Effect of spatial and temporal
urban isolation on the genetic diversity, acoustic variation, and
morphological characteristics of an urban survivor bird species
Luis Cueva1,2*, Eric J. Fuchs1,
Gilbert Barrantes1, Ruth
Madrigal-Brenes1 & Luis Sandoval1
1Universidad de Costa Rica, Escuela de Biología,
Ciudad Universitaria, Rodrigo Facio, San Pedro de Montes de Oca, San
José, 11501-2060
2Programa de Posgrado en Biología, Universidad de
Costa Rica, Escuela de Biología, Sistema de Estudios de Posgrado, Ciudad
Universitaria, Rodrigo Facio, San Pedro de Montes de Oca, San José,
11501-2060
*Contact information: Luis Cueva, San Pedro, San José 11501-2060, +506
62784357,luisfer_cueva@hotmail.com
Abstract
Urbanization
alters ecosystems, fragmenting natural habitats, and hence, increasing
isolation between populations. Therefore, a reduction in gene flow among
isolated populations is expected with greater distance and time since
fragmentation. Changes in the structure, density, or community
composition in the remaining habitats often result in species‘
modifications of vocalizations and morphological traits. However, the
relationship between genetic, vocalizations and morphological divergence
in urban areas over time remains poorly understood. We analyzed ten
years of genetic, acoustic, and morphological data from isolated
populations of the white-eared ground-sparrow. We used seven
microsatellites (SSRs), recorded, and measured five acoustic traits, and
measured six morphological traits, and compared them over a period of
ten years. We found an increase in inbreeding, increase in song
duration, number of elements, and frequency of maximum amplitude, but a
reduction in female body size and changes in male beak. However, we only
identified a significant correlation between genetic diversity and the
acoustic characteristics of song.
Keywords: Melozone leucotis, Costa Rica, genetic structure,
Bioacoustic
Introduction
Urban development results in rapid environmental changes (Grimm et al.,
2008; Johnson & Munshi-South, 2017) which transform natural habitats
into small patches of vegetation surrounded by a matrix of man built
constructions such as buildings and roads. These fragments with
depauperate resources are also frequently highly contaminated by noise,
lights, or solid debris (Fahrig & Rytwinski, 2009; Biamonte et al.,
2011). This reduction in natural habitats isolates populations, as urban
development restricts or eliminates the movement of individuals across
populations (Lynch & Baker, 1994; Crooks et al., 2004), lowering gene
flow and, consequently, the genetic diversity of populations (Lynch &
Baker, 1994; Johnson & Munshi-South, 2017). In birds, changes in
behavior (e.g., vocalizations, nest construction, predator responses)
and morphology (e.g., bill and body size) have also frequently been
reported as a consequence of different selective pressures acting on
isolated populations (e.g., sexual selection, predation, environment)
(Slabbekoorn & Smith, 2002; Brumm, 2004; Warren et al., 2006; Foster et
al., 2008; Laiolo & Arroyo, 2011; Luther & Derryberry, 2012; Sandoval
et al., 2014; Geffroy et al., 2020; Corrales-Moya et al., 2021; Méndez
et al., 2021)
The increase in distance between populations and the effect of drift and
inbreeding in populations of small size are the primary causes of the
loss of genetic diversity within populations and the increase in spatial
genetic structure in urban isolated bird populations (Keller & Waller,
2002; Whitlock, 2004; Johnson & Munshi-South, 2017; Miles et al.,
2019). In wren-tits Chamaea fasciata , significant genetic
structure was found when populations were separated by a highway and a
strip of residential development, in southern California (Delaney et
al., 2010b). In the song sparrow Melospiza melodia, researchers
found lower genetic diversity in populations where urban development had
been extensive and had occurred over long periods of time (Unfried et
al., 2013). These results indicate that urbanization has the potential
to lower genetic diversity within fragmented populations inside
urbanized matrices and increase the geographic genetic structure of bird
populations.
