Uncovering a hidden diversity: a new species of freshwater shrimp Macrobrachium (Decapoda: Caridea: Palaemonidae) from Neotropical region (Brazil) revealed by morphological review and mitochondrial genes analyses Rossi, Natalia Magalhães, Célio Mesquita, Elis R. Mantelatto, Fernando L. Zootaxa 2020 2020-02-11 4732 1 177 195 6424dade-8c68-4c20-9aa1-bce09e026fca Rossi & Magalhães & Mesquita & Mantelatto, 2020 Rossi & Magalhães & Mesquita & Mantelatto 2020 [151,481,1402,1429] Malacostraca Palaemonidae Macrobrachium Animalia Decapoda 5 182 Arthropoda species veredensis sp. nov.  ( Figs. 2and 3) http://zoobank.org/ urn:lsid:zoobank.org:act: D86A5A10-08BB-419F-9341-5724E8CB7723   Material examined:   Holotype: Male(CL 38.67 mm), MZUSP 39527, Brazil, Minas Gerais state: Chapada Gaúcha, Grande Sertão Veredas National Park, prainha do Rio Preto(little beach of the Black river), 15º08.97’S, 45°45.16’W,  14/vii/2011, Carvalho, F.C.& Souza-Carvalho, E.    Paratypes: Brazil, Minas Gerais state: 1 male( 38.30 mm), MZUSP 39528and  5 males(CL 23.10–38.00 mm), CCDB 535, same data as holotype.  Others:  9 males(CL 26.59–49.92 mm), 1 ovigerous female (CL 34.85 mm), CCDB 543, Chapada Gaúcha, Grande Sertão Veredas National Parknear to Cariranha river, 15°07.25’S, 45°44.43’W,  13/vii/2011, Carvalho, F.C.& Souza-Carvalho, E.;  2 males(CL 27.46 mm, 31.92 mm), CCDB 464, Brazil, Chapada Gaúcha, Grande Sertão Veredas National Park, Black river, 15°10.92’S, 45°46.17’ W,  13/vii/2011, Carvalho, F.C., Jacobucci, G.& Souza- Carvalho, E.;  3 males(CL 31.90–35.10 mm), CCDB 5456, Povoado de Pandeiros, 15°30’46.0” S, 44° 45’ 12.4” W,  12/ii/2014, Bueno, A.; 3 males(CL 28.11–32.00 mm),  1 female(CL 21.29 mm), CCDB 534, Comunidade S. Joaquim, Jabuticaba river, Januária/ Chapada Gaúcha,  12/vii/2011, Carvalho, F.C.& Souza-Carvalho, E.;  2 males(CL 23.14 and 28.77 mm), CCDB 922, Córrego do Bom Jantar, Januária, 15°33.36’S, 44° 44’ 29.46’S W,  12/vii/2011, Carvalho, F.C.& Souza-Carvalho, E.  Additional comparative material examined:  Macrobrachium ferreirai:  Brazil, Amazonasstate: 1 male(CL 50.85 mm), CCDB 2587, Jaú National Park,  25/xi/2000, Magalhães, C.; 2 males(CL 42.18 and 52,70 mm), CCDB 2588, Jaú National Park,  25/xi/2000, Magalhães, C.; Tocantins state: 1 male(CL 41.03 mm) and 1 ovigerous female (CL 33.67 mm), CCDB 6204, Alta Mateiros,  19/v/2013, Mantelatto, F.L. et al .   Macrobrachium nattereri:  Brazil, Amazonasstate: 1 male(CL 47.97 mm) CCDB 2130,  15/01/2005, Biotupé project;  Tocantins state: 2 males(CL 33.69 and 36.06 mm) CCDB 5023, Ponte Alta,  19/v/2013, Mantelatto, F.L. et al .   Diagnosis:Rostrum straight, almost reaching end of antennular peduncle and scaphocerite; upper margin with 8–10 teeth, usually 9, regularly arranged; 2–3 postorbital, usually second one on postorbital margin; lower margin of rostrum with 2–3 teeth. Antennule peduncle 3–segmented, with inner and outer flagella, outer flagellum biramous. Carapace smooth. Second pereopods (P2) similar in shape, different in size, overreaching scaphocerite with all carpus length; dactylus longer than propodus half-length. Dactylus with 1–2 large teeth, sometimes with smaller ones on cutting edges, row of small tubercles along inner margin. Propodus flattened, smooth with sparse spinules and setae on external faces. Fifth pleonal somite with rectangular pleuron and peaked lower margin. Males with endopod of second pleopod bearing appendix masculina smaller than appendix internae. Telson with posterior margin ending in distinct acute tip, bearing 2 spines with setae between them; inner pair of spines reaching or overreaching apex of telson.   