MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

https://doi.org/10.47193/mafis.3322020301107 163

ORIGINAL RESEARCH

Metamorphosis of whitemouth croaker Micropogonias furnieri

(Pisces, Sciaenidae)

MARA S. BRAVERMAN1, *, DANIEL BROWN1and E. MARCELO ACHA1, 2

1Instituto Nacional de Investigación y Desarrollo Pesquero (INIDEP), Paseo Victoria Ocampo Nº 1, Escollera Norte, B7602HSA -

Mar del Plata, Argentina. 2Instituto de Investigaciones Marinas y Costeras (IIMyC), Universidad Nacional de Mar del Plata (UNMdP),

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

ABSTRACT. Fish metamorphosis is an important ontogenetic process with a key role on early

stages survival and on successful recruitment to adult populations. The whitemouth croaker

(Micropogonias furnieri) is an important commercial resource for the coastal fisheries of Argentina

and Uruguay. Metamorphosis was studied using morphometric and morphological analysis during

larval development. Changes in morpho-meristic characters before and after metamorphosis were

employed to determine the length interval of this transition by employing Principal Component

Analysis. Individuals (n =430) from 4 to 41 mm standard length (SL) were collected in the Río de

la Plata estuary (35.45° S, 56.35° W) in March 2006. Length ranges of individual’s developmental

stages were associated with the presence of key morphological characters. During early life stages,

M. furnieri changes from a big-headed, robust shape larva to a slender and more elongated body

form. Most of the morphometric variables showed an inflexion point at 15.2 mm SL, with a 95%

confidence interval of 14.0-16.4 mm. The anterior part of the body grows faster during early stages,

probably related to an intense feeding activity strategy. The completion of pectoral fin rays and the

onset of squamation determine the beginning of metamorphosis at 11-12 mm SL. At around 18 mm

SL, squamation ends, first barbels develop and the sagittae otoliths primordium is closed. The

length-at-metamorphosis for M. furnieri was established between 9 to 18 mm SL, since all devel-

opmental characters studied highly overlapped at that interval. All those processes are indicative of

the beginning of the juvenile period associated to the settlement and the start of a bottom-oriented

life-style.

Key words: Sciaenidae, larvae transformation, ontogeny, morphology, morphometry.

La metamorfosis de la corvina rubia Micropogonias furnieri (Pisces, Sciaenidae)

RESUMEN. La metamorfosis de los peces es un proceso ontogenético importante con un papel

clave en la supervivencia de las primeras etapas y en el reclutamiento exitoso a las poblaciones adul-

tas. La corvina rubia (Micropogonias furnieri) es un recurso comercial importante para las pesque-

rías costeras de la Argentina y Uruguay. Se estudió su metamorfosis mediante análisis morfométri-

cos y morfológicos durante el desarrollo larvario. Se utilizaron los cambios en los caracteres morfo-

merísticos antes y después de la metamorfosis para determinar el intervalo de duración de esta tran-

sición mediante el Análisis de Componentes Principales. Se colectaron individuos (n =430) de 4 a

41 mm de longitud estándar (LE) en el estuario del Río de la Plata (35,45° S, 56,35° W) en marzo

de 2006. Los rangos de longitud de las etapas de desarrollo de los individuos se asociaron con la

presencia de caracteres morfológicos clave. Durante las primeras etapas de vida, M. furnieri cambia

de una larva de cabeza grande y forma robusta a una forma corporal más delgada y alargada. La

mayoría de las variables morfométricas mostraron un punto de inflexión a los 15,2 mm LE, con un

Marine and

Fishery Sciences

MAFIS

*Correspondence:

mbraverman@inidep.edu.ar

Received: 25 June 2020

Accepted: 15 September 2020

ISSN 2683-7595 (print)

ISSN 2683-7951 (online)

https://ojs.inidep.edu.ar

Journal of the Instituto Nacional de

Investigación y Desarrollo Pesquero

(INIDEP)

This work is licensed under a

Creative Commons Attribution-

NonCommercial-ShareAlike 4.0

International License

INTRODUCTION

The way on that ontogeny of fishes proceed

has been broadly discussed leading to two antag-

onistic visions. One states that ontogeny is a grad-

ual process during which small and inconspicu-

ous changes in form and structure accumulate

continuously (Kováč and Copp 1999). The sec-

ond view, represented by the ‘theory of saltatory

ontogeny’ (Balon 1984), considers development

as a sequence of longer stabilized steps (during

which all the structures and organs develop grad-

ually and continuously) alternating with thresh-

olds. A threshold is a short interval where rapid

changes occur from one steady state to the next.

Nevertheless, early life stages of fish have been

recognized by both ‘gradualists’ and ‘saltation-

ists’ (see Kováč and Copp 1999) as being charac-

terized by different fish capabilities, require-

ments, ecological interactions and growth/mortal-

ity events (e.g. Smith 1985; Koumoundouros et

al. 2009; Nikolioudakis et al. 2010). The begin-

ning of the larval period (Balon 1984) occurs

when the individual is capable of feed orally,

while the juvenile stage implies the disappear-

ance of all larval characters and the appearance of

nearly all the adult ones (Pavlov 1999). The tran-

sit between those periods consists on a remodel-

ing process called ‘metamorphosis’ (Balon 1989),

which represents an important ontogenetic event

for fish with consequences for the survival of

early stages and strong consequences for recruit-

ment to adult populations (Govoni 2004).

