Research Article |
Corresponding author: Felipe Polivanov Ottoni ( fpottoni@gmail.com ) Academic editor: Peter Bartsch
© 2019 Felipe Polivanov Ottoni, José L. O. Mattos, Axel M. Katz, Pedro H.N. Bragança.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ottoni FP, Mattos JLO, Katz AM, Bragança PHN (2019) Phylogeny and species delimitation based on molecular approaches on the species of the Australoheros autrani group (Teleostei, Cichlidae), with biogeographic comments. Zoosystematics and Evolution 95(1): 49-64. https://doi.org/10.3897/zse.95.31658
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Three distinct and independent molecular-based species delimitation analyses were performed among the species and populations included within the Australoheros autrani group, based on sequences of the mitochondrial gene Cytochrome b: a tree-based method proposed by Wiens and Penkrot (WP), a Character-based DNA Barcoding (CBB) and coalescent species delimitation method termed the Bayesian Implementation of the Poisson tree processes (bPTP). The congruence of WP and CBB delimited 11 independent lineages (species), while the bPTP delimited just nine lineages. We did not favour any of the methods, and we considered the possibility of two slightly variant scenarios. A time-calibrated phylogenetic analysis is proposed based on the predominant congruence of the results of these three species delimitation methods herein applied. The monophyly of the A. autrani species group was highly supported with maximum node support value and diagnosed by 11 nucleotide substitutions. The sister clade of the A. autrani species group is the clade comprising A. sp. Timbé do Sul and A. minuano. The phylogenetic analysis supports three main clades within the A. autrani species group, supported by maximum node support value, with the Southern Mata Atlântica clade as the most basal clade. Divergence time estimates indicate that the diversification of the Australoheros originated during the early Neogene, but only in the late Neogene did the processes of diversification in the southeast and north regions occur. Diversification within the Australoheros autrani species group occurred synchronically for the three main clades during the beginning of the Quaternary. It is demonstrated that molecular characters are valuable tools for species recognition, particularly in speciose groups with inconspicuous or difficult to record morphological characters. The resulting phylogeny of the Australoheros autrani group is highly compatible with the geological and biogeographic scenarios proposed for the Neogene and Quarternary shaping of the extant river basins of eastern Brazil. Despite the origin of the A. autrani group being dated to the late Miocene, species level diversification occurred in the Pleistocene and was probably driven by headwater capture events and sea-level fluctuations.
Atlantic rain forest, Biodiversity, Biogeography Cichlinae, Heroini , Ichthyology, mitochondrial DNA, Neotropical region
Over the past two decades, research on cryptic species have exponentially increased, mainly due to the improvement of molecular methods and availability of DNA sequences (
The ongoing destruction and disturbance of natural ecosystems and the resultant increase in extinction rates, makes it urgent to catalogue and describe biodiversity, as well as to develop approaches directed to the study of species complexes (
The Neotropical region, known for its rich species diversity, has inspired classical studies on evolutionary biology. In this region the most diverse fauna of freshwater fishes in the world is found, exhibiting intriguing and unique specializations (e.g.
Australoheros Říčan & Kullander, 2006 is a South American cichlid genus of the tribe Heroini, which was described to include species previously placed in Cichlasoma Swainson, 1839. Before 1995, all the 29 nominal species presently contained in Australoheros were considered to belong to a single species, Cichlasoma facetum, which was thought to be geographically widespread between southeastern Brazil and northeastern Argentina (
Presently, the A. autrani group includes 16 nominal species (
Specimens of the A. autrani group were fixed in absolute ethanol immediately after collection and later preserved in the same solution (see Suppl.material
Map of the samples obtained for the present work. Circles = Australoheros autrani species group; Red circles = Southern Mata Atlântica clade; Yellow circles = Upper/middle Paraíba do Sul river basin and adjacent drainages clade; Green circles – Northern Mata Atlântica clade; and Square = A. sp. Timbé do Sul. Localities: A. autrani = 1 and 18, A. barbosae = 2, 3, 10, 11 and 19, A. ipatinguensis = 4, A. macacuensis = 5, A. macaensis = 6, A. muriae = 9, A. perdi = 12, A. ribeirae = 14, A. robustus = 7, 8, 15, 16 and 20, A. sanguineus = 17, A. cf. capixaba = 13, and A. sp. Timbé do Sul = 21.
