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Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 423-437, March 2021 (Published Mar. 30, 2021)

Morphological variability of recent species of the order Cassiduloida

(Echinodermata: Echinoidea) of Mexico

Andrea Alejandra Caballero-Ochoa

1,2

Blanca E. Buitrón-Sánchez

Carlos A. Conejeros-Vargas

Brenda L. Esteban-Vázquez

Mariana P. Ruiz-Nava

José Carlos Jiménez-López

Francisco A. Solís-Marín

Alfredo Laguarda-Figueras

1. Facultad de Ciencias, Universidad Nacional Autónoma de México. Circuito Exterior, C.P. 04510, Ciudad de México,

México; a.caballero.ochoa@ciencias.unam.mx (*Correspondence), brenda.estebanv@gmail.com

2. Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, C.P. 04510, Ciudad de México,

México.

3. Instituto de Geología, Departamento de Paleontología, Universidad Nacional Autónoma de México, Circuito Exterior,

C.P. 04510, Ciudad de México, México; blancab@unam.mx

4. Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, C.P. 04510, Coyoacán,

Ciudad de México, México; conejeros@ciencias.unam.mx

5. Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México. Av. de Los Barrios 1,

Tlalnepantla de Baz, C.P. 54090, Estado de México, México; mpaola.ruiznava@gmail.com

6. Posgrado en Ciencias de la Tierra, Instituto de Geología, Universidad Nacional Autónoma de México. Circuito

Exterior, C.P. 04510, Ciudad de México, México; jcjl1712@gmail.com

7. Colección Nacional de Equinodermos “Dra. Ma. Elena Caso Muñoz”, Laboratorio de Sistemática y Ecología de

Equinodermos, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, C.P. 04510,

Ciudad de México, México; fasolis@cmarl.unam.mx, laguarda@cmarl.unam.mx

Received 15-VII-2020. Corrected 09-IX-2020. Accepted 15-XI-2020.

ABSTRACT

Introduction: In Mexico, there are two recorded living species of Cassiduloida: Cassidulus caribaearum

and Rhyncholampas pacifica. Most of the taxonomic studies on cassiduloids have used external morphology,

pedicellariae and morphometric characters; however, the intraspecific variation of quantitative and qualitative

characters has been poorly evaluated. Objective: To compare the basic morphology of R. pacifica and C. carib-

aearum. Methods: We examined a total of 2 158 specimens of R. pacifica and C. caribaearum, selecting 50 to

evaluate shape and size with linear regression and Principal Component analysis. We selected an additional 62

specimens per species to identify significant character correlations and morphological groups within species.

Results: There is a direct relationship between Test length and Test width. Test height/Test width, and Total

length (oral view)/Distance from the ambitus to the peristome apex, are the two main ratios to distinguish both

species. C. caribaearum is more dorsoventrally compressed and has a round peristome base; versus R. pacifica

has a tall and triangular one. There are four morphological groups of C. caribaearum and two groups for R.

pacifica. Conclusions: These two species can be distinguished with reliable morphological characters, in which

peristome shape suggests that R. pacifica is more adapted to burrowing deeper into certain types of substratum.

Key words: Neognathostomata; Cassiduloida; morphometry; Mexico.

Caballero-Ochoa, A.A., Buitrón-Sánchez, B.E., Conejeros-

Vargas, C.A., Esteban-Vázquez, B.L., Ruiz-Nava,

M.P., Jiménez-López, J.C., Solís-Marín, F.A., &

Laguarda-Figueras, A. (2021). Morphological

variability of recent species of the order Cassiduloida

(Echinodermata: Echinoidea) of Mexico. Revista de

Biología Tropical, 69(S1), 423-437. DOI 10.15517/

rbt.v69iSuppl.1.46382

DOI 10.15517/rbt.v69iSuppl.1.46382

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The order Cassiduloida (sensu Souto,

Mooi, Martins, Menegola, & Marshall, 2019)

includes the families Cassidulidae, Eurhodi-

idae, Faujasiidae, Neolampadidae, and Pliol-

ampadidae (Souto et al., 2019), is represented

by 800 species, most of them fossils and a

few living ones (Kier, 1962). One of the most

characteristic aspects of these families is that

their fossil record, and not the living represen-

tatives, contains most of their morphological

diversity. Their variable morphology has made

their taxonomic study complicated and pos-

sibly denotes that the group is in the process of

extinction (Suter, 1988).

