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Ibis (2023) doi: 10.1111/ibi.13181

Phylogenetic relationships and systematics of the

bamboo-specialist Amaurospiza blue seedeaters
JUAN I. ARETA,*1 MAR
IA JULIANA BEN
ITEZ SALD
IVAR,1 MIGUEL LENTINO,2 JHONATHAN MIRANDA,3,4
MATEUS FERREIRA,5 JOHN KLICKA6,7 & JORGE PEREZ-EM

2,8
AN
1
Laboratorio de Ecolog
ıa, Comportamiento y Sonidos Naturales (ECOSON), Instituto de Bio y Geociencias del
Noroeste Argentino (IBIGEO-CONICET), Salta, Argentina
2
Coleccio
n Ornitolo
gica Phelps, Caracas, Venezuela
3
Provita, Caracas, Venezuela
4

n Venezolana de Ornito
Unio
logos, Caracas, Venezuela
5
Centro de Estudos da Biodiversidade (CBio), Universidade Federal de Roraima (UFRR), Boa Vista, Roraima, Brazil
6
Burke Museum of Natural History, University of Washington, Seattle, Washington, USA
7
Department of Biology, University of Washington, Seattle, Washington, USA
8
Instituto de Zoolog
ıa y Ecolog
ıa Tropical, Universidad Central de Venezuela, Caracas, Venezuela

The Amaurospiza ‘seedeaters’ are bamboo-specialized mixed strategists, most often found
in bamboos in vegetative state, feeding on buds, shoots, petioles and insects. As bamboos
die after flowering, birds may wander in search of live vegetative bamboo. The three
currently recognized species of Amaurospiza are allopatrically distributed: the Blackish-
blue Seedeater Amaurospiza moesta in the Atlantic Forest of Argentina, Brazil and Para-
guay, and in forest enclaves in the Cerrado; the recently described Carrizal Seedeater
Amaurospiza carrizalensis known from a few localities in southeastern Venezuela; and
the Blue Seedeater Amaurospiza concolor distributed patchily from Mexico to Peru.
Three subspecies are recognized within A. concolor: relicta in southwest Mexico, concolor
from southern Mexico to Panama and aequatorialis from southwest Colombia to nor-
thwest Peru. Full species status has been advocated for relicta and aequatorialis but evi-
dence supporting their recognition is weak, while relicta was described in the
monospecific genus Amaurospizopsis. Here we (1) test the monophyly of Amaurospiza,
(2) reconstruct the phylogenetic relationships of all its constituent taxa using mitochon-
drial and nuclear markers, (3) re-assess species limits in Amaurospiza with the aid of
vocalizations and genetic and plumage data, and (4) discuss the link between bamboo
life history, biogeographical patterns and extent of genetic differentiation. Amaurospiza
was found to be monophyletic in both the ND2 and multilocus analyses. In the ND2
tree A. moesta and A. carrizalensis were sister to each other, A. concolor was found to be
paraphyletic because aequatorialis was placed as sister to moesta–carrizalensis, and a clade
including nominate concolor and relicta was sister to all the other taxa. The multilocus
tree showed the same relationships, but lacked nuclear samples of relicta. Mean ND2
pairwise distance between concolor and aequatorialis (8.3%) was greater than that
between moesta and carrizalensis (5.7%), while relicta diverged on average 1.0% from
nominate concolor. The South American clade has more slender bills and white unde-
rwing coverts, while the Central American clade has thicker bills and bluish underwing
coverts. All taxa exhibited typical Amaurospiza songs with quickly delivered, warbled,
pure and fairly high-pitched musical notes. Number of inflections/second exhibited a ste-
pped pattern, with concolor and relicta on the lower end and carrizalensis, aequatorialis

*Corresponding author.
Email: esporofila@yahoo.com.ar
Twitter: @Nachornis

© 2023 British Ornithologists' Union.

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2 J. I. Areta et al.

and moesta on the upper end. Similarly, moesta, carrizalensis and aequatorialis had overall
more inflections per note than concolor and relicta. Linear discriminant analysis using nine
acoustic variables correctly assigned all 62 songs to the correct taxon. Morphology, plu-
mage, vocalizations and phylogenetic data indicate that aequatorialis should be afforded
full species status as the Ecuadorian Seedeater (A. aequatorialis), suggest keeping relicta
as a subspecies of A. concolor and support continued recognition of A. carrizalensis. Our
data support merging Amaurospizopsis into Amaurospiza.
Keywords: Cardinalidae, Chusquea, Merostachys, Neotropical, speciation.

The Neotropical Region harbours over 100 bird
species that specialize on the fluctuating resources
provided by bamboos (Parker III et al. 1996, Krat-
ter 1997, Cockle & Areta 2013, Lebbin 2013).
Bamboo-specialist birds have been classified into
three main life history categories: insectivores, seed
specialists and mixed strategists (Hilty et al. 1979,
Parker III et al. 1997, S
anchez 2005, Areta &
Cockle 2012). The population dynamics of bam-
boo specialists is causally tied to that of the bam-
boos they inhabit (Neudorf & Blanchfield 1994,
Olmos 1996, Sick 1997, Areta et al. 2009, 2013,
Areta & Cockle 2012). Neotropical woody bam-
boos tend to flower massively and die after a fixed
number of years (typically c. 30 years for Merosta-
chys and Guadua bamboos), creating a temporally
ephemeral food resource for seed specialists at the
same time that habitat for mixed strategists and
insectivores is decimated. Therefore, bamboo habi-
tats are highly dynamic through time, creating a
complex mosaic of spatiotemporal conditions that
are difficult-to-impossible to predict for individual
birds (Areta & Cockle 2012).
The genus Amaurospiza is a member of the Car-
dinalidae, sister to a clade including all the
Cyanoloxia grosbeaks (Todd 1923, Klicka
et al. 2007, Bryson Jr et al. 2014, Garc
ıa
et al. 2016). Despite their vernacular name, the
Amaurospiza ‘seedeaters’ are considered bamboo-
specialized mixed strategists: they are most often
found in bamboos in vegetative state feeding on
buds, shoots and petioles (Bertoni 1919, Areta
et al. 2016, J. I. Areta & A. Bodrati unpubl. data)
and insects, and can take advantage of the super-
abundance of seeding bamboos (Restall et al. 2006).
The unique flared basal tomia of the maxilla in
Amaurospiza (Lentino & Restall 2003) may help to
smash vegetable matter and seems to constitute an
adaptation to bud and shoot foraging. As mixed-
strategists, Amaurospiza species may take advantage
of episodic events of massive bamboo seeding, but
regardless of their ability to exploit mastings, popu-
lation numbers should decrease locally after such
masting events, prompting individuals to wander in
search of vegetative bamboo habitats (Areta &
Cockle 2012, Areta et al. 2016).
The three currently recognized species of Amau-
rospiza are allopatrically distributed from Mexico
to Argentina (Fig. 1). The Blackish-blue Seedeater
Amaurospiza moesta dwells in the Atlantic Forest
of Argentina, Brazil and Paraguay, and is appar-
ently patchily distributed across the forest enclaves
of the Cerrado of Brazil (Partridge 1954, Sick 1997,
Curcino & Feraboli 2010, Lopes et al. 2011); the
recently described Carrizal Seedeater Amaurospiza
carrizalensis is known from just a handful of locali-
ties in southeastern Venezuela (Lentino &
Restall 2003); and the geographically variable Blue
Seedeater Amaurospiza concolor is distributed
patchily in forested environments from Mexico to
Peru (Fig. 1, Howell & Webb 1995, Ridgely &
Greenfield 2001, Lentino & Restall 2003). Three
subspecies have been consistently recognized
within A. concolor: concolor from Panama to south-
ern Mexico, relicta in southwest Mexico and aequa-
torialis extending from southwest Colombia to
northwest Peru (Hellmayr 1938, Howell &
Webb 1995, Lentino & Restall 2003).
The species-level taxonomy and generic bound-
aries of Amaurospiza blue seedeaters are not set-
tled. Species and subspecies limits in Amaurospiza
have primarily been based on subtle colour differ-
ences and variations in bill shape and size (Caba-
nis 1861, Sharpe 1888, Hellmayr 1904,
Griscom 1934, Orr & Ray 1945). Yet these same
characters have been considered conserved in the
genus, resulting in a fairly homogeneous group
(Lentino & Restall 2003). As a consequence, the
taxonomy of Amaurospiza has been largely idiosyn-
cratic and based strictly on museum specimen
data, with no reference to natural history or vocal-
izations of the forms involved. A recent molecular

© 2023 British Ornithologists' Union.


Figure 1. Geographical distribution and sonograms of Amaurospiza taxa recognized in this study. Small black points are raw data
from eBird (https://ebird.org), and large circles show localities of genetic samples included in this study (Table 1). Darker shading
indicates higher relief.
phylogenetic study found A. moesta to be closely
related to A. carrizalensis and a paraphyletic A.
concolor, where a single sample of the subspecies
aequatorialis was more closely related to A. moesta
and A. carrizalensis than to nominate A. concolor
(Bryson Jr et al. 2014), and the enigmatic taxon re-
licta remains unsampled. Full species status has
been advocated for the taxa relicta and aequatori-
alis (Hellmayr 1938, Howell & Dyer 2022, Howell
& Webb 1995, Lentino & Restall 2003) but evi-
dence supporting their recognition is weak. Fur-
thermore, relicta was originally described in the
monospecific genus Amaurospizopsis based on
structural features such as a relatively short bill,
large rounded nostrils and long rictal bristles,
whereas in plumage it was considered to be a ‘rep-
resentative’ of Amaurospiza (Griscom 1934).
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Amaurospiza systematics 3
In this paper we construct a hypothesis of molec-
ular phylogenetic relationships for all currently rec-
ognized taxa in the bamboo-specialized genus
Amaurospiza to: (1) test the monophyly of the
genus; (2) reconstruct the phylogenetic relation-
ships of its constituent taxa; (3) re-assess species
limits in Amaurospiza with the aid of vocalizations,
and genetic and plumage data; and (4) discuss the
link between bamboo life history, biogeographical
patterns and extent of genetic differentiation.
METHODS
Molecular phylogenetics
To understand phylogenetic relationships within
Amaurospiza and to test the monophyly of the
© 2023 British Ornithologists' Union.

