Ethics and Permissions
Data collection received ethics clearance from Nelson Mandela
University: Research Ethics Committee (Animal; A15-SCI-ZOO-007) as well
as from the Ethics Committee of the Department of Zoology & Entomology,
Rhodes University (RU-DZE-2017-10-028). Capture permits have been
received from both Western Cape Province: Cape Nature (AAA041-00565),
and Eastern Cape Province: Department of Economic Development and
Environmental Affairs and Tourism (CRO55/17CR and CRO56/17CR). Birds
were ringed with permission from SAFRING (Ringer no: 17059), and blood
sampling technique was confirmed by Dr. Tarryn Fick (BVSc, Newton Park
Animal Hospital, Port Elizabeth, SA).
Results
Sequence variation
In total we produced 74 cytb sequences (380 bp; C. frenatus N =
59, C. aurantius N = 15), 85 ND2 sequences (505 bp; C.
frenatus N = 70, C. aurantius N = 15), and 55 RAG1 sequences
(455 bp; C. frenatus N = 45, C. aurantius N = 10). For the
cytb marker we found more genetic variation within C. aurantius(N = 5 haplotypes) compared to C. frenatus (N = 4 haplotypes).
For the ND2 marker we found five haplotypes in C. aurantius , and
15 haplotypes for C. frenatus (see below for details).
Uncorrected p -distances within C. frenatus varied from
0.0–0.002, within C. aurantius varied from 0.0–0.003, and
between C. frenatus and C. aurantius varied from
0.0–0.043 (Table 2).
Tajima’s D for both the cytb and ND2 markers in C.
frenatus were significantly negative (cytb: Tajima’s D = −1.70,p = 0.020; ND2: Tajima’s D = −1.77, p = 0.023),
while significantly negative for cytb but not significant for ND2 inC. aurantius (cytb: Tajima’s D = −0.78, p = 0.028;
ND2: Tajima’s D = 1.08, p = 0.893).
Population genetic
structure
We found no evidence for genetic differentiation among C.
frenatus localities, with low fixation indices showing strong evidence
for interbreeding among the various populations (cytb:
FST = 0.035, DF = 6; ND2: FST = 0.057,
DF = 6). Despite the proximity of C. aurantius sample populations
to one another, we found little evidence for interbreeding among them,
with higher fixation indices than those for C. frenatus (cytb:
FST = 0.277, DF = 1; ND2: FST = 0.470,
DF = 1). The phylogenetic networks did not show any clear pattern of
genetic structure among the sky islands (Figure 2).
The haplotype network for cytb showed little variation or pattern, while
we found a ”starburst” pattern for C. frenatus in the ND2 network
(Figure 2). When visualized by frequency per locality, ND2 showed little
evidence for spatial genetic structure (see inset Figure 3 below). The
low number of haplotypes within C. frenatus at the cytb locus (N
= 4) resulted in a haplotype network which did not have the variability
to visualize frequency by locality
Overall, most of genetic variation was within populations of C.
frenatus (cytb: 96.48 %, ND2: 94.26 %; see Table 3 for summary of
AMOVA), with similar variation partitioned between speciesC. frenatus and C. aurantius (cytb: 93.86 %; ND2: 93.77
%). We also found that while some variation existed within individual
populations of both C. frenatus and C. aurantius (cytb:
5.59 %; ND2: 5.35 %), there was little variation among populations
(cytb: 0.55 %; ND2: 0.88 %). High fixation indices for both cytb and
ND2 showed there was little evidence for interbreeding betweenC. frenatus and C. aurantius (cytb: FCT =
0.939, DF = 1; ND2: FCT = 0.938, DF = 1).
Our consensus tree based on ML inference resulted in stronger predictive
weight of node probabilities compared to the Bayesian tree (see Appendix
Figure A1 for Bayesian inference tree), and while we found no obvious
structuring within C. frenatus , we did find strong support for
two clear clades within family Chaetopidae being C. frenatus andC. aurantius (Figure 3).
