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Continuous Osteological Characters in the Reconstruction of PhylogeneticRelationships of the Six Euro-Mediterranean Mullet Species (Mugilidae)Author(s): Ivanka AntovićSource: Zoological Science, 30(9):754-759. 2013.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.30.754URL: http://www.bioone.org/doi/full/10.2108/zsj.30.754

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2013 Zoological Society of JapanZOOLOGICAL SCIENCE 30: 754–759 (2013)

Continuous Osteological Characters in the Reconstruction of

Phylogenetic Relationships of the Six Euro-Mediterranean

Mullet Species (Mugilidae)

Ivanka Antovic*

Department for Biomedical Sciences, State University of Novi Pazar,Vuka Karadzica b.b., 36 300, Novi Pazar, Serbia

Sixty-three continuous osteological characters (18 skull continuous characters and the total length

of neurocranium, 45 continuous characters of 15 elements of the viscerodermal skeleton) were ana-

lyzed and included in the reconstruction of phylogenetic relationships of the six Euro-Mediterranean

mullet species from the South Adriatic Sea: Mugil cephalus Linnaeus, 1758; Liza saliens Risso, 1810;

Liza aurata Risso, 1810; Liza ramada Risso, 1826; Chelon labrosus Risso, 1826 and Oedalechilus labeo Cuvier, 1829. The study reveals that Sphyraenidae was separated clearly from Mugilidae, C. labrosus and three Liza species form a common cluster (L. ramada and L. saliens being the closest),

while O. labeo and M. cephalus cluster together.

Key words: Mugilidae, Adriatic Sea, continuous osteological characters, phylogenetic relationships, phy-

logenetic tree

INTRODUCTION

Various approaches have been adopted in analyzing

relationships among mullet species. Morphology has often

been used (Schultz, 1946; Pillay, 1951; Farrugio, 1977;

Capanna et al., 1984; Drake et al., 1984; Harrison and

Howes, 1991; Turan et al., 2011), as have biochemical,

genetic, and molecular characters (Cataudella et al., 1974;

Autem and Bonhomme, 1980; Rizzotti, 1993; Caldara et al.,

1996; Papasotiropoulos et al., 2001, 2002, 2007; Gornung

et al., 2001, 2004; Rossi et al., 2004; Turan et al., 2005;

Semina et al., 2007).

In recognition of the importance of strong morphology-

based phylogenies (e.g., Wiens, 2004), which can help

understanding of systematic status of species, this study

presents the reconstruction of phylogenetic relationships of

the six mullet species, obtained from continuous osteologi-

cal characters. A traditional method is adopted in the pres-

ent study, but it is important to point out that this could be

problematic in light of the issues discussed in many studies

(e.g., Felsenstein, 1988; Swiderski et al., 1998; Stevens,

2000, Wiens, 2001; Guerrero et al., 2003) and related to dif-

ficulties in converting continuous characters into discrete

states, states overlapping, etc.

A key to Mugilidae species in the Northeastern Atlantic

and Mediterranean with explanatory notes was given by

Trewavas and Ingham (1972), while Tortonese (1972)

reported on the Mediterranean mullets. The mullet species

of Euro-Mediterranean distribution occurring in the South

Adriatic Sea, i.e., Mugil cephalus Linnaeus, 1758, Chelon

labrosus Risso, 1826, Oedalechilus labeo Cuvier, 1829,

Liza aurata Risso, 1810, Liza saliens Risso, 1810 and Liza ramada Risso, 1826, are considered here, with the objective

of presenting continuous osteological characters as informa-

tive phylogenetically, and contributing to the understanding

of systematic status of these species.

Differing analyses and results in regard to phylogenetic

relationships among these mullets have left many questions

unsolved, and make this systematic problem still actual. For

example, the Chelon-Liza relation is an important issue in

mullet systematics. Allozyme data (Papasotiropoulos et al.,

2001) clustered the three Liza species (with L. saliens as

more distinct) and C. labrosus as the second group, sup-

porting the traditional view for Liza genus monophyly, as

well as the phyletic relations of the five mullet species given

by Autem and Bonhomme (1980) – with L. aurata as more

distinct, and L. ramada and L. salines grouped together. The

possible non-monophyly of the Liza genus was suggested

by Harrison and Howes (1991) – from morphological data.