To define the onset of the juvenile period, dif-

ferent approaches are traditionally used. From a

functional and structural perspective, this event is

associated with a change in allometric growth or

shape (e.g. Copp and Kováč 1996; Sagnes et al.

1997; Kováč et al. 2006), coupled with a series of

changes (abrupt or gradual) in morphological and

meristic characters. Such changes are the acquisi-

tion of adult complement of fins spines and rays,

adult pigmentation, onset or end of squamation,

ossification of the axial skeleton and disappear-

ance of larval characters (McCormick et al. 2002;

Urho 2002; Ditty et al. 2003). From an ecological

perspective, in demersal and benthic fish, meta-

morphosis is usually linked to settlement (Werner

2002) i.e., the ontogenetic shift by which pelagic

larvae colonize benthic habitats (McCormick et

al. 2002). Metamorphosis is a preparation for the

colonization of a new habitat and the integration

into a new trophic web, as larvae abandon the

plankton to become part of the demersal-benthic

communities (e.g. Secor 2015). That shift can be

detected in otoliths of several fish species (Wilson

and McCormick 1997, 1999) as a transition mark

or ‘check’ (Campana and Neilson 1985). For tem-

perate and cold water fishes, formation of acces-

sory nuclei in their otoliths has been associated

with metamorphosis and settlement processes

(Sogard 1991; Morales-Nin and Aldebert 1997;

Morioka et al. 2001; Buratti and Santos 2010).

Setting a threshold value as the ‘length-at-

metamorphosis’ (Ljuv from Fuiman’s ontogenetic

index) is highly complex due to individual vari-

ability and ontogenetic stage of individual char-

acters. This is why in several studies a single

164 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

intervalo de confianza del 95% de 14,0-16,4 mm. La parte anterior del cuerpo crece más rápidamente durante las primeras etapas, pro-

bablemente relacionada con una estrategia de actividad alimentaria intensa. La adquisición del número definitivo de radios de la aleta

pectoral y el inicio de la escamación determinan el comienzo de la metamorfosis a los 11-12 mm LE. Alrededor de los 18 mm LE, ter-

mina la escamación, se desarrollan las primeras barbillas y se cierra el primordio de los otolitos sagittae. La longitud-de-metamorfosis

para M. furnieri se estableció entre 9 y 18 mm LE, dado el alto grado de superposición que presentaron los caracteres de desarrollo estu-

diados. Todos esos procesos son indicativos del inicio del período juvenil asociado al asentamiento y al comienzo de un estilo de vida

orientado hacia el fondo.

Palabras clave: Sciaenidae, transición larva-juvenil, ontogenia, morfología, morfometría.

character (e.g., the onset or end of squamation,

definitive fin radios acquisition, etc.) is consid-

ered, although this approach results inadequate

for a precise definition of metamorphosis (Urho

2002; Ditty et al. 2003), where a series of mor-

phological, morphometric, physiological and

ethological events should converge. In that sense,

Urho (2002) stated that ‘the use of a single mor-

phological trait to infer metamorphosis is as inad-

equate as using a single character for the identifi-

cation of a species’. A clear determination of lar-

vae-juvenile transition is particularly important to

identify ontogenetic changes in the use of

resources (Juanes and Conover 1994; Boglione et

al. 2003). Most of the characters associated to

metamorphosis undergo changes in a synchro-

nized way (Ditty et al. 2003), suggesting the need

to analyze the metamorphosis process as a holis-

tic approach.

The whitemouth croaker, Micropogonias

furnieri (Desmarest 1823), is the dominant

species in terms of biomass in the Río de la Plata

region and the main target of the coastal fisheries

of Argentina and Uruguay, representing ca. 20%

of coastal species landings in Argentina (Carozza

et al. 2004). It is a demersal species with a long

lifespan (up to 39 years in the region). In Argenti-

na and Uruguay it reproduces from November to

April, spawning several batches of pelagic eggs

with an indeterminate annual fecundity. Main

reproduction ground covers a narrow band across

the inner Río de la Plata estuary between Monte-

video (34° 50′ S-56° 10′ W) and Punta Piedras

(35° 25′ S-57° 10′ W), at depths between 6 and 8

m (Acha et al. 1999; Carozza et al. 2004). The

area is characterized by a bottom salinity front

(Mianzan et al. 2001) in which whitemouth

croaker eggs were only present below the halo-

cline where salinity ranges from 9.7 to 27.3

(Acha et al. 1999). During warmest months

(October through May), there is evidence of

retention of whitemouth croaker larvae in the

inner part of the Río de la Plata estuary. This area

coincides with the location of the bottom salinity

front and the maximum turbidity zone (Braver-

man et al. 2009). Larvae retention in this area

would ensure closeness to the main nursery

ground: Samborombón Bay (Mianzan et al.