The cassiduloids first appeared and

changed from infaunal to epifaunal habits

during the Lower Jurassic (Boivin, Saucède,

Laffont, Steimetz, & Neige, 2018; Souto et

al., 2019). They diversified in the Early Creta-

ceous and survived the K-Pg mass extinction,

were common in the Late Cretaceous and

Early Cenozoic, being more successful in the

Eocene (> 40 % of the echinoid diversity), and

finally have dramatically declined in number

since then (Kier, 1962; Kier, 1974; Suter,

1988; McNamara, Pawson, Miskelly, & Byrne,

2017). Kier (1962) noticed several morpho-

logical changes within the cassiduloids: abrupt

reduction from two pores to one pore in each

ambulacral plate beyond the petal and the intro-

duction of buccal pores (Cenomanian), due to

a radical change in the living habits (began to

borrow shallowly into the substratum); and a

change in the structure of the apical system

from tetrabasal to monobasal (Maastrichtian),

probably produced by parallel mutations and

parallel selections. Recent studies have shown

that their evolutionary history has been domi-

nated by high levels of homoplasy and a dearth

of unique, novel traits (Souto et al., 2019).

In Mexico, there exist two living species

of cassiduloids: Cassidulus caribaearum and

Rhyncholampas pacifica (Buitrón-Sánchez,

Solís-Marín, Conejeros-Vargas, & Caballero-

Ochoa, 2019). Cassidulus caribaearum inhab-

its warm, shallow waters (26-28 °C), from

less than 1 to 18 m in depth, buried up to 20

cm in calcareous sand (± 2 000-44 μm) or

Fig 1. Distribution map of Cassidulus caribaearum and Rhyncholampas pacifica in Mexico.

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carbonate substratum of the tropical Atlantic

West coast, and it is probably endemic to the

Caribbean Sea (Fig. 1) (Kier, 1975; Gladfel-

ter, 1978; Borrero-Pérez, Benavides-Serrato,

& Diaz-Sanchez, 2012; Solís-Marín et al.,

2013; Solís-Marín, Caballero-Ochoa, Laguar-

da-Figueras, & Durán-González, 2017; Souto

& Martins, 2018). Rhyncholampas pacifica

lives in warm, shallow waters (26-30 °C) of the

Tropical Eastern Pacific Ocean (Fig. 1). This

species lives gregariously and partially buried

to the level of the petaloids on sandy beaches

at a depth of 2 to 130 m (Agassiz, 1872; Clark,

1925; Mooi, 1990; Solís-Marín et al., 2013;

Caballero-Ochoa, Martínez-Melo, Conejeros-

Vargas, Solís-Marín, & Laguarda-Figueras,

2017; Schultz, 2017).

Morphometrics evaluates the size and

shape variation of biological forms through sta-

tistical analysis (Ocakoglu & Ercan, 2013). The

traditional approach involves two-dimensional

linear measurements such as lengths, widths

and distances, and angles or ratios; it has been

used in taxonomy since it is useful for mak-

ing morphological comparisons and establish-

ing specific boundaries, as well as assessing

growth changes (Ocakoglu & Ercan, 2013;

Remagnino, Mayo, Wilkin, Cope, & Kirkup,

2016; MacLeod, 2017). The application of

morphometrics on cassiduloid taxonomy dates

back to McKinney (1986), who discussed the

heterochronic-ecological relationships between

fossil irregular echinoids, including Rhynchol-

ampas species. Although Carter and Beisel

(1987) did not perform any statistical analysis,

they also considered width/length ratios of the

test for separating Eurhodia, Rhyncholampas

and Cassidulus species. In addition, Ciampa-

glio and D’Orazio (2007) and Martínez-Melo

(2008) provided insights into the growth tra-

jectories and heterochronic processes between

Eurhodia appendiculata, Rhyncholampas caro-

linensis and Eurhodia rugosa, and between

C. caribaearum and R. pacifica, respectively.

Recently, Martínez-Melo, De Luna and Buit-

rón-Sánchez (2017) evaluated the contours of

the tests (lateral, aboral and posterior) of Cas-

sidulidae species through geometric analyses,

being the first study focused on this computa-

tional approach for cassiduloids.

The test shape is the most important char-

acteristic to distinguish between species of

cassiduloids (Souto et al., 2019). In most of

the studies, the morphological diversity of the

order Cassiduloida has been described (Souto et

al., 2019); however, the intraspecific variation

of the morphological characters of the recent

cassiduloids in Mexico has not been evalu-

ated. The objective of this work is to compare

the basic morphological and morphometric

aspects, as well as to evaluate the intraspecific

variation of the morphometric characters of R.

pacifica and C. caribaearum.

MATERIALS AND METHODS

Data collection: A total of 2 158 speci-

mens of recent cassiduloids were examined: C.

caribaearum and R. pacifica; these are housed

at Colección Nacional de Equinodermos “Dra.