In molecular phylogenies not supporting monophyly of the

Liza genus, the position of C. labrosus (in regards to the

Liza species) was found to be different, but also positions of

the three Liza species (though often poorly supported): L. ramada in the sister group with L. aurata and L. saliens-C. labrosus lineage (Caldara et al., 1996), L. aurata-L. ramadaas one and L. saliens-C. labrosus as the other lineage

(Papasotiropoulos et al., 2002), L. saliens in the sister group

with L. ramada and C. labrosus-L. aurata lineage (Rossi et

al., 2004; Papasotiropoulos et al., 2007; Semina et al.,

2007), etc. So, e.g., Semina et al. (2007) reported Chelonand Liza as paraphyletic, and suggested taxonomic revision

and synonymization of Chelon and Liza genus. In addition,

Durand et al. (2012) concluded that considerably more

research is required to clarify the taxonomy of Mugilidae at

the species level. Therefore, new information and various

* Corresponding author. Tel. : +381-20-317-754;

Fax : +381-20-337-669;

E-mail: Ivanka_Antovic@yahoo.com

doi:10.2108/zsj.30.754

Phylogeny of the Six Mullet Species 755

approaches (and the use of various characters, including

morphological ones) are warranted, and should help resolve

the systematic status of the mullets.

MATERIALS AND METHODS

All specimens (Table 1) were collected in the South Adriatic

Sea, along the Coast of Montenegro (Bar and Tivat mainly, but also

Petrovac and Budva), using a trawl net. The analyzed sample also

contained Sphyraena sphyraena Linnaeus, 1758 (fam. Sphyraeni-

dae) – considered as outgroup taxa.

The measurements were taken by millimeter caliper (precision:

0.1 mm), under a binocular at 4 × magnification. The osteological

complex encompassed 63 characters: 18 skull continuous charac-

ters and the total length of neurocranium, 45 continuous characters

of 15 elements of the viscerodermal skeleton (Fig. 1).

The intraspecies variability was tested using descriptive analy-

sis (DA), with an arithmetic mean and standard deviation – as indi-

Table 1. Number of specimens examined.

Species Number of examined specimens

M. cephalus 16

L. aurata 16

L. saliens 16

L. ramada 24

C. labrosus 16

O. labeo 16

S. sphyraena 16

Fig. 1. (A) Model of continuous characters of skull: C–C' – Smeseth

(neurocranium width at mesethmoid level), D–D' – Sexoeth (neurocra-

nium width at exoethmoid level), E–E' – Sf (neurocranium width at

frontal level), F–F' – Sspo (neurocranium width at sphenotic level),

G–G' – Spto (neurocranium width at pterotic level), H–H' – Sepo (neu-

rocranium width at epiotic level), I–I' – Lpepo (length of posterior epi-

otic process), N–N' – Sopo (neurocranium width at opisthotic level),

J'–D' –Lvexoeth (vomer-exoethmoid distance), D'–F' – Lexospo (exoeth-

moid-sphenotic distance), F'–G' – Lspopto (sphenotic-pterotic dis-

tance); A–B – Lcranium, A–A' – Lv (vomer length), A"–B' – Lp

(parasphenoid length), J–J' – Sv (vomer width), K–K' – Lorbit (longitu-

dinal length of orbit), L–L' – Sbf (neurocranium width at lateral frontal

processes (bases level)), M–M' – Sp (parasphenoid width), B–O –

Hboc (basioccipital height). (B) Model of continuous osteological

characters of viscerodermal skeleton; Lpm –premaxillar length, Hpm –

premaxillar height, lpm – length of the front process of premaxillar,

Lm – maxillar length, l1m – maxillar length at level of the process

which attaches to palatine, l2m – length of the front joint surface of

maxillar which attaches to the head of premaxillar, Lpal – palatine

length, lpal – length of the front joint surface of palatine, Ld – dental

length, l1d – length of shorter process of dental which does not artic-

ulate with angular, l2d – length of tooth-line of dental, La – angular

length, la – length of angular process which is not attached to dental,

Lq – quadrate length, Sq – quadrate width, l1q – length of the quad-

rate process which is not attached to ectopterygoid and mesoptery-

goid, l2q – length of the quadrate cutting, Lhy – hyomandibular length

at level of the process which attaches to pterotic, S1hy – hyomandib-

ular width at level of the process which attaches to pterotic and the

process which attaches to sphenotic, S2hy – hyomandibular width at

level of the process which attaches to symplectic, l1hy – hyomandib-

ular length at level of the process that attaches to pterotic and the

process that attaches to opercle, l2hy – hyomandibular length at level

of the process that attaches to sphenotic and the process that

attaches to symplectic, Lu – urohyal length, Lbs1 – branchiostegal I

length, lbs1 – length of the branchiostegal I cutting, Hop – opercle

height, Lop – opercle length, Lpop – preopercle length, l1pop – length

of ventral preopercular process, l2pop – length of dorsal preopercular

process, Liop – interopercle length, Hiop – interopercle height, hiop –

interopercul height at level joint that attaches to interhyal, Lcl –

cleithrum length, Scl – cleithrum width, l1cl – cleithrum length at level

of the process that attaches to postcleithrum and the process that

attaches to scapula, l2cl – length of cleithrum lateral process in the

direction of the branchiostegals, l3cl – cleithrum length at lateral pro-

cesses bases level, l4cl – cleithrum length at lateral process bases

level and at dorsal process that attaches to supracleithrum, Lpcl –

postcleithrum length, l1pcl – length of process of postcleithrum that

attaches to cleithrum, l2pcl – length of process of postcleithrum which

does not attach to cleithrum, Lpt – posttemporal length, l1pt – post-

temporal length at level of the process which attaches to opisthotic,

l2pt –posttemporal length between the process which attaches to epi-

otic and the process which attaches to opisthotic.

