This study analyzed genetic data from Leporinus copelandii fish collected from four river basins to identify potential cryptic species. Several species delimitation analyses found evidence for two distinct lineages - one containing fish from the Doce, Mucuri, and Jucuruçu river basins, and another containing fish from the Paraíba do Sul river basin and an aquaculture farm. Molecular dating estimated that the two lineages diverged around 1 million years ago during the Pleistocene, likely due to headwater capture events between coastal rivers. The results suggest L. copelandii represents two cryptic species and warrants a taxonomic review.

1. Supporting evidence for a cryptic species within the
Neotropical freshwater fish group
Leporinus copelandii
Izabela Santos MENDES1,2 ; Bruno Francelino de MELO3; Júnio Damasceno SOUZA2; Daniel Fonseca TEIXEIRA1,2 ;
Daniel Cardoso CARVALHO1,2
1 Postgraduate Program in Genetics, Federal University of Minas Gerais, Brazil
2 Conservation Genetics Laboratory, Post-Graduate Program in Vertebrate Biology, Pontifical Catholic University of Minas Gerais, Brazil
3 instituto de Biociências, Universidade Estadual Paulista, São Paulo, Brazil
Email: izabelasantosmendes@hotmail.com

2. Introduction
Leporinus copelandii (Anostomidae) is a freshwater fish species described
from the Northeastern Atlantic Forest and the Paraíba do Sul ecoregions.
Considering its wide distribution and morphological and genetic variations
previously found among populations from distinct river basins, L. copelandii may
be considered a complex of species represented by several Molecular
Operational Taxonomic Units (MOTUs). Therefore, we aim to shed light on the L.
copelandii phylogeographic based on two mitochondrial (COI gene and rRNA 12S
region) and one nuclear (the first intron of the S7 protein) markers and four
species delimitation methods to identify MOTUs within L. copelandii from four
coastal drainages: Doce (DRB), Mucuri (MRB), Jucuruçu (JRB) and Paraíba do Sul
(PSRB and an aquaculture farm - EHAP) (Figure 1). In addition, using a time
calibrated analysis we tested biogeography hypothesis such as paleodrainages
connections and headwater capture events that may have contributed to species
diversification and distribution along the coastal basins of the Northeastern
Atlantic Forest and the Paraíba do Sul freshwater ecoregions.
Figure 1. Map showing sampled locations surveyed around the Northeastern
Atlantic Forest and the Paraíba do Sul ecoregions.

3. Sample collection was conducted during June 2014 and August 2016, across drainages from the
Northeastern Mata Atlântica and the Paraíba do Sul freshwater ecoregions. Specimens of L. copelandii were
obtained from the Doce River basin (DRB) (n=13); Mucuri River basin (MRB) (n=3); Jucuruçu River basin (JRB)
(n=4); Paraíba do Sul River basin (PSRB) (n=6); and from one aquaculture farm (Estação de Hidrobiologia e
Aquacultura de Paraibuna - EHAP) (n=7) (Figure 1).
We applied single-locus cluster delimitation analyses based on Bayesian [Generalized Mixed Yule Coalescent
(GMYC) and Bayesian Poisson Tree Processes (bPTP)] and genetic distance methods [Automatic Barcode Gap
Discovery (ABGD) and based on Kimura-2-parameter model] to test the hypothesis that L. copelandii consists
of distinct MOTUs correlated with each drainage.
The estimation of divergence time in the ML phylogeny was carried out using BEAST on a COI dataset that
included sequences from Anostomidae members, totaling 74 sequences.
Materials and Methods

4. Using COI dataset, all species delimitations analyses, except bPTP, clearly indicate three MOTUs within L. copelandii: (a) DRB+MRB; (b) JRB
and (c) PSRB+EHAP (Figure 2b). Similar to the COI gene, species delimitation analyses using the 12S gene indicate the presence of two
major groups within L. copelandii: DRB+MRB+JRB and PSRB+EHAP (Figure 2c). In this case, Bayesian analyses, GMYC and bPTP,
overestimated the number of MOTUs, resulting in 10 and 17 lineages, respectively. Differently from the other markers, the phylogenetic
tree constructed using S7 grouped L. copelandii DRB+PSRB and segregated L. copelandii MRB and JRB (Figure 2d).
Our molecular dating indicated that at around 1 Ma (0.7–3.2 Ma HPD), during the middle/late Pleistocene epoch L. copelandii PSRB+EHAP
split from L. copelandii DRB+MRB+JRB (Figure 3).
Figure 2. (a) Map showing sampled locations surveyed around the Northeastern Mata Atlântica and the Paraíba do Sul ecoregions and Bayesian phylogenetic ultrametric tree for COI , 12S and S7 genes with all species
analyzed. MOTUs are represented by different sizes and colors in accordance with each different species delimitation methods (GMYC, bPTP, ABGD and interspecific genetic distances thresholds based on K2P).
COI 12S S7
Results

5. Our results reveal hidden biodiversity and suggest a taxonomic
review for L. copelandii from these four river basins.
Thus, the two recovered MOTUs (Figure 3) may be explained by
headwater captures between these coastal rivers during the
Late Pleistocene, rejecting the hypothesis of paleodrainages
connection that occurred during earlier Pleistocene.
The recovering of cryptic species help to better describe and
thus manage the Neotropical fish megabiodiversity inhabiting
highly degraded basins, threatened by pollution, mining
activities, habitat fragmentation and invasive species.
Figure 3. Timetree of Leporinus copelandii and relatives obtained from maximum likelihood relaxed clock analysis of
the reduced dataset using †Leporinus scalabrinii (Anostomidade) as calibration point. Horizontal node bars indicate
dating estimations with 95% posterior probability densities (HPD).
Leporinus
copelandii
DRB+MRB+JRB
Leporinus
copelandii
PSRB+EHAP
Discussion and Conclusions