Ma. Elena Caso Muñoz” (ICML-UNAM) in

Mexico (Appendix 1).

Morphometric analyses: We selected 50

specimens of different sizes of C. caribaearum

and R. pacifica, from 3.3 to 51 mm in length;

these were randomly selected and were pho-

tographed from the aboral, oral and lateral

views. ImageJ software was used to obtain nine

measurements for each specimen: TLa, TW,

Da-ppa, TLo, Da-pta, THl, TWl, PpL, and PpW

(Fig. 2, Table 1). A linear regression analysis

was performed to test the relationship between

the length and width of the test in GraphPad

Prism. A Principal Component analysis using

Primer-6 software was performed; five ratios

were considered: 1) test length (aboral view)/

test width (at the level of the apical system),

2) test length (aboral view)/distance from the

ambitus to the periproct apex, 3) test length

(oral view)/distance from the ambitus to the

peristome apex, 4) test height (lateral view)/test

length (lateral view), and 5) periproct length/

periproct width. The data were transformed

through square roots (Lawrence & Cobb, 2017)

(Table 1).

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Additionally, we randomly selected 62

other specimens of C. caribaearum and R.

pacifica in order to have representatives from

other localities and to consider all possible

sizes; and to observe which characters yield

significant information for species identifica-

tion. Eleven traits (TWl, THl, PW, PL, AIL,

AIIIL, MAW, PIW, PB, AFFP, AFTP) were

measured three times each, using an electronic

Vernier caliper. We also considered two quali-

tative data: the peristome base shape and apical

system position. All measurements, qualitative

data, and ratio abbreviations are detailed in

Table 1 (Fig. 3).

The Pearson’s Correlation Coefficient was

used to identify the greatest number of signifi-

cant correlations between the characters of the

species R. pacifica and C. caribaearum. A dis-

tribution analysis was performed to analyze the

shape of the peristome using CRAN R’s facto-

extra and FactoMineR packages (Lê, Josse, &

Husson, 2008; R Core Team, 2019). To com-

pare the average values between different mea-

surements, a F-test was run to check that there

were similar variances between the species; the

results of these F-test were then used in t-tests

to analyze whether there are specific differenc-

es. Normality of measurements was verified by

TABLE 1

Abbreviations and definitions of measurements and ratios used in this study

Abbreviation Definition

TLa Test length (aboral view)

TW Test width (at the level of the apical system)

Da-ppa Distance from the ambitus to the periproct apex

TLo Test length (oral view)

Da-pta Distance from the ambitus to the peristome apex

THl Test height (lateral view)

TWl Test width (lateral view)

PpL Periproct length

PpW Periproct width

A Ambitus

PW Peristome width

PL Peristome length

AIIIL Third ambulacrum length

AIL First ambulacrum length

MAW Maximum width ambulacra

PIW Maximum width of the outer poriferous zone of petal I

PB Petaloid beginning (%)

AFFP

AFTP

Angle between the first and fifth petaloids (°)

Angle between the first and third petaloids (°)

TLa / TW Test length (aboral view) / Test width (at the level of the apical system)

TLa / Da-ppa Test length (aboral view) / Distance from the ambitus to the periproct apex

TLo / Da-pta Test length (oral view) / Distance from the ambitus to the peristome apex

THl / TWl Test height (lateral view) / Test width (lateral view)

PpL / PpW Periproct length / Periproct width

PBS Peristome base shape (rect, rounded, trapezoidal and triangular)

ASP Apical system position (subcentral, lateral)

PL / PW Peristome length / Length of posterior side of peristome

AIL / AIIIL Ambulacrum I length / Ambulacrum III length

MAW / PIW Maximum ambulacral I width / Maximum width of the outer poriferous zone of petal I

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a Shapiro-Wilk test. We considered a test with

a P-value < 0.05 to be statistically significant.

To determine whether morphological

groups exist within the analyzed species, a

cluster analysis was carried out using Ward’s

minimum variance method. For this analysis,

all the data from the specimens were used, and

it was found that in both species the topology

of the variables presents a similar ordering.

To define the number of groups, the “Average

Silhouette” method of the fviz_nbclust func-

tion, included as part of CRAN R’s factoextra

and FactoMineR packages (Lê et al., 2008; R

Core Team, 2019) was used. This also allowed

us to report the average values of the non-

standardized numerical variables by group, and

to decide what separation distance to accept

between different clusters.

Linked to the “Average Silhouette”, k-mean

analysis was performed to check if there were

differences between the parameters in assigned

groups. These analyses use the algorithm of

Hartigan and Wong (1979), assigning the vari-

ables to the fixed number of clusters.

