Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e49359, enero-diciembre 2024 (Publicado Ene. 29, 2024)

Phylogenetic relationships of the Mangrove Hummingbird,

“Amazilia” boucardi (Apodiformes: Trochilidae) of Costa Rica

Federico J. Albertazzi1*; https://orcid.org/0000-0002-8488-3941

Ghisselle Alvarado2; https://orcid.org/0009-0001-0041-4073

F. Gary Stiles3; https://orcid.org/0000.0002.8707.3987

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

federico.albertazzi@ucr.ac.cr (Correspondence*)

2. Museo Nacional de Costa Rica, Costa Rica; galvarado@museocostarica.go.cr

3. Instituto en Ciencias Naturales, Universidad Nacional de Colombia, Colombia; fgstilesh@unal.edu.co

Received 23-X-2023. Corrected 20-XI-2023. Accepted 25-I-2024.

ABSTRACT

Introduction: A recent revision of the generic classification of the Trochilidae based on DNA sequences revealed

many inconsistencies with the current generic classification, largely based on plumage characters subject to

homoplasy, especially in the Trochilini, the largest tribe. A thorough generic reorganization brought the classifi-

cation into accord with the phylogeny, but due to lack of genetic data, two species remained unclassified. One of

these was the Mangrove Hummingbird, “Amazilia” boucardi, endemic to Costa Rica and included in the IUCN

red list of threatened species.

Objective: To obtain molecular evidence to clarify the generic relationships of “A.” boucardi.

Methods: We isolated DNA from tissues of this species and amplified 4 nuclear and 4 mitochondrial fragments

and compared these with homologous fragments from 56 species in the Trochilini, constructing phylogenetic

trees with maximum likelihood and Bayesian methods.

Results: Our phylogenetic analyses confirmed the placement of boucardi in the Trochilini and definitely excluded

it from Amazilia but placed it with high confidence in the genus Chrysuronia Bonaparte, 1850, within which its

closest relative is C. coeruleogularis, which also inhabits mangroves.

Conclusions: Our genetic data based on nuclear and mitochondrial regions clearly indicate the relationship of A.

boucardi and L. coeruleogularis. Moreover, it is also supported by their habitat distribution in the mangroves of

the Pacific coast of Costa Rica and Western Panama. Therefore, we suggested to exclude A. boucardi as “incertae

sedis”.

Key words: endemic hummingbird; mangroves; nuclear and mitochondrial genes; phylogeny; taxonomy.

RESUMEN

Relaciones filogenéticas del Colibrí manglero “Amazilia” boucardi

(Apodiformes: Trochilidae) de Costa Rica

Introducción: Una revisión reciente de la clasificación de la familia Trochilidae con base en secuencias de ADN

demostró muchas incongruencias con la clasificación genérica previa, que había sido hecho con base en caracte-

res del plumaje muy sujetos a homoplasia, especialmente en la tribu más grande, Trochillini. Una reorganización

de los géneros logró llevar su clasificación genérica a la concordancia con la filogenia, pero debido a la ausencia

de datos genéticos, dos especies permanecieron sin clasificar. Una de estas fue el colibrí de manglar Amazilia

boucardi, una especie endémica de Costa Rica, considerada como amenazada en la lista roja de la UICN.

https://doi.org/10.15517/rev.biol.trop..v72i1.49359

VERTEBRATE BIOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e49359, enero-diciembre 2024 (Publicado Ene. 29, 2024)

INTRODUCTION

The Mangrove Hummingbird Amazilia

boucardi is an endemic species of Costa Rica,

and has been considered Near Threatened

(IUCN, 2021). Males are bronze-green above,

the throat and breast are bright bluish green,

the abdomen and undertail coverts white; the

tail is blackish and shallowly forked with the

outer two rectrices notably narrow. The female

is mostly white below, spotted with bronze-

green laterally, the tail is blackish, the outer

two rectrices narrowly tipped grayish-white

(Stiles & Skutch 1989). Its habitat is mangrove

swamps, mainly those where the piñuela man-

grove Pelliciera rhizophorae abounds, though

it sometimes ventures into the adjoining forest

edge or secondary growth. It feeds on nectar

from Pelliciera flowers and insects, as well

as visiting flowers of Lonchocarpus trees and

epiphytes. In Costa Rica it is found along the

Pacific coast and is now rare around the Golfo

de Nicoya but more common southward, espe-

cially around the Golfo Dulce.

The species boucardi was described in the

monotypic genus Arena by Mulsant and Ver-

reaux (1877) but the genus name was changed

to Arinia and finally Arenella because the first

two names were preoccupied. Ridgway (1911)

and Cory (1918) placed boucardi in the genus

Lepidopyga because of similarity of its tail and

plumage pattern to those of this genus, but

Peters (1945) subsumed it into the large genus

Amazilia Lesson, 1843. The study of McGuire

et al. (2014) derived a phylogeny of the Tro-

chilidae based upon DNA sequencing of ca.