2001). Early life stages of this species have been

described for identification purposes with a clas-

sical approach (Sinque 1980; Weiss 1981), not

intending to outline critical moments of the early

life history, such as the larvae-juvenile transition.

Thus, to define metamorphosis more accurately

we employed a multivariate approach using mor-

phometric and morphological analyses during lar-

val development.

The multivariate approach consists of a mor-

phometric analysis based on the principles of

multivariate allometry described by Shea (1985)

and a morpho-meristic analysis based on Ditty et

al. (2003). The former studied the relationship

between a group of morphometric measures and

the individual’s total length. The later consisted

on the assignation of scores to individual charac-

ters, where the sum of them for each individual

represents an index of morphological change

associated to metamorphosis. Hence, the objec-

tive of this study was to investigate the larvae-

juvenile transition (hereafter metamorphosis),

looking for changes in morpho-meristic charac-

ters before and after the metamorphosis to deter-

mine the length interval for these changes.

MATERIALS AND METHODS

M. furnieri larvae and juveniles (less than a

year-old) were collected during a scientific cruise

to Río de la Plata spawning area in March 2006.

A vertically stratified plankton sampling along

transects perpendicular to bathymetry was per-

formed during daylight. Samples were taken at

stations separated 10-12 km at three depth levels.

Water column was sampled above (or c.a. 2.5 m

depth) and below (or c.a. 6.2 m depth) the halo-

cline with a Motoda sampler equipped with a

165

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

mechanical opening-closing device and a 200 µm

mesh span net. The third level was sampled by an

epibenthic sledge with a 500 µm mesh span net

(see Braverman et al., 2009).

A total of 249 M. furnieri individuals were

identified on board and frozen for a later otoliths

extraction; the rest of the samples were fixed in

4% formalin. Some specimens from the ichthy-

oplankton collection of the Instituto Nacional de

Investigación y Desarrollo Pesquero (INIDEP)

were also used to complete the length classes (n

= 15). Those individuals from the collection

come from the same study area. An alizarin dying

technique (modified from Potthoff 1984 and Tay-

lor and Van Dyke 1985) was performed onto the

specimens for a better observation of structures.

Morphometric and morphological analyses

were performed on an ontogenetic series from 4 to

41 mm standard length (SL). For morphometric

analysis, images of each individual (n = 71) were

taken using a digital camera mounted in a com-

pound microscope. A series of measurements

(Figure 1; Table 1) were taken from those images

with the Axio Vision Software (Carl Zeiss). The

shortening correction due to the fixation method

was not considered assuming that it was similar in

all measured individuals. For morphological

analysis, a group of events related to the external

morphology was analyzed on the ontogenetic

series (n = 120). Development stage was deter-

mined following Moser (1996) and Fuiman and

Werner (2002): larvae at pre-flexion, flexion, post-

flexion, in transformation and early juveniles.

Distinctive characters of each individual were

registered: fin rays appearance (pectoral, anal,

dorsal and pelvic); scales coverage was classified

into 4 categories: (a) on the caudal peduncle, (b)

from the caudal peduncle to the anus, (c) from the

caudal peduncle to the head, (d) totally scaled;

and presence/absence of mentonian barbels.

Finally, otoliths were extracted from frozen spec-

imens (n = 85) using a NaClO solution for tissue

disintegration. They were mounted in glass slides

with a transparent mounting medium and pol-

ished when necessary with lapping film paper in

a decreasing order of porosity (12, 9 or 3 μ) for a

better visualization. Otolith development was

studied by registering the occurrence of accessory

nuclei and the primordium (polygonal area cen-

ter-enclosed by the accessory nuclei).

166 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

Figure 1. Morphometric measurements taken from Micropogonias furnieri individuals. SL: standard Length, ED: eye diameter,

HL: head length, PreAL: pre-anal length, HD1: head depth 1, HD2: head depth 2, AD: anal depth, CD: caudal depth,

Pre-OL: pre-orbital length, Mid-BL: mid-body length, CL: caudal length. Illustration taken from Weiss, 1981.

HD2 HD1

PreOL

MidBL

1 mm

PreAL

Data analysis

Morphometry and bi-variate allometry

Firstly, morphometric relationships were stud-

ied using linear regression analysis between the

logarithms of each variable (morphometric meas-

urement) versus length (SL) (Table 1). We

employed standard length for post-flexion indi-

viduals and notochord length for pre-flexion and

flexion specimens. Allometric growth pattern of

each variable in relation to SL was studied by

using the logarithmic form of the allometric

growth model (Huxley 1932): log (Y) = log (a) +

b log (SL), where Yis the variable examined and

bis the allometric coefficient. Secondly, regres-

sions’ residuals (Y) were analyzed to identify pos-

sible allometric changes as follows: if allometric

growth of the variables was constant (slope with-

out changes) then residuals would show a random

pattern; alternatively, if variables showed changes

in their growth they would have a residual distri-

bution (Y versus SL) with a specific form as it was

shown by Sagnes et al. (1997), Gozlan et al.

(1999) and Nikolioudakis et al. (2010).

