Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

Evaluating the reliability of DNA Barcoding for Central American Pacific

shallow water echinoderms identification: a molecular taxonomy

and database accuracy analysis

José Leonardo Chacón-Monge1, 2, 4*; https://orcid.org/0000-0002-9754-1254

Juan Ignacio Abarca-Odio1; https://orcid.org/0000-0002-3155-8264

Kaylen González-Sánchez1, 3; https://orcid.org/0000-0002-7208-9302

1. Centro de Investigación en Ciencias del Mar y Limnología, Universidad de Costa Rica, 11501-2060, San José, Costa

Rica; jose.chaconmonge@ucr.ac.cr (*corresponding author), jiao24@gmail.com, kaylen.gonzalez@ucr.ac.cr

2. Museo de Zoología, Centro de Investigación en Biodiversidad y Ecología Tropical, Universidad de Costa Rica,

Costa Rica.

3. Centro de Investigación en Biología Celular y Molecular Universidad de Costa Rica, Costa Rica.

4. Escuela de Biología, Universidad de Costa Rica, Costa Rica.

Received 11-VII-2023. Corrected 20-XI-2023. Accepted 15-I-2024.

ABSTRACT

Introduction: Molecular divergence thresholds have been proposed to distinguish recently separated evolutive

units, often displaying more accurate putative species assignments in taxonomic research compared to traditional

morphological approaches. This makes DNA barcoding an attractive identification tool for a variety of marine

invertebrates, especially for cryptic species complexes. Although GenBank and the Barcode of Life Data System

(BOLD) are the major sequence repositories worldwide, very few have tested their performance in the identifica-

tion of echinoderm sequences.

Objective: We use COI echinoderm sequences from local samples and the molecular identification platforms

from GenBank and BOLD, in order to test their accuracy and reliability in the DNA barcoding identification for

Central American shallow water echinoderms, at genus and species level.

Methods: We conducted sampling, tissue extraction, COI amplification, sequencing, and taxonomic identifica-

tion for 475 specimens. The 348 obtained sequences were individually enquired with BLAST in GenBank as well

as using the Identification System (IDS) in BOLD. Query sequences were classified depending on the best match

result. McNemar’s chi-squared, Kruskal-Wallis’s and Mann-Whitney’s U tests were performed to prove differ-

ences between the results from both databases. Additionally, we recorded an updated list of species reported for

the shallow waters of the Central American Pacific.

Results: We found 324 echinoderm species reported for Central American Pacific shallow waters. Only 118 and

110 were present in GenBank and BOLD databases respectively. We proposed 325 solved morphology-based

identities and 21 provisional identifications in 50 putative taxa. GenBank retrieved 348 molecular-based identi-

fications in 58 species, including twelve provisional identifications in tree taxa. BOLD recovered 170 COI iden-

tifications in 23 species with one provisional identification. Nevertheless, 178 sequences retrieved unmatched

terms (in 34 morphology-based taxa). Only 86 sequences (25 %) were retrieved as correct identifications and

128 (37 %) as identification errors in both platforms. We include 84 sequences for eleven species not represented

in GenBank and 65 sequences for ten species in BOLD Echinoderm COI databases. The identification accuracy

using BLAST (175 correct and 152 incorrect identifications) was greater than with IDS engine (110 correct and

218 identification errors), therefore GenBank outperforms BOLD (Kruskal-Wallis = 41.625, df = 1, p < 0.001).

https://doi.org/10.15517/rev.biol.trop..v72iS1.58997

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

INTRODUCTION

The use of DNA barcoding with COI

sequences is proposed as a highly efficient

identification tool for a diverse range of

marine invertebrates. Given that interspecific

divergence gaps tend to be wider than intra-

specific divergences, divergence thresholds

demonstrate the capability to effectively dis-

tinguish recently diverged groups, leading to

Conclusions: Additional echinoderm sample references are needed to improve the utility of the evaluated DNA

barcoding identification tools. Identification discordances in both databases may obey specific parameters used

in each search algorithm engine and the available sequences. We recommend the use of barcoding as a comple-

mentary identification source for Central American Pacific shallow water echinoderm species.