4 J. I. Areta et al.
genus, we expanded on previous work (Bryson Jr
et al. 2014) by adding more samples of each of the
three species as well as from subspecies concolor,
aequatorialis (including nuclear markers for this
last taxon) and the little-known taxon relicta for
the first time (Table 1). Our ingroup dataset con-
sisted of 19 specimens comprising all currently rec-
ognized taxa in Amaurospiza, nine of which were
newly sequenced; species in the genera Cyano-
compsa, Cyanoloxia, Passerina and Spiza were used
as outgroups (Table 1), rooting in the more dis-
tantly related Rose-breasted Grosbeak Pheucticus
ludovicianus based on previous phylogenetic work
in Cardinalidae (Klicka et al. 2007).
We used published sequences (Bryson Jr
et al. 2014; Dryad: doi: 10.5061/dryad.tb50m) and
our nine new samples, for which total genomic
DNA was extracted using a DNeasy tissue extrac-
tion kit (Qiagen, Valencia, CA, USA). All samples
were tissues with the exception of two samples of
A. c. relicta from Morelos, Mexico, from which we
extracted DNA from toe-pads. DNA from toe-pad
clips was extracted in a laboratory used exclusively
for historical DNA, to avoid cross-contamination
with other samples. We sequenced the complete
subunit 2 of the mitochondrial NADH dehydroge-
nase gene (ND2; 1041 base pairs (bp)) using pri-
mers L5215 (Hackett 1996) and TrC (Miller
et al. 2007). For toe-pads we amplified fragments
of 200 bp that overlapped by at least 50 bp using
polymerase chain reaction (PCR; Table S1). PCR
amplifications were performed in 12.5-lL reac-
tions under the following conditions: denaturation
at 94 °C, followed by 35 cycles of 94 °C for 30 s,
54 °C for 45 s and 72 °C for 1 min. This was fol-
lowed by a 10-min extension at 72 °C. For a sub-
set of samples (Cyanoloxia cyanoides, A. concolor,
A. c. aequatorialis, A. moesta, A. carrizalensis;
Table 1), we used published or obtained new
sequences of three nuclear introns, including
951 bp of Aconitase 1 intron 9 (ACO1), 519 bp
of b-fibrinogen intron 5 (FGB5) and 685 bp of
Myoglobin intron 2 (MB), using primers specified
in Kimball et al. (2009). PCR amplifications were
performed under similar conditions as for ND2
but annealing temperatures were changed to
60 °C for all three nuclear genes. PCR products
were sent to the High-Throughput Genomics Unit
(HGTU) of the University of Washington for all
subsequent DNA sequencing steps (in both direc-
tions). Some samples were kindly sequenced at
other laboratories (see Acknowledgements). For
© 2023 British Ornithologists' Union.
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nuclear genes, we used International Union of Pure
and Applied Chemistry ambiguity codes to repre-
sent apparent heterozygosities. New sequences
were deposited in GenBank (accession numbers
OP380634–OP380668). Uncorrected pairwise dif-
ferences (p-distance), both within and among cur-
rently recognized Amaurospiza taxa, were
calculated for the ND2 dataset using MEGA 7
(Kumar et al. 2016).
Phylogenetic analyses for both the ND2 and the
multilocus datasets were performed using both
Maximum Likelihood and Bayesian Inference
methods. We ran the Maximum Likelihood analy-
sis in RAxML 8.2.9 (Stamatakis 2014) using the
Cipres Portal (Miller et al. 2010; http://www.
phylo.org/). This program implements a general
time reversible model of nucleotide substitution
with c-distributed heterogeneity among sites
(GTR + G model) and support was determined via
1000 bootstrap pseudoreplicates based on a rapid
bootstrap algorithm (Stamatakis et al. 2008). We
ran the Bayesian inference analysis in MrBayes
3.2.6 (Ronquist et al. 2012). The DNA substitution
model was selected using jModeltest2 (Darriba
et al. 2012) under the corrected Akaike Informa-
tion Criterion (AICc). The selected model was
GTR with substitution rates following a c distribu-
tion with a proportion of invariable sites
(GTR + G + I). We conducted our search using
four independent runs, each one conducted using
four chains (one cold, three heated) of Metropolis-
coupled Markov chains running simultaneously for
10 million generations each, with trees sampled
every 1000 generations (total of 40 000 trees). We
discarded the first 10% of generations of each run
as burn-in. Similar strategies were used for our
multilocus data but we applied a partitioned model
by gene in RAxML (GTR + G for each partition).
In MrBayes, we used the following mixed model,
unlinking all parameters: TIM2 + I for ND2;
TPM1uf (Kimura three-parameter (K81) with
unequal base frequencies (uf)) for ACO1;
HKY + G for FGB5; and TPM1 (K81) for MB. As
model selection based on different information cri-
teria might have an effect on tree topology (Hoff
et al. 2016), we ran an analysis using models
selected by the Bayesian Information Criterion,
TrN + I for ND2; F81 for both ACO1 and FGB5;
and JC for MB, which were less complex than
those found using AICc, but no differences were
found in tree topology or statistical support of dif-
ferent branches. We examined convergence of
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Amaurospiza systematics 5

Table 1. Samples of Amaurospiza and related taxa that were included in phylogenetic analyses in this study. See Table S4 for Gen-
Bank accession numbers.

Speciesa Museum Tissue no.b Country Locality [state/province/department]

Amaurospiza concolor concolor UNAM YACH302 Mexico Monumento Natural Yaxchilan [Chiapas]
Amaurospiza concolor concolor * UWBM 109 865(JK02-012) Guatemala San Felipe Retalhuleu, 5 km S [Retalhuleu]
Amaurospiza concolor concolor FMNH 434 109 El Salvador Municipio San Francisco Mene
ndez [Ahuachapan]
Amaurospiza concolor concolor # STRI MJM4762 Panama Boquete [Chiriqu
ı]
Amaurospiza concolor relicta # AMNH 778 553 Mexico ~o
Can
n de Lobos [Morelos]
Amaurospiza concolor relicta # AMNH 778 554 Mexico ~o
Can
n de Lobos [Morelos]
Amaurospiza [concolor] ANSP 183 549(18386) Ecuador Nanegalito [Pichincha]
aequatorialis
Amaurospiza [concolor] LSUMNS B67239 Peru PN Cerros de Amotape; El Barco [Tumbes]
aequatorialis*,#
Amaurospiza [concolor] LSUMNS B67308 Peru PN Cerros de Amotape; El Barco [Tumbes]
aequatorialis #
Amaurospiza [concolor] LSUMNS B67314 Peru PN Cerros de Amotape; El Barco [Tumbes]
aequatorialis #
Amaurospiza carrizalensis * COP 80 450(IC594) Venezuela R
ıo Caron
ı, frente Rio Claro, Isla Carrizal [Bol
ıvar]
Amaurospiza carrizalensis COP 80 449(IC569) Venezuela R
ıo Caron
ı, frente Rio Claro, Isla Carrizal [Bol
ıvar]
Amaurospiza carrizalensis COP 80 451(IC589) Venezuela R
ıo Caron
ı, frente Rio Claro, Isla Carrizal [Bol
ıvar]
Amaurospiza carrizalensis # COP 82 235(ML1319) Venezuela Sector Santa Rosa; Comunidad 5 de Marzo
[Bol
ıvar]
Amaurospiza carrizalensis COP 82 236(ML1320) Venezuela Sector Santa Rosa; Comunidad 5 de Marzo
[Bol
ıvar]
Amaurospiza carrizalensis # COP 82 270(ML1354) Venezuela Sector Santa Rosa; Comunidad 5 de Marzo
[Bol
ıvar]
Amaurospiza moesta*,# ZMUC 119 601(JMS10) Brazil Fazenda Harmonia; Bonito [Mato Grosso do Sul]
Amaurospiza moesta DZUFMG 5771 Brazil Fazenda Ba
ıa de Pedra [Mato Grosso do Sul]
Amaurospiza moesta LGEMA 11 440 Brazil Ortigueira [Parana
]
Cyanoloxia brissonii LSUMNS B18658 Bolivia Cordillera [Santa Cruz]
Cyanoloxia rothschildii LSUMNS B12708 Bolivia Velasco [Santa Cruz]
Cyanoloxia cyanoides * COP 81 377(IC968) Venezuela Sierra de San Luis; Cerro Galicia [Falco
n]
Cyanoloxia glaucocaerulea ANSP 22 361(10255) Uruguay [Rocha]
Cyanocompsa parellina Unknownc CWT082 Mexico Chivela [Oaxaca]
Passerina caerulea LSUMNS B20958 USA Cameron Pa. [Louisiana]
Spiza americana MMNH JK95-047 USA Cameron Co. [Texas]
Pheucticus ludovicianus MMNH X7253 USA Brown Co. [Minnesota]

AMNH, American Museum of Natural History; ANSP, Academy of National Sciences of Philadelphia; COP, Coleccio
n Ornitolo

gica
Phelps; DZUFMG, Museo del Departamento de Zoologia da Universidade Federal de Minas Gerais; FMNH, Field Museum of Natural
History; LGEMA, Laborato
rio de Gene
tica e Evolucßa
~o Molecular de Aves do Instituto de Biocie
^ ncias da Universidade de Sa~o Paulo;
LSUMNS, Louisiana State University Museum of Natural Science; MMNH, University of Minnesota Bell Museum of Natural History;
STRI, Smithsonian Tropical Research Institute; UNAM, Universidad Nacional Auto
noma de Mexico; UWBM, University of Washington
Burke Museum; ZMUC, Zoological Museum University of Copenhagen. aMitochondrial ND2 was sequenced for all listed samples; *
indicates specimens for which nuclear ACO1 (intron 9), MB (intron 2) and FGB (intron 5) were sequenced, # indicates specimens
sequenced for this study. bWhen two numbers are associated with a tissue, the first refers to the voucher (as it appears on the insti-
tutional online catalogue) and the second to the tissue. cThis tissue was associated with UWBM in previous publications but neither
tissue nor voucher are held by this institution or any other collection where we have requested information. We included it here as it
is the reference for a sequence (deposited in GenBank) we used as one of the outgroups in the ND2 gene phylogenetic analysis.

log-likelihood and all parameter values using TRA-

CER 1.5 (Rambaut & Drummond 2007), allowing
for effective sample size values for each parameter
to be larger than 200. We also examined the phylo-
genetic information provided by each nuclear gene
running independent gene analyses using
Paup*4.0b10 (Swofford 2002).
Divergence dating
We tested for clock-like evolution of our ND2 data-
set using a likelihood ratio test in Paup*4.0b10
(Swofford 2002). This test rejected the hypothesis
of rate homogeneity (P < 0.05); consequently, we
performed an Uncorrelated Lognormal Relaxed

© 2023 British Ornithologists' Union.