Discussion
We found no evidence for genetic structure among populations ofC. frenatus . The results of population substructure among
populations (F ST < 0.05) indicated only
a slight to moderate genetic differentiation among the populations,
similar to two other studies on genetic structuring in sky island
endemic birds (Bech et al., 2009; Sittenthaler et al., 2018). Instead,
our study seems to provide support for the hypothesis that sky island
species with specialized niche requirements may experience selective
pressure which limits their genetic diversity [see Orsini et al.,
(2013)]. We also found evidence for interbreeding among individuals
from different mountain ranges, suggesting that birds may be able to
effectively disperse between mountain ranges. This suggests C.
frenatus is not currently experiencing inbreeding depression. However,
the lack of structure may also indicate the chosen loci are under
positive selective pressure and so are similar due to mutations relevant
for alpine species. The shape of our mtDNA haplotype networks suggestC. frenatus experienced a bottleneck or founder effect in their
recent genetic past. Tajima’s D also showed support for a past
bottleneck event in C. frenatus at both mtDNA markers. In
addition, overall low genetic diversity within C. frenatusindicates the potential for negative effects from a reduced effective
population size.
Population variation,
structure, and dispersal
Our finding that within C. frenatus most genetic variation was
from within populations, as opposed to among populations, suggests some
genetic structuring, and our haplotype frequency network implies this is
not geographically based (Figure 3). The genetic composition of C.
frenatus , along with the lack of geographic distribution of genetic
populations, suggest that although mountain populations ofC. frenatus are separated from one another by unsuitable lowland
habitat, the actual degree of separation does not hamper effective
dispersal. The question then remains as to how C. frenatus are
managing to effectively disperse across large tracts of unsuitable
habitat, presenting an interesting avenue for future research. While in
some sky island species low occurrence rates in lowland habitat implied
low dispersal, as in Mexican Jays Aphelocoma ultramarine(McCormack, Bowen, & Smith, 2008), according to SABAPs 1 and 2
(1987–1991, and 2007–present, respectively) there is currently a
complete lack of observations for C. frenatus in the lowland
habitat separating the mountain ranges of the Cape Fold Belt
(http://sabap2.adu.org.za; Harrison et al. 1997).
We also found evidence that while populations may be interbreeding,C. frenatus have low overall genetic diversity among populations
for both cytb and ND2 markers (i.e. genetic variation <5 %).
While we found no variation for our nDNA marker, we decided against the
addition of a different marker; the little variation we found for both
faster-evolving mtDNA markers suggests the addition of a second
(slower-evolving) nDNA marker, or a faster-evolving nDNA marker (i.e.
microsattelites) was unlikely to provide additional insight,. Moreover,
our choice of nDNA marker (RAG1) remains relevant for determining
population structure in birds (e.g. Dantas et al., 2019; Stervander,
Ryan, Melo, & Hansson, 2019). Low genetic variation among populations
may be from low diversity within the species, or close inbreeding within
an otherwise diverse population (Ceballos, Joshi, Clark, Ramsay, &
Wilson, 2018); since the results of the AMOVA suggest C. frenatuspopulations are interbreeding, there may be low diversity within the
species itself. Low diversity can arise from a recent demographic
expansion (e.g. bottleneck), here possibly tracking the mountain Fynbos
expansion ~37 KYA. A bottleneck may have extirpated many
individuals, reducing overall genetic variability. Because such a
population expansion would be recent in evolutionary terms, there has
not been time enough to accumulate measurable mutations. Alternatively,
as the loci analyzed are protein-coding, natural selection may be
purging all but a few advantageous alleles (e.g. strong stabilising
selection due to habitat specialisation; Orsini et al., 2013), asTajima’s D ’s for the mtDNA loci were negative, indicating a
higher frequency of rare alleles than would be expected under
neutrality. Although C. frenatus had a greater number of ND2
haplotypes than C. aurantius (N = 15 and 5, respectively), the
number is not proportionally greater considering the difference in
distribution coverage (~750 km and ~20
km respectively).