Key words: taxonomy; universal primers; mitochondrial DNA; vouchers; scientific collections; BioMar-ACG;

Costa Rica.

RESUMEN

Evaluando la fiabilidad del Código de Barras de ADN para la identificación de equinodermos

en aguas poco profundas del Pacífico de América Central: un análisis de taxonomía molecular

y precisión de bases de datos

Introducción: Se han propuesto los umbrales de divergencia molecular para distinguir unidades evolutivas

recientemente separadas, que a menudo muestran asignaciones de especies putativas más precisas en la investi-

gación taxonómica en comparación con los enfoques morfológicos tradicionales. Esto hace que los Códigos de

Barras de ADN sean una herramienta de identificación atractiva para una variedad de invertebrados marinos,

especialmente para complejos de especies crípticas. Aunque GenBank y Barcode of Life Data System (BOLD)

son los principales repositorios de secuencias en todo el mundo, muy pocos han probado su desempeño en la

identificación de secuencias de equinodermos.

Objetivo: Utilizamos secuencias de equinodermos COI de muestras locales y las plataformas de identificación

molecular de GenBank y BOLD, para probar su precisión y confiabilidad en la implementación de códigos de

barras de ADN para equinodermos de aguas someras de Centroamérica, a nivel de género y especie.

Métodos: Realizamos muestreo, extracción de tejido, amplificación de COI, secuenciación e identificación taxo-

nómica de 475 especímenes. Las 348 secuencias obtenidas fueron consultadas individualmente con BLAST en

GenBank así como utilizando el Sistema de Identificación (IDS) en BOLD. Las secuencias consultadas se clasi-

ficaron según el mejor resultado de coincidencia. Se realizaron las pruebas chi-cuadrado de McNemar, Kruskal-

Wallis y U de Mann-Whitney para comprobar diferencias entre los resultados de ambas bases de datos. Además,

registramos una lista actualizada de especies reportadas para las aguas someras del Pacífico Centroamericano.

Resultados: Encontramos 324 especies de equinodermos reportadas para aguas someras (< 200 m) del Pacífico

centroamericano. Sólo 118 y 110 estaban presentes en las bases de datos GenBank y BOLD respectivamente.

Propusimos 325 identidades resueltas basadas en morfología y 21 identificaciones provisionales en 50 taxones

putativos. GenBank recuperó 348 identificaciones de base molecular en 58 especies, incluidas doce identificacio-

nes provisionales en tres taxones. BOLD recuperó 170 identificaciones de COI en 23 especies con una identifi-

cación provisional. Sin embargo, 178 secuencias recuperaron términos no coincidentes (en 34 taxones basados

en morfología). Sólo 86 secuencias (25 %) se recuperaron como identificaciones correctas y 128 (37 %) como

errores de identificación en ambas plataformas. Incluimos 84 secuencias para once especies no representadas en

GenBank y 65 secuencias para diez especies ausentes en las bases de datos BOLD Echinoderm COI. La precisión

de la identificación usando BLAST (175 identificaciones correctas y 152 incorrectas) fue mayor que con el motor

IDS (110 correctas y 218 errores de identificación), por lo tanto, GenBank supera a BOLD (Kruskal-Wallis =

41.625, df = 1, p < 0.001).

Conclusiones: Se necesitan muestras adicionales de equinodermos de referencia para mejorar la utilidad de las

herramientas de identificación de códigos de barras de ADN evaluadas. Las discordancias de identificación en

ambas bases de datos pueden obedecer a parámetros específicos utilizados en cada algoritmo de búsqueda y a las

secuencias disponibles. Recomendamos el uso de códigos de barras como fuente de identificación complementa-

ria para las especies de equinodermos de aguas someras del Pacífico centroamericano.

Palabras clave: taxonomía; primers universales; ADN mitocondrial; vouchers; colecciones científicas; BioMar-

ACG; Costa Rica.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

more accurate putative species assignments in

taxonomic research compared to morphologi-

cal approaches, especially for cryptic complexes

(Laakmann et al., 2016; Layton et al., 2016).