6 J. I. Areta et al.
Clock analysis using BEAST 1.8.4 to determine
divergence times (Drummond et al. 2012). The
commonly used calibration rate of 2.1% substitu-
tions/site/million years is based on cytochrome b
(Cyt b) data (Weir & Schluter 2008). However,
our comparisons of pairwise genetic distances for
some of the taxa in our dataset from which we had
comparable Cyt b data, as well as more general
published comparisons with larger data sets (Smith
& Klicka 2010), indicated that the ND2 gene
evolves at a higher rate than Cyt b (1.17–1.25
times). Consequently, we used a lognormal prior
for mutation rate of 1.25 9 10 2 substitutions/lin-
eage/site/million years (equivalent to 2.5% substitu-
tions/site/million years between lineages) with a
standard deviation of 0.1, which accounts for varia-
tion around such estimates. We assumed a Yule
process for the speciation prior and ran the analysis
for 5 9 107 generations, sampling every 1000 gen-
erations. We used TRACER v1.5 to assess the mix-
ing of Metropolis-coupled Markov chains in the
analysis (achieving reliable effective sample size val-
ues >200) and discarded 10% of the generations as
burn-in. For the multilocus data, we used one indi-
vidual for each currently recognized species in the
genus (including subspecies aequatorialis, which is
potentially a different lineage from concolor; Bryson
Jr et al. 2014) to reconstruct a time-calibrated mul-
tilocus species tree using *BEAST (Heled & Drum-
mond 2010) in BEAST 1.8.4. Our goal was to
contrast these results with previous ones obtained
with just ND2 data for aequatorialis (Bryson Jr
et al. 2014). We ran the analysis by 5 9 107 gener-
ations (sampled each 1000 generations), with simi-
lar models used in previous MrBayes analyses
(based on AICc), constraining the node including
all Amaurospiza as monophyletic and using uncor-
related lognormal relaxed clocks (with similar rates
as used in Bryson Jr et al. 2014). Samples were
summarized on the maximum clade credibility tree
using TREE ANNOTATOR1.8.4 (included with
BEAST). Effective sample sizes were low for some
parameters, particularly those associated with sub-
stitution models and molecular clock rates (ucld.-
mean). Consequently, we selected simpler models
(based on Bayesian Information Criteria as previ-
ously indicated) and a strict molecular clock model
for nuclear genes, a strategy that improved conver-
gence on all parameters, kept tree topology
unchanged (compared to previous analyses) and
produced similar ages of divergence among taxa.
© 2023 British Ornithologists' Union.
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Vocalizations
We assembled a database of songs of all currently
recognized taxa in Amaurospiza (Table S2). Sam-
ple sizes were uneven, with single recordings avail-
able for carrizalensis (containing two songs) and
relicta (containing four songs), being the only
known recordings for these taxa. For all taxa, we
analysed a single song from each individual male
after finding that songs had high repeatability
based on visual examination of consistency of
spectrograms of all available songs. As Amau-
rospiza can utter partial songs, we chose the song
with the larger number of notes in each sample
(i.e. the most complete song per individual).
To compare the warbling quality of the songs of
all taxa we calculated the number of inflections per
second and average number of inflections per note.
An inflexion was defined as a point in a note in which
the previous frequency/time slope was swapped (i.e.
each time a note switched from descending to
ascending or vice versa). The ratio of the number of
inflections per second for each species was obtained
by dividing the sum of effective sound (i.e. adding
the duration of all notes in all songs) by the sum of
the total number of inflections in all songs. The aver-
age number of inflections per note for each species
was obtained by dividing the sum of the total num-
ber of inflections in all songs by the total number of
notes analysed per species. These data were checked
for normality with a Shapiro–Wilk test and for
homogeneity of variance with Levene’s test. As data
on the number of inflections were not normally dis-
tributed, we applied non-parametric tests.
All recordings were resampled at 44.1 kHz and
16 bits irrespective of their initial recording parame-
ters to facilitate standard measurements. Given the
spectral features of Amaurospiza vocalizations and
the parameters measured, the effect of mp3 com-
pression and posterior re-sampling should not have
a noticeable effect on our dataset. All measurements
were made with Raven Pro 1.4 (http://www.birds.
cornell.edu/raven, Cornell Lab of Ornithology,
Ithaca, NY, USA) using default spectrogram param-
eters (Window-Type: Hann, Size: 256 samples
(= 5.8 ms), 3-dB Filter Bandwidth: 248 Hz; Time
grid-Overlap: 50%, Hop size: 128 samples
(= 2.9 ms); Frequency grid-DFT size: 256 samples,
Grid spacing: 172 Hz). Note and song measure-
ments were derived from onscreen selections
including the whole time and frequency domain
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Amaurospiza systematics 7

expanses. We obtained measurements of 11 vari-
ables directly from the spectrograms (minimum fre-
quency, maximum frequency, bandwidth 90%,
centre frequency, frequency 5%, frequency 95%,
peak frequency (i.e. frequency with the highest
energy), first quartile frequency, third quartile fre-
quency, song duration and interquartile frequency)
and computed values for 11 derived variables con-
structed by us (minimum frequency average of all
notes per song, maximum frequency average of all
notes per song, bandwidth 90% frequency average
of all notes per song, peak frequency average of all
notes per song, centre frequency average of all notes
per song, minimum frequency of first three notes,
maximum frequency of first three notes, peak fre-
quency of first three notes, centre frequency of first
three notes, bandwidth of first three notes, song
duration of three first notes). We chose the first
three notes because this increased the sample size
and guaranteed the comparability of allegedly
homologous notes across taxa. These measurements
were used to test for differences in vocalizations
among taxa with univariate and multivariate meth-
ods. To maximize our ability to uncover differences
and to evaluate vocal diagnosability of all taxa we
performed a Linear Discriminant Analysis after scal-
ing and testing for multivariate normality (Royston
test, H = 9.93, P = 0.30) and retaining variables that
were normally distributed and had normally dis-
tributed variances (Table S3). All vocalization anal-
yses were performed using R 4.0.5 (R Development
Core Team 2021).
RESULTS
Phylogenetic analyses
Amaurospiza was found to be monophyletic in
both the ND2 and multilocus analyses (100%
bootstrap; 1.0 posterior probability; Fig. 2). In the

Figure 2. Phylogenetic hypothesis of relationships within the genus Amaurospiza based on the (a) mtDNA and (b) multilocus data-
sets. Numbers on nodes represent maximum likelihood bootstrap support followed by Bayesian posterior probabilities. Asterisks indi-
cate 100% bootstrap or posterior probabilities equal to 1.0. Individuals are labelled based on sample numbers (Table 1). Illustration
of Blackish-blue Seedeaters Amaurospiza moesta by A. Chiappe from Pearman and Areta (2020); reproduced with permission.

© 2023 British Ornithologists' Union.


8 J. I. Areta et al.
ND2 mitochondrial gene tree A. moesta and A.
carrizalensis were recovered as sister to each other
(86% bootstrap support, but with a low posterior
probability in the Bayesian analysis, posterior prob-
ability = 0.82). As currently delineated, A. concolor
was found to be paraphyletic because aequatorialis
was placed as sister to the moesta-carrizalensis
clade (100%; 1.0) and a clade including both nom-
inate concolor and relicta was sister to all the other
taxa (Fig. 2a). A tree enforcing monophyly of A.
concolor including aequatorialis was less likely than
the phylogenetic hypothesis found in this study
(S-H test: Diff –lnL = 20.11, P < 0.02, Shimodaira
& Hasegawa 1999). Haplotypes of relicta were
recovered as monophyletic, either within a poly-
tomy of concolor haplotypes in the ND2 gene tree
(Fig. 2), or as sister to concolor in the BEAST tree
(Fig. 3). Nuclear genes varied in their phylogenetic
information content: although the monophyly of
Amaurospiza was largely supported by each locus
analysed (five synapomorphic characters in ACO1,
six in FGB5 and nine in MB), relationships among
species were less resolved. Although no phyloge-
netic structure was recovered by both FGB5 and
MB, the clade including moesta, carrizalensis and
aequatorialis was supported by three synapomor-
phic characters in ACO1. Moreover, multilocus
data highly supported the sister relationship of
Figure 3. Bayesian phylogenetic reconstruction of Amau-
rospiza based on the mtDNA (ND2 gene) sequence data
showing mean divergence times. Bars represent 95% highest
probability density of divergence time estimates. Scale at the
bottom shows time in million years before the present.
Plio = Pliocene, Pleist = Pleistocene. Black dots indicate 0.95–
1.00 posterior probabilities (if lower, those are indicated by a
number and an arrow directed to the node).
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aequatorialis to the moesta-carrizalensis clade and
showed these three taxa as sister to nominate con-
color, and also enhanced the support for the sister
relationship of the moesta-carrizalensis clade
(Fig. 2b).
Mitochondrial genetic divergences among taxa
were an order of magnitude larger than those found
within taxa (Table 2), with the exception of relicta
in comparison to nominate concolor samples. Mean
ND2 pairwise distances between concolor and aequa-
torialis were greater (8.3%) than those found
between moesta and carrizalensis (5.7%). In compar-
ison, the two available relicta samples from Morelos
diverged on average by 1.0% from nominate con-
color. Though we could not obtain complete ND2
sequences from these relicta samples
(AMNH778553: 571 bp, AMNH778554: 289 bp),
the longest sequence from this taxon differed by four
or five substitution changes from samples of concolor
collected from Mexico (Chiapas) to Panama.
The shallow genetic distances between geo-
graphically distant samples of A. moesta from Mato
Grosso do Sul and Paran
a (Brazil) (c. 680 km, p-
distance = 0.0–0.7% for ND2) and those recovered
between the distant samples of nominate concolor
from Chiapas (Mexico) to Chiriqu
ı (Panama) were
similarly divergent (c. 1250 km, p-distance = 0.0–
0.7% for ND2; Table 2). These genetic distances
are comparable to those recovered between sam-
ples of A. carrizalensis that come from a set of clo-
sely placed localities (c. 40 km, p-distance = 0.0–
0.7% for ND2). Interestingly, samples of relicta
from Morelos (Mexico) and of concolor from Chia-
pas (Mexico), c. 900 km away, showed a slightly
larger divergence (p-distance = 0.7–1.0% for
ND2), similar to the divergence between the same
Morelos samples of relicta to those of concolor from
Guatemala and Panama c. 1800 km away (p-
distance = 0.7–1.3% for ND2; Table 2).
Temporal estimates of divergence from our
relaxed-clock model using the mitochondrial
(ND2) dataset indicated a split of Amaurospiza
from its closest relatives (Cyanocompsa and
Cyanoloxia) during the Middle to Late Miocene
ranging from 11.6 to 6.0 million years ago (Ma)
(mean 8.7 Ma, Fig. 3). Diversification within
Amaurospiza began in the Late Miocene to Plio-
cene, between 8.4 and 4.0 Ma (mean 6.1 Ma,
Fig. 3), with the divergence of Central and South
American groups, and continued during the Late
Pliocene–Pleistocene with further differentiation of
South American lineages (mean 3.4 Ma, 95%
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Amaurospiza systematics 9

Table 2. Mean percentage uncorrected genetic distances (ND2, p-distance) among and within currently recognized Amaurospiza
taxa.