Similarly, morphometric indexes (Im) showing

representative changes in the larvae-juvenile tran-

sition were analyzed. Indexes were calculated as

the percentage of the morphometric variable (Vm)

with respect to length (SL): Im=Vm/SL * 100.

The length at which the change in oblique orien-

tation of Imoccurred (Li) was estimated by using

a piecewise linear regression fitted with a non-

linear estimation procedure: Im=b0+b1SL +

b2(SL − Li) (SL ≥ Li); where b0is the y-intercept,

b1is the slope of the relationship of the values ≤

Li, b2is the change in the slope (b1) that results in

the slope when SL ≥Li, and Liis the length at

which slope changes (Nikolioudakis et al. 2010),

considered here to be related to the transforma-

tion process (end of larval period).

Morphometry and multivariate approach

To determine the length-at-metamorphosis

(Lm), log-transformed morphometric measure-

ments with respect to SL using a Principal Compo-

nent Analysis (PCA) with covariance matrices

were studied (Jolicoeur 1963a, 1963b; Shea 1985).

In a PCA of a logarithmic covariance matrix of

groups of animals with different growth patterns,

the first component (PC1) summarizes the varia-

tion in shape as a result of a common pattern of

allometric growth; while the second component

167

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

Table 1. Description of morphometric characters measured in Micropogonias furnieri and its abbreviations (adapted from

Nikolioudakis et al. 2010).

Character Abbreviation Description

Standard length SL From the tip of the snout to the caudal rays insertion

Eye diameter ED Parallel to the longitudinal axis of the body

Head length HL From the tip of the snout to the margin of the gill cover

Pre-anal length PreAL From the tip of the snout to the anus

Head depth 1 HD1 From the dorsal to the ventral margin of the body at the operculum

Head depth 2 HD2 From the dorsal to the ventral margin of the body at the center of the eye

Anal depth AD From the dorsal to the ventral margin of the body at the anus

Caudal depth CD From the dorsal to the ventral margin of the body at the caudal peduncle

Pre-orbital length PreOL From the tip of the snout to the anterior margin of the eye

Mid-body length MidBL From the margin of the gill cover to the anus

Caudal length CL From the margin of the gill cover to the insertion of the caudal fin

(PC2) and the rest summarize the variations of

shape as a result of divergent growth trajectories

(Shea 1985). Then, the Lmat which a change in

PC2 orientation occurs and the lengths of possible

inflexion points (Li) were statistically estimated

using a quadratic function PC2 =a +b(PC1) +

c(PC1)2of the Non-linear estimation Module of

STATISTICA software v.7 2004 (StatSoft Inc.).

Morphology

Length ranges of developmental stages of M.

furnieri were determined. These ranges were

associated with the appearance/disappearance of

several morphological characters (Table 2).

Moreover, the pectoral fin ray’s definitive num-

ber acquisition was analyzed with respect to

length (pectoral fin tends to be the last one in

acquiring the whole set of rays (Urho 2002)).

Then, the squamation process was described. For

morpho-meristic analysis, each specimen was

scored for a suite of characters where each score

represented a discrete ontogenetic event or a

character stage of development (Table 2). Every

character had equal weight, i.e. three possible

states 0, 1 and 2. The sum of character scores of

each individual as an index of overall change in

morphology associated with metamorphosis was

calculated and expressed as percentage of the

maximum total score. Total scores for the charac-

ter set were analyzed with respect to length class-

es performing a cluster analysis with complete

linkage, and to the Manhattan (city-block) dis-

tances method (Ditty et al. 2003). Finally, the

length at which 50% of morphological change

occurs (L50) was obtained from the relationship

between the accumulative scores percentage (P)

and SL described by the logistic function: P

= 100/(1 +exp(-a*(SL-b))). Length at which P

= 100% constitutes the juvenile stage length.

RESULTS

Developmental morphometry

Residual distribution of each log-transformed

variable fitted against SL with an allometric func-

tion showed different responses (see Appendix).

With few exceptions (ED and MidBL, Figure 1;

Table 1), residuals revealed a non-random distri-

bution indicating changes in the relative growth of

morphometric variables evidenced by inflexion

points in the distribution of morphometric indexes

(Figure 2). Thus, ED grew isometrically while

MidBL showed a positive allometry in the entire

168 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

Table 2. Scores assigned to each Micropogonias furnieri individual according to the developmental stage of considered charac-

ters. The scoring of rays was performed separately for each fin (pectoral, anal, dorsal and pelvic).

Character Score Observed pattern Length ranges (mm SL)

Scales cover 0 No scales 4-10

1 Scales do not cover the entire body 10.1-16.9

2 Scales covering the entire body, including head 18-34

Fin rays 0 No rays 4-6.3

1 Rays in formation 6.7-11.9

2 Definitive rays 12-34

Otolith development 0 No accesory nuclei 4-10.9

1 Accesory nuclei not closing the primordium 6.8-17

2 Closed primodium 15-34.9

169

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

Figure 2. Relationships of morphometric indexes with standard length (SL) of individuals. Fitted lines came from non-linear

regressions used to determine inflexion lengths (Li). CI: 95% confidence interval for Li, Vm: morphometric variable.