Therefore, molecular taxonomy is a valuable

alternative for solving conflicts related to the

application of morphology-based taxonomic

diagnosis, particularly when open-access bar-

code reference libraries are available. There

are 23 297 echinoderm COI entrances in Gen-

Bank and 37 267 public records in BOLD for

at least 2 115 echinoderm species (verified 18

Nov. 2023). However, only few studies have

tested the accuracy of public sequence reposi-

tories in DNA identification (Laakmann et al.,

2016; Meiklejohn et al., 2019) and none have

explored it for Central American echinoderm

molecular taxonomy.

Many Central American countries share

a common origin in echinoderm research,

marked initially by North American and Euro-

pean expeditions in the late nineteenth century,

leading to the description of the majority of

Central American echinoderm species. Subse-

quently, there was an increase in investigations

conducted by regional scientists or research

institutes in the second half of the twentieth

century (Alvarado et al., 2013; Alvarado & Fab-

regat-Malé, 2021). Historically, samples have

been kept in foreign museums, limiting access

to valuable vouchers and type material for local

scientists (Alvarado et al., 2017). Nevertheless,

the Colección Nacional de Equinodermos Ma.

Elena Caso Muñoz at Instituto de Ciencias

del Mar y Limnología, Universidad Nacional

Autónoma de México, hosts one of the largest

specimen collections in Latin America (Alvara-

do & Solís-Marín, 2013). Furthermore, the

Museo de Zoología (MZ-UCR) at the Centro

de Investigación en Biodiversidad y Ecología

Tropical (CIBET, Universidad de Costa Rica)

has consistently expanded its echinoderm col-

lection, facilitating local scientists’ access to

more in-depth voucher scrutinizing and creat-

ing opportunities for developing collaborative

networks in regional integrative research (Cor-

tés & Joyce, 2020; Kress, 2014; Miralles et al.,

2020; Schilthuizen et al., 2015).

While there have been ongoing efforts in

Central American echinoderm research with a

consistent focus on ecological and taxonomic

issues, the exploration of related areas as molec-

ular taxonomy has been limited (Alvarado et

al., 2013; Alvarado & Fabregat-Malé, 2021;

Varela-Sánchez et al., 2020). Since GenBank

and the Barcode of Life Data System (BOLD)

are the main molecular public repositories, we

tested their accuracy and reliability for COI

echinoderm sequences identification at genus

and species level (i.e. barcoding). This study

started the establishment of a validated and

comprehensive echinoderm reference library,

comprising morphological, and molecular data,

as well as additional metadata for samples col-

lected at Área de Conservación Guanacaste,

Costa Rica (Cortés & Joyce, 2020). However,

the biogeographic and bathymetric distribu-

tion of many of the present species extends the

applicability of this library to Central American

Pacific shallow waters (< 200 m depth) (Alvara-

do et al., 2010; Solís-Marín et al., 2013). We

matched morphological identifications with the

molecular species assignments (correct identi-

fications or identification errors) as a reference

method for database improvement in further

taxonomic and systematic studies in the region.

MATERIALS AND METHODS

Sample preparations: Sampling and tis-

sue extraction for 475 vouchers were con-

ducted during the BioMar-ACG echinoderm

surveys in 25 sample sites (Fig. 1). Different

body parts were dissected for DNA extraction

depending on the taxonomic group, gener-

ally dermis, podia, or arm tips were dissected

and stored in absolute ethanol (96 %) on lysis

plates, then frozen until its processing. Iden-

tifications were done by direct observation,

using optic stereoscopes and light microscopes.

Taxonomic identifications were based on pro-

posed diagnostic characters from external

and internal anatomy or skeletal elements (i.e.

ossicles) sourced in taxonomic specialized lit-

erature (Borrero-Pérez & Vanegas-González,

2019, Borrero-Pérez & Vanegas-González,

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

2020; Granja-Fernández et al., 2014, Granja-

Fernández et al, 2020; Lessios, 2005; Martín-

Cao-Romero et al, 2017; Massin et al., 2002;

Solís-Marín et al., 2009, Solís-Marín et al.,

2014, Solís-Marín et al., 2020; Woo et al., 2015).

The nomenclature for taxonomic designations

follows the World Register of Marine Species

(WoRMS Editorial Board, 2024). When identi-

fications could not be established at the species

level, provisional identifications were given to

the lowest possible taxon.