Taxa concolor relicta aequatorialis moesta carrizalensis

concolor 0.5 (0.2)

[0.0–0.7]
relicta 1.0 (0.4) 0.0 (0.0)
[0.7–1.3] [0.0]
aequatorialis 8.3 (0.8) 8.8 (1.3) 0.2 (0.1)
[8.1–8.5] [8.0–9.6] [0.0–0.4]
moesta 9.2 (0.9) 9.0 (1.4) 6.0 (0.7) 0.5 (0.2)
[8.6–9.4] [8.2–9.7] [5.9–6.1] [0.0–0.7]
carrizalensis 9.5 (0.9) 10.0 (1.4) 5.8 (0.7) 5.7 (0.7) 0.4 (0.1)
[9.1–9.9] [9.1–11.0] [5.6–6.0] [5.3–6.2] [0.0–0.7]

Diagonal values represent genetic differentiation within each taxon. Bold values represent mean percentage uncorrected genetic dis-
tance, values within parentheses represent standard errors estimated with 500 bootstrap replicates and values within square brackets
provide the range of individual genetic distance values found in our dataset.

Table 3. Quantitative characterization of the inflections in the songs of Amaurospiza taxa: average values are followed by  stan-
dard deviation and ranges are given within parentheses.

Number of inflections/ Average number of Sample size (notes/inflections/

Taxon second inflections per note individuals)

A. moesta 21.51 2.76  1.37 (0–9)b 301/830/42

A. carrizalensis 17.28 2.83  0.75 (2–4)a,b 6/17/1
A. aequatorialis 20.09 2.10  1.26 (0–6)a 78/164/8
A. concolor concolor 7.90 1.22  1.14 (0–4)c 51/62/10
A. concolor relicta 13.85 1.57  1.13 (0–3)a,b,c 7/11/1

Different lower case letters indicate statistically significant differences (P < 0.05) between taxa in the pairwise Wilcoxon’s rank-rank
tests with Bonferroni correction.

highest posterior density (HPD) 4.7–2.1 Ma). Pop-
ulations of relicta (based on the current number of
samples) diverged from concolor about 1 million
years ago (0.2–1.1 Ma), representing the youngest
divergence of a currently recognized Amaurospiza
taxon (Fig. 3). Our multilocus species tree recov-
ered an identical tree topology compared with the
ND2 and concatenated multilocus datasets, but
divergence times were younger (Fig. S1). Accord-
ing to these estimates, the Amaurospiza split from
its closest relatives occurred at 4.6 Ma (95% HPD
6.1–3.3 Ma), diversification within the genus
begun at c. 3.2 Ma (95% HPD 4.4–1.5 Ma) and
divergence within South American lineages started
about 1.9 Ma (95% HPD 2.7–1.0 Ma).
Vocalizations
All taxa examined exhibited typical Amaurospiza
songs constituted by quickly delivered, warbled,
pure and fairly high-pitched musical notes,
suggesting a strong conservatism in overall pattern
in the genus (Fig. 1, Table S3). Aural examination
of songs suggested that songs of moesta, carrizalen-
sis and aequatorialis were more warbled than those
of concolor and relicta. The number of inflections
per second exhibited a stepped pattern, with con-
color and relicta on the lower end and carrizalensis,
aequatorialis and moesta on the upper end, corrob-
orating our subjective impression of a notably less
warbled song in concolor and relicta (Table 3). The
same overall pattern was found with the average
number of inflections per note, where moesta, car-
rizalensis and aequatorialis had overall more inflec-
tions per note than concolor and relicta (Table 3).
Statistical comparisons lend partial support to this
idea, depending on the number of samples avail-
able for each taxon. Pairwise comparisons of aver-
age number of inflections per note showed that
the better sampled taxa (with nine or more
recordings) moesta, aequatorialis and concolor dif-
fered significantly from each other (P < 0.001),

© 2023 British Ornithologists' Union.


10 J. I. Areta et al.
whereas for the taxa for which only one recording
was available, relicta was indistinguishable from
other taxa (P range 0.226–1) and carrizalensis dif-
fered only from concolor (P = 0.023; Table 3).
The linear discriminant analysis using nine
acoustic variables (Table S3) indicated that all taxa
can be successfully diagnosed (Fig. 4). Broadly,
two fairly contiguous groups can be separated
along the first axis: one with the South American
taxa and the other with the Central American taxa
(Fig. 4). In the South American clade, moesta (the
taxon with the larger number of samples) exhib-
ited the largest dispersion, the single sample of car-
rizalensis fell within data of moesta along the
values in the first axis, whereas most aequatorialis
samples were clearly segregated along both axes
(Fig. 4). In the Central American clade, the single
sample of relicta was closer to concolor than to any
South American taxon (Fig. 4). Importantly from
a taxonomic perspective, samples of the taxon ae-
quatorialis were the most distant from samples of
concolor and relicta along the first axis (Fig. 4). The
first linear discriminant consisted mainly of maxi-
mum frequency (Coefficient = 1.10), peak fre-
quency average of all notes per song
(Coefficient = 1.01) and song duration of the first
three notes (Coefficient = 1.04). Two of these
variables also contributed greatly to the second lin-
ear discriminant: maximum frequency (Coeffi-
cient = 1.03), peak frequency average of all notes
per song (Coefficient = 1.54), as well as the aver-
age of maximum frequency of all notes per song
(Coefficient = 1.45). Predicted performance was
high; linear discriminant analysis correctly assigned
Figure 4. Comparison of songs of Amaurospiza taxa through
linear discriminant analysis (LDA). Plot of recordings according
to the first and second linear discriminant functions obtained
from nine song variables (see Tables S2 and S3).
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all 62 songs to the correct taxon. Hence, the over-
all predictive accuracy was 100% and the jack-
knifed/leave-one-out cross-validation for this data-
set only had a slight decrease to 95%. This last
procedure correctly classified 95.2% of moesta indi-
viduals, 90% of concolor individuals and all eight
aequatorialis individuals; one concolor individual
(MLNS30440961) was misclassified as moesta
whereas two moesta individuals were misclassified
as aequatorialis or concolor (XC55844 and
XC60845, respectively). The cross-validation tech-
nique accurately classified a high percentage of
individuals for three taxa (moesta, aequatorialis,
concolor), while the method itself was unable to
attribute group membership to instances of a sin-
gle sample such as the cases of relicta and
carrizalensis.
DISCUSSION
Our study shows that the genus Amaurospiza is
monophyletic and that genetic differentiation
among species is well marked. We identified two
largely divergent clades, one in South America
(aequatorialis-moesta-carrizalensis) and one in Cen-
tral America (concolor-relicta), a structure congru-
ent with geography, vocalizations, plumage and
morphology. The taxon aequatorialis was allied to
the South American species A. moesta and A. car-
rizalensis based on geography, vocalizations, plu-
mage and morphology, instead of to A. concolor,
indicating an inaccurate species-level taxonomy as
presently recognized (Remsen et al. 2023). These
results suggest that underwing coloration, the ratio
of bill depth to bill width and general patterns of
vocalizations are underappreciated characters car-
rying phylogenetic signal in Amaurospiza (Table 4).
Our molecular phylogenetic hypothesis largely
reflects evolutionary relationships based on our
mitochondrial gene data (ND2) due to its greater
phylogenetic signal. It might be argued that this
can limit our ability to make inferences on species
limits and evolutionary processes. Indeed, one
mitochondrial gene tree hypothesis might differ
from the species tree because of sex-biased disper-
sal (mitochondrial DNA is maternally inherited),
natural selection, introgression and/or hybridiza-
tion, or most importantly because of the uncer-
tainty associated with gene trees as representatives
of the historical relationships of populations due to
the random nature of the coalescence process
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Amaurospiza systematics 11

Table 4. Characterization of the two clades of Amaurospiza taxa uncovered in this study.

South American clade [aequatorialis,

Variable moesta and carrizalensis] Central American clade [concolor and relicta]

Vocalizations More warbled (faster) song with more Less warbled (slower) song with fewer
inflections per note inflections per note
Underwing coverts White Bluish
Bill depth/bill width ratio < 0.89 > 0.92
Genetic data Monophyletic group with three species One polytypic species with two subspecies

Bill depth/bill width ratio from Lentino and Restall (2003), plumage data from Griscom (1934), Hellmayr (1938), Howell and
Webb (1995) and Restall et al. (2006), and vocal and genetic data from this study.