The observed patterns of genetic
structure caused by urbanization are analogous to differences in songs
reported for several bird species: white-bellied short wingBrachypteryx major , rufous-and-white wrens Thryophilus
rufalbus , and wedge-tailed sabrewing Campylopterus pampa . (Shaw
& Lesnick, 2009; Gonzalez et al., 2011; Purushotham & Robin, 2016;
Graham et al., 2017). Variation or polymorphism in the acoustic traits
of songs among populations occurs in response to different habitat
characteristics (e.g., vegetation density or noise levels), and they are
aimed at improving sound transmission and communication across different
environments (Morton, 1975; Boncoraglio & Saino, 2007; Ey & Fischer,
2009; Hardt & Benedict, 2021). Acoustic variation may also occur via
female selection, differences in female choice among populations may
lead to selective changes in song characteristics by males (Searcy &
Andersson, 1986; Laiolo & Arroyo, 2011). Acoustic variations may also
be the result of acoustic drift, which occurs when a small group of
birds randomly develops distinct repertoires. (Baker & Cunningham,
1985; Wilkins et al., 2013). For example, in bird species that inhabit
both urban and rural environments, songs from urban populations had
higher frequencies than populations from birds inhabiting rural
environments to cope with anthropogenic noise (Hu & Cardoso, 2009;
Luther & Derryberry, 2012; Méndez et al., 2021). Differences in song
traits may have evolved through acoustic drift as in populations of
Darwin’s finches (B. R. Grant & Grant, 1996). These changes in songs
can be maintained or increase over time, because songs are either
learned or transmitted over generations (Boyd & Richerson, 1985; Warren
et al., 2006). Hence, the correlation between genetic and vocal
polymorphism may arise due to the relationship between song pattern
diversity and the survival and reproductive success of individuals, as
well as the presence of distinct genotypes within each population
(Danielson-François et al., 2006; Irwin et al., 2008).
Variation in song acoustic characteristics (e.g., frequency and
duration), have also been related with the body and beak size of
individuals (Podos, 2001, 2010), because larger birds and larger beaks
tend to produce lower frequency songs (Wallschläger, 1980; Bradbury &
Vehrencamp, 1998; Fletcher & Tarnopolsky, 1999; Slabbekoorn & Smith,
2002a; Brumm, 2009 ). For ex ample, in purple-crowned fairy-wrensMalurus coronatus , barn swallows Hirundo rustica , and
black swans Cygnus atratus; individuals with large body sizes had
lower song frequencies (Galeotti et al., 1997; Patel et al., 2010; Hall
et al., 2013). In House finches Haemorhous mexicanus from
Arizona, urban individuals have longer and deeper beaks than rural birds
and emit songs with slower trills and wider frequency range (Badyaev et
al., 2008).
White-eared ground-sparrows, Melozone leucotis, inhabit the
central valley of Costa Rica in isolated populations embedded inside the
greater urban area (Sandoval et al., 2015; Juárez et al., 2020;
Rodríguez-Bardía et al., 2022). Previous work on this species reported
that urbanization limits the local movement of individuals and gene flow
between populations, which resulted in significant genetic structuring
among Costa Rican populations (Rodríguez-Bardía et al., 2022). Previous
research has also identified changes in song dialects among different
populations (Sandoval et al., 2014, 2016; Bonilla-Badilla 2021).
Additionally, it has been observed that differences in the frequency and
duration of songs among populations are associated with levels of
anthropogenic noise. Specifically, males in populations exposed to
higher levels of noise tend to exhibit an increase in both the minimum
frequency and duration of their songs. (Sandoval et al., 2014, 2015,
2016; Juárez et al., 2020; Méndez et al., 2021; Rodríguez-Bardía et al.,
2022). These studies suggest that genetic and cultural traits are under
intense selective pressures imposed by urbanized environments. However,
it is still unknown how these traits change over time and how they track
population dynamics.
The main objective of this study is to describe the changes in genetic
diversity, vocalization, morphology, and their relationship, over a
10-year period in populations of white-eared ground-sparrow isolated by
urban development in Costa Rica. We hypothesize that differences in
genetic diversity, song acoustics, and morphology will correlate with
the length of time communities have been separated, as population
divergence increases with isolation.
Material and Methods
We conducted this study in four populations of white-eared
ground-sparrows in Costa Rica: (1) Estación Biológica Monteverde (MTV),
Puntarenas province (10° 18ʹ N, 84° 48ʹ W, 1600 m), characterized by
coffee plantations and large forest patches that connect with more
extensive mature forests (Rodríguez-Bardía et al., 2022) without an
urban isolation effect yet. (2) Getsemani (HDA), Heredia province (10 °
01’N, 84 ° 05’W, 1350 m); it is a non-urbanized site, with secondary
forest patches and abandoned coffee plantations, and dense thickets with
little human presence (Juárez et al. 2020). Urbanization started to
expand from the edges of the study area since 2000. (3) Jardín Botánico
Lankester (JBL), Cartago province (9 ° 50’N, 83 ° 53’W, 1370 m); it
includes secondary forests, gardens, and buildings, with little
disturbance and human presence, but immerse in a urban matrix that had
rapidly extended since 1990 (Juárez et al. 2020). (4) The Universidad de
Costa Rica (UCR), San José province (09 ° 56’N, 84 ° 05’W, 1200 m); is a
highly urbanized site, with a secondary forest reserve surrounded by
buildings, open areas and gardens, and it is exposed to intense human
disturbance (Juárez et al., 2020). Urban development began before the
1900’s but most fragmentation occurred after the 1970´s (Biamonte et
al., 2011).