I. Antovic756

cators of characters variability in the

sample. Univariate statistics (ANOVA)

and multivariate analysis (MANOVA)

were used to check statistical signifi-

cance of variability for individual charac-

ters and all characters, respectively

(Sokal and Rohlf, 1981).

Percentages for coding of continu-

ous characters to whole numbers are

calculated in regard to the total length of

skull for each character. The coding of

continuous characters for the phyloge-

netic analysis was carried out using cod-

ing procedures (Thorpe, 1984). The

determination of states and coding are

performed by the help of standardized

discontinuities in the ranges of variation:

with the complete standard deviation

(“gap coding A”) for 25 osteological

characters; with half of the pooled

within-group standard deviation (“gap

coding B”) for 30 osteological charac-

ters. The determination of states and

coding had to be performed by the help

of arbitrary determined segments (“range

coding”) for eight characters (Table 2).

Because of its contribution to estab-

lishing phylogenetic relationships of

mullets, in the sense of the affirmation of

transformation series of characters and

polarity of character states in the frame

of the transformation series (direction of

transformation from plesiomorphic to

apomorphic character state), the “out-

group comparison” was carried out using

European barracuda, S. sphyraena, as

mentioned above.

The reconstruction of phylogeny by

method parsimony was performed using

PAUP v. 4.0b1 (Swofford, 1988). As pro-

posed by Farris (1972), Swofford (1985),

and Abbot et al. (1985), the most parsi-

monious Wagner tree was generated

between OTUs on the Manhattan dis-

tances between their coded character

states.

RESULTS

The results of DA analysis

showed similarity in the high

intraspecies variability of individual

characters (Antovic, 2006), while

interspecies analyses by ANOVA

and MANOVA respectively showed

statistically significant variability of

individual characters and all the

characters (< 0.001).

The phylogenetic analysis was

carried out on the base of osteolog-

ical character states given in Table

2. Of 63 osteological characters

included in the analysis, 33 have

information significant for recon-

struction of mullet phylogenetic

relationships.

Table 2. Number and name of characters, with the method coding applied for each, and the coded character states; HTUs: 12, 11, 10, 9, 8; OTUs: Mc-Mugil cephalus, Ol-Oedalechilus labeo,Lr-Liza ramada, La-Liza aurata, Ls-Liza saliens, Cl-Chelon labrosus, Ss-Sphyraena sphyraena.

