Study of morphology has been used to infer evolutionary relatedness among species. Sometimes species living in similar environmental conditions independently evolve similar morphological features. The use of molecular sequencing can be a fine tool to detect relatedness and to distinguish morphologically very similar species. Differences in the rate of evolution and separation of gene fragments can be used to construct phylogenetic trees. Phylogenetic trees can be constructed using significant DNA or protein sequences and applying various methods to analyse them. Genetic distances are calculated from multiple sequence alignments but do not always invoke an evolutionary model. Multiple sequence alignment computer programs such as Clustal W use simple algorhythms such as those based on distance. Some methods estimate phylogenies by incorporating evolution models. For example, maximum parsimony selects the tree that requires the least evolutionary change to explain the data and other methods incorporate the optimum criterion of maximum likelihood and apply this to a particular evolutionary model. Although phylogenetic trees can be valuable in suggesting evolutionary lines, they do have limitations and care must be taken in their interpretation and in the selection of data used to create them.
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Crustaceans are a large subphylum of the Arthropods and are characterised by having two-part limbs and the nauplius form of their initial larval stage. Like other arthropods they have an exoskeleton, which they need to moult in order to grow.
The crustacean hyperglycemic hormone (CHH) family polypeptides are the most abundant hormones produced by the X organ sinus gland complex in the crustacean eyestalk. CHH are neurohormones involved in the regulating endogenous blood glucose (Santos and Keller, 1993) and are particularly important for energy metabolism during environmental and physiological stress (Chung, 2009). They are used in other physiological processes including moulting (Chung et al., 1999) reproduction (Khayat et al., 1998) osmoregulation (Serrano et al., 2003) and lipid metabolism (Santos et al. 1997). Duplication, insertion or deletion events have shaped the evolution of the CHH family giving rise to neuropeptides with a wide variety of functions ( Chen et al. 2005, Montagne et al. 2010). This and the structural conservation of these peptides make them a suitable homone for phylogenetic investigation.
MOIH, GIH, VIH:
Moult-inhibiting hormone (MOIH), gonad-inhibiting hormone,(GIH) mandibular organ-inhibiting hormone (MOIH ), vitellogenesis- inhibiting hormone (VIH) are all neuropeptides produced in the eyestalk X-organ sinus gland complex. The amino acid chain MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in moult suppression. GIH is involved in gonad maturation and plays a complex role in the control of reproduction and moulting In female lobsters it inhibits vitellogenesis. Structural similarities among these neuropeptides in the CHH/MIH/GIH gene family in crustaceans (Keller, 1992, Lacombe et al.,1999, Chan et al, 2005) suggest that these neuropeptides probably diverged from a common ancestor. Several isoforms of this gene family are expressed in different tissue (Chan et al., 2003, Fanjul-Moles, 2006). Gene duplication followed by functional divergence of duplicated genes is one of the most important underlying mechanisms for the evolution of new gene functions (Ohno, 1970, Zhang, 2003) Positive natural selection is the likely cause of amino acid substitutions among duplicated genes. (Padhi et al 2006) CHH, MOIH, GIH and VIH are therefore useful for constructing phylogenetic trees.
Crustaceans can make rapid reversible colour changes and movements of the eye pigment. The colour changes are produced by concentration or dispersal of pigment within epithelial chromatophores. The eye pigment movements, associated with adaption to light or dark, may be caused by photoreceptor cells within retinular cells or may also involve extraretinular ommatidial pigment cells. The extra-retinular ommatidial pigment cells and also the epithelial chromatophores are controlled by neurosecretory hormones,the pigment-dispersing hormone, (PDH) and the pigment-dispersing factor (PDF) ( Rao, 2001).
The family of pigment-dispersing hormones (PDH) are a group of related neuropeptides common to arthropods. In this peptide family, at least 50% of the sequence is conserved and amino acid chain length (18 residues) and termini (N-terminal Asn, C-terminal Ala-NH2) (Kleinholtz et al., 1986).The primary structures are known for the major form of PDH in several crustacean species. The primary structures of related pigment-dispersing factors (PDF) are also known from some insects The hormones of this family cross-react, but structure-function studies of the hormones demonstrate that different parts of their structures are involved in bonding to the various receptors (Joseffson, 2009). This makes them useful for constructing phylogenetic trees.