Historical and current sampling
Historical acoustic and morphological data was collected in 2011 and
2012 and was deposited in the Laboratorio de Ecología Urbana y
Comunicación Animal (LEUCA), Universidad de Costa Rica. These data are
part of a long-term research focus on analyzing the effect of
urbanization on behavior, morphology, and genetic diversity of bird
populations. To obtain historic genetic diversity estimates, we used
blood samples collected in 2011 – 2012. We will refer for the
historical 2011 – 2012-year sampling as “period 1”.
Current acoustic and morphological data was collected from 2018 to 2022.
We recorded each banded pair using a Marantz PMD661 digital recorder and
a Sennheiser ME66/K6 unidirectional microphone. The recordings were
stored in WAV format with a sample rate of 44.1 kHz and a precision of
24 bits. All recording were conducted between 4:55 - 6:00 h when this
species is most vocally active (Sandoval et al., 2016). We captured
individuals using mist nets (12 x 2.5m, 15 mm mesh eye) inside each pair
territory. We provided each captured individual with a unique numbered
metal ring and a color combination of plastic rings. From each bird, we
obtained 10 µL of blood from the brachial vein and stored it in 95%
ethanol or Lysis buffer for molecular analyses (Seutin et al., 1991;
Rodríguez-Bardía et al., 2022). We will refer for the period 2018 –
2022-year sampling as “period 2”.
All procedures were conducted in accordance with Costa Rican law. The
Research Committee of the Biology School and the Animal Care Committee
(ACC) of the University of Costa Rica granted to LS, the principal
investigator, research permits and animal care protocols.
DNA extraction and SSR amplification
We extracted DNA from blood samples using the DNeasy blood and tissue
Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s
protocol. We used seven primers: Mme2, Mme7, Mme8, Asµ15, Escµ6, Gf01 y
Gf05 (Petren, 1998; Jeffery et al., 2001; Bulgin et al., 2003) which
have been previously shown to be polymorphic and amplified well for this
species (Rodríguez-Bardía et al., 2022). We followed the Qiagen
Multiplex master kit ((Qiagen Inc., Valencia, CA) procedure to amplify
the aforementioned SSRs. All markers were amplified through Polymerase
Chain Reactions (PCR) using 10 μL reaction, containing 2 μL of 0.4 µM
primer mix, 1.5 μL of 20 ng of template DNA, 5 μL of Multiplex Master
Kit (Qiagen) for mixed primers and Top Taq Master Kit (Qiagen) for Gf01
and 1.5 μL of water nuclease free (Qiagen). We followed the PCR thermal
profiles and mixes described in Rodriguez et al. (2022). The PCR was
performed in a Veriti™ thermocycler (Applied Biosystems, Foster City,
CA, USA). Capillary electrophoresis was performed on a 3500 genetic
analyzer (Applied Biosystems) using Hi-Di™ formamide and GeneScan™ 500
LIZ™ dye size standard (Applied Biosystems). Multilocus genotypes were
scored using GeneMarker 1.91 (SoftGenetics, State College, PA, USA).
Acoustic analyses
We analyzed all songs using Raven pro 1.6 software (Cornell Lab of
Ornithology, Ithaca, NY, USA). We classified each song according to its
structure in different types following the classification of the sound
library for white-eared ground-sparrow available in LEUCA and used in
previous studies (Sandoval et al., 2014, 2015, 2016; Méndez et al.,
2020). In addition, we measured the following acoustic characteristics:
duration (s), minimum frequency
(kHz), maximum frequency (kHz), frequency of maximum amplitude (kHz),
and the number of elements. We used different windows to obtain the
measurements: the spectrogram window was used to identify and classify
sounds, the power spectrum was used to measure frequency characteristics
with a threshold of -30dB relative to vocalization peak, and the
oscillogram to measure the temporal characteristics of sounds.
Spectrograms were constructed using a Hann window with a size of 512
samples, 3 dB bandwidth of 124 Hz; a Time grid with an overlap of 50%
and hop size of 5.80 ms; and a frequency grid spacing with DFT size of
512 samples and grip spacing of 86.1 Hz.
Morphometric measurements
We measured six morphological traits: tarsus length (from the
intertarsal joint to the middle of the foot sole), tail length, wing
chord length (unflattened), exposed culmen length (from the tip of the
beak to base of skull), beak width (at the beak gape), and beak depth
(measured at right angles to the point on the lower mandible where the
feathers end) following the protocol described in Sandoval & Mennill,
(2013). We used a dial caliper (model: SPI Plastic Caliper 150mm,
AVINET, NY, USA) to get the bill and tarsus measurements, a metal wing
ruler (model: WING15ECON, AVINET, NY, USA) to measure wing chord and
tail length.
Statistical analyses