Fish display a staggering variation of prey capturing strategies, from suction feeding to biting
and crushing of food (Westneat 2006). Different feeding strategies may place contrasting biomechanical and functional demands on the feeding apparatus (Bellwood et al. 2006). For example, a stronger bite force may trade-off with the maximal extend of the mouth's gape. Therefore, similar traits are expected to re-evolve in trophically-similar species, and the morphology is expected to reflect the trophic guild of the fish. These trophic groups vary from herbivores grazing on sessile algae, planktivores feeding from the open water column, to piscivores pursuing active and mobile prey (Price et al. 2011).
Much of the diversity in the form and structure of the viscerocranium that is a prominent characteristic in adult fish is not observed in their larvae at the earliest stages of ontogenetic development. Majority of tropical reef fishes experience a critical phase of metamorphosis in their life cycle when transforming from larvae into adult form. The metamorphosis coincides with a transition in habitat, from living in the plankton-rich pelagial, to recruitment to the coral reef community and settlement in the benthic or demersal zones where the adults of the species dwell (McCormick et al. 2002; Leis 2007; Dufour et al. 2012). During this phase the larvae undergo many morphological changes, including a massive ossification and allometric growth of the feeding apparatus, the creation of the vertebral column from the cartilaginous notochord and major alterations in appearance (McCormick et al. 2002). Additionally, the metamorphosis is accompanied by a transition in feeding strategies, from the ubiquitous feeding on plankton particles to the diverse feeding strategies of adults (Anto & Turingan 2010).
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I have chosen the marine paraphyletic family of Labridae (Wrasse) for my research as they are morphologically and ecologically diverse (Westneat & Alfaro 2005). In this monophyletic family, a wide range of diets and modes of feeding is observed- from grazing on algae and sea grass, scraping coral mucus, to crushing and picking on various hard shelled Arthropods, Gastropods and Polychaetes worms (Randall 1967). While gross morphology has been recorded in the larvae of this family (e.g., Kimura et al. 1998) there hasn't been a description of internal structures and its relation to functional morphology in an ecological context.
A set of morphological measurement of feeding-related traits will be taken for adults and
several stages of larvae are required for inter- and intra-species comparison. These parameters will be overlaid on two dimensional morphospace by using principle component analysis (PCA). In this analysis, clusters of points represent groups of individuals with similar attributes (see appended).
Using this dataset, I will address these following questions in my thesis:
Does the degree of morphological disparity within coral reef Labrids change throughout their ontogeny? Specifically, is there a lower level of disparity between larvae of different species than between adults? Does functional clustering of feeding-related traits appear in larvae belonging to a convergent trophic group on the morphospace? And, are larvae from subsequent life stages of the same species more similar to one another than expected in
Methods and Materials
In my research I will focus on three major life stages for intraspecies comparison for every
species: planktonic larvae, recruiting larvae and adults. The intended sample size is twenty five to thirty five species, with five individuals per every larval life stage, and two individuals for the adult stage. Measurements of performance-determining morphologies will as specified in Price et al. (2011). These include gross morphological characters such as total length and snout angle, as well as more detailed measurements of the viscerocranium, such as hyoid and maxilla length.
Adult collection, identification and measurement
Identified adults will be acquired from existing preserved specimen in the Holzman lab at the IUI Eilat. Adults will be cleared and stained in double-staining protocol for whole mount specimen (Noback & Noback 1944). The clear and stain procedure results in a complete transparency of the tissues, leaving only a stain in cartilaginous parts and ossified bones. Subsequently, a set of morphological measurements shall be taken.
Larvae collection, identification and measurement
Fish larvae will be collected during the autumn of 2014 with two complementary collection methods preformed in the northern Gulf of Aqaba. For collecting pre-settlement larvae I will
Always on Time
Marked to Standard
use MOCNESS (Multiple Opening Closing Nets and Environmental Sampling System) tows.
Recruitment stages larvae will be caught as they enter the reef in light traps deployed in the IUI reef over shallow bottom depths (20-50 m), at a depth of ~1m below the surface (Hickford & Schiel 1999). All larvae will be identified to the family level based on external morphology, and all labridae larvae will be stored separately in individually-tagged vials. The larvae will be photographed for whole body morphological measurements. The posterior half of the larvae will be removed and sent to DNA barcoding for species-level identification (Herbert et al. 2003, Hajibabaei et al. 2005, Ward et al. 2005, Hubert et al. 2010, Steinke & Hanner, 2011). The anterior part of the larvae will be cleared and stained in with modified protocol for small organisms (Gavaia et al. 2000, Walker & Kimmel 2006). More
measurements shall be taken. Finally, the larvae will be stored for future reference.
Statistical analysis & PCA for morpho-space
I will use phylogenetic principle component analysis (PCA) in order to reduce the multiple dimensionalities of the continuous and categorical variables (the size corrected measurement parameters). Only the first few axes explaining >70% of the variance will be used. From these axes, trajectories for the three life stages for every species will be extracted. The mean morphological distances between life-stages will be calculated for intra-species disparity, and between contemporary age groups for inter-species disparity. I will examine if clustering appears according to trophic groups in adults and whether this applies to larval stages as well. As I have only recently started my graduate work (Apr. 2014), much of the further statistical analysis and methods for calculating evolutionary rates remains to be decided and adjusted according to the data structure.
April - July 2014: Testing the clear and stain protocol on Sparus aurata larvae for needed modifications and adjustments. Testing photographing on pre- and post-staining individuals. August - October 2014: Larvae collection, pre-stain photographing, total sample measurements and identification through DNA barcoding.
November 2014 - May 2015: Clearing and staining the larvae, post-stain photographing and feeding-apparatus measurements. Clearing and staining adults, execution of all needed measurements.
May - July 2015: Time reserved for repeating clearing, staining and measuring of problematic individuals or additional individuals for a larger sample size. August - February 2015: Data analysis, Thesis writing.
(A) (B) (C)
Representation of performance-determining morphological measurements mapped on morphospace. Each number represents a species from a unique trophic group, and the three stages presented are planktonic stages larvae (square, filled-in backgrounds), recruitment stages larvae (square, open backgrounds), and Adults (circle, open backgrounds).
Some possible outcomes of the PCA analysis
(A) Random pattern. All life stages are not in a closer proximity to one another than
expected in random and no clustering appears in any stage.
(B) No functional clustering of feeding-related traits appears in larvae or adults. All life
stages are in a closer proximity to one another than expected in random. Additionally, all life stages are in a directional trajectory according to ontogenesis, every species occupies a distinct space within the morphospace and no trophic mixing occurs.
(C) Functional clustering is present in larvae; all larvae exhibit lesser disparity than
subsequent life stages of the same species.