sequence-secondary

Structure Alignment

Apart from the ITS 2 sequences initially used for the

primary sequence analysis, some more sequences

were included for secondary structure alignment

with the primary sequence. Multiple sequence-

structure alignment from MARNA was used in

ProfDistS program to build phylogenetic tree (Fig.

6); though secondary structure information helped to

improve alignment, the proposed phylogeny showed

slight differences. However, the monophyletic

character of different groups was retained with few

exceptions. The Echinococcus species were placed

close to species of Hymenolepis. Taenia saginata

and Taenia crassiceps were placed in the upper

branch of the phylogenetic tree away from the basal

group of Taenia species. These slight differences in

tree topology may be due to specific ITS2 rate

matrix used in the analysis. As most of the studies

related to ITS2 have been carried out pertaining to

plants and fungi, the specific rate matrix developed

largely depends on those data. The ITS2 region of

cestodes may follow different rates of evolution and

thus ITS2 rate matrix specific to cestode may

provide better results. Overall there was

considerable similarity between the ITS analysis and

the consensus of previous phylogenetic

reconstruction using other DNA loci.

Figure 6: Multiple sequence-structure alignment.

4 DISCUSSION

The ITS2 region is a requisite in ribosome

biogenesis (Cote et al., 2001) and its gradual

removal from mature rRNA is driven by its specific

secondary structure. Using the secondary structure

of ITS2 sequences from various cyclophyllidean

cestodes covering six important families in this

study, we aimed to pursue three consecutive queries

concerning their systematic relationships (i) whether

the cyclophyllidean cestodes retain the same

taxonomic positions at higher levels and can be

regarded as monophyletic considering ITS2 as

molecular markers for drawing the phylogeny; (ii)

how does the secondary structure of ITS2 sequences

contribute to the Cyclophyllidea phylogeny using

molecular morphometrics approach, and whether the

latter would corroborate the monophyletic

characteristics at the family level?

The order Cyclophyllidea has 15 families. Of

these 6 cestode species belonging to families

Hymenolepididae, Dipylidiidae, Taeniidae,

Anoplocephalidae Mesocestoididae and Davaineidae

were considered for the analysis. Using the ITS2

sequence data of Diphyllobothrium latum that

represents the Order Pseudophyllidea as an out

group, we constructed phylogenetic trees using

distance-based, character-based and Bayesian

methods. Besides, molecular morphometrics

approach was employed taking sequence-structure

alignment into consideration. Our study shows that

all the taxa were clearly monophyletic within their

families and principally correspond to earlier

classifications based upon morphology and biology

(Khalil et al., 1994).

The genus Mesocestoides has a complicated

taxonomy owing to its high degree of nonsignificant

morphological variations; the genital pore is median,

scolex armature and rostellum are lacking, the ovary

and vitellaria both consist of two compact masses,

and a paruterine organ develops at the posterior end

of the tube like uterus. The Order Mesocestoididea is

placed between the Trypanorhyncha and the

Tetrabothridea (Wardle et al., 1974), while the two

known genera (Mesocestoides and Mesogyna) are

raised to family rank, with an uncertain relationship

with Cyclophyllidea (Khalil et al., 1994). The

families Mesocestoididae and Taeniidae share a

common origin as tentatively suggested on the basis

of tegumental hairs of their metacestodes, in contrast

to cysticercoids of other cyclophyllideans, have

series of fibrous layers instead (Brooks et al., 1991).

Moreover, the entire lifecycle of Mesocestoides is

quite aberrant and the number of intermediate hosts

remains enigmatic. Our dendrograms, predicted

through several in-silico approaches, demonstrate

that Mesocestoides spp from USA are closer to the

families Anoplocephalidae and Taeniidae. Due to

the lack of ITS 2 sequences of other Mesocestoidae

genera in the public domain, we could not build a

better dataset for accurate resolution of the family

with high precision. Nevertheless our data supported

Khalil et al’s (1994) arrangement of Mesocestoides

into Cyclophyllidea and a narrow relationship

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between Mesocestoididae, Taeniidae,

Hymenolepididae and Anoplocephalidae.

Taeniids are the best-known cestodes. The

various phylogenetic methods applied to Taenia and

Echinococcus corroborates the monophyletic

grouping of the family Taeniidae. The present

analysis agrees to the monophyly of other families

under Cyclophyllidea; further analysis can be done

once more and more molecular markers are

deposited in public gene bank databases.

5 CONCLUSIONS

Molecular morphometrics approach that uses

combined features both from anatomical and

quantitative morphometrics and molecular primary

sequence comparison was the basis of our study. The

approach differentiates significant features between

anatomical and molecular characters that make

molecular morphometrics a strong predictive tool for

phylogenetic resolution. There is always more than

one gene involved in anatomical variations and most

importantly the genetic sites responsible for

morphological characters are usually not known. On

the contrary, molecular structural variations are

because of identifiable mutations that can be

characterized at the single mutational level. The

observed anatomical characters are the outcome of

both the genetic characters as well as epigenetic

effects (environmental influences) whereas the

molecular morphometrics method takes advantage of

the fact that molecular characters are independent of

their somatic expression (Smith, 1992).

The analysis corroborated strong results for

phylogenetic relationships of cyclophyllidean

cestodes and this was so because of using ITS2 data

as phylogenetic molecular markers and the inclusion

of secondary structure information that offers a

resolution power for relationships from the level of

sub species up to the order level.

ACKNOWLEDGEMENTS

This study was carried out under the DIT sponsored

project, “Northeast Parasite Information and

Analysis Centre” sanctioned to VT by the Ministry

of Communication & Information Technology

(Government of India) and Bioinformatics Centre

NEHU with partial support from UGC-supported

UPE-Bioscioences Programme in the School of Life

Sciences at NEHU.

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