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The Phylogeny.fr platform was used to analyse the data. Sequences alignment was performed using ClustalW (v2.0.3) (Thompson et al 1994). After alignment,G blocks (version 0.91b) was used to remove ambiguous parts of the sequence that contained gaps or were poorly aligned. This used the following parameters: minimum length of a block after removal of gaps is10; no gaps were allowed in the final alignment; all segments with contiguous nonconserved positions bigger than 8 were rejected and the minimum number of sequences for a flank position is 85% (Castresana, 2000).
The phylogenetic tree was reconstructed using the maximum likelihood method (Anisimova and Gascuel, 2006, Guindon and Gascuel, 2003) implemented in the PhyML program (v 3.0 aLRT). The default substitution model was used assuming an estimated proportion of invariant sites ( 0.117) and 4 gamma-distributed rate categories to account for heterogeneity across sites (Dreepner et al. 2008). The gamma shape parameter was estimated directly from the data (gamma=1.082). Reliability for internal branches were assessed using the aLRT test (SH-Like).
Graphical representation and edition of the phylogenetic tree were performed with TreeDyn (v 198.3) (Chevenet, 2006). Multi sequence alignments show the sequences used to create the phylogenetic trees and illustrates the differences and similarities between sequences.
Phylogenetic trees can be considered a general estimate of genetic relationships. In these trees (Fig 1 CHH tree, Fig 2 PDH, PDF tree and Fig 3 MIH,GIH, VIH tree) species are grouped based on relatedness of these hormones. This generally appears to agree with taxanomic grouping.
The CHH tree (Fig.1) encompasses the most species. These species are all Decapods of the Malacostraca class except for Armadillium vulgare, the pill woodlouse which is an Isopod of the Malacostraca class. The fresh water crayfish species are all closely grouped at the bottom of the tree. Crab species are grouped, lobsters are grouped and so are salt water shrimps and prawns which are separated from the fresh water Macrobranchium species.
The PDHF tree (Fig 2) groups species of the Malacostraca and Insect classes. Within the Malacostraca group, the Decapod shrimp and prawn specis are grouped, the Isopod species Pill Woodlouse Armadillium vulgare and the Speckled Sealouse Eurydice puchra share the same branch and the Decapod crab and crayfish species are grouped with the fresh water crayfish Cambarellas montezumae and Oronectes limosus sharing the same branch. Four Insect species are grouped at the top of the tree. The Diptera species Musca domestica and Anophelos gambiae show a greater degree of separation than the grasshopper, Romalea microptera and the cricket Gryllus bimaculatus which share the share the same branch.The other two insect species in this tree are widely separated from these and plot at the bottom of the tree. These Diptera species, the black blowfly, Phornia regina and the fruitfly, Drusophila melangaster share the same branch but have a significant morphological separation.
The MIH/GIH/VIH tree (Fig 3) orders Decapod species of the Malacostraca class and one Isopod,
Armadillium vulgare, the pill woodlouse which is separated from the other species and loosely grouped with the spider crab Libinia emarginata. The freshwater crayfish Oronectes limosus and Procambarus clarkii share a branch and are also separated from the other species.The rest of the species on the tree comprise crab species which are grouped together, one lobster species and shrimps and prawns which are also grouped together.
One of the problems in interpreting phylogenetic trees is that the data on which they are based may n exhibit random natural fluctuations which are not neccessarily evolutionary trends. Moreover, convergent evolution and conserved sequences can complicate the evolutionary interpretation. Also analyses based on a single hormone or a limited combination of hormones, can produce spurious results because trees constructed from other unrelated data sources can differ considerably. For this reason, inferrences about phylogenetic relationships among species must be made cautiously particularly where genetic material may be involved in lateral gene transfer and recombination or where separate haplotype blocks have different histories.