Abbreviations as in Figure 1.

0510 15 20 25 30 35

0.40

0.50

0.60

0.70

0510 15 20 25 30 35

PreAL

0510 15 20 25 30 35

preOL

0510 15 20 25 30 35

0.22

0.25

0.27

0.30

0.32

0510 15 20 25 30 35

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0510 15 20 25 30 35

0.03

0.05

0.07

0.09

0.11

0510 15 20 25 30 35

0.06

0.07

0.08

0.09

0.10

0.02

0.04

0.06

0.08

0.10

0.20

0.23

0.25

0.28

0.30

0.20

0.25

0.30

0.35

0.40

0.20

0.23

0.26

0.29

0.32

0.35

0510 15 20 25 30 35

MidBL

Morphometric ndex ( /SL)iV

SL (mm)

0.60

0.63

0.66

0.69

0.72

0.75

0510 15 20 25 30 35

R 0.79=

L 14.23 mm

CI [12.96-15.5]=

R 0.70=

L 14.48 mm

CI [13.03-15.93]=

R 0.65=

L 13.15 mm

CI [11.47-14.84]=

R 0.80=

L 14.52 mm

CI [13.36-15.72]=

R 0.84=

L 11.23 mm

CI [9.93-12.52]=

R 0.53=

L 9.29 mm

CI [7.95-10.63]=

R 0.83=

L 7.34 mm

CI [5.78-8.90]=

R 0.75=

L 9.67 mm

CI [8.10-11.23]=

length range. In turn, HL, PreOL and HD1 grew

faster than SL (positive allometry) to a maximum

and then got slower (negative allometry), while

CL showed an opposite pattern growing slower

than SL until a minimum value when it started to

grew faster. In addition, variables related with

body depth (HD2, AD, CD) and PreAL grew

faster than SL to a point where they remained con-

stant (or slightly grew). Inflexion points (Li) and

their confidence intervals were determined by

non-linear regressions of each variable (expressed

as the Im) with respect to SL (Figure 2).

Principal Components Analysis (Table 3) of

log-transformed morphometric measurements

using the covariance matrix revealed an impor-

tant change in the oblique orientation of PC2 val-

ues of transformed variables when plotted

against PC1 or SL (Figure 3). A highly significant

quadratic relationship (R = 0.62, p <0.001)

allowed to determine an Lm=15.18 mm SL [CI

=14.01-16.37]. When residuals of allometric

regressions were separately examined for indi-

viduals <15.18 mm SL and ≥15.18 mm SL, we

observed they were randomly distributed.

Because no pattern or structure were shown, it

could be assumed that bi-variate allometric equa-

tions were appropriate to describe relative

growth of individuals before (SL <Lm) or after

(SL ≥Lm) the metamorphosis length.

Morphological events

The end of post-flexion stage and the beginning

of the transition to juvenile (metamorphosis) was

determined considering the completion of pec-

toral fin rays and the onset of squamation process,

which occurred at 11 mm SL and 12 mm SL,

respectively (Table 4; Figure 4 A: arrows 3 and 4).

Meanwhile, at ca. 18 mm SL barbels’ appearance

and the end of squamation process established the

end of metamorphosis according to morphologi-

cal events (Figure 4 A: arrows 5 and 6).

First pectoral rays appeared between 5 to 7 mm

SL and the fin rapidly completed its rays at 12 mm

170 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

Table 3. Principal Component results of the first (PC1) and second factor (PC2) for the log-transformed morphometric characters

studied (abbreviations in Table 1).

Eigenvectors Factor score Factor-variable

coefficients correlations

Morphometirc PC1 PC2 PC1 PC2 PC1 PC2 Eigenvalues % total

character variance

SL -0.275 0.163 -0.357 1.955 -0.996 0.063 0.595 97.872

ED -0.284 0.284 -0.369 3.412 -0.986 0.106 0.007 1.137

PreAL -0.305 0.048 -0.396 0.574 -0.998 0.017 0.002 0.385

HL -0.292 -0.126 -0.379 -1.516 -0.995 -0.046 0.001 0.194

HD1 -0.291 0.075 -0.377 0.897 -0.997 0.028 0.001 0.155

AD -0.339 0.063 -0.349 -1.474 -0.992 0.020 0.001 0.101

CD -0.340 -0.056 -0.440 0.762 -0.990 -0.018 0.000 0.068

PreOL -0.324 -0.821 -0.441 -0.674 -0.963 -0.264 0.000 0.058

HD2 -0.269 -0.123 -0.419 -9.876 -0.993 -0.049 0.000 0.030

MidBL -0.317 0.289 -0.411 3.478 -0.990 0.097 0.000 0.001

CL -0.268 0.298 -0.347 3.579 -0.990 0.119 0.000 0.000

SL (Figure 4 B). Squamation occurred in the

transformation stage in a posterior-anterior direc-

tion (Figure 5). First scales appeared in the caudal

peduncle separated from each other. Its morphol-

ogy was rather simple: oval-shaped, with smooth

edges and a few concentric rings (Figure 5.1). As

squamation process progressed, scales morpholo-

gy became more complex developing projections

in its posterior margin (ctenii) and scallops in the

anterior margin, and starting to overlap each other

(Figure 5.3). The head was the last part to be cov-

ered (Figure 5.2).

