6 AMERICAN MUSEUM NOVITATES NO. 3963 
et al. (2014) and were given early access to sequence data subsequently published in Pat- 
terson et al. (2019); see appendix 2. Data for an outgroup, Submyotodon latirostris, were 
also obtained from GenBank (appendix 2). Genomic DNA from the new species of Myotis 
was extracted at CIBIO-InBIO, University of Porto, Portugal, using Qiagen DNeasy kits 
(Qiagen, Crawley, UK) and stored at -20° C. Mitochondrial cytochrome b (cyt b) gene 
was amplified by polymerase chain reaction (PCR) using the primers MOLCIT-F 
(5’-AATGACAT-GAAAAATCACCGTTGT-3’; Ibafez et al., 2006) and MVZ16-R 
(5'-AAATAGGAARTATCAYTCTGGTTTRAT-3’; Smith and Patton, 1993), PCR’s were 
performed in a 10 uL volume, which included 1 uL of DNA extract, 0.4 uL of each primer 
(10 uM), 5 pL of Qiagen Master Mix, and double-distilled water was added until final 
volume was reached. Reactions were performed under the following conditions: 95° C for 
15 min; 40 cycles of 95° C for 30 s, 50° C for 45 s, 72° C for 1 min; 60° C for 10 min, and 
DNA sequencing performed on an ABI3700 DNA sequencer (Applied Biosystems). 
Sequences were aligned using Geneious Prime v.2020.0.2 and edges of incomplete 
sequences were trimmed to reduce missing data, The final alignment included 102 indi- 
viduals with 634 BP and 277 variables sites, Models of sequence evolution were explored 
in jModel test v.2.1.10 (Darriba et al., 2012) and likelihood scores calculated using PhyML 
v.3.0 (Guindon et al., 2010). The model that best fits the data, TrN+I+G (Tamura and Nei, 
1993) was identified using the Akaike Information Criterion (wAIC = 0.9915). Phyloge- 
netic inferences were performed using two approaches. First, we used a Bayesian approach 
as implemented in BEAST v.2.6.0 (Bouckaert et al., 2014). A Yule prior was used with a 
uniform distribution. Two independent chains were run, each for 50 million steps sampled 
every 5000, and a burn-in equivalent to 25%, Convergence was assessed using effective 
sampling size in Tracer v.1.7.1 (Rambaut et al., 2018), Trees from the posterior distribution 
were summarized using a maximum clade credibility tree using mean node heights and a 
burning equivalent to 25% as implemented in Tree Annotator v.2.6.0 (Drummond and 
Rambaut, 2007), Second, we use a maximum likelihood (ML) approach as implemented in 
RAXML v.8 (Stamatakis, 2014). The best scoring ML tree was estimated using 20 replicates. 
Nodal support was calculated by 100 bootstrap replicates. Finally, the percentage of genetic 
divergence among species of the subgenus Chrysopteron was calculated using Neis dA 
index (Nei and Kumar, 2000) as implemented in strataG v.2.4.905 (Archer et al., 2017). 
Only taxa with more than two samples were included in the analysis. 
MORPHOLOGICAL ANALYSES 
We examined 34 specimens of adult Myotis (15 males, 16 females, 3 of unknown sex; 
appendix 1) as well as literature descriptions of all Chrysopteron species, and evaluated external 
and osteological characters including but not restricted to those discussed by Hill and Morris 