No. Name Coding method Character states for HTUs and OTUs

1 Lv Gap coding A 12:1; 11:1; 10:1; 9:1; 8:2; Ls:2; La:1; Lr:2; Ol:1; Mc:1; Cl:1; Ss:3

2 Lp Gap coding B 12:3; 11:3; 10:1; 9:2; 8:2; Ls:3; La:2; Lr:2; Ol:3; Mc:3; Cl:1; Ss:4

3 Smeseth Gap coding A 12:3; 11:3; 10:3; 9:3; 8:3; Ls:2; La:3; Lr:3; Ol:3; Mc:4; Cl:3; Ss:1

4 Sexoeth Gap coding B 12:3; 11:3; 10:3; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

5 Sf Gap coding A 12:3; 11:3; 10:3; 9:2; 8:2; Ls:3; La:2; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

6 Sspo Gap coding B 12:4; 11:4; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:4; Mc:4; Cl:3; Ss:1

7 Spto Gap coding B 12:3; 11:3; 10:3; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

8 Sepo Gap coding A 12:3; 11:3; 10:3; 9:3; 8:2; Ls:2; La:3; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

9 Lpepo Gap coding B 12:1; 11:2; 10:2; 9:2; 8:2; Ls:3; La:3; Lr:3; Ol:1; Mc:4; Cl:3; Ss:2

10 Sv Gap coding A 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:2; Lr:3; Ol:2; Mc:3; Cl:3; Ss:1

11 Lorbit Gap coding A 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:1; Lr:2; Ol:2; Mc:2; Cl:2; Ss:1

12 Sp Gap coding A 12:1; 11:1; 10:1; 9:1; 8:1; Ls:1; La:3; Lr:1; Ol:3; Mc:2; Cl:1; Ss:1

13 Sbf Gap coding A 12:1; 11:2; 10:3; 9:3; 8:3; Ls:3; La:2; Lr:3; Ol:4; Mc:2; Cl:3; Ss:1

14 Sopo Gap coding A 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:2; Cl:2; Ss:1

15 Hboc Gap coding B 12:1; 11:5; 10:5; 9:5; 8:5; Ls:5; La:4; Lr:3; Ol:2; Mc:5; Cl:5; Ss:1

16 Lvexoeth Range coding 12:1; 11:1; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:2; Mc:1; Cl:1; Ss:3

17 Lexospo Range coding 12:3; 11:3; 10:2; 9:2; 8:1; Ls:3; La:2; Lr:1; Ol:4; Mc:3; Cl:2; Ss:3

18 Lspopto Gap coding A 12:1; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:4; Cl:2; Ss:1

19 Lpm Gap coding B 12:1; 11:1; 10:1; 9:1; 8:2; Ls:2; La:1; Lr:2; Ol:4; Mc:3; Cl:1; Ss:5

20 Hpm Range coding 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:1; Cl:2; Ss:3

21 lpm Range coding 12:3; 11:3; 10:3; 9:3; 8:2; Ls:4; La:3; Lr:2; Ol:3; Mc:4; Cl:3; Ss:1

22 Lm Gap coding A 12:2; 11:2; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:2; Mc:2; Cl:1; Ss:3

23 l1m Gap coding A 12:3; 11:1; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:3; Mc:2; Cl:1; Ss:3

24 l2m Gap coding B 12:1; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:2; Cl:2; Ss:1

25 Lpal Gap coding A 12:1; 11:1; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:1; Mc:1; Cl:1; Ss:2

26 lpal Gap coding A 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:1; Mc:2; Cl:2; Ss:2

27 Ld Gap coding B 12:2; 11:2; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:2; Mc:2; Cl:1; Ss:3

28 l1d Gap coding A 12:1; 11:1; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:1; Mc:2; Cl:1; Ss:3

29 l2d Gap coding B 12:1; 11:1; 10:1; 9:1; 8:1; Ls:1; La:1; Lr:1; Ol:2; Mc:3; Cl:1; Ss:4

30 La Gap coding B 12:1; 11:1; 10:1; 9:1; 8:1; Ls:2; La:1; Lr:1; Ol:3; Mc:2; Cl:1; Ss:4

31 la Gap coding B 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:1; Ol:2; Mc:2; Cl:2; Ss:3

32 Lq Gap coding A 12:3; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:3; Mc:2; Cl:3; Ss:1

33 Sq Gap coding B 12:1; 11:5; 10:5; 9:2; 8:2; Ls:4; La:3; Lr:2; Ol:6; Mc:5; Cl:5; Ss:1

34 l1q Gap coding B 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:4; Mc:3; Cl:3; Ss:1

35 l2q Gap coding B 12:1; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:2; Cl:2; Ss:1

36 Lhy Gap coding B 12:4; 11:4; 10:2; 9:2; 8:2; Ls:3; La:2; Lr:2; Ol:4; Mc:4; Cl:3; Ss:1

37 S1hy Gap coding A 12:3; 11:3; 10:3; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

38 S2hy Gap coding A 12:1; 11:3; 10:3; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:3; Cl:3; Ss:1

39 l1hy Gap coding B 12:4; 11:4; 10:3; 9:3; 8:2; Ls:2; La:3; Lr:2; Ol:4; Mc:4; Cl:3; Ss:1

40 l2hy Gap coding B 12:1; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:2; Cl:2; Ss:1

41 Lu Range coding 12:4; 11:4; 10:1; 9:1; 8:1; Ls:1; La:2; Lr:3; Ol:2; Mc:4; Cl:1; Ss:4

42 Lbs1 Gap coding B 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:3; Cl:2; Ss:1

43 lbs1 Range coding 12:4; 11:4; 10:3; 9:3; 8:3; Ls:2; La:3; Lr:3; Ol:4; Mc:4; Cl:3; Ss:1

44 Hop Gap coding B 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:5; Mc:4; Cl:3; Ss:1

45 Lop Gap coding B 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:3; Cl:2; Ss:1

46 Lpop Gap coding B 12:1; 11:1; 10:1; 9:2; 8:1; Ls:2; La:1; Lr:1; Ol:1; Mc:3; Cl:2; Ss:1

47 l1pop Gap coding B 12:3; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

48 l2pop Gap coding B 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:4; Mc:5; Cl:3; Ss:1

49 Liop Range coding 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:1; Ol:1; Mc:2; Cl:3; Ss:2

50 Hiop Gap coding B 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:3; Ol:2; Mc:4; Cl:2; Ss:1

51 hiop Gap coding B 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:3; Ol:2; Mc:3; Cl:3; Ss:1