RESULTS

Evaluation of test length and width: The

linear regression (Fig. 4) showed a positive

correlation between the variables Test length

(aboral view) and Test width (at the level of the

apical system). In this regard, R. pacifica reach-

es test length values of 51.5 mm and test width

values of 42.6 mm, whereas C. caribaearum

does not exceed sizes of 22.9 mm and 19.4 mm.

The eigenvalues for PC1 and PC2 were

1.21×10

-2

and 4.41×10

-3

, whereas the percent-

ages of variation were 59.3 % and 21.6%,

respectively (Table 2); therefore, both PC1 and

PC2 explain 80.9 % of the cumulative varia-

tion of the data. The greatest eigenvalues for

the first component occurred in THl/TWl and

PpL/PpW, whereas in the second component,

TLo/Da-pta and THl/TWl had the greatest

Fig. 2. Rhyncholampas pacifica, ICML-UNAM 4.48.3. A. Aboral view. B. Oral view. C. Lateral view. D. Posterior

view. Cassidulus caribaearum, ICML-UNAM 4.96.6. E. Aboral view. F. Oral view. G. Lateral view. H. Posterior view.

Abbreviations refer to measurements defined in Table 1.

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eigenvalues. Considering both components,

the THl/TWl (0.917) and TLo/Da-pta (0.925)

were the two ratios which most contribute to

the variation of the data, and separation of R.

pacifica from C. caribaearum (Fig. 5).

Morphometric evaluation of other

characters of taxonomic importance: In R.

pacifica the highest Pearson´s Correlations

Coefficients were found between PW and PL,

the lengths of ambulacra I and III; and MAW

and PIW. Pearson’s Correlation Coefficient

values for this species ranged from 0.71 (test

width (lateral view) vs. length of ambulacrum

III; peristome length vs. peristome width and

0.99 (test width (lateral view) vs. lengths of

ambulacra I/III and test height (lateral view)).

In the case of C. caribaearum, the strongest

correlations had values of 0.60 (length of

ambulacrum I vs. length of peristome) and

0.94 (test width (lateral view) vs. test height

(lateral view)).

When analyzing the peristome base shape

along with PW and PL, it was found that, in R.

pacifica, it was wider and higher and the shape

was triangular, while in C. caribaearum, the

most predominant shape was round (Fig. 6).

In the specimens of R. pacifica, the largest

correlation values ranged between 0.45 and

0.50, while in C. caribaearum the correlation

values were between 0.40 and 0.45, which

indicates a weak correlation (Fig. 7). When

comparing the average values of the relation-

ships between the lateral height and length for

both species, a significant difference was found

between the analyzed species (F test for vari-

ances: F = 0.5072, g.l.

numerator

= 59, g.l.

denominator

= 58, P = 0.0104; t-test: t = 6.2751, d.f. =

104.69, P = 8.022×10

-9

When linear regression models were used

to compare the relationships of TWl and PW

with respect to other variables as possible intra-

specific differentiators, no significant covaria-

tions were found. Covariates that were analyzed

and showed values of lower significance were:

1) PBS (estimated parameter = 1.7148, S.E. =

1.2320, t = 1.392, P = 0.17), 2) THl/TWl ratio,

and 3) PBS (estimated parameter = -4.3054,

S.E. = 2.7814, t = 1548, P = 0.128).

Fig. 3. Measurements used in the analysis. A. Aboral view and ambulacra I and II. B. Lateral view. C. Oral view.

Abbreviations refer to measurements defined in Table 1.

Fig 4. Linear regression of Test width (at the level of

the apical system) vs Test length (aboral view) between

Rhyncholampas pacifica and Cassidulus caribaearum.

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Relationships between THl vs. TWl of

R. pacifica tend to be slightly lower in the

specimens with an ambitus between 21 and

40 mm; this was also verified with a regres-

sion model (estimated parameter Ambitus

21-40

= -0.2008, S.E. = 0.8861, t = -2.266, P =

0.0276). This observation should be tested

further with a larger sample to determine if it

occurs in a general way in the species. Cassidu-

lus caribaearum specimens with larger ambitus

perimeter sizes (> 40 mm) tended to present a

relationship between TWl and PW independent

of the relationship between THl vs. TWl, while

specimens of smaller size (up to 40 mm perim-

eter) tended to have a PL/PW ratio that cor-

relates positively with the relationship between

Fig. 5. Ordination (Principal Components Analysis) of Rhyncholampas pacifica and Cassidulus caribaearum. See Table 1

for acronyms.

Fig. 6. Peristome base shape distribution analysis of Cassidulus caribaearum and Rhyncholampas pacifica.

