2.4 Putative species estimation
The two single-locus DNA barcoding methods consisted of two
fundamentally different approaches. First, we implemented three
distance-based approaches: (1) the Automatic Barcode Gap Discovery –
ABGD (Puillandre et al., 2012) performed using the online version of the
software
(https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html);
(2) the Assemble Species by Automatic Partitioning – ASAP (Puillandre
et al., 2021), an updated implementation of the ABGD hierarchical
clustering algorithm, performed with the ASAP web version
(https://bioinfo.mnhn.fr/
abi/public/asap/). Both methods used the MUSCLE aligned matrix as the
input file and adopted the Kimura model, following the default settings
for all parameters. (3) The Barcode Index Number (BIN), a method
implemented in BOLD, in which newly submitted and already available
sequences clustered in unique BINs using a refined single linkage
analysis in which records with high sequence similarity and connectivity
are clustered and separated from those with lower similarity and sparse
connectivity (Ratnasingham & Hebert, 2013).
Second, we applied four tree-based approaches, which models speciation
along the branches of an inferred phylogenetic: (1) the Single Poisson
Tree Processes – sPTP (Zhang et al., 2013), implemented using the PTP
online version
(http://species.h-its.org/ptp);
(2) the Bayesian Poisson tree process – bPTP (Zhang et al., 2013), also
conducted on the PTP web-server. Both analyses were conducted with
500,000 MCMC generations and other parameters as default. (3) the
Multi-rate Poisson Tree Processes – mPTP (Kapli et al., 2017),
performed with the mPTP web-server
(https://mcmc-mptp.h-its.org/mcmc/),
using the multi-rate Poisson tree process model and following default
settings. All PTP analyses (sPTP, mPTP and bPTP) used the ML trees
calculated with Iq-Tree. (4) the Generalized Mixed Yule Coalescent –
GMYC (Pons et al., 2006), performed by submitting the single ultrametric
MCC tree resulting from BI obtained from BEAST to the online version of
the GMYC software
(https://species.h-its.org/gmyc/),
following default single-threshold (sGMYC). We also tested the
multiple-threshold model (mGMYC); however, it did not perform well,
overestimating putative species (data not shown). Similar species
estimates with the mGMYC algorithm were also observed in previous
studies (Fujisawa & Barraclough, 2013; Schwarzfeld & Sperling, 2015).