52 Lcl Gap coding B 12:3; 11:3; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:3; Cl:2; Ss:1

53 Scl Gap coding A 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:2; Lr:1; Ol:2; Mc:3; Cl:3; Ss:1

54 l1cl Gap coding A 12:1; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:4; Mc:3; Cl:3; Ss:1

55 l2cl Gap coding B 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:3; Lr:2; Ol:2; Mc:3; Cl:2; Ss:1

56 l3cl Gap coding B 12:1; 11:2; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:4; Mc:2; Cl:3; Ss:1

57 l4cl Gap coding A 12:1; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:4; Cl:2; Ss:1

58 Lpcl Gap coding B 12:4; 11:4; 10:4; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:4; Mc:4; Cl:4; Ss:1

59 l1pcl Gap coding A 12:3; 11:3; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:3; Mc:3; Cl:2; Ss:1

60 l2pcl Range coding 12:3; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:2; Ol:3; Mc:3; Cl:3; Ss:1

61 Lpt Gap coding A 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:2; Cl:2; Ss:1

62 l1pt Gap coding A 12:2; 11:2; 10:2; 9:2; 8:2; Ls:2; La:2; Lr:2; Ol:2; Mc:3; Cl:2; Ss:1

63 l2pt Gap coding A 12:3; 11:3; 10:3; 9:3; 8:3; Ls:3; La:3; Lr:3; Ol:3; Mc:2; Cl:3; Ss:1

Phylogeny of the Six Mullet Species 757

In addition to the seven OTUs

(operational taxonomic units)

included, five HTUs (hypothetical

taxonomic units) were also

involved in the phylogenetic tree,

with the same number of “extra

steps.”

The three most parsimonious

trees were inferred, and were fol-

lowed with character states matrix,

lists of exchanges of character

states with consistency index, the

lists of apomorphic character

states for each OTU and HTU.

Moreover, from the above-

mentioned most parsimonious

three, a consensus tree has been

inferred (and its probability was

tested by the bootstrap method –

1000 replicates). The rooted phy-

logenetic tree (with total length L =

171, total consistency index CI = 0.888, and retention index

RI = 0.525) was also established, with the total amount of

homoplasy fvalue = 2, f–ratio = 0.058 (Farris, 1972).

The autapomorphic and synapomorphic character

states were obtained in a posteriori analysis (Table 3). This

cladistic analysis revealed the following as the most signifi-

cant characters: length and height of some skull parts;

length of some visceral skeleton elements (premaxillar,

maxillar, dental, angular); length and width of some visceral

skeleton elements (quadrate and hyomandibular); length

and height of some gills lid bones and their processes (pre-

opercle, interopercle); and length and width of some shoul-

der zone elements (cleithrum, postcleithrum).

An illustration of the phylogenetic tree (with bootstrap

support values) is shown in Fig. 2, and has three main

branches: in the first – S. sphyraena as outgroup taxa, in the

second – M. cephalus and O. labeo, and in the third one –

C. labrosus and the three species of the Liza genus.

DISCUSSION

The analyses of continuous osteological characters indi-

cate that Sphyraenidae was separated clearly from Mugilidae,

with 37 autapomorphic character states, and only one syna-

pomorphic for S. sphyreana and the six mullet species (see

Table 3). In contrast, 17 character states are found to be syn-

apomorphic for the lineage encompassing M. cephalus and

O. labeo, and the other four mullet species lineage (Table 3).

In addition to the numerous autapomorphic character

states for O. labeo and M. cephalus (see Table 3), this anal-

ysis clusters them in the sister group (Fig. 2), not confirming

in this way the phenetic relationships of mullets based on

continuous characters of viscerodermal skeleton, in which

M. cephalus was clearly separated from the other five mullet

species (Antovic and Simonovic, 2006). At the same time,

this is also different from the results of phylogenetic recon-

struction, in which the phylogenetic position of O. labeo has

been studied for the first time (by cytochrome b and 12s rRNA

analysis) (Caldara et al., 1996) and indicated that O. labeois in the sister group with the Liza-Chelon lineage and pres-

ents the most divergent species (considering Liza, Chelon

and Oedalechilus). The same conclusion (O. labeo as the

most divergent) was given by Rossi et al. (2004) on the

basis of allozymes and 16S mt-rDNA analyses. However, in

the cytochrome b and 16S rDNA analysis using ML (maxi-

mum likelihood) method (Aurelle et al., 2008), O. labeo was

grouped with L. ramada (cytb), but also in the sister group

with the lineage containing C. labrosus-L. aurata and L. ramada – all of which together form the sister group to L. salines (16S rDNA). In the tree of the nine Mediterranean

mullet species obtained from allozyme analysis (Turan et al.,

2005), O. labeo is together with C. labrosus in the first main

lineage, and in the sister group with L. ramada. Turan et al.