(Funk & Omland 2003, Edwards & Bensch 2009,
Toews & Brelsford 2012). However, our nuclear
dataset provides phylogenetic signal (though weak)
and phylogenetic hypotheses that are congruent
with, and never contradict, our mitochondrial
hypothesis (i.e. there is no nuclear–mitochondrial
discordance). Moreover, enhanced node support
values on our concatenated nuclear–mitochondrial
reconstruction, and congruence among our phylo-
genetic hypotheses (including the coalescent multi-
locus phylogenetic reconstruction) and patterns
derived from other independent characters such as
morphology and vocalizations indicate that our
hypothesis of species relationships is strong.
Species limits in Amaurospiza
The lack of monophyly of A. concolor, with aequato-
rialis being deeply divergent from concolor and more
closely related to other South American forms,
together with the morphological and vocal differ-
ences between nominate concolor and aequatorialis,
strongly indicates that aequatorialis should not be
retained as a subspecies of concolor (Figs 1–4,
Table 4). Based on phylogenetic position (sup-
ported by mitochondrial and nuclear genes), mor-
phology, genetic differentiation and vocalizations
(Lentino & Restall 2003, Bryson Jr et al. 2014, this
study), we recommend affording full species status
to A. aequatorialis. We propose adopting the com-
mon English name Ecuadorian Seedeater for A.
aequatorialis on account that most of its range
occurs inside Ecuador and that an alternative name
Equatorial Seedeater (see Hellmayr 1938) conveys
a sense of lowland tropicality that this species lacks,
being a montane bird. We also propose to retain
Blue Seedeater for A. concolor, a name with a long
tradition of use.
Our finding that the taxon relicta is probably
sister to concolor (Figs 2 and 3), together with our
vocal analysis of the only known recording of this
taxon (Fig. 4), allows us to provide some thoughts
on the systematics of this mysterious taxon. Some
authors considered relicta as a distinct Amaurospiza
species (Orr & Ray 1945, Navarro-Sig€ uenza &
Peterson 2004) and even as a species in the mono-
typic genus Amaurospizopsis (Griscom 1934, but
see Hellmayr 1938). This taxon occurs in Mexico
in the states of Guerrero, Morelos and Oaxaca, to
the west of the Isthmus of Tehuantepec, and is
associated with the Sierra Madre del Sur (even
though it is not a strictly montane bird), as
opposed to concolor, whose populations occur to
the east of the Isthmus of Tehuantepec. The Sierra
Madre del Sur is known for its high avian
endemicity (Hern
andez-Ba~ nos et al. 1995) and
phylogeographical studies have shown large diver-
gences among bird populations (Garc
ıa-Moreno
et al. 2004, Bonaccorso et al. 2008, Rocha-M
endez
et al. 2019). Genetic differentiation between relicta
and concolor is well below the typical species-
specific distances in other Amaurospiza. The near-
est samples of relicta and concolor separated by
around 900 km showed a slightly smaller diver-
gence than that between relicta and the southern-
most samples of concolor separated by around
1800 km (Table 2), suggesting that the Isthmus of
Tehuantepec depression is a relatively minor bar-
rier for this taxon. The isolating role of the Isth-
mus of Tehuantepec has been shown to be
pervasive in many avian taxa distributed across this
lowland barrier, but although some lineages show
deep phylogeographical structure across the barrier
(e.g. Cort
es-Rodr
ıguez et al. 2008, Barrera-
Guzm
an et al. 2012, Mendoncßa et al. 2021),
others exhibit small genetic differentiation or a
lack of it (e.g. Arbel
aez-Cort
es et al. 2010,
Zamudio-Beltr
an & Hern
andez-Ba~ nos 2015,
Zamudio-Beltr
an et al. 2020, Castillo-Chora
et al. 2021). Comparative phylogeographical

© 2023 British Ornithologists' Union.

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12 J. I. Areta et al.

studies have shown multiple waves of colonization A. concolor concolor Cabanis 1861
across the Isthmus of Tehuantepec (Barber & A. concolor relicta (Griscom 1934)
Klicka 2010, Ornelas et al. 2013), suggesting dif- Ecuadorian Seedeater Amaurospiza aequatorialis
ferent factors such as lineage age and/or ecology as Sharpe 1888
potential drivers of such divergence time differ- Blackish-blue Seedeater Amaurospiza moesta
ences as shown across other important geographi- (Hartlaub 1853)
cal barriers in the Neotropics (Smith et al. 2014). Carrizal Seedeater Amaurospiza carrizalensis
The close geographical proximity of relicta (west Lentino & Restall 2003
of the isthmus) and concolor (east of the isthmus)
It has been suggested that the genus Amau-
in southern Mexico (Fig. 1) could be interpreted
rospiza be subsumed under Cyanoloxia (Klicka
as evidence of phenotypically divergent conspecific
et al. 2007). However, the morphological distinc-
populations or alternatively as evidence that a
tions (smaller bills with lateral flares and smaller
strong barrier may have interrupted genetic flux
overall size), faster vocalizations and bamboo spe-
between these populations, ending in speciation.
cialization of Amaurospiza, coupled to their long
Our small sample size does not allow us to test if
period of independent evolution (sharing a most
the small genetic divergence found is a product of
recent common ancestor around 9 Ma, Fig. 3),
a recent allopatric divergence, or if there is current
support the recognition of Amaurospiza and
gene flow among these populations, and a more
Cyanoloxia as separate genera (Lentino &
thorough study is required to understand the cur-
Restall 2003, Bryson Jr et al. 2014). Our data
rent status of these taxa.
unambiguously support the placement of relicta in
Previously reported differences in vocalizations
Amaurospiza instead of in the monotypic genus
between concolor and relicta (Howell & Webb 1995)
Amaurospizopsis (Griscom 1934, Hellmayr 1938).
were based on limited data but are currently
It is clear that Griscom (1934) oversplit genera,
thought to be minor, with relicta now being consid-
and subspecies-level taxa when describing the
ered a typical Amaurospiza with song and calls very
genus Amaurospizopsis for relicta and two sub-
similar to those of nominate concolor in southern
species within A. concolor (grandior and australis),
Mexico (S. Howell in litt. 2012). Our quantitative
which were later lumped with the nominate sub-
results (based on a single individual) suggest that
species (Hellmayr 1938, Orr & Ray 1945).
relicta is vocally similar to concolor (Figs 1 and 4).
The reduced genetic differentiation and similarities
in plumage, morphology and vocalizations suggest Diversification in Amaurospiza
that relicta should not be afforded species status,
Divergence times based on our multilocus species
and lead us to consider relicta as a subspecies of A.
tree reconstruction were younger than those
concolor. We highlight the need for further studies
obtained based on the mitochondrial ND2 gene.
including critical comparative examination of speci-
These results are congruent with previous biogeo-
mens, sound recordings and fresh genetic samples
graphical reconstructions in blue cardinalids (Bry-
to rigorously assess its taxonomic status.
son Jr et al. 2014), and are a recurrent pattern in
The deep genetic differentiation of the recently
other bird studies (McCormack et al. 2010,
described A. carrizalensis (Lentino & Restall 2003)
Schultz et al. 2019). Younger ages are expected
in comparison to its sister A. moesta (Figs 2 and
as multilocus analyses take into account random
3), and the single sample of the identifiable yet
coalescence events of gene trees and avoid poten-
relatively similar song (Table 3, Fig. 4), support its
tial overestimation of divergence times, as gene
continued recognition as a separate species.
divergence pre-dates speciation events (Edwards
We therefore propose the recognition of four
& Beerli 2000, Carstens & Knowles 2007). Fol-
species in Amaurospiza following the Biological
lowing our younger temporal estimates of diversi-
Species Concept (Mayr 1963) and the Recognition
fication, the origin of Amaurospiza occurred
Species Concept (Paterson 1980, 1985), including
between the Miocene and Pliocene, with a split
one polytypic species with two subspecies and
between Central and South American groups dur-
three monotypic species.
ing the Pliocene and Pleistocene, and most differ-
Blue Seedeater Amaurospiza concolor Caba- entiation within groups is dated as of Pleistocene
nis 1861 age.

© 2023 British Ornithologists' Union.


Regardless of what divergence time estimation
we select for the diversification of the Amaurospiza
lineage, our results indicate that this genus has had
a long evolutionary history in the Neotropics. Pre-
vious biogeographical reconstructions of the com-
plete lineage of blue cardinalids suggested
colonization of this group into South America,
with a split of the Amaurospiza and Cyanoloxia lin-
eages in the Late Miocene (about 6 Ma, Bryson Jr
et al. 2014). Our estimates are younger (4.6 Ma)
but still indicate that divergence across the Pana-
manian Isthmus is placed well before the suggested
formation of the landbridge between these conti-
nents (3.1–3.5 Ma; Coates & Obando 1996),
though temporal estimates overlap when consider-
ing the uncertainty in our estimate of timing of
Amaurospiza diversification (6.1–3.3 Ma). Older
estimates for the Panamanian Isthmus have been
published pointing to Miocene landbridge connec-
tions (Farris et al. 2011, Montes et al. 2012), allow-
ing for congruent biogeographical scenarios of
dispersal in some groups (e.g. Copernicia palms,
Bacon et al. 2012). The debate about these tempo-
ral estimates is ongoing (O’Dea et al. 2012, 2016,
Jaramillo et al. 2017), and geological and phyloge-
netic evidence points toward a continuous rather
than a discrete event with periods of discrete con-
nection–disconnection between both continents
(Cody et al. 2010, Smith & Klicka 2010, Pinto-
S
anchez et al. 2012, Jaramillo 2018). Moreover,
several studies have suggested a Late Pliocene colo-
nization of South America from Middle America,
particularly in birds (Weir & Schluter 2008, Smith
et al. 2014, Guti
errez-Pinto et al. 2012), indicating
the potential for some geological, ecological or cli-
matic factors favouring such movements at the end
of formation of the landbridge. Regardless of these
alternative scenarios, either the ancestor of Amau-
rospiza had a more continuous distribution across
the Panamanian landbridge around the Miocene or
dispersal occurred by that time as a result of geo-
logical or ecological factors.
Diversification of Amaurospiza within South
America occurred during the Late Pliocene and
Early Pleistocene. These temporal estimates coin-
cide with a period of intense climatic and land-
scape changes in the Neotropics, including the
glacial–interglacial cycles, and the final uplift of
the northern Andes with its consequences for the
regional climate and the river network system
(Hoorn et al. 2010, Pupim et al. 2019).
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Amaurospiza systematics 13
Ecology and speciation in mixed-
bamboo strategists
The ecology of Amaurospiza blue seedeaters is
poorly known, but all species seem to be locally
scarce and appear to be tightly associated with
vegetative and seeding bamboo stands. Amau-
rospiza moesta is known to use different bamboo
species across its distribution, involving small tan-
gling species (e.g. Chusquea ramosissima) and tall
erect species (e.g. Guadua trinii, Guadua tagoara,
Guadua paniculata, Merostachys claussenni) and
feeds on seeds, flowers, petioles and buds of Chus-
quea and Guadua bamboos in the Atlantic Forest
(Bertoni 1919, Belton 1985, Della-Flora
et al. 2010, J. I. Areta pers. obs.). In Misiones,
Argentina, populations of A. moesta seem to have
peaked during the massive flowering and seeding
of M. claussennii, and at a specific locality, Parque
Provincial Cruce Caballero, their numbers
dropped down after the end of the masting with
the disappearance of green stands of M. claussenni,
which seeds once every 30 years (Areta
et al. 2009, Bodrati et al. 2010, A. Bodrati & J. I.
Areta unpubl. data). How far the birds involved
may have moved is not known, but this distance
should be largely dependent upon the contingent
setting of bamboo patches at any given time (see
Areta & Cockle 2012). Reports that A. moesta has
a ‘marked predilection for a single habitat type:
the understorey formed by the lower strata of for-
est vegetation in the more covered and shady
places’ (Partridge 1953: 88; our translation) may
have stemmed from overlooking the presence of
bamboos in such forests or from sampling birds at
times in which bamboos were dead. The local and
little-known A. carrizalensis is known from riverine
areas (but also away from rivers) with vegetative
bamboo stands of Guadua venezuelae, Guadua lat-
ifolia and Rhipidocladum sp., and has been
observed feeding on seeds of G. latifolia and Chro-
molaena odorata and on insects from the leaves of
an unidentified tree (Lentino & Restall 2003, M.
Lentino and J. Miranda pers. obs.). Both sub-
species of A. concolor in Mexico (concolor and re-
licta) show predilection for bamboo stands
(Howell & Webb 1995) and although a nest of re-
licta was outside bamboo, a group was observed
nearby feeding ‘on bunches of dry seeds of a
climbing vine’ (a climbing bamboo?) (Row-
ley 1962: 266). At El Imposible in El Salvador, A.
© 2023 British Ornithologists' Union.