Data collection: In order to assess the

whole representation of the Central American

shallow water Pacific echinoderm richness,

we registered an updated list of species report-

ed from the Central American Pacific coast

(excluding Panama) from 0 to < 200 m depth

(Appendix 1). The DNA extraction, ampli-

fication, and sequencing of COI fragments

were conducted by the Center for Biodiversity

Genomics, Guelph University, Canada (Cortés

& Joyce, 2020). Throughout an automatized

process developed and improved since the

early 2000’s (Ivanova et al., 2006; deWaard et

al., 2008; for detailed protocols). The obtained

sequences were analyzed with the GenBank and

BOLD identification tools.

DNA barcode analyses: The obtained

sequences were individually searched with the

Nucleotide Basic Local Alignment Search Tool

(BLAST Best Match) in GenBank’s identifica-

tion core (Altschul et al., 1990) and also com-

pared with the Identification System (IDS)

engine available in the Barcode of Life Data

System, BOLD (Meiklejohn et al., 2019; Ratnas-

ingham & Hebert, 2007). Setting the “standard

nucleotide BLAST” and the “Public Record

Barcode Database’’ for nucleic species identifi-

cation respectively, each obtained sequence was

requested under GenBank and BOLD built-in

search tools. Query sequences were considered

“Correctly identified” (C) if a record with the

same taxonomic name had the best match

statistic or highest percent identity, otherwise

they were classified as an Identification error

(IE) (Laakmann et al., 2016; Meiklejohn et al.,

2019). Morphologically unsolved taxa were

not included in identity comparisons, instead

Fig. 1. Echinoderm sample sites in the BioMar-ACG project at Área de Conservación Guanacaste, north Pacific of Costa

Rica. Peninsular sites (blue dots) and Archipelago sites (red dots) are shown. Modified from Chacón-Monge et al. (2021),

published with permission.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

were labeled as No Apply (NA). Given our

data nature, McNemar’s Chi-squared, Kruskal-

Wallis’s and Mann-Whitney’s U tests were per-

formed to prove the differences between these

databases and their identification accuracy.

RESULTS

Overall representativeness: About 466

echinoderm species have been registered in

Central America, and around 364 (78 %) have

been listed for Costa Rica (Alvarado & Fabre-

gat-Malé, 2021; Alvarado et al., 2017; Alvarado

et al., 2022; Chacón-Monge et al., 2021; Solís-

Marín et al., 2013). We listed a total of 324 echi-

noderm species reported for Central American

Pacific shallow waters (< 200 m) (Appendix

1), and about 236 (73 %) should be present in

Costa Rica (Alvarado et al., 2017; Borrero-Pérez

& Vanegas-González, 2020; Chacón-Monge et

al., 2021; Granja-Fernández et al., 2020; Solís-

Marín et al., 2020). Nevertheless, only 118

(36 %) and 110 (34%) of Central American

echinoderm fauna were represented in the

GenBank nucleotide search and BOLD public

databases respectively, with sequences for only

96 shared species and sharing 192 absences

(Appendix 1). Our database includes 348 COI

samples successfully sequenced in the BioMar-

ACG project for 44 morphology-based species

in 325 sequences (Appendix 1), and 21 provi-

sional identifications in seven taxa (Appendix

2). Representing only 19 % of Costa Rican and

14 % of Central American Pacific shallow water

echinoderm species, but 37 % and 40 % of the

total representativeness available for the region

in each database (GenBank and BOLD).