The collected specimens were euthanized in a buffered solution of tricaine methane sulphonate (MS-222) at a concentration of 250 mg/L, for a period of 10 min, following the guidelines of the Journal of the American Veterinary Medical Association (AVMA Guidelines) (
The genomic material was extracted from muscle tissue of the caudal peduncle region through the commercial DNeasy Blood and Tissue Kit (Qiagen). We used the primers CytB-F (
Three distinct operational criteria (species delimitation methods), based on molecular data, were performed: a tree-based method as proposed by
The WP is based on the direct inspection of haplotype trees generated from the phylogenetic analyses having as terminals at least two individuals (haplotypes) of each focal species. In this method, the term “exclusive” is used instead of monophyletic, since the term monophyly is considered inapplicable below the species level (
The topology for the WP approach was generated by Bayesian inference analysis performed in the MrBayes 3.2.5 program (
Phylogenetic haplotype tree based on Bayesian Inference (BI). Numbers above branches are posterior probability values, and below branches are numbered nodes which represent the combination of nucleotide substitution which define the species (in CBB) or clades. The nucleotide substitutions (CBB) can be checked in box1. Posterior probability value supporting the Australoheros autrani group is indicated in blue, as well as, the three clades herein proposed within this species group are indicated in green. Species of the in-group delimited though the tree based method (WP) are indicated with red bars, as well as, the species of the in-group delimited by nucleotide substitution method (CBB) have their nodes marked in red.
The CBB is similar to the population aggregation analysis proposed by
List of nucleotide substitutions from each lineage (species) and some crucial points of the cladogram of the Fig.
CBB: 1 (exclusive combination of nucleotide substitution of A. barbosae) - Cytb258 (C→T*), Cytb474 (T→C), 2 (exclusive combination of nucleotide substitution of A. robustus) - Cytb348 (G→A**), Cytb612 (G→A), Cytb 684 (C→T**),Cytb 978 (G→A),Cytb 1.032 (A→G*), 3 (exclusive combination of nucleotide substitution of A. macacuensis) - Cytb120 (T→C), Cytb 127 (G→A**), Cytb 561 (T→C), Cytb 822 (T→C**), Cytb 939 (A→G*), 4 (exclusive combination of nucleotide substitution of A. ipatinguensis) - Cytb 78 (T→C), Cytb 1.070 (T→C*), 5 (exclusive combination of nucleotide substitution of A. ipatinguensis + A. perdi. Australoheros perdi differs from A. ipatinguensis by the absence of the nucleotide substitutions listed in number 4) - Cytb 700 (A→G*), 6 (exclusive combination of nucleotide substitution of A. cf. capixaba) - Cytb 837 (C→T**), 7 (exclusive combination of nucleotide substitution of A. muriae) - Cytb 435 (C→T**), Cytb 519 (T→C), Cytb 653 (A→G*), Cytb 960 (A→G**), 8 (exclusive combination of nucleotide substitution of A. macaensis + A. muriae. Australoheros macaensis differs from A. muriae by the absence of the nucleotide substitutions listed in number 7) - Cytb 1.041 (T→ C**), 9 (exclusive combination of nucleotide substitution of A. autrani) - Cytb 270 (C→T*), Cytb 357 (T→C**), Cytb 519 (T→C), Cytb 564 (C→T**), Cytb 864 (A→G*), 10 (exclusive combination of nucleotide substitution of A. sanguineus) - Cytb 63 (C→A*), Cytb 120 (T→C), Cytb 204 (C→T*), Cytb 219 (A→G*), Cytb 405 (A→G**), Cytb 474 (T→C), Cytb 967 (T→C*), Cytb 1.044 (T→C**), 11 (exclusive combination of nucleotide substitution of A. ribeirae) - Cytb 114 (A→C*), Cytb 364 (C→T), Cytb 390 (C→T*), Cytb 408 (A→G), Cytb 528 (A→C*), Cytb 741 (T→C**), Cytb 786 (G→A**), Cytb 813 (A→G*), Cytb 870 (G→A**), Cytb 897 (T→C**), Cytb 1.003 (T→C*). Other relevant nucleotide substitutions: 12- Cytb 465 (C→T**), Cytb 846 (A→G*), Cytb 917 (T→C**), 13 – Cytb 364 (C→T), Cytb 408 (A→G), Cytb 540 (C→T*), Cytb 552 (C→T*), Cytb 784 (T→C**), Cytb 825 (A→G*), Cytb 867 (A→G**), Cytb 954 (G→A**), Cytb 978 (G→A), Cytb 993 (T→C**), 14- Cytb 352 (G→A**), Cytb 354 (T→C*), Cytb 438 (T→C*), Cytb 630 (T→C*), Cytb 688 (T→C**), Cytb 690 (A→G*), Cytb 726 (T→C*), Cytb 906 (A→G*), Cytb 916 (G→A**), Cytb 945 (T→C**), Cytb 1.020 (A→G**), Cytb 1.047 (T→C**), Cytb 1.074 (T→C), 15 - Cytb 345 (T→C), Cytb 474 (C→T),Cytb 589 (C→T*), Cytb 723 (C→T), Cytb 741 (C→T),Cytb 795 (G→A), Cytb 807 (A→G*), Cytb 852 (T→C), Cytb 897 (C→T), Cytb 978 (A→G), Cytb 1.038 (A→C), 16 – Cytb 303 (G→A), Cytb 768 (C→T*), Cytb 954 (A→G), Cytb 960 (G→A*), Cytb 1.026 (C→A*), 17 – Cytb 585 (T→C**), Cytb 721 (G→A**), 18- Cytb 561 (T→C), 19 – Cytb 114 (A→G**), Cytb 612 (G→A), 20 – Cytb 78 (C→T**), Cytb 141 (A→G*), Cytb 174 (T→C*), Cytb 351 (T→C*), Cytb 600 (T→C**), Cytb 750 (G→A**), Cytb 891 (C→T*), Cytb 924 (C→T**). |