(2011), in reconsidering systematic status of the same nine

mullet species by morphological characters, concluded that

the first branch contains O. labeo and C. labrosus (as the

closest taxa), in the sister group with L. aurata (according to

meristic data); but also – C. labrosus is the closest to L. ramada, being the sister group to O. labeo (morphometric

analysis).

Table 3. List of autapomorphic and synapomorphic character states.

Autapomorphic character states

OTU Ordinal number of character

S. sphyraena 1-8, 14, 16, 19-22, 25, 27-32, 36-37, 39, 42-43, 45, 47, 50, 52, 55, 58-63

M. cephalus 3, 9, 12, 18, 19, 20, 23, 28-29, 42, 44-46, 48, 50, 57, 62, 63

O. labeo 9, 13, 15-19, 23-24, 26, 29-30, 33-35, 40, 44, 48, 51, 54, 56-57

C. labrosus 6, 49

L. aurata 15, 33

L. saliens 3, 33, 43

L. ramada 15, 31, 34, 41, 44, 47-48, 50, 54, 58, 60

Synapomorphic character states

HTU/OTU, HTU/HTU, OTU/OTU Ordinal number of character

11/10/S. sphyraena 9

11 and 10 9, 10, 15, 18, 23, 24, 33, 34, 35, 38, 40, 44, 48, 51, 53, 54, 57

9 and C. labrosus 46

8 and L. aurata 46

L. saliens and L. ramada 9

Fig. 2. Phylogenetic tree of the six Euro-Mediterranean mullet

species, with frequencies of occurrence, i.e. bootstrap support val-

ues for particular clades (L = 171, CI = 0.888, RI = 0.525).

I. Antovic758

Many previous genetic analyses (allozyme, mtDNA) of

mullet species have shown that the most distinct is M. cephalus (e.g., Papasotiropoulos et al., 2001, 2002, 2007;

Semina et al., 2007). Using allozyme analyses, Turan et al.

(2005) concluded that M. cephalus with M. soiuy clustered

together and were clearly separate from the other three gen-

era (Liza, Chelon and Oedalechilus). Meristic analysis of the

same species (Turan et al., 2011) showed the highest level

of divergence of M. cephalus, which was found to be close

to its sister species M. soiuy and L. abu. Moreover, from

morphometrical data, L. aurata and M. cephalus were found

more divergent from the other three species (C. labrosus, L. ramada, O. labeo), and L. saliens was found to be morpho-

metrically most divergent (Turan et al., 2011).

In regard to Chelon-Liza relations, Durand et al. (2012),

in “molecular phylogenetic evidence challenges two centu-

ries of morphology-based taxonomy” resulted – C. labrosusgrouped with the seven species of the Liza genus (including

three considered here) to form a monophyletic subclade.

The present study shows C. labrosus as clustered with

the three Liza species in the third main branch (as one of

two subclades) (Fig. 2). The character states found to be

autapomorphic and synapomorphic for these two lineages

are given in Table 3 (can be also seen from Table 2), as well

as for the second subclade containing L. aurata, and the lin-

eage L. ramada-L. saliens.From the chromosomes and karyotypes analyses of

the six Mediterranean mullet species (Cataudella et al.,

1974), it was concluded – there are no substantial differ-

ences between the karyotype of C. labrosus and the three

Mediterranean Liza species. Semina et al. (2007) reported

that all the Mediterranean Liza species have approximately

the same genetic distances among each other (8–10% of

nucleotide substitutions), and Chelon and Liza representa-

tives are close genetically. However, Autem and Bonhomme

(1980) had found appreciable genetic differences between

Chelon and Liza.

As is abovementioned, results of some previous studies

did not support monophyletic origin of the Mediterranean Lizaspecies (e.g., Gornung et al., 2001, 2004; Turan et al., 2005,

2011; Papasotiropoulos et al., 2007), giving different cluster-

ing of C. labrosus – with L. aurata (Rossi et al., 2004), but

also with L. saliens (Caldara et al., 1996, Papasotiropoulos

et al., 2002), or the L. ramada-L. aurata lineage (Murgia et

al., 2002). Here presented results (i.e., closeness of the Lizaand Chelon) are in accordance with the phenetic relation-

ships of mullets based on continuous characters of viscero-

dermal skeleton (Antovic and Simonovic, 2006), while the

positions of C. labrosus and the three Liza species (not con-

sidering the clades support) are in accordance with the posi-

tions given by Autem and Bonhomme (1980).