14 J. I. Areta et al.
concolor is almost always associated with vegetative
bamboo in the understorey and its population
numbers seem to have increased in the years fol-
lowing a massive flowering and seeding event of
Rhipidocladum racemiflorum, which seeds once
every 13 years (Fagan 2009, J. Fagan in litt. 2010).
The association of A. aequatorialis with bamboo
has been proposed to be less strict than in other
members of the genus, being at least as often in
bamboo as away from it in Ecuador (Ridgely &
Greenfield 2001). However, most recent records
in Ecuador pertain to birds feeding on seeds and
insects in Chusquea and unidentified bamboos (D.
Brinkhuizen in litt., A. Solano-Ugalde in litt.) and
on Guadua bamboos (Restall et al. 2006), while
records from Peru are also from Chusquea bamboo
stands (Angulo Pratolongo et al. 2012, S
anchez
et al. 2012). To sum up, the available evidence
shows that all the Amaurospiza taxa are tightly
linked to bamboo stands.
The high mobility that mixed-strategist bamboo
specialists would need to have to cope with peri-
ods of food scarcity during bamboo die-offs (Areta
et al. 2009, Areta & Cockle 2012) and their tight
dependence on bamboo habitats must impact the
genetic architecture and mode of speciation of
mixed-strategists. We uncovered low levels of
intraspecific genetic differentiation between distant
populations that contrast markedly with the deep
divergences between the widely allopatric Amau-
rospiza species. Genetic differentiation among pop-
ulations within species for which we had widely
distant samples (A. moesta and A. concolor; Fig. 1)
was low and comparable to variation within the
local population of A. carrizalensis (Table 2).
Comparative phylogeographical studies are needed
to further understand how the presumed patterns
of mobility of mixed-strategists affect the spatial
genetic structure in comparison with other co-
distributed taxa with other life histories.
The deep genetic divergences and widely allo-
patric distributions of species of Amaurospiza lead
us to propose that their speciation could be trig-
gered by the appearance of exceptional distribu-
tional discontinuities. Such discontinuities could in
principle arise through the appearance of strong
extrinsic geographical barriers separating peripheral
populations (a precursor of this situation could be
the outlying Cerrado populations of A. moesta) or
might arise through unusual long-distance dispersal
events in search of bamboos, thereby creating
effective long-term isolation between populations
© 2023 British Ornithologists' Union.
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that can subsequently differentiate. Our phyloge-
netic reconstructions of the South American lin-
eage resulted either in a polytomy of three species,
moesta from the Atlantic Forest and isolated
patches of Cerrado forest, carrizalensis from the
Guianan Shield at the Orinoco River, and aequato-
rialis from the Andes (results based on Bayesian
analyses of the ND2 gene or the species tree,
Fig. 2 and Fig. S1), or suggested a sister relation-
ship between moesta and carrizalensis. Although
there is no evidence to propose a biogeographical
hypothesis for the current distributions of these
taxa, either a scenario of widespread distribution
and further extinction in most of the Amazonian
basin or dispersal from one region to another asso-
ciated with geological or climate changes as well as
ecological factors could apply to these organisms.
The differentiation of Amaurospiza could be
related to different scenarios of dispersal patterns
but the tight association with bamboo cycles adds
yet another dimension to be taken into considera-
tion when thinking about vicariance versus disper-
sal models. Future work integrating models of past
distribution of bamboo habitats and more exten-
sive geographical sampling should help understand
the interplay between climatic and habitat fluctua-
tions, dispersal and evolutionary differentiation in
Amaurospiza and other bamboo-specialist birds.
We thank A. Solano-Ugalde, D. Brinkhuizen and J.
Fagan for sharing field observations, and S. Howell for
sharing the only known sound recording of the taxon re-
licta. We thank Paul van Els for sequencing Peruvian
samples of aequatorialis at Louisiana State University
Museum of Natural Science facilities, Matt Miller for
sequencing a sample of A. concolor from Chiriqu
ı at
Smithsonian Tropical Research Institute of Panama, and
Brian Smith and Paul Sweet who were indispensable in
obtaining toe-pads of relicta at the American Museum of
Natural History. We are also grateful to one anonymous
reviewer for appreciating the importance of our work,
and to a second anonymous reviewer for making useful
suggestions. M. Pearman and N. K. Krabbe provided
feedback on the usage of the name Ecuadorian Seedea-
ter. Gary Voelker contributed editorial comments that
improved our work.
AUTHOR CONTRIBUTIONS
Juan I. Areta: Conceptualization; data curation;
formal analysis; investigation; methodology; visual-
ization; writing – original draft; writing – review
and editing. Mar
ıa Juliana Ben
ıtez Sald
ıvar: Data

curation; formal analysis; visualization; writing –
review and editing. Miguel Lentino: Investigation;
writing – review and editing. Jhonathan Miranda:
Data curation; formal analysis; visualization; writ-
ing – review and editing. Mateus Ferreira:
Methodology; resources. John Klicka: Funding
acquisition; resources. Jorge P
erez-Em
an: Concep-
tualization; data curation; formal analysis; investi-
gation; methodology; visualization; writing –
original draft; writing – review and editing.
CONFLICT OF INTEREST
The authors have no conflicts of interest to
declare.
FUNDING
This work was funded in part by CONICET (to
JIA), NSF DEB 0315469 (to JK) and by CVG-
EDELCA (to ML).
ETHICAL NOTE
Samples used in this study were obtained via loans
from museums.
Data Availability Statement
Vocalizations analysed in this study can be found
in Table S2. Genetic sequences are available from
GenBank (accession numbers OP380634–
OP380668) and are summarized in Table S4.
REFERENCES
Angulo Pratolongo, F., Flanagan, J.N.M., Vellinga, W.P. &
Durand, N. 2012. Notes on the birds of Laquipampa wildlife
refuge, Lambayeque, Peru. Bull. Brit. Orn. Club 132: 162–174.
Arbela
ez-corte
s, E., Nya


ri, A.S. & Navarro-Sigu€ enza, A.G.
2010. The differential effect of lowlands on the
phylogeographic pattern of a Mesoamerican montane
species (Lepidocolaptes affinis, Aves: Furnariidae). Mol.
Phylogenet. Evol. 57: 658–668.
Areta, J.I. & Cockle, K.L. 2012. A theoretical framework for
understanding the ecology and conservation of bamboo-
specialist birds. J. Ornithol. 153: S163–S170.
Areta, J.I., Bodrati, A. & Cockle, K. 2009. Specialization on
Guadua bamboo seeds by three bird species in the Atlantic
Forest of Argentina. Biotropica 41: 66–73.
Areta, J.I., Bodrati, A., Thom, G., Rupp, A.E., Velazquez,
M., Holzmann, I., Carrano, E. & Zimmermann, C.E. 2013.
Natural history, distribution, and conservation of two
1474919x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ibi.13181 by University Of Montana Mansfield Library-Serials, Wiley Online Library on [04/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Amaurospiza systematics 15
nomadic Sporophila seedeaters of bamboo in the Atlantic
forest. Condor 115: 237–252.
Areta, J.I., Bodrati, A. & Cockle, K.L. 2016. Un panorama de
las aves especialistas en bambu
es de la Argentina. In
Ru
golo de Agrasar, Z. (ed) Bambu
es len ~ osos de la
Argentina, nativos y exo
ticos: 221–236. Buenos Aires,
Argentina: Instituto de Bota
nica Darwinion.
Bacon, C., Baker, W.J., Gardens, R.B. & Simmons, M.P.
2012. Miocene dispersal drives Island radiations in the palm
tribe Trachycarpeae (Arecaceae). Syst. Biol. 61: 426–442.
Barber, B.R. & Klicka, J. 2010. Two pulses of diversification
across the Isthmus of Tehuantepec in a montane Mexican
bird fauna. Proc. R. Soc. B 277: 2675–2681.
Barrera-Guzma
n, A.O., Mila
, B., Sa
nchez-Gonza
lez, L.A. &
Navarro-Sigu € enza, A.G. 2012. Speciation in an avian
complex endemic to the mountains of middle America
(Ergaticus, Aves: Parulidae). Mol. Phylogenet. Evol. 62:
907–920.
Belton, W. 1985. Birds of Rio Grande do Sul, Brazil. Part 2.
Formicariidae to Corvidae. Bull. AMNH 180: 1–242.
Bertoni, A.W. 1919. Especies de aves nuevas para el
Paraguay. Hornero 1: 255–258.
Bodrati, A., Cockle, K., Segovia, J.M., Roesler, I., Areta, J.I.
& Jordan, E.A. 2010. La avifauna del Parque Provincial
Cruce Caballero, Provincia de Misiones, Argentina. Cotinga
32: 41–64.
Bonaccorso, E., Navarro-Sigu € enza, A.G., Sa
nchez-
Gonza
lez, L.A., Peterson, A.T. & Garcia-Moreno, J. 2008.
Genetic differentiation of the Chlorospingus ophthalmicus
complex in Mexico and Central America. J. Avian Biol. 39:
311–321.
Bryson, R.W. Jr., Chaves, J., Smith, B.T., Miller, M.J.,
Winker, K., Pe
rez-Ema

n, J.L. & Klicka, J. 2014.
Diversification across the new world within the ‘blue’
cardinalids (Aves: Cardinalidae). J. Biogeogr. 41: 587–599.
Cabanis 1861. Uebersicht der im Berliner Museum
befindlichen Vogel von Costa Rica. J. Fu € r Ornithol. 49: 1–11.
Carstens, B.C. & Knowles, L.L. 2007. Shifting distributions
and speciation: species divergence during rapid climate
change. Mol. Ecol. 6: 619–627.
Castillo-Chora, V.D., Zamudio-Beltra
n, L.E., Pozo, C. &
Herna
ndez-Ban ~ os, B.E. 2021. Phylogeography of Habia
fuscicauda (Cardinalidae) indicates population isolation,
genetic divergence and demographic changes during the
quaternary climate shifts in the Mesoamerican rainforest. J.
Ornithol. 162: 961–976.
Coates, A. & Obando, J. 1996. The geologic evolution of the
central American isthmus. In Jackson, J.B.C., Budd, A.F. &
Coates, A.G. (eds) Evolution and Environment in Tropical
America: 21–56. Chicago, IL: The University of Chicago
Press.
Cockle, K. & Areta, J.I. 2013. Specialization on bamboo by
neotropical birds. Condor 115: 217–220.
Cody, S., Richardson, J.E., Ellis, C. & Pennington, R.T.
2010. The great American biotic interchange revisited.
Ecography 33: 326–332.
Corte
s-Rodr
ıguez, N., Herna
ndez-Ban ~ os, B.E., Navarro-
Sigu € enza, A.G., Peterson, A.T. & Garcia-Moreno, J. 2008.
Phylogeography and population genetics of the amethyst-
throated hummingbird (Lampornis amethystinus). Mol.
Phylogenet. Evol. 48: 1–11.
© 2023 British Ornithologists' Union.