284 species representing nearly all the genera

in the family and indicated that numerous

reorganizations of the current generic classi-

fication (Schuchmann, 1999) were necessary.

Over half of the incongruences between the

previous generic taxonomy and the phylogeny

occurred in the largest tribe, the Trochilini or

Emeralds. A high proportion of the Emerald

species show very limited morphological varia-

tion, with many genera having been based on

homoplastic characters of the plumage. Follow-

ing an extensive review of the many problems

of nomenclature (Stiles et al., 2017a), Stiles et al.

(2017b) proposed a new generic classification

of the Emeralds; one of their major changes was

the dismemberment of the genus Amazilia, spe-

cies of which appeared in nine different genera

based on molecular data. However, due to the

lack of genetic material (DNA), the generic

placement of two species including boucardi

could not be determined and given the wide-

spread homoplasy in plumage characters, Stiles

et al. (2017b) were obliged to leave “Amazilia”

boucardi as “incertae sedis” (uncertain place).

Therefore, the present study aims to provide the

first molecular data based on DNA segments

from the nucleus and mitochondria to deter-

mine the generic relationships “A”. boucardi

within the Trochilini.

Objetivo: Obtener evidencia molecular para esclarecer las relaciones genéricas de A. boucardi.

Métodos: Se aisló ADN de tejidos de esta especie y se amplificaron 4 fragmentos de ADN del núcleo y 5 de la

mitocondria, y se compararon con fragmentos homólogos de 56 especies en la tribu Trochillini, generando árbo-

les filogenéticos con métodos de máxima verosimilitud y bayesiano.

Resultados: Los análisis filogénticos obtenidos confirmaron la ubicación de boucardi en Trochilini y definiti-

vamente la excluyó del género Amazilia, pero la ubicó con un alto grado de confianza en el género Chrysuronia

Bonaparte, 1850, dentro los cuales su pariente más cercano es C. coeruleogularis, que también habita manglares.

Conclusiones: Nuestros datos genéticos basados en regiones nucleares y mitocondriales indican claramente la

relación entre A. boucardi and L. coeruleogularis. Es más, lo anterior se sustenta por su distribución en los man-

glares de la costa Pacífica de Costa Rica y oeste de Panamá. Por lo tanto, sugerimos excluir a A. boucardi como

“incertae sedis”.

Palabras clave: colibrí endémico; filogenia; genes nucleares y mitocondriales; manglares; taxonomía.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e49359, enero-diciembre 2024 (Publicado Ene. 29, 2024)

MATERIALS AND METHODS

Tissue material, DNA extraction, PCR

amplification and sequencing, DNA sequence

data: A total of 3 tissue samples (heart, liver,

and breast muscle) were included in the analy-

ses. Because “A”. boucardi is an endemic species

and it has been included in the red list of UICN,

only one immature male specimen was collect-

ed by GA in Sierpe, Puntarenas Province, Costa

Rica (8°52’06” N & 83°29’29” W). The speci-

men was identified by GA and FGS and depos-

ited in the ornithology collection of the Museo

Nacional de Costa Rica (voucher MN29285).

Total DNA was isolated from each tissue using

a modified CTAB 2 % protocol (Doyle &

Doyle, 1990). DNA sequences for amplifica-

tion were 4 nuclear fragments (intron 7 of beta

fibrinogen “FGB”, intron 5 of adenylate kinase 1

“AK1”; a fragment of the ornithine decarboxyl-

ase gene from end exon 6 and beginning exon 8

“ODC”; a fragment of Z-linked muscle skeletal

receptor tyrosine “MUSK”) and 4 mitochon-

drial segments (complete gene ND2, partial

ND4, complete tRNA-His and tRNA-Ser). PCR

amplifications and sequencing were generated

using the primers described by McGuire et

al. (2007). PCR reactions (25 µl) containing

80 ng (1.5 µl) were performed according to

standard protocols. PCR products were puri-

fied using the kit NucleoSpin Gel and PCR

clean up (Macherey-Nagel, USA). Sequencing

reactions were performed in both directions

using Big Dye Termination Cycle Sequenc-

ing kit ver. 3.1 (Applied Biosystems, USA)

following the manufacturer’s protocol. Cycle

sequencing products were visualized on an ABI

3730 automated sequencer (Applied Biosys-

tem, USA). Sequences from 6 loci have been

deposited in GenBank NCBI for FGB (acces-

sion number MT332711), AK1 (MT332712),

ODC (MT332708), MUSK (MT332707), ND2

(MT332709) and ND4/tRNA-His/tRNA-Ser

(MT332710). DNA sequences from 6 loci were

retrieved directly from GenBank.