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THl vs. TWl (Fig. 7); however the increased

ratios were not significant (P = 0.170). In R.

pacifica, some of the forms that had higher PL/

PW ratios also had elevated and triangular peri-

stome base shapes. In addition, the specimens

that had perimeters of the ambitus between 40

and 59 mm generally tended to present a lower

PL/PW ratio as the first of these relationships

decreases (Table 3); there was also no signifi-

cant correlation (P = 0.0991).

To describe other morphometric features,

we analyzed the distributions of the angle

between the ambulacra I and V vs. the THl/

TWl ratio. In this case, we recognized two

patterns: 1) The larger specimens of C. carib-

aearum (ambitus 59-78.1 mm) had larger

TABLE 2

Eigenvalues (Principal Components Analysis) of Rhyncholampas pacifica and Cassidulus caribaearum

Eigenvalues Principal Component Eigen-values Explained variation (%) Cumulative variation (%)

PC1 1.21×10

-2

59.3 59.3

PC2 4.41×10

-3

21.6 80.9

PC3 2.3×10

-3

11.3 92.2

PC4 1.21×10

-3

5.9 98.1

PC5 3.89×10

-4

1.9 100

Eigenvectors Variable PC1 PC2 PC3 PC4 PC5

TLa / TW -0.138 -0.011 0.212 -0.224 0.941

TLa / Da-ppa 0.020 -0.085 0.370 -0.878 -0.290

TLo / Da-pta -0.366 0.925 0.001 -0.077 -0.061

THl / TWl 0.197 0.364 -0.053 -0.081 0.132

PpL / PpW 0.079 0.058 0.903 0.408 -0.094

TABLE 3

K-means (Silhoutte) cluster analysis in Cassidulus caribaearum (Dissimilar averages are marked in bold.

Minimum and maximum averages are marked in black)

Variable

SW test

P-value

Centers (Average)

ANOVA

P-value

Group 1 Group 2 Group 3 Group 4

Lateral length = TWl

0.0579 16.155 24.484 22.567 24.532 0.162

Lateral height = THl

0.0439

7.1400 10.315 9.6115 10.810 0.324

THl/TWl ratio

0.0591 0.4424 0.4212 0.4280 0.4423 0.222

Peristome length = PL

0.4174 1.6975 2.2000 1.8321 2.1550 0.625

Peristome width = PW

0.0539 1.5550 2.0600 1.9029 1.9675 0.256

PW/PL ratio

0.0316

0.9349 0.9519 1.0691 0.9592 0.117

Ambulacral I length = AIL

0.3911 5.6587 9.1062 8.2250 9.5600 0.368

Ambulacral III length = AIIIL

0.8939 7.4375 11.629 10.205 12.212 0.696

AIL/AIIIL ratio

0.1204 0.7714 0.8131 0.8173 0.7845 0.429

Ambulacral I-III angle

0.1449 66.250 63.750 70.429 73.750 0.926

Ambulacral I-V angle

0.0297

64.750 60.000 67.714 77.250 0.069

Max. ambulacral width = MAW

0.8554 1.8562 3.6925 2.5100 2.8100 0.345

Maximum width of the outer poriferous zone

of petal I = PIW

0.6090 0.4500 0.4837 0.4014 0.5750

0.015

MAW/PIW ratio

0.0010

4.2321 5.9318 6.8442 4.9714

0.008

Size of group

8 8 14 4

Within Sum of Squares

176.9432 246.7969 387.0671 66.1314

Between_SS / Total_SS

63.50 %

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angles and larger distances between ambulacra,

from 59-79 mm long and more than 70° (acute

angle), with straight and rounded peristome

base shape, and were independent from the test

ratio compared with the rest of the sizes; and

2) In R. pacifica there was a relation between

the variables: large specimen size (ambitus

45-69 mm) tended to have ratio values from

0.45-0.50, the angles were between 50 and

70° (more acute than C. caribaearum) and the

peristome was tall and triangular. These two

patterns were noticeable in other clusters of

morphological test variations. In terms of the

general test morphology of C. caribaearum, the

ambitus and the bulge were slightly ovoid and

elongated, and dorsoventrally flattened, with

low ambitus sizes between 21 and 78.91 mm.

We observed within the different groups and

sizes low, intermediate and high ratios between

the MAW and PIW, in combination with the

lowest values of the distances between ambu-

lacra, and that specimens had an ambitus of

intermediate to large size. This suggests that C.

caribaearum does not have specific variations

between the different sizes; intermediate mor-

phology may exist, meaning a continuous vari-

ability. Rhyncholampas pacifica, however, has

a gibbous test and shows several kinds of varia-

tions; sizes (ambitus measurements) showed

that the specimens with the largest perimeters

of the ambitus had the lowest THl, TWl, AIL,

AIIIL, angle between ambulacra I and V and

low relationships between the MAW and PIW,

AIL and AIIIL measurements; conversely, the

medium-sized specimens had the highest val-

ues of these features and the smallest sizes of

the species (ambitus 1.92-21 mm) did not have

a particular relation between them.