The bPTP is a coalescent phylogeny-based species delimitation method intended to delimit species based on single locus molecular data (
The bPTP analysis was performed in the Exelixis Lab’s web server http://species.h-its.org/ptp/, following the default parameters except for a 20% burn-in and the tree was rooted on A. facetus. The results are presented in Figure
Species delimitation tree generated by the Bayesian Poisson Tree Processes (bPTP) model, using a fragment of the mitochondrial gene CYTB. Black lines indicate branching processes among species, red lines indicate branching processes within species. Species of the A. autrani species group delimited through bPTP are indicated with grey bars.
After performing the different species delimitation methods, only one haplotype from each species recovered by the congruence of all species delimitation methods was included (Suppl. material
The protein-coding sequences were partitioned by codon position. The Jmodeltest 2.1.7 program (
The divergence time analysis was performed in Beast v.1.8 (
WP and CBB
These species delimitation analyses produced identical results, delimiting 11 lineages (species) within the A. autrani group (Fig.
bPTP
This species delimitation analysis indicates nine lineages (species) within the A. autrani group (Fig.
The monophyly of the A. autrani species group was highly supported with maximum node support value and diagnosed by 11 nucleotide substitutions (Fig.
Time-scaled phylogeny obtained from the Bayesian analysis in BEAST. Values above nodes are mean average ages of the nodes, followed below blue bars representing the 95% highest posterior densities intervals for estimated ages; numbers indicated by arrows are the posterior probability obtained from the Bayesian analysis in MrBayes followed by the node number, corresponding to the node numbers of the Figure
The phylogenetic analyses support three main clades within the A. autrani species group. First an Upper/middle Paraíba do Sul river basin and adjacent drainages clade: including A. barbosae, A. macacuensis, and A. robustus, sister group of the Northern Mata Atlântica clade, supported by maximum value, and three synapomorphic nucleotide substitutions (Fig.
Second, a Northern Mata Atlântica clade: including A. autrani, A. ipatinguensis, A. macaensis, and A. muriae; sister group to the aforementioned clade, corroborated by maximum node support value and by 10 synapomorphic nucleotide substitutions (Fig.
Third, a Southern Mata Atlântica clade: including A. ribeirae and A. sanguineus, the most basal clade within the A. autrani species group, corroborated by maximum node support value and by 13 synapomorphic nucleotide substitutions (Fig.
Divergence time estimates (Fig.
Many species of Australoheros can be definitely considered cryptic species for three reasons. This is especially true of the species which are distributed in eastern Brazil. (1) In previous studies, these were identified and classified as Australoheros facetus, a species whose type locality is in the north of Uruguay. Even now, in some cases, species and populations of the A. autrani group have been erroneously classified and considered as populations of A. facetus. (2) According to
The coastal river basins of eastern Brazil are characterized by a low diversity of fish groups when compared to other areas, such as the Amazon basin. However, in spite of this relatively low diversity, the groups that occur in this region present a high degree of endemism along the river drainages, and speciation events along eastern Brazil are probably related to a complex palaeogeographical history (
The speciation events within the A. autrani species group occurred about 2.6–0.6 MYA during the Pleistocene (Fig.
Similar to the A. autrani group, in that several species occur along the latitudinal zonation of eastern Brazil, a congruent distribution pattern and endemism is also shared by other freshwater fish groups in this region, such as: Delturinae (family Loricariidae) (
In addition to the complex paleogeographical history of eastern Brazil, which probably promoted speciation in Australoheros, we also cannot ignore the behavioural characteristics of cichlids. Species from this family are usually territorial, not carrying out extensive migratory movements, forming pairs during the reproductive periods, and with different strategies of parental care, from protection of eggs to juveniles in their early stages of life (
The origin of the genus Australoheros lineage is dated herein from about 46 MYA, during the middle Eocene (Fig.
