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.
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, “Amaziliaboucardi, 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
Key words: endemic hummingbird; mangroves; nuclear and mitochondrial genes; phylogeny; taxonomy.
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.
2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e49359, enero-diciembre 2024 (Publicado Ene. 29, 2024)
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)
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. BecauseA”. 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 manufacturers 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
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).
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.
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.
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