Cluster analysis over the scores of morpholog-

ical characters defined two main groups at a dis-

tance of 50% approximately (Figure 6 A); a group

of small larvae from 4 to 9 mm SL, and a group

of individuals larger than 9 mm SL. From the

later, individuals bigger than 16 mm SL were

grouped in an approximate distance of 15%,

remaining an intermediate interval from 9-16 mm

SL of non-homogeneous groups. On the other

hand, cumulative scores showed a highly signifi-

cant fit (R =0.9845; p <0.001) to a logistic func-

tion with L50 =9.24 mm SL (Figure 6 B) and

fully metamorphosed individuals larger than 18-

20 mm SL.

DISCUSSION

Throughout the early life history, M. furnieri

changed from a larval big-headed and robust

shape to a slender and more elongated body

171

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

Figure 3. Multivariate analysis (PCA) showing the relation between SL (representing PC1) and PC2, and the quadratic fit (lines)

assessed for the length-at-metamorphosis (Lm). Dashed lines represent 95% of confidence.

Table 4. Length intervals of developmental stages of

Micropogonias furnieri. Transition length was calcu-

lated as the mean value between the highest value of

a stage and the lowest value of the previous stage.

Length (mm SL)

Stage N Min Max Transition

Pre-flexion 19 - 6.2 5.6

Flexion 26 4.9 8.4 7.4

Post-flexion 26 6.4 11.3 11.5

Transformation 32 11.7 19.2 18.6

Juvenile 17 18 -

PC SL2 2.3044-0.3767*SL 0.0124*=+

R 0.62=

Standard length (mm)

0 5 10 15 20 25 30 35

-2.0

-1.0

0.0

1.0

2.0

PC2

Conﬁdence interval 95%=

〉

shape. Most of the morphometric variables

showed an inflexion point in their development

and the compound analysis showed an inflexion

length of 15.2 mm SL [CI =14-16.4]. Multivari-

ate morphometric analysis (PCA) indicated a

change in the allometric growth of ontogenetic

series evidenced by a change in the slope orienta-

tion of the PC2 versus PC1 (or SL) as stated by

Shea (1985). Relative changes in the growth of

whitemouth croaker were seen in the residuals of

allometric relations as well as in the estimated

Lm, resulting in a random distribution of residuals

of the two size groups detected (i.e., SL <Lmand

SL >Lm). These results constitute a valid estima-

tion of the length-at-metamorphosis from a mor-

phometric point of view.

172 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

Figure 4. Summary of morphological events occurred throughout Micropogonias furnieri early ontogeny. A) Scheme of length

intervals per stage. Arrows show important events: 1- caudal, dorsal and anal fins occurrence (rays in formation), 2- pel-

vic fin appearance, 3- onset of squamation, 4- pectoral fin definitive set of rays, 5- barbels occurrence, 6- ending of squa-

mation process (scales in the head). B) Development of pectoral fins: a- rays in formation, b- definitive set of rays.

Illustrations taken from Weiss, 1981.

Flexion

Post flexion-

Transformation

Juvenile

Pre flexion-

611 12 18

51015

20 25 30 35

Number of pectoral rays

Length (mm SL)

200 mm

Length (mm SL)

Two well-defined groups were found in the

scoring analysis (morphology): one with individ-

uals <9 mm SL and other with individuals

>16 mm SL, matching the length at which 50%

and 100% of the body shape change occurs (L50

= 9.2 mm SL and L100 =17 mm SL, respectively).

Individuals between 9 to 16 mm SL did not form

a homogeneous group probably due to they were

undergoing a shape transition. In accordance to

Weiss (1981), morpho-meristic analysis during

this length interval showed the definitive pectoral

ray’s acquisition and the onset of squamation at

11 mm and 12 mm SL, respectively. These events

occurred close to the appearance of accessory

nuclei in the otoliths (Braverman et al. 2015),

coincidently with the change in body proportions.

Finally, squamation ended at 18 mm SL approxi-

mately, when first barbels appeared and the

otolith primordium was closed (c.a. 16 mm SL,

Braverman et al. 2015).

Early in larval development, variables related

to the head showed a higher relative growth rate

than the rest of the body. A higher growth of the

anterior part of the body during the first ontoge-

netic stages has been related to feeding (Fuiman

1983; Osse et al. 1997), because an early devel-

opment and differentiation of nervous (brain),

sensorial (neuromasts, photoreceptors and olfac-

tory receptors) and digestive (mandible struc-

tures) systems would contribute to improve prey

detection and capture. Fin formation would

increase swimming efficiency and the appearance

of barbel during metamorphosis would constitute

an important character helping juveniles to colo-

nize a new habitat (settlement) and start a bottom-

oriented lifestyle.

173

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

Figure 5. Sequence of squamation (A-D) observed in Micropogonias furnieri. Different colors indicate degree of squamation,

from lesser (A) to greater (D) complexity. Boxes 1-4: details of coverage and complexity of scales throughout the body.