Data analysis: All sequences were man-

ually edited using Geneious Prime version

2021.0.3 (BIOMATTERS Ltd, New Zealand)

(Kearse et al., 2012) applying the alignment

method MUSCLE ver. 3.8.425 using default set-

tings, and then adjusted manually.

Phylogenetic analysis: Data partition and

nucleotide substitution models were identi-

cal to those mentioned by McGuire et al.

(2007) and McGuire et al. (2014). The selec-

tion of the best-fitting model for each region

was: FGB GTR+I+G, AK1 GTR+G, ODC

GTR+I+G, MUSK GTR+I+G, ND2 GTR+I+G,

ND4 GTR+I+G, and tRNAs HKY+I. For the

RAxML ver. 8.2.11 (Stamatakis, 2014) analyses,

the GTRGAMMA model was used for both tree

inference and bootstrapping, with 1 000 non-

parametric bootstrap pseudoreplicates. Bayes-

ian analyses applied to the concatenated matrix

were conducted with the program MrBayes ver.

3.2.6 (Ronquist et al., 2011), 100 000 000 gen-

erations were iterated, and sampled every 1 000

generations, for two runs each with four chains;

the first 25 % trees were discarded as burn-in.

The final trees were visualized using FigTree

ver. 1.4.4 (Rambault, 2018).

RESULTS

PCR amplification, sequencing, and

alignment: DNA amplification from three dif-

ferent tissues was successful and the sequences

were the same, therefore we performed all

analyses with DNA from the liver. The PCR

products of the 6 loci, FGB (979 bp), AK1 (529

bp), ODC (603 bp), MUSK (596 bp), ND2 (945

bp), ND4/tRNA-His/tRNA-Ser/tRNA-Leu par-

tial (885 bp), and BLAST analysis confirmed

the identity. The same DNA fragments were

obtained from GenBank for 56 Emerald spe-

cies (Trochilini) according to McGuire et al.

(2014) (SMT1). Alignments with MUSCLE of

the concatenated data (5 009 positions) did not

reveal any significant topological differences by

visual comparison.

Phylogenetic analysis: The concatenated

alignment of 6 loci for 56 species was used to

generate a rooted Bayesian and RAxML tree

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e49359, enero-diciembre 2024 (Publicado Ene. 29, 2024)

(Fig. 1). The robustly resolved phylogeny of

the tribe Trochilini achieved in our study sub-

divided the tribe Trochilini into four groups in

accordance with the classification proposed by

Stiles et al. (2017b). Analyses of the complete

concatenated molecular data produced con-

gruent phylogenies with two methodological

approaches (Fig. 1). Our analysis confirmed the

placement of boucardii in group D with strong

support. This group has the greatest number

of Emerald species, including those formerly

in Amazilia but now separated in seven dif-

ferent genera. The analysis placed boucardi

closest to “Lepidogyga” coeruleogularis (Gould,

1851), with both now included within the

genus Chrysuronia Bonaparte 1850, as circum-

scribed by Stiles et al.(2017b), also with strong

support. The genus Lepidopyga Reichenbach,

1855 is now considered a synonym of Chry-

suronia. We therefore place boucardi in the

genus Chrysuronia.

DISCUSSION

Our conclusion that the closest relative of

C. boucardi is coeruleogularis (possibly they are

sister species) is interesting, because both are

habitants of mangrove swamps along the Pacific

coast of Middle America, boucardi in Costa

Rica and coeruleogularis in Western Panama. In

Colombia, the distribution of C. coeruleogularis

then follows the Caribbean slope in mangroves

and adjacent habitats, while its place in the

Pacific mangroves southward from extreme

Eastern Panama is occupied by C. humboldtii

(deLattre & Bourcier, 1846). Our results also

agree with the classification of boucardi in Lepi-

dopyga by Ridgway (1911) and Cory (1918):

both species are now included in Chrysur-

onia. This leaves only the Honduran Emerald

“Amazilia” luciae (Lawrence, 1867) still unclas-

sified due to lack of a genetic sample. Although

Schuchmann (1999) considered this species

Fig. 1. Bayesian consensus tree inferred from a dataset of emerald hummingbirds group using six loci. Letters represent

subgroup classifications according to Stiles et al. (2017b) and are highlighted in different colors. The numbers above the

nodes correspond to percent of posterior probabilities values and maximum likelihood bootstrap. Amazilia boucardi is

denoted by an asterisk.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e49359, enero-diciembre 2024 (Publicado Ene. 29, 2024)

and boucardi to be possibly sisters (perhaps

because of their geographical proximity), there

are differences in their plumages that leave

room for uncertainty, hence we prefer to leave

luciae as “incertae sedis” until a genetic sample

can be analyzed.

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

a07v72n1-MS1

ACKNOWLEDGMENTS

Special thanks to Museo Nacional de Costa

Rica. This research was supported by VI-UCR

grants (VI-801-B8-269).

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