In the case of the angles between the ambu-

lacra I and V, it was found that C. caribaearum

tended to present values that range between

59 and 79 mm (average value = 66.76 mm,

S.D. = 4.0869 mm), while for R. pacifica these

values oscillated between 45 and 69 mm (aver-

age value = 59.8 mm, S.D. = 5.6382). Also, C.

caribaearum tended to present rounded forms

of the peristome base shape, and the forms that

had larger ambitus also had angles between

ambulacra I and V that were much greater than

the rest of the specimens in the same species

(above 70°); this was independent of their rela-

tionships between THl and TWl. Meanwhile, in

R. pacifica the specimens had peristome base

shape that are triangular, and the specimens

with higher values of ambitus tended to present

THl/TWl ratio values that were concentrated

between 0.45 and 0.50 mm (Fig. 7).

In R. pacifica the organisms had a size

range that can exceed 50 mm in length (largest

specimen: 51.6 mm) and 40 mm (largest speci-

men: 42.6 mm) in width; in C. caribaearum the

sizes did not exceed 25 mm in length (largest

specimen: 22.9 mm) and 20 mm in width (larg-

est specimen: 19.4 mm) (Table 3, Table 4).

For C. caribaearum, a first cluster includes

the specimens with smaller ambitus combined

with some specimens with ambitus up to 59

mm in perimeter, with a considerable number

of specimens showing low ratios between

MAW/PIW. A particular case for this variable

is presented by a second group, where the spec-

imens reach the highest ratios between MAW/

PIW in combination with the lowest values of

PIW and whose specimens have ambitus of

intermediate to high size, with some specimens

being in the range of 59 to 91 mm in perim-

eter. A third group is formed by specimens in

which the ratios between MAW/PIW generally

have intermediate values, in combination with

slightly low values for THl, TWL, AIL and

MAW. The fourth group was represented by

specimens with the highest average of ambu-

lacral angles, in combination with slightly

greater distance between PIW than in the other

groups. The groups and average values of the

analyzed measurements are shown in Fig. 8.

Two groups were observable for R. paci-

fica, the first of which includes the specimens

with the largest perimeters of the ambitus with

generally low measurements of THl, TWl, AIL,

AIIIL, and angle between ambulacra I and V,

these specimens also presented weak relation-

ships between the MAW and PIW; and AIL and

AIIIL. Some of these specimens also showed

slightly large values for the PIW and the rela-

tionship between THl/TWl of the organism.

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Fig. 7. Scatterplot of angle between ambulacra I and V vs. lateral height/lateral length ratio.

TABLE 4

K-means (Silhoutte) cluster analysis in Rhyncholampas pacifica (Dissimilar averages are marked in bold.

Minimum and maximum averages are marked in black)

Variable

S-W test

P-value

Centers (Average)

Welch t-test

P-value

Group 1 Group 2

Lateral length = TWl

0.7440 30.658 45.4996

3.89 × 10

-12

Lateral heigh = THl

0.7038 14.478 21.0444

8.12 × 10

-11

THl/TWl ratio

0.6036 0.4726 0.4626 0.118

Peristome length = PL

0.0021

2.4559 3.4030 1.49 × 10

-5

Peristome width = PW

0.4988 2.9733 3.5485

0.008

PW/PL ratio

0.4730 1.2277 1.0724

0.041

Ambulacral I length = AIL

0.9538 11.709 18.523

1.54 × 10

-11

Ambulacral III length = AIIIL

0.9589 13.082 20.309

2.58 × 10

-11

AIL/AIIIL ratio

0.8015 0.8943 0.9132 0.254

Ambulacral I-III angle

0.0002

72.296 69.815 0.240

Ambulacral I-V angle

0.1038 62.074 59.741 0.064

Maximum ambulacral width = MAW

0.8296 3.2211 4.6459

1.66 × 10

-9

Maximum width of the outer poriferous zone of petal I = PIW

0.9724 0.5644 0.6681

0.031

MAW/PIW ratio

0.0317

6.1316 7.4129

0.026

Size of group

27 27

Within Sum of Squares

3905.777 3869.790

Between_SS / Total_SS

39.70 %

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The second group is composed by specimens

with small to medium sized ambitus, in this

case the specimens of greater THl and TWl,

with one of them showing even the largest sizes

on the PW, another with the lowest value of

the angle between I and V ambulacra and one

more presenting the lowest values of PW that

also corresponds to the lowest ratio between

PL/PW. The groups and average values of the

analyzed measurements are shown in Fig. 9.