Geological evidence suggests that the continental margin of south-eastern Brazil passed through instability events during three main phases (90–75 MYA; 50–40 MYA; and 25–0 MYA), separated by quieter intervals, coinciding with phases of Andean orogeny (
This geological instability of the region resulting in headwater stream capture events and connections of drainage systems was first observed by
The results of the present study corroborate the idea of this complex biogeographic history. The Paraíba do Sul is the river basin of south-eastern Brazil revealing the highest diversity for the genus Australoheros. The present paper demonstrates three lineages (species) of Australoheros occurring along that river basin: A. barbosae, occurring in its upper/middle portion, A. robustus, occurring in the middle portion, and A. muriae, occurring in the lower portion. This fact is not common along the river basins of south-eastern Brazil, most of which only include one Australoheros species (Fig.
Australoheros barbosae, besides occurring in the upper/middle Paraíba do Sul river basin, also occurs in the upper Tietê river drainage, corroborating the suggested hypothesis of the existence of an ancient connection between these river basins. The species also occurs in the upper Grande river drainage (another river drainage of the Paraná river basin), which also suggests a possible ancient connection between these river drainages. Australoheros robustus, besides occurring in the middle portions of the Paraíba do Sul river basin, also occurs in the river drainages of the Paraopeba and Rio das Velhas (tributaries of the upper São Francisco river basin) (Fig.
However, the geologically documented separation of the Tietê river basin from the Upper Paraíba do Sul river basin, which in ancient times were connected discharging directly into the Atlantic ocean, occurred between 20–11.8 MYR (
However, in the A. autrani group some species could have been affected by the sea-level fluctuation. One example is A. autrani, which nowadays forms isolated populations in small isolated coastal river systems (São João river basin and Saquarema lagoon system). These isolated populations had some degree of contact during the Pleistocene regression of the sea level. Similarly, we cannot discard the role of sea-level fluctuations in the Pleistocene diversification patterns among the Northern Mata Atlântica clade, opposing to the idea presented by
This study confirms the importance of integrating different methods for species delimitation, as suggested by several proponents of “Integrative Taxonomy” (e.g.
Accurate estimates of biodiversity are of utmost importance due to the rapid loss, degradation, intense destruction and modification of natural environments by anthropic actions, especially in tropical areas (
The present study recognizes nine lineages (species) or 11 within the A. autrani species group, depending on the species delimitation method, grouped into three distinct and highly supported clades: the Southern Mata Atlântica clade, the Upper/middle Paraíba do Sul river basin and adjacent drainages clade, and the Northern Mata Atlântica clade. Four to five formerly described species are not resolved with the genetic species delimitation methods used in this study, but the majority of the species of the A. autrani group are well supported. However, it does not mean that knowledge of the number of lineages (species) occurring along the coastal river basins of eastern Brazil is complete. If more haplotypes of other populations are included in these species delimitation tests more lineages (species) may be recovered, whereas some lineages could be coalesced. Therefore, analyses including more populations and more genes are encouraged, in particular for the Northern Mata Atlântica clade, which is the clade within the A. autrani species group that presents incongruences related to the number of delimited species between methods here employed.
According to the present study, is clear that the A. autrani species group represents a valid and monophyletic group. It also contrasts the hypothesis that all species represent a widely distributed A. facetus. In fact, A. facetus was not even recovered as sister to the A. autrani group. This study thus represents a major contribution towards the knowledge and conservation of Australoheros. Finally, the time calibrated analysis provides dates of origin and diversification of Australoheros for the first time. Despite the origin of the A. autrani group in the late Miocene, species level diversification occurred in the Pleistocene and was probably driven by headwater capture events and sea-level fluctuations.
Thanks are due to Maria Anais Barbosa, Pedro Amorim, Filipe Rangel-Pereira, Wilson Costa, and Orlando Simões for the help in the field expeditions. We also thank Fernanda Estrella for the help with the map design. Valter M. Azevedo-Santos and Mauro Triques are gratefully acknowledged for provision of some material, Pedro Amorim for laboratory assistance. Wilson Costa critically red an earlier version of the manuscript and Jason Dunlop corrected the english language and style. This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (Finance Code 001), and the German Cichlid Society (DCG).
Taxon sampling, voucher catalogue number, GenBank accession number, gene sequenced country, drainage and new sequence in the present study
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