Sequence recovery: In terms of sequence

recovery, our study achieved a sequencing suc-

cess rate of 73 %, resulting in 348 sequences

out of 475 samples. Not too far from the

78 % obtained in a major Canadian echino-

derm database (1 285 vouchers), where the

sequencing failure was attributed to primer

effectiveness (Layton et al., 2016). Sequence

length ranges from 200 to 680 bp (average 629

bp). We proposed 325 solved and 21 unsolved

morphology-based identities in 50 putative

taxa (Appendix 2). All query sequences were

identified using the GenBank nucleotide iden-

tification engine. There were 56 species pro-

posed for 336 sequences and twelve provisional

identities for three taxa, with correspondences

from 81.2 % to 100 %. BOLD recovered 169

solved identifications in 22 taxa and one pro-

visional identification, with correspondences

from 97.06 % to 100 %. But 178 sequences were

retrieved as unmatched terms (in 33 morphol-

ogy-based taxa), last verification October 9th,

2023 (Appendix 2). The general taxonomic

representativeness, success and numbers of

proposed genera and species identities using

each identification method is summarized by

class (Appendix 3). Our study contributes to

the inclusion of sequences from local vouchers

in molecular databases. Specifically, we provide

84 sequences for eleven species not previously

represented in GenBank and 65 sequences for

nine species absent in BOLD (Appendix 2).

Barcoding identification: Although 39 of

the putative morphology-based taxa were rep-

resented at GenBank COI database (in 265 seq.)

and 41 morphology-based taxa were found

at BOLD sequence database (in 283 seq.),

not always the query sequence retrieved the

expected identification. For genus and species

level, identifications success differs (McNemar’s

chi-squared = 32.84, df = 1, p < 0.01; McNe-

mar’s chi-squared = 35.43, df = 1, p < 0.01)

between identification engines, and was greater

for GenBank at genus (Kruskal-Wallis = 52.44,

df = 1, p < 0.01) and species level (Kruskal-

Wallis = 41.625, df = 1, p < 0.01). According

to the GenBank research engine, there were

175 sequences correctly identified and 152

identification errors, while in the BOLD public

record barcode, there were 111 correct identifi-

cations and 216 identification errors (Table 1).

Otherwise 21 Vouchers did not apply for this

comparison, since they correspond to mor-

phology-based unsolved identities (Appendix

2). Only 87 (24 %) and 128 (37 %) sequences

were simultaneously correctly and incorrectly

identified respectively (Appendix 2). Despite

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

the identity percentage was higher for BOLD

compared to GenBank (Mann-Whitney U =

2110, p < 0.01), the accuracy for genus and spe-

cies identification using the BLAST Best Match

was better than the obtained by the IDS engine,

therefore GenBank outperforms BOLD identi-

fication at both taxonomic categories (Fig. 2).

Tabl e 1

Classification of query sequences for Central American

Pacific shallow water echinoderms using GenBank

(BLAST) and BOLD (IDS) identification tools

ID BLAST IDS

Correct (C) 175 111

Identification Errors (IE) 152 216

NA 21 21

No match 0 178

DISCUSSION

Identification success: Low species iden-

tification success was not expected, given the

relatively well representation of the proposed

taxa related to Central American Pacific shal-

low water echinoderms in GenBank and BOLD

libraries. It is also possible that the discrimina-

tory power for species-level identification may

be diminished on short sequences (< 430 bp),

but similar results have been obtained using

COI fragments of 650 bp and 130 bp for insect

identification (Grzywacz et al., 2017).

Misidentifications: Identification discor-

dances in both databases may obey specif-

ic parameters used in each search algorithm

engine, and sequences available for barcod-

ing under comparison (Layton et al., 2016).

GenBank compares the query sequence to the

nucleotide database, the Mega BLAST search

tool (established by default) is optimized with

similar sequences to search against all oth-

ers using the command-line interface. While

in BOLD, when public database records are

chosen, a selection from the published proj-

ects section in BOLD (including all public

COI records from BOLD and GenBank with

a minimum sequence length of 500 bp) is

automatically selected to compare the query

sequence (Meiklejohn et al., 2019). Therefore,

each requested sequence is compared against

the appropriate group of barcode sequences

according to the specific parameters for each

database. General discordances can appear

because of the specific evolutionary history of

species, imperfect species delineation and over-

looked diversity (“bad taxonomy” sensu Ebach

et al., 2006; Hörandl, 2007). In consequence,

putative species are more reliable when sup-

ported by various delimitation methods and

COI based techniques need to be evaluated

under integrative taxonomy (Laakmann et al.,

2016; Sonet et al., 2022).