DISCUSSION

Cassidulus caribaearum and R. pacifica

have been considered very closely related

species, but Souto et al. (2019) proved that the

genera Rhyncholampas and Cassidulus have

been separated for more than 60 million years.

However, the interspecific morphological dif-

ferences between these two extant species are

still being analyzed.

Souto et al. (2019) studied the intraspecific

variations of the morphological features of C.

caribaearum and R. pacifica, tests noticed a

great variation in the number of phyllopores

plates and of occluded plates in all phyllodes,

and in the number of additional pore pairs in

the unequal (paired) petals. Regarding test

length and width (lateral view), C. carib-

aearum is a small to medium-sized species

(neotype test measurements from Souto &

Fig. 8. Heatmap coupled to cluster diagrams for Cassidulus caribaearum.

Centered variables were used for the construction of the heatmap.

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Martins, 2018: total length 26.68 mm, total

width 22.65 mm, and total height 11.71 mm)

with oval test; the total width is approximately

85 % of the total length. The lateral edges are

straight with round margins; the greatest height

is at the apical disc; it has a triangular trans-

verse section and a concave oral region (Souto

& Martins, 2018). Schultz (2017) mentioned

that length of C. caribaearum scarcely reaches

25 mm. In this study we found that C. carib-

aearum from the Mexican Caribbean Sea (50

specimens from Punta Nizuc to Punta Maroma,

Quintana Roo, Mexico) does not exceed sizes

of 22.9 mm length and 19.4 mm width; indi-

vidual variation ranges of test length are from

3.312-22.959 mm and width are from 2.708-

18.995 mm. The genus Rhyncholampas have

been described as small to large of varying test

shape with maximum length up to 70 mm in the

living species (Agassiz, 1869; Schultz, 2017;

Souto et al., 2019). This analysis showed speci-

mens of R. pacifica from the Mexican Pacific

(50 specimens from Punta Barron, Sinaloa to

Acapulco, Guerrero, Mexico) clearly reach test

length values of 51.5 mm and test width values

of 42.6 mm; individual variation ranges of test

length are from 5.01-51.59 mm and width are

from 4.549-43.222 mm.

Martínez-Melo (2008) stated that test

height, peristome length and distance from the

peristome to the anterior edge of the test are the

most significant measurements that separate

C. caribaearum from R. pacifica, supporting

that test dimensions are different between both

species. Here we also used the test height and

distance from the peristome to the anterior edge

Fig. 9. Heatmap coupled to cluster diagrams for Rhyncholampas pacifica.

Centered variables are used for the construction of the heatmap.

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of the test, here referred as Da-pta, as variables

of the ratios TH1/TWI and TLo/Da-pta which

primarily contributes to the discrimination of

C. caribaearum from R. pacifica in the PCA

analysis. Therefore, we confirm that these

measurements, as variables of such ratios,

allow us to distinguish between both species.

Martínez-Melo (2008) also mentioned that R.

pacifica had a taller test and longer peristome

than C. caribaearum. In this regard, we found

test height values of 6.919 to 15.67 mm for R.

pacifica, and 5.976 to 6.088 mm for C. carib-

aearum, agreeing with R. pacifica having a

taller test. In addition, Martínez-Melo (2008)

mentioned that C. caribaearum had a lower

distance from the peristome to the anterior edge

of the test. We confirm her statement because

the Da-pta values for C. caribaearum range

from 1.354 to 8.235 mm, and 1.55 to 15.789 for

R. pacifica. The author explained these lower

values of Da-pta for C. caribaearum as a result

of the higher growth of its skeleton.

When analyzing the intraspecific varia-

tion in the test length and width in proportion,

we found that R. pacifica had 1:1.047, which

means it is slightly longer than it is wide; in

C. caribaearum the proportion is 1:1.185, also

being longer than wide. From the measure-

ments of TWl, THl, AL, PL, PW, AIL, AIIIL,

MAW, PIW, PB, AFFP, TLa/TW, TLa/Da-ppa,

TLo/Da-pta, THl/TLl, PpL/PpW, A, PBS, PS,

ASP, PL/PW, AIL/AIIIL, and MAW/PIW, we

conclude that the small specimens developed a

more inflated form during their juvenile stage,

with longer and wider ambulacra; then, when

they reach the adult form, they developed a

more flat test gibbosity inside their own trian-

gular, inflated shape, unlike C. caribaearum

which keep almost the same shape during

their transition from small to large size (3.313-

22.959 mm).