In conclusion, the results of the first reconstruction of

mullet phylogenetic relationships obtained from continuous

osteological characters could help understanding systematic

status of the six Euro-Mediterranean mullet species (from

the South Adriatic Sea), and contribute to already estab-

lished knowledge about the mullets relationships. This is

particularly because the Chelon-Liza relations and mono-

phyletic origin of the Liza genus are still under discussion.

In the present study, a monophyly of M. cephalus and O. labeo was found with the probability of 57%, while that for

C. labrosus and the three Liza species ~ 54%. In regards to

the Liza genus, the probability to be monophyletic group (the

three species considered in the present study) was found to

be less than 50% (i.e., 37.4%). This support (bootstrap

value) indicates that the Liza could be paraphyletic in

regards to Chelon (as suggested by some morphological

and molecular phylogenies), as well as that a further

research in this field is needed. A further research should

include another coding procedure of the continuous charac-

ters, but also greater number of both specimens and spe-

cies from the Chelon and Liza genus.

ACKNOWLEDGMENTS

The author thanks Prof. K. Hensel (Komenius University,

Bratislava) and Prof. P. Simonovic (Faculty of Biology, University in

Belgrade) for helpful instructions, and two anonymous reviewers for

improving the manuscript.

REFERENCES

Abbott LA, Bisby FA, Rogers DJ (1985) Taxonomic Analysis in

Biology: Computers, Models, and Databases, Columbia Univer-

sity Press, New York

Antovic I (2006) Reconstructing the phylogenetic relationships of the

mullet species (Mugilidae) of Euro-Mediterranean distribution

from the genera Mugil, Chelon, Liza and Oedalechilus. PhD

thesis, University in Belgrade, Faculty of Biology (in Serbian)

Antovic I, Simonovic P (2006) Phenetic relationships of six mullet

(Mugilidae) species from South Adriatic on their visceral and

dermal skeleton. Russ J Мar Biol 32: 250–254

Aurelle D, Barthelemy RM, Quignard JP, Trabelsi M, Faure E (2008)

Molecular phylogeny of Mugilidae (Teleostei: Perciformes).

Open Mar Biol J 2: 29–37

Autem M, Bonhomme F (1980) Elements de Systematique Biochi-

mique chez les Mugilides de Mediterranee. Biochem Syst Ecol

8: 305–308

Caldara F, Bargellone L, Ostellari L, Penzo E, Colombo L, Patarnello

T (1996) Molecular phylogeny of grey mullets based on mito-

chondrial DNA sequence analysis: Evidence of a differential rate

of evolution at the intrafamily level. Mol Phylogenet Evol 6:

416–424

Capanna E, Cataudella S, Monaco G (1984) The pharyngeal struc-

ture of Mediterranean Mugilidae. Monit Zool Ital 8: 29–46

Cataudella S, Civitelli MV, Capanna E (1974) Chromosome comple-

ments of the Mediterranean mullets (Pisces, Perciformes).

Caryologia 27: 93–105

Drake P, Arias AM, Gallego L (1984) Biologia de los mugilidos

(Osteichthyes, Mugilidae) en los esteros de las salinas de San

Fernando (Cadiz). III. Habitos alimentarios y su relacion con la

morfometria del aparato digestivo. Investigacion Pesquera 48:

337–367

Durand JD, Shen KN, Chen WJ, Jamandre BW, Blel H, Diop K, et al.

(2012) Systematics of the grey mullets (Teleostei: Mugiliformes:

Mugilidae): Molecular phylogenetic evidence challenges to cen-

turies of morphology-based taxonomy. Mol Phylogenet Evol 64:

73–92

Farris JS (1972) Estimating phylogenetic trees from distance matri-

ces. Am Nat 106: 645–668

Farrugio H (1977) Les muges (Poissons, Teleosteens) de Tunisie.

Repartition et peche, Contribution a leur etude systematique et

biologique. Le grade de docteur d’universite. Academie de

Montpellier. Universite des Sciences et Techniques du

Languedoc

Felsenstein J (1988) Phylogenies and quantitative characters. Ann

Rev Ecol Syst 19: 445–471

Gornung E, Cordisco C, Rossi A, Innocentiis DeS, Crosetti D, Sola L

Phylogeny of the Six Mullet Species 759

(2001) Chromosomal evolution in Mugilidae: karyotipe charac-

terization of Liza saliens and comparative localization of major

and minor ribosomal genes in the six Mediterranean mullets.