Fig. 2. COI sequence identification accuracy (%) for Central American Pacific shallow water echinoderms using GenBank

(BLAST) and BOLD (IDS) identification tools.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

Considering all identification errors and

unmatched terms, after a consequent taxo-

nomic revision and morphologic comproba-

tion, we found 23 morphology based vouchers

in four putative species erroneously identified

or unsolved before the BLAST query (five

Holothuria arenicola previously identified as H.

impatiens; six H. impatiens identified as H. par-

dalis, two Ophioderma panamense identified as

O. cf. panamense and ten O. hendleri identified

as Ophioderma sp.). Seven of them were also

correctly identified by BOLD in two species

(five H. arenicola and two O. panamense, the

other 16 were retrieved as unmatched terms.

The rest of morphology-based unsolved taxa

correspond to juvenile stages. Based on the

molecular databases compared and employed

analysis, the suggested misidentifications may

correspond to real barcoding identification

errors. Rather than possible morphology based

errors, contrary to the expected rate of identi-

fication success using COI molecular DNA for

echinoderm identification. We strongly recom-

mend taking precautions when using public

sequence databases to identify morphologically

undetermined species of Central American

Pacific shallow water echinoderms. With spe-

cial awareness if they include not verified taxa,

juvenile stages or information from “unpub-

lished projects”, which might include unsolved

taxonomy. In contexts where no prior taxo-

nomic information about echinoderm samples

is known, accurate and reliable interpretations

of discordant identities may impose a challenge

(Sonet et al., 2022).

Regional representativeness: This effort

also marks a crucial step towards systematically

evaluating the representativeness and accu-

racy of the GenBank and BOLD identification

engines for echinoderm species in Central

American Pacific shallow waters. Not only

refining molecular identification tools, but also

increasing the overall regional representative-

ness of COI sequences databases. This work

is a first step in the DNA barcode library con-

struction for Central American Pacific shallow

water echinoderms, but additional echinoderm

sample references are needed to improve the

utility of DNA barcoding as an identification

tool in the region.

Applicability: DNA barcoding is a com-

plementary approach to biodiversity studies. It

is possible to obtain robust results using these

databases in combination, as well as to improve

data sets for systematic purposes and further

taxonomic research, by applying methods for

putative species identification based on COI

sequences for previously known taxonomic

identities. Nevertheless, we do not recommend

the use of molecular repositories as the main

source for Central American Pacific shallow

water echinoderm species identification. Echi-

noderm systematics could benefit from addi-

tional integrative taxonomy approaches and

molecular data analysis only if it is keeping

connection between voucher morphology and

sequences. It directly highlights the importance

of increasing the sequence’s representativeness

for many common and rare species in global

public scientific molecular repositories and

holding vouchers in local natural history collec-

tions with corroborated taxonomy.

Ethical statement: the 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 fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

See supplementary material

a18v72s1-MS1

ACKNOWLEDGEMENTS

We thank the Universidad de Costa Rica,

specially to the Vicerrectoría de Investigación,

the Centro de Investigación en Ciencias del

Mar y Limnología (CIMAR), the Museo de

Zoología (MZ-UCR) at Centro de Investig-

ación en Biodiversidad y Ecología Tropical

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58997, marzo 2024 (Publicado Mar. 01, 2024)

(CIBET) and to Escuela de Biología. For sup-

porting the BioMar-ACG project (808-B9-508)

and guaranteeing the availability of materials,

equipment, workspace, voucher and sample

storage that made possible this research. We

also thanks the Sistema Nacional de Áreas de

Conservación (SINAC) and the Ministerio de

Ambiente y Energía (MINAE) for gave the

research and collection permits (R-SINAC-

PNI-SE-002-2018 and R-SINAC-ACG-PI-004-

2019-BIOMAR). And to the Guanacaste Dry

Forest Conservation Fund for financing the

BioMar-ACG project. We are grateful to the

Cuajiniquil Diving Center, Gilberth Ampié,

Yelba Vega, Génesis Coto, Carolina Sheridan-

Rodríguez, Cristian Mora-Barboza, Juan José

Alvarado, Frank Joyce, Daniel Jansen, Winnie

Hallwachs and Jorge Cortés. For their help

during or before field sampling and facilities

for tissue processing. We greatly appreciate the

invitation from the scientific editors to par-

ticipate in this volume and to two anonymous

lecturers that peer reviewed our manuscript.

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