Species limits and phylogeography
The species status of both lava lizards and geckos from Floreana and
surrounding islets is strongly supported by morphological evidence (Van
Denburgh, 1912; van Denburgh & Slevin, 1913; but see Discussion). To
further test that populations from Floreana and surrounding islets
represent the same species, we examined species limits of both M.
grayii and P. baurii with phylogenetic analyses and genetic
distances. We assume that monophyly and genetic distances relative to
closely related species are strong criteria for delimiting species.
Sequences were assembled and aligned in Geneious v9.1.8
(http://www.geneious.com/) under default settings for MAFFT (Algorithm:
Auto, Scoring Matrix: 200PAM/k=2, Gap open penalty: 1.53, Offset value:
0.123; Katoh & Standley, 2013). Alignments were verified by translating
sequences into amino acids to check for misplaced stop codons. After
partitioning each dataset (i.e., Microlophus andPhyllodactylus ) by codon position, we conducted maximum
likelihood analyses using RAxML-HPC2 v.8.2.9 with a heuristic search
that included 100 random addition replicates and 1000 thorough bootstrap
pseudo-replications. We applied the GTR+GAMMA model to all partitions.
Analyses were performed using the CIPRES platform (Miller, Pfeiffer, &
Schwartz, 2010). The phylogenetic trees were visualized and edited using
FigTree v1.4.2 (Rambaut, 2014). In addition, for each dataset we
calculated uncorrected genetic distances in MEGA 7 (Kumar, Stecher, &
Tamura, 2016) to address genetic differentiation within M.
grayii , P. baurii , and their corresponding radiations.
The relationship among haplotypes from Floreana, Champion and Gardner
was evaluated for both lizard species by inferring TCS networks
(Templeton, Crandall, & Sing, 1992) in PopART (Leigh & Bryant, 2015).
This method has been used widely with nucleotide sequence data to infer
population level genealogies when divergences are low (Georgiadis et
al., 1994; Gerber & Templeton, 1996; VilĂ et al., 1999). Because
haplotype networks can be misleading in the presence of missing data
(Joly, Stevens, & van Vuuren, 2007), two datasets were analyzed for
each species: one including all sampled sequences, and the other
removing those sequences containing significantly more undefined states
than others.