Regarding the variations of the peristome,

although in both species the relationship

between the measurements of the top of the test

and the shape of the base of the peristome is not

very clear, it is possible to identify the variation

of the base of the peristome between species; in

C. caribaearum, the smaller specimens (lateral

height and lateral width) have a straight shape,

while in larger specimens the base is rounded.

We observed a similar behavior in R. paci-

fica, since in the smaller specimens the base

of the peristome is triangular and tall; on the

other hand, in the larger specimens the base

is perceived as completely triangular. Souto

and Martins (2018) assigned a neotype of C.

caribaearum (with measurements of test length

= 26.68 mm and test height = 22.65 mm) men-

tioning that the shape of the peristome is pen-

tagonal; this probably indicates that the growth

of the test is related with the change in the

shape of the peristome base and, consequently,

of the complete shape of the peristome.

Rhyncholampas pacifica shows a test with

slender form, which corresponds to a lon-

ger and pointier interambulacral basicoronal

plate V towards the peristome while in C.

caribaearum the test shape is broader with a

flattened plate. As suggested by Saitoh and

Kanazawa (2012), slender forms tend to dig

deeper into the substratum whereas robust

forms dig shallow; the anterior suggests that

R. pacifica is more adapted to dig deeper in

certain types of substratum (e.g. sandy) than C.

caribaearum.

We confirm that R. pacifica has a taller

test than C. caribaearum, while the latter has

a lower distance from the peristome to the

anterior edge of the test. We also recognize two

intraspecific patterns between the ambulacra

length and angles, and the peristome shape, for

each living species.

In sum, it is demonstrated that morphomet-

ric data of the tests and peristome are useful

to address taxonomical issues on recent cas-

siduloids, suggesting that more morphometric

studies including other species could be carried

out. Moreover, new morphometric analysis of

the species studied here adding specimens from

the rest of their geographic distribution range

would be useful to compare these results.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we

436

Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 423-437, March 2021 (Published Mar. 30, 2021)

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We are thankful to Camilla Souto (Smith-

sonian Institution, Washington D.C., USA),

Sergio A. Martínez (Universidad de la Repúbli-

ca de Uruguay, Montevideo, Uruguay) and

an anonymous reviewer for providing feed-

back that improved the quality of the manu-

script. Thanks to Ma. Esther Diupotex Chong

and Alicia Durán González (ICML, UNAM)

for their technical support at the Colección

Nacional de Equinodermos (ICML, UNAM).

We would like to thank M.G. Lovegrove for

his valuable comments on the manuscript’s

English. CACV (scholarship holder 666781)

thanks the Consejo Nacional de Ciencia y Tec-

nología (CONACyT) for his doctorate grant

722925. DGAPA-UNAM, PAPIIT Project No.

IN108717 “Tasas de evolución y paleobiogeo-

grafia de la familia Cassidulidae (Echinoder-

mata: Echinoidea)”.

RESUMEN

Variabilidad morfológica de especies recientes del

orden Cassiduloida (Echinodermata: Echinoidea)

de México

Introducción: En México, hay dos especies vivientes

registradas de Cassiduloida: Cassidulus caribaearum y

Rhyncholampas pacifica. La mayoría de los estudios taxo-

nómicos sobre casiduloides han utilizado morfología exter-

na, pedicelarios y caracteres morfométricos; sin embargo,

la variación intraespecífica de caracteres cuantitativos y

cualitativos ha sido poco evaluada. Objetivo: Comparar

la morfología básica de R. pacifica y C. caribaearum.

Métodos: Examinamos un total de 2 158 especímenes de

R. pacifica y C. caribaearum, seleccionando 50 para eva-

luar la forma y el tamaño con regresión lineal y análisis de

componentes principales. Seleccionamos 62 especímenes

adicionales por especie para identificar correlaciones sig-

nificativas de caracteres y grupos morfológicos dentro de

las especies. Resultados: Existe una relación directa entre

la longitud de la testa y el ancho de la testa. La Altura de

la testa / Anchura de la testa y la Longitud total (vista oral)

/ Distancia desde el ambitus hasta el ápice del peristoma,

son las dos proporciones principales para distinguir ambas

especies. C. caribaearum está más comprimido dorsoven-

tralmente y tiene una base del peristoma redonda; versus

R. pacifica que tenía una alta y triangular. Hay cuatro

grupos morfológicos de C. caribaearum y dos grupos de

R. pacifica. Conclusiones: Estas dos especies se pueden

distinguir con caracteres morfológicos confiables, en los

que la forma del peristoma sugiere que R. pacifica está

más adaptada para excavar más profundamente en ciertos

tipos de sustratos.

Palabras clave: Neognathostomata; Cassiduloida; morfo-

metría; México.

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