16 J. I. Areta et al.
Curcino, A. & Feraboli, A. 2010. Primeiro registro de
Cyanoloxia moesta (Hartlaub, 1853) (Aves, Cardinalidae)
para o Estado de Goia
s, Brasil. Rev. Bras. Orn. 18:
136–137.
Darriba, D., Taboada, G.L., Doallo, R. & Posada, D. 2012.
jModelTest 2: more models, new heuristics and parallel
computing. Nat. Methods 9: 772.
Della-Flora, F., Brodt, M.S.C., Bastos, G.D., Silva, C.T. &
Canto-Dorow, T.S. 2010. Blackish-blue seedeaters
(Cyanoloxia moesta) and red-crested finches
(Coryphospingus cucullatus) foraging in bamboo Chusquea
ramosissima. Rev. Bras. Orn. 18: 344–346.
Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A.
2012. Bayesian phylogenetics with BEAUti and the BEAST
1.7. Mol. Biol. Evol. 29: 1969–1973.
Edwards, S.V. & Beerli, P. 2000. Perspective: gene
divergence, population divergence, and the variance in
coalescence time in phylogeographic studies. Evolution 54:
1839–1854.
Edwards, S. & Bensch, S. 2009. Looking forwards or looking
backwards in avian phylogeography? A comment on Zink
and Barrowclough 2008. Mol. Ecol. 18: 2930–2933.
Fagan, J. 2009. Birding El Salvador. Neotrop. Birding 5:
61–70.
Farris, D.W., Jaramillo, C., Bayona, G., Restrepo-Moreno,
S.A., Montes, C., Cardona, A., Mora, A., Speakman, R.J.,
Glascock, M.D. & Valencia, V. 2011. Fracturing of the
Panamanian Isthmus during initial collision with South
America. Geology 39: 1007–1010.
Funk, D.J. & Omland, K.E. 2003. Species-level paraphyly
and polyphyly: frequency, causes, and consequences, with
insights from animal mitochondrial DNA. Ann. Rev. Ecol.
Evol. Syst. 34: 397–423.
Garc
ıa, N.C., Barreira, A.S., Lavinia, P.D. & Tubaro, P.L.
2016. Congruence of phenotypic and genetic variation at the
subspecific level in a neotropical passerine. Ibis 158:
844–856.
Garc
ıa-Moreno, J., Navarro-Sigu € enza, A.G., Peterson, A.T.
& Sa
nchez-Gonza
lez, L.A. 2004. Genetic variation
coincides with geographic structure in the common bush-
tanager (Chlorospingus ophthalmicus) complex from Mexico.
Mol. Phylogenet. Evol. 33: 186–196.
Griscom, L. 1934. The ornithology of Guerrero, Mexico. Bull.
Mus. Comp. Zool. 75: 367–422.
Gutie
rrez-Pinto, N., Cuervo, A.M., Miranda, J., Pe
rez-Ema
n,
J.L., Brumfield, R.T. & Cadena, C.D. 2012. Non-
monophyly and deep genetic differentiation across low-
elevation barriers in a neotropical montane bird
(Basileuterus tristriatus; Aves: Parulidae). Mol. Phylogenet.
Evol. 64: 156–165.
Hackett, S.J. 1996. Molecular phylogenetics and
biogeography of tanagers in the genus Ramphocelus
(Aves). Mol. Phyl. Evol. 5: 368–382.
Hartlaub, G. 1853. Beitra € ge zur exotische Ornithologie. J. Fu €r
Ornithol. 1: 30–44.
Heled, J. & Drummond, A.J. 2010. Bayesian inference of
species trees from multilocus data. Mol. Biol. Evol. 27:
570–580.
Hellmayr, K.E. 1904. Uber € neue und wenig bekannte
Fringilliden Brasiliens, nebst Bemerkungen u €ber notwendige
€
Anderungen in der Nomenklatur einiger Arten.
© 2023 British Ornithologists' Union.
1474919x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ibi.13181 by University Of Montana Mansfield Library-Serials, Wiley Online Library on [04/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Verhandlungen Zoologische und Botanische Wien 54:
516–537.
Hellmayr, C.E. 1938. Catalogue of birds of the Americas and
adjacent islands. F. Museum Nat. Hist. Publ. Zool. 11:
1–662.
Herna
ndez-Ban ~ os, B.E., Townsend Peterson, A.T.,
Navarro-Sigu € enza, A.G. & Escalante-Pliego, P. 1995. Bird
faunas of the humid montane forests of Mesoamerica:
Biogeographic patterns and priorities for conservation. Bird
Conserv. Int. 5: 251–277.
Hilty, S., Parker, T.A. III & Silliman, J. 1979. Observations
on plush-capped finches in the Andes with a description of
the juvenal and immature plumages. Wilson Bull. 91:
145–148.
Hoff, M., Orf, S., Riehm, B., Darriba, D. & Stamatakis, A.
2016. Does the choice of nucleotide substitution models
matter topologically? BMC Bioinformatics 17: 143.
Hoorn, C., Wesselingh, F.P., Ter Steege, H., Bermudez,
M.A., Mora, A., Sevink, J., Sanmantin, I., Sanchez-
Meseguer, A., Anderson, C.L., Figueiredo, J.P.,
Jaramillo, C., Riff, D., Negri, F.R., Hooghiestra, H.,
Lundberg, J., Stadler, T., Sa €rkinen, T. & Antonelli, A.
2010. Amazonia through time: Andean uplift, climate
change, landscape evolution, and biodiversity. Science 330:
927–931.
Howell, S.N.G. & Dyer, D. 2022. Costa Rica even richer than
we thought? N. Am. Birds 73: 22–33.
Howell, S.N.G. & Webb, S. 1995. A Guide to the Birds of
Mexico and Northern Central America. Oxford: Oxford
University Press.
Jaramillo, C. 2018. Evolution of the Isthmus of Panama:
biological, paleoceanographic and paleoclimatological
implications. In Hoorn, C., Perrigo, A. & Antonelli, A. (eds)
Mountains, climate and biodiversity: 323–338. London: John
Wiley & Sons Ltd.
Jaramillo, C., Montes, C., Cardona, A., Silvestro, D.,
Antonelli, A. & Bacon, C.D. 2017. Comment (1) on “
Formation of the Isthmus of Panama” by O’ Dea et al. Sci.
Adv. 3: 1–9.
Kimball, R.T., Braun, E.L., Barker, F.K., Bowie, R.C.K.,
Braun, M.J., Chojnowski, J.L., Hackett, S.J., Han, K.,
Harshman, J., Heimer-torres, V., Holznagel, W.,
Huddleston, C.J., Marks, B.D., Miglia, K.J., Moore, W.S.,
Reddy, S., Sheldon, F.H., Smith, J.V., Witt, C.C. & Yuri,
T. 2009. A well-tested set of primers to amplify regions
spread across the avian genome. Mol. Phylogenet. Evol. 50:
654–660.
Klicka, J., Burns, K. & Spellman, G.M. 2007. Defining a
monophyletic Cardinalini: a molecular perspective. Mol.
Phylogenet. Evol. 45: 1014–1032.
Kratter, A.W. 1997. Bamboo specialization by Amazonian
birds. Biotropica 29: 100–110.
Kumar, S., Stecher, G. & Tamura, K. 2016. MEGA 7:
Molecular evolutionary genetics analysis version 7.0 for
bigger datasets. Mol. Biol. Evol. 33: 1870–1874.
Lebbin, D.J. 2013. Nestedness and patch size of bamboo-
specialist bird communities in southeastern Peru. Condor
115: 230–236.
Lentino, M. & Restall, R. 2003. A new species of
Amaurospiza blue seedeater from Venezuela. Auk 120:
600–606.