White arrows indicate scales position. Box 3: scheme of a scale.

1mm

1 mm

ACD

500 mm

1 mm

500 mm

In terms of body height and pre-anal length,

larvae changed their growth rate at smaller

lengths (9-11 mm SL) while the head and the rest

of the body showed an opposite pattern changing

their growth rates at larger lengths (13-15 mm

SL). We suggest that as the head reduces its rela-

tive growth the rest of the body has to grow faster

to maintain proportions. Our findings could indi-

cate a sequence of growth for these stages of

development, giving priority first to the feeding

function and then to locomotion and predators

escape instead of digestive system development

(Osse and van den Boogaart 1995). This could be

possible because of the high digestibility of

croaker larvae’s prey (tintinnids, copepod nauplii

and small copepods, Rodríguez-Graña et al.

2018) and the powerful digestive enzymes of fish

larvae as shown in herring Clupea harengus (Ped-

ersen et al. 1987). Moreover, it has been suggest-

ed that fish larvae can utilize exogenous enzymes

from their prey to improve digestion (Lauff and

Hoffer 1984; Kolkovski et al. 1993).

174 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

Figure 6. Morphological analysis of Micropogonias furnieri ontogenetic development. A) Cluster analysis of morphological cha-

racters based on Manhattan distances’ method (City-Block) with complete linkage. B) Logistic graph and function with

cumulative scores of characters with respect to length.

100

5101520253035

Cumulative scores (%)

Length (mm SL)

400

L50

y 100/(1 e )=+

-(0.432)*(x-9.181)

45679810

11 12 13 1415 16 17 18 20 21 23 25 27 28 29 30 31 32 36 38 41

100

(Dlink/Dmax)*100

The squamation process in demersal species

has been considered an indicator of transforma-

tion between larval and juvenile stages (Miller et

al. 2003), along with the process of primordium

formation in otoliths, with drastic changes in

early life history of fish as observed in Trachurus

japonicus (Xie et al. 2005) and Merluccius hubbsi

(Buratti and Santos 2010). Similar results were

reported in blennids (Ditty et al. 2003), consider-

ing a group of morphological characters in which

the ontogenetic development of different struc-

tures seemed to overlap during metamorphosis

(see also Kanou et al. 2004; Nikolioudakis et al.

2010). Those findings are consistent with the high

level of synchronization achieved in certain onto-

genetic thresholds (Balon 1984) to get to the next

step in development (e.g., from larval to juvenile

period) (Kováč 2002). Differences in timing and

developmental rates of individual characters

make difficult the recognition of thresholds that

could be in the root of the ‘saltationist-gradualist’

debate (Kováč and Copp 1999). Hence, multi-

variate approach performed in our study helps to

a better identification of thresholds based on the

degree of coincidence of changing variables.

Multivariate approach showed that develop-

mental characters studied on the whitemouth

croaker highly overlapped, even though morpho-

logical or morphometric characters could begin

the transformation at different ontogenetic times

(lengths, sensu Fuiman et al. 1998) and at differ-

ent speeds. Although not all the inflexion points

were detected at the same size, 10 morphometric

measurements demonstrated significant changes

in relative growth within a length interval similar

to that found in the study of morphological

events. Considering all the events together, it is

strongly suggested a developmental interval of 9

to 18 mm LS as the moment of greatest change in

body shape, i.e., metamorphosis.

Most marine fishes tend to spawn at specific

times and places within predictable and distinc-

tive circulation features. The most common life

history of fishes that use estuaries involves

spawning of planktonic eggs at sea and the subse-

quent recruitment to estuaries as post-larvae or

juveniles. Due to the net seaward movement of

estuarine waters, the export of early life-history

stages from estuaries has been argued to be a

major problem for estuarine spawners (Boehlert

and Mundy 1988), and the lack of retention

mechanisms has been proposed to explain why

fishes do not typically spawn inside estuaries

(e.g., Dando 1984; Haedrich 1992). Unlike most

estuaries, spawning activity is rather common in

the Río de la Plata, in which the existence of

retention mechanisms has been proposed (Acha

et al. 1999; Simionato et al. 2008; Braverman et

al. 2009). This estuary is very shallow and essen-

tially dominated by the wind. Retention process

is a consequence of the estuarine response to nat-

ural wind variability at the scale of 3-5 days act-

ing over bathymetric features (Simionato et al.

2008). Moreover, river discharge fluctuations

modulate retention variability at interannual

scales (Acha et al. 2008). Whitemouth croakers

spawn well inside the estuary at the bottom salin-

ity/turbidity front (Macchi and Christiansen

1996; Acha et al. 1999), where their larvae

remain retained (Braverman et al. 2009). Reten-

tion is by no manner a 100% effective mechanism

and recruitment success of M. furnieri shows

strong fluctuations that would be linked to the

dynamics of the estuarine waters (Acha et al.

