Phylogenetic relationships of the Mangrove Hummingbird, “ Amazilia” boucardi (Apodiformes: Trochilidae) of Costa Rica

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 classification 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 ”.


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 bronzegreen 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 mangrove 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, especially around the Golfo Dulce.
The species boucardi was described in the monotypic genus Arena by Mulsant and Verreaux (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 Trochilidae 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 classification (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 variation, with many genera having been based on homoplastic characters of the plumage.Following 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, species 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 widespread 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 determine the generic relationships "A".boucardi within the Trochilini.

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 analyses.Because "A".boucardi is an endemic species and it has been included in the red list of UICN, only one immature male specimen was collected by GA in Sierpe, Puntarenas Province, Costa Rica (8°52'06" N & 83°29'29" W).The specimen was identified by GA and FGS and deposited 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 amplification were 4 nuclear fragments (intron 7 of beta fibrinogen "FGB", intron 5 of adenylate kinase 1 "AK1"; a fragment of the ornithine decarboxylase gene from end exon 6 and beginning exon 8 "ODC"; a fragment of Z-linked muscle skeletal receptor tyrosine "MUSK") and 4 mitochondrial 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 purified using the kit NucleoSpin Gel and PCR clean up (Macherey-Nagel, USA).Sequencing reactions were performed in both directions using Big Dye Termination Cycle Sequencing kit ver.3.1 (Applied Biosystems, USA) following the manufacturer's protocol.Cycle sequencing products were visualized on an ABI 3730 automated sequencer (Applied Biosystem, USA).Sequences from 6 loci have been deposited in GenBank NCBI for FGB (accession 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.

RESULTS
PCR amplification, sequencing, and alignment: DNA amplification from three different 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 partial (885 bp), and BLAST analysis confirmed the identity.The same DNA fragments were obtained from GenBank for 56 Emerald species (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 (Fig. 1).The robustly resolved phylogeny of the tribe Trochilini achieved in our study subdivided 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 congruent 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 different genera.The analysis placed boucardi closest to "Lepidogyga" coeruleogularis (Gould, 1851), with both now included within the genus Chrysuronia Bonaparte 1850, as circumscribed by Stiles et al.(2017b), also with strong support.The genus Lepidopyga Reichenbach, 1855 is now considered a synonym of Chrysuronia.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 Lepidopyga by Ridgway (1911) and Cory (1918): both species are now included in Chrysuronia.This leaves only the Honduran Emerald "Amazilia" luciae (Lawrence, 1867) still unclassified due to lack of a genetic sample.Although Schuchmann (1999) considered this species 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 conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements.All financial sources are fully and clearly stated in the acknowledgments section.A signed document has been filed in the journal archives.

Fig. 1 .
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.