Lopes, L.E., De Pinho, J.B. & Benfica, C.E.R.T. 2011.
Seasonal distribution and range of the blackish-blue
seedeater (Amaurospiza moesta): A bamboo-associated
bird. Wilson J. Ornithol. 123: 797–802.
Mayr, E. 1963. Animal Species and Evolution. Cambridge,
MA: Harvard University Press.
McCormack, J.E., Heled, J., Delaney, K.S., Peterson, A.T.
& Knowles, L.L. 2010. Calibrating divergence times on
species trees versus gene trees: Implications for speciation
history of Aphelocoma jays. Evolution 65: 184–202.
Mendonc ß a, P., Dias, C., Aleixo, A., Silva, L.C., Araripe, J. &
Re ^ go, P.S. 2021. Diversification across the isthmus of
Tehuantepec explains the phylogeographic arrangement of
the widespread bright-rumped Attila (Attila spadiceus;
Tyrannidae) and reveals the existence of two major
lineages. J. Ornithol. 163: 327–332.
Miller, M.J., Bermingham, E. & Ricklefs, R.E. 2007.
Historical biogeography of the New World solitaires
(Myadestes spp.). Auk 124: 868–885.
Miller, M.A., Pfeiffer, W. & Schwartz, T. 2010. Creating the
CIPRES science gateway for inference of large phylogenetic
trees. In Proceedings of the Gateway Computing
Environments Workshop (GCE): 1–8. New Orleans, LA.
Montes, C., Bayona, G., Cardona, A., Buchs, D.M., Silva,
C.A. & Moro
n, S. 2012. Arc-continent collision and Orocline
formation: closing of the central American seaway. J.
Geophys. Res. 117: 1–25.
Navarro-Sigu € enza, A.G. & Peterson, A.T. 2004. An
alternative species taxonomy of the birds of Mexico. Biota
Neotrop. 4: 1–32.
Neudorf, D.L. & Blanchfield, P.J. 1994. The slate-colored
seedeater (Sporophila schistacea): a bamboo specialist?
Ornitol. Neotrop. 5: 129–132.
O’Dea, A., Hoyos, N., Rodr
ıguez, F., Degracia, B. & De
Gracia, C. 2012. History of upwelling in the tropical eastern
pacific and the paleogeography of the isthmus of Panama.
Palaeogeogr. Palaeoclimatol. Palaeoecol 348–349: 59–66.
O’Dea, A., Lessios, H.A., Miller, A.G., Eytan, R.I., Restrepo-
Moreno, S.A., Cione, A.L., Collins, L.S., De Queiroz, A.,
Farris, D.W., Norris, R.D., Stallard, R.F., Woodburne,
M.O., Aguilera, O., Aubry, M., Berggren, W.A., Budd,
A.F., Cozzuol, M.A., Coppard, S.E., Duque-caro, H.,
Finnegan, S., Gasparini, G.M., Grossman, E.L., Johnson,
K.G., Keigwin, L.D., Knowlton, N., Leigh, E.G., Leonard-
pingel, J.S., Marko, P.B., Pyenson, N.D., Rachello-
dolmen, P.G., Soibelzon, E., Soibelzon, L., Todd, J.A.,
Vermeij, G.J. & Jackson, J.B.C. 2016. Formation of the
isthmus of Panama. Sci. Adv. 2: 1–12.
Olmos, F. 1996. Satiation or deception?: mast-seeding
Chusquea bamboos, birds and rats in the Atlantic Forest.
Rev. Bras. Biol. 56: 391–401.
Ornelas, J.F., Sosa, V., Soltis, D.E., Daza, J.M., Gonza
lez,
C., Soltis, P.S., Gutie
rrez-Rodr
ıguez, C., Espinosa de los
Monteros, A., Castoe, T.A., Bell, C. & Ruiz-Sanchez, E.
2013. Comparative phylogeographic analyses illustrate the
complex evolutionary history of threatened cloud forests of
northern Mesoamerica. PLoS One 8: e56283.
Orr, R.T. & Ray, M.S. 1945. Critical comments on seed-eaters
of the genus Amaurospiza. Condor 47: 225–228.
Parker, T.A. III, Stotz, D.F. & Fitzpatrick, J.W. 1996.
Ecological and distributional databases. In Stotz, D.F.,
Fitzpatrick, J.W., Parker, T.A. III & Moskovits, D.K. (eds)
1474919x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ibi.13181 by University Of Montana Mansfield Library-Serials, Wiley Online Library on [04/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Amaurospiza systematics 17
Neotropical Birds: Ecology and Conservation: 113–436.
Chicago, IL: University of Chicago Press.
Parker, T.A. III, Stotz, D.F. & Fitzpatrick, J.W. 1997. Notes
on avian bamboo specialists in southwestern Amazonian
Brazil. Ornithol. Monogr. 48: 543–547.
Partridge, W.H. 1953. Notas breves sobre aves de Paraguay.
Hornero 10: 86–88.
Partridge, W.H. 1954. Estudio preliminar sobre una coleccio
n
de aves de Misiones. Rev. Mus. Arg. Cs. Nat. Bernardino
Rivadavia 3: 87–153.
Paterson, H.E.H. 1980. A comment on “mate recognition
systems”. Evolution 34: 330–331.
Paterson, H.E.H. 1985. The recognition concept of species. In
Vrba, E.S. (ed) Species and Speciation: 21–29.
Johannesburg: Transvaal Museum Monographs, Transvaal
Museum.
Pearman, M. & Areta, J.I. 2020. Birds of Argentina and the
South-West Atlantic. London: Christopher Helm.
Pinto-Sa
nchez, N.R., Iba
n~ ez, R., Madrin
~a
n, S., Sanjur, O.I.,
Bermingham, E. & Crawford, A.J. 2012. The great
American biotic interchange in frogs: Multiple and early
colonization of Central America by the south American
genus Pristimantis (Anura: Craugastoridae). Mol.
Phylogenet. Evol. 62: 954–972.
Pupim, F.N., Sawakuchi, A.O., Almeida, R.P., Ribas, C.C.,
Kern, A.K., Hartmann, G.A., Chiessi, C.M., Tamura, L.N.,
Mineli, T.D., Savian, J.F., Grohmann, C.H. Jr., Bertassoli,
D.J., Stern, A.G., Cruz, F.W. & Cracraft, J. 2019.
Chronology of Terra Firme formation in Amazonian lowlands
reveals a dynamic Quaternary landscape. Quat. Sci. Rev.
210: 154–163.
R Core Team 2021. R: A Language and Environment for
Statistical Computing. Vienna, Austria: R Foundation for
Statistical Computing.
Rambaut, A. & Drummond, A.J. 2007. Tracer v1.5. Available
at: http://beast.bio.ed.ac.uk/Tracer (accessed 1 December
2020).
Remsen, J.V. Jr., Areta, J.I., Bonaccorso, E., Claramunt,
S., Jaramillo, A., Lane, D.F., Pacheco, J.F., Robbins,
M.B., Stiles, F.G., & Zimmer, K. J. 2023. A classification of
the bird species of South America. American Ornithological
Society. Available at: http://www.museum.lsu.edu/~Remsen/
SACCBaseline.htm (accessed 12 September 2022).
Restall, R., Rodner, C. & Lentino, M. 2006. Birds of Northern
South America. London, UK: Christopher Helm.
Ridgely, R.S. & Greenfield, P.J. 2001. The Birds of Ecuador.
Vol. 1, Status, Distribution and Taxonomy. London:
Christopher Helm.
Rocha-Me
ndez, A., Sa
nchez-Gonza
lez, L.A., Gonza
lez, C. &
Navarro-Sigu € enza, A.G. 2019. The geography of
evolutionary divergence in the highly endemic avifauna from
the Sierra Madre del Sur, Mexico. BMC Evol. Biol. 19: 237.
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L.,
Darling, A., Ho € hna, S., Larget, B., Liu, L., Suchard, M.A.
& Huelsenbeck, J.P. 2012. MrBayes 3.2: efficient Bayesian
phylogenetic inference and model choice across a large
model space. Syst. Biol. 61: 539–542.
Rowley, J.S. 1962. Nesting of the birds of Morelos, Mexico.
Condor 64: 253–272.

nchez, C. 2005. First description of the nest and eggs of
Sa
the Slaty finch (Haplospiza rustica) and observations on
song and breeding behavior. Ornitol. Neotrop. 16: 493–501.
© 2023 British Ornithologists' Union.

18 J. I. Areta et al.

nchez, C., Saucier, J.R., Benham, P.M., Lane, D.F.,
Sa
Gibbons, R.E., Valqui, T., Figueroa, S.A., Schmitt, C.J.,
Sa
nchez, C., Schmidt, B.K., Milensky, C.M., Garc
ıa
Bravo, A. & Garc
ıa Olaechea, D. 2012. New and
noteworthy records from northwestern Peru, Department of
Tumbes. Bol. UNOP 7: 18–36.
Schultz, E.D., Pe
rez-Ema
n, J., Aleixo, A., Miyaki, C.Y.,
Brumfield, R.T., Cracraft, J. & Ribas, C.C. 2019.
Diversification history in the Dendrocincla fuliginosa complex
(Aves: Dendrocolaptidae): Insights from broad geographic
sampling. Mol. Phylogenet. Evol 140: 106581.
Sharpe, R.B. 1888. Amaurospiza. In Catalogue of the Birds in
the British Museum, Vol. 12: 156–158. London: Taylor &
Francis.
Shimodaira, H. & Hasegawa, M. 1999. Multiple comparisons
of log- likelihoods with applications to phylogenetic
inference. Mol. Biol. Evol. 16: 1114–1116.
Sick, H. 1997. Ornitologia Brasileira. Rio de Janeiro, Brazil:
Nova Fronteira, Ed.
Smith, B.T. & Klicka, J. 2010. The profound influence of the
late Pliocene Panamanian uplift on the exchange,
diversification, and distribution of New World birds.
Ecography 33: 333–342.
Smith, B.T., McCormack, J.E., Cuervo, A.M., Hickerson,
M.J., Aleixo, A., Cadena, C.D., Pe
rez-Ema
n, J., Burney,
C.W., Xie, X., Harvey, M.G., Faircloth, B.C., Glenn, T.C.,
Derryberry, E.P., Prejean, J., Fields, S. & Brumfield, R.T.
2014. The drivers of tropical speciation. Nature 515: 406–
409.
Stamatakis, A. 2014. RAxML version 8: a tool for
phylogenetic analysis and post-analysis of large
phylogenies. Bioinformatics 30: 1312–1313.
Stamatakis, A., Hoover, P. & Rougemont, J. 2008. A rapid
bootstrap algorithm for the RAxML web servers. Syst. Biol.
57: 758–771.
Swofford, D.L. 2002. PAUP*: phylogenetic analysis using
parsimony (*and other Methods). Version 4.0b10.
Todd, W.E.C. 1923. A review of the genus Cyanocompsa.
Auk 40: 58–69.
Toews, D.P.L. & Brelsford, A. 2012. The biogeography of
mitochondrial and nuclear discordance in animals. Mol. Ecol.
21: 3907–3930.
Weir, J.T. & Schluter, D. 2008. Calibrating the avian
molecular clock. Mol. Ecol. 17: 2321–2328.
© 2023 British Ornithologists' Union.
1474919x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ibi.13181 by University Of Montana Mansfield Library-Serials, Wiley Online Library on [04/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Zamudio-Beltra
n, L.E. & Herna
ndez-Ban~ os, B.E. 2015. A
multilocus analysis provides evidence for more than one
species within Eugenes fulgens (Aves: Trochilidae). Mol.
Phylogenet. Evol. 90: 80–84.
Zamudio-Beltra
n, L.E., Ornelas, J.F., Malpica, A. &
Herna
ndez-Ban ~ os, B.E. 2020. Genetic and morphological
differentiation among populations of the Rivoli’s
Hummingbird (Eugenes fulgens) species complex (Aves:
Trochilidae). Auk 137: 1–20.
Received 19 January 2022;
Revision 10 September 2022;
revision accepted 3 January 2023.
Associate Editor: Gary Voelker.
SUPPORTING INFORMATION
Additional supporting information may be found
online in the Supporting Information section at
the end of the article.
Figure S1. Bayesian time-calibrated multilocus
species tree of Amaurospiza based on *BEAST
reconstruction.
Table S1. Primers designed for PCR amplifica-
tion and sequencing of the NADH dehydrogenase
subunit gene (ND2) from toe-pads of Amaurospiza
concolor relicta.
Table S2. Vocalizations of Amaurospiza taxa
examined for this study.
Table S3. Mean and standard deviation of the
variables measured from the songs and notes of
Amaurospiza taxa.
Table S4. GenBank accession numbers for sam-
ples of Amaurospiza and related taxa included in
phylogenetic analyses in this study (see Table 1 for
localities).