2008). For many marine fishes habitat shifts often

occur at metamorphosis when larvae undergoes a

striking change from a pelagic, planktonic organ-

ism, to a demersal one (Werner 2002). This seems

to be the case with M. furnieri, whose smaller lar-

vae (<10 mm SL) appear at the whole water col-

umn but larger sizes (>10 mm SL) probably

undergo the settlement process inhabiting near

the bottom (Braverman et al. 2009).

During settlement, larvae and young juveniles

move from pelagic to demersal food webs with

attendant changes to their foraging success and

predation risk. However, settlement means not

only the encounter of a new array of prey and

175

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

predators, it also means a shift from a pelagic and

more dispersive stage to a demersal and more

sedentary one (Secor 2015). Retention would

improve when larvae or early juveniles remain in

the bottom boundary layer (Mann and Lazier

1996), in this way settled croakers would diminish

their chances of being exported from the estuary.

A fast metamorphosis and successful settlement

could enhance survival, and consequently improv-

ing recruitment. So identifying the processes that

allow juveniles to attain retention inside adequate

habitats is important to effectively understand

marine species population dynamics.

ACKNOWLEDGEMENTS

We are grateful to Dr Daniela Alemany and to

Dr Marina Diaz for their support and useful com-

ments on the manuscript. We also thank the

reviewers for their valuable suggestions that

helped us to improve the quality of our original

manuscript. This study was supported by Agencia

PICT 2003 no 07-13659; CONICET PIP 2009,

Universidad Nacional de Mar del Plata EXA

355/06 and by a grant from the Inter-American

Institute for Global Change Research (IAI) CRN

2076 sponsored by the US National Science

Foundation (Grant GEO-0452325). This is an

INIDEP contribution no 2143.

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179

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

APPENDIX

Linear regressions of log-transformed morpho-

metric variables and residuals’ charts used to

identify changes in variables meaning changes in

individual’s shape.

180 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.60 0.80 1.00 1.20 1.40 1.60

Log ( )ED

-0.1

0.1

0.2

0.6 1.1 1.6

Residuals

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.60 0.80 1.00 1.20 1.40 1.60

Log ( )HL

-0.1

-0.05

0.05

0.1

0.6 1.1

Residuals

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.60 0.80 1.00 1.20 1.40 1.60

Log (PreOL)

Log (SL)

-0.3

-0.2

-0.1

0.1

0.2

0.6 0.8 1.0 1.2 1.4 1.6

Residuals

Log(ED) 1.0316*Log(SL) 1.1279-=

R 0.9768 Isometry

Log(HL) 1.0525*Log(SL) 0.5528=-

R 0.9822 Positive allometry

Log(PreOL) 1.147*Log(SL) 1.2655=-

R 0.8905 Positive allometry

1.6

Appendix. Continued.

181

BRAVERMAN ET AL.: METAMORPHOSIS OF MICROPOGONIAS FURNIERI

0.60 0.80 1.00 1.20 1.40 1.60

Log ( )HD1

-0.1

-0.05

0.05

0.1

0.6 0.8 1.0 1.2 1.4 1.6

Residuals

0.0

0.2

0.4

0.6

0.8

1.0

0.60 0.80 1.00 1.20 1.40 1.60

Log (HD2)

-0.1

-0.05

0.05

0.1

0.6 1.1 1.6

Residuals

0.0

0.2

0.4

0.6

0.8

1.0

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

0.60 0.80 1.00 1.20 1.40 1.60

Log ( )AD

-0.2

-0.15

-0.1

-0.05

0.05

0.1

0.15

0.6 1.1 1.6

Residuals

1.0

1.2

1.4

Log ( )preAL

0.8

0.4

0.6

0.05

Residuals

0.2 -0.05

0.6 1.0 1.6

Log(HD1) 1.0507*Log(SL) 0.6168=-

R 0.9907 Positive allometry

Log(HD2) 0.9699*Log(SL) 0.5606=-

R 0.9761 Negative allometry

Log (AD) 1.22*Log (SL) 0.995=-

R 0.969 Positive allometry

Log(PreAL) 1.1024*Log(LE) 0.3486=-

2=Positive allometryR 0.9921

0.0

-0.1

0.60 0.80 1.00 1.20 1.40 1.60

Log (SL)

Appendix. Continued.

182 MARINE AND FISHERY SCIENCES 33 (2): 163-182 (2020)

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.60 0.80 1.00 1.20 1.40 1.60

Log (MidBL)

-0.15

-0.1

-0.05

0.05

0.1

0.6 1.1 1.6

Residuals

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.60 0.80 1.00 1.20 1.40 1.60

Log (C )D

-0.3

-0.2

-0.1

0.1

0.2

0.6 1.1 1.6

Residuals

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0.60 0.80 1.00 1.20 1.40 1.60

Log ( )CL

Log (SL)

-0.04

-0.02

0.02

0.04

0.6 0.8 1.0 1.2 1.4 1.6

Residuals

Log(MidBL) 1.1482*Log(SL) 0.7454=-

Positive allometryR 0.9811

Log(CD) 1.2172*Log(SL) 1.3573=-

Positive allometryR 0.9577

Log(CL) 0.9795*Log(SL) 0.146=-

Negative allometryR 0.995