Mar Biol 139: 55–60

Gornung E, Mannarelli ME, Rossi AR, Sola L (2004) Chromosomal

evolution in Mugilidae (Pisces, Mugiliformes): FISH mapping of

the (TTAGGG)n telomeric repeat in the six Mediterranean mul-

lets. Hereditas, 140: 158–159

Guerrero JA, de Luna E, Sanchez-Hernandez C (2003) Morphomet-

rics in the quantification of character state identity for the

assessment of primary homology: an analysis of character vari-

ation of the genus Artibeus (Chiroptera: Phyllostomidae). Biol J

Linn Soc 80: 45–55

Harrison IJ, Howes GJ (1991) The pharyngobranchial organ of

mugilid; its structure, variability, ontogeny, possible function

and taxonomic utility. Bull Br Mus Nat Hist (Zool) 57: 111–132

Murgia R, Tola G, Archer SN, Vallerga S, Hirano J (2002) Genetic

identification of grey mullet species (Mugilidae) by analysis of

mitochondrial DNA sequence: Application to identify the origin

of processed ovary products (Bottarga). Mar Biol 4: 119–126

Papasotiropoulos V, Klossa-Kilia E, Alahiotis S (2001) Genetic

divergence and phylogenetic relationships in grey mullets

(Teleostei: Mugilidae) using allozyme data. Biochem Genet 39:

155–168

Papasotiropoulos V, Klossa-Kilia E, Alahiotis S (2002) Genetic

divergence and phylogenetic relationships in grey mullets

(Teleostei: Mugilidae) based on PCR-RFLP analysis of mtDNA

segments. Biochem Genet 40: 71–86

Papasotiropoulos V, Klossa-Kilia E, Alahiotis SN, Kilias G (2007)

Molecular phylogeny of grey mullets (Teleostei: Mugilidae) in

Greece: Evidence from sequence analysis of mtDNA seg-

ments. Biochem Genet 45: 623–636

Pillay TVR (1951) Structure and development of the scales of five

species of grey mullets of Bengal. Proc Nat Inst Sc India 17:

413–424

Rizzotti M (1993) Fish hemoglobins: The family Mugilidae (Perci-

formes). Trends Comp Biochem Physiol 1: 385–392

Rossi AR, Ungaro A, De Innocentiis S, Crosetti D, Sola L (2004)

Phylogenetic analysis of Mediterranean mugilids by allozymes

and 16S mt-rRNA genes investigation: are the Mediterranean

species of Liza monophyletic? Biochem Genet 42: 301–315

Schultz LP (1946) A revision of the genera of mullets, fishes of the

family Mugilidae, with descriptions of three new genera. Proc

US Natl Mus 96: 377–395

Semina AV, Polyakova NE, BrykovVlA (2007) Analysis of mitochon-

drial DNA: taxonomic and phylogenetic relationships in two fish

taxa (Pisces: Mugilidae and Cyprinidae). Biochemistry (Moscow)

72: 1349–1355

Sokal RR, Rohlf FJ (1981) Biometry. W.H. Freeman & Co, San

Francisco, CA

Stevens PF (2000) On characters and character states: do overlap-

ping and non-overlapping variation, morphology, and molecules

all yield data of the same value? In: “Homology and systematics:

coding characters for phylogenetic analysis” Ed by RW Scotland,

RT Pennington, Taylor and Francis Inc, London, pp81–105

Swiderski DL, Zelditch ML, Fink WL (1998) Why morphometics is

not special: coding quantitative data for phylogenetic analysis.

Syst Biol 47: 508–519

Swofford DL (1985) PAUP-Phylogenetic Analysis Using Parsimony

(manual for Version 2.4.). Illinois Natural History Survey,

Champaign

Swofford DL (1988) PAUP-Phylogenetic Analysis Using Parsimony

(and Other Methods). Version 4, Sinauer Associates, Sunderland,

MA

Tortonese E (1972) I Mugilidi del Bacino Mediterraneo (Pisces,

Perciformes). Natura Societa Italiano di Scienze Naturale,

Museo Civico di Storia Naturale e Acquario Civico, Milano 63:

21–36

Trewavas E, Ingham SE (1972) A key to the species of Mugilidae

(Pisces) in the North Eastern Atlantic and Mediterranean, with

explanatory notes. J Zool (London) 167: 15–29

Turan C, Caliskan M, Kucuktas H (2005) Phylogenetic relationships

of nine mullets species (Mugilidae) in the Mediterranean Sea.

Hydrobiologia 532: 45–51

Turan C, Gürlek M, Ergüden D, Yaglıoglu D, Öztürk B (2011)

Systematic status of nine mullet species (Mugilidae) in the

Mediterranean Sea. Turk J Fish Aquat Sc 11: 315–321

Thorpe RS (1984) Coding morphometric characters for constructing

distance Wagner networks. Evolution 38: 241–255

Wiens JJ (2001) Character analysis in morphological phylogenetics:

Problems and solutions. Syst Biol 50: 680–699

Wiens JJ (2004) The role of morphological data in phylogeny recon-

struction. Syst Biol 53: 653–661

(Received March 14, 2013 / Accepted May 12, 2013)