Description:Fully grown male ( Fig. 2A). Body moderately robust. Rostrum straight, extending beyond or as long as antennular peduncle and almost reaching end of scaphocerite, armed dorsally with 8–10 teeth, usually 9, regularly arranged, 2–3 teeth postorbital, usually the second one on postorbital margin; lower margin of rostrum straight, not enough pronounced; lower margin of rostrum with 2–3 teeth. Rostral formula 8–10 (2–3)/2–3. Antennular spine acute, placed slightly below orbital angle, hepatic spine of similar size or smaller than antennal spine and located slightly behind and lower than antennal spine; paired hepatic spines parallel to antennal spines situated between upper first and second teeth of rostrum on carapace. Carapace smooth, some individuals with spinules sparsely scattered at anterior and ventral region. Eyes well developed. Antennule ( Fig. 3I) three-jointed protopodite bearing three multiarticulated flagella at distal end and statocyst at base. Inner flagellum of antennule unique and longer outer flagellum. Antennular peduncle almost reaching distal margin of scaphocerite. Basal segment of antennular peduncle moderately broad, lateral margin straight, with small proximal tooth, ventral edge tapering posteriorly; stylocerite short and sharp. Antenna ( Fig. 3H) with scaphocerite longer than wide, outer lateral side straight. Antennal flagellum multiarticulated, as long as body length. Mandible ( Fig. 3G) with slender, 3-articulated palp; incisor process robust, with 3 strong teeth; molar process robust, truncate distally, with four principal peripheral teeth. Maxilla ( Fig. 3D) with basal endite bearing setae on apical margin; endopodite and exopodite (scaphognathite) with numerous short setae distally; scaphognathite broad, anterior lobe large, posterior lobe moderately narrow, both lobes rounded. Maxillula ( Fig. 3E) with apical margin of endopodite weakly bilobed; coxa and basis surface with stiff setae. First maxilliped ( Fig. 3F) with setose palp, basal and coxal endites distinct, flagellum of exopod with numerous plumose setae distally, epipod deeply bilobed. Second maxilliped ( Fig. 3C) with normal endopod, flagellum with numerous setae distally, epipod simple, with well-developed podobranch. Third maxilliped ( Fig. 3B) with robust endopod, exopod with numerous plumose setae distally, reaching to distal margin of ischio-merus. First pereopod ( Fig. 3J) slender, smooth, reaching beyond scaphocerite by entire distal margin of carpus, equal in length, similar in form. Palm as long as finger, carpus 1.9 times as long as chela, merus shorter than carpus; both fingers ending in small apical claw. Second pereopod—P2 ( Fig. 2B) similar in shape, different in size, longer than total length; overreaching scaphocerite with all carpus length. P2, dactylus with 1–2 large teeth, sometimes with smaller ones on cutting edges, row of small tubercles along inner margin; gaping when fingers closed; dactylus longer than propodus half-length; propodus flattened, smooth with sparce spinules and setae on external faces; upper spinules obtuse; chela with small spines, with lower ones smaller than upper ones and bending towards legs ( Fig. 3A). Last three pereopods slender, smooth, similar in form; propodus, carpus, merus, with few setae. Pleon with second somite broad with lateral overlapping the first and third; fifth somite with rectangular pleuron and peaked lower margin; sixth somite slightly shorter than telson. Male second pleopod with appendix masculina longer, stouter than appendix internal, with numerous stiff setae. Telson ( Fig. 3C) almost three times as long as wide, mostly triangular, with posterior margin ending in distinct acute tip, bearing two pairs of spines with setae between them; inner pair of spines reaching or overreaching apex of telson.   Type locality:  Brazil, Minas Gerais state, Grande Sertão Veredas National Park(PARNA—Grande Sertão Veredas), Chapada Gaúcha, prainha do Rio Preto(little beach of the Black river), 15º08.97’S, 45°45.16’W.   Distribution:Only known from part of the middle São Francisco river basin, in the state of Minas Gerais, Brazil.   Etymology:The new species name is derived from geographical name where the holotypewas collected, the Grande Sertão Veredas National Park, in Minas Gerais state, Brazil.  Molecular data.We aligned 43 sequences (23 news), with 528 base pairs (bp) to 16S rDNA, and 16 new sequences with 581bp, without pseudogenes, for COI mtDNA. The genetic distance among the sequences of specimens previously identified as  M. brasilienseranged from 0.0 to 8.5% for 16S, and 0.0 to 12.7% for COI. When individuals were separated into two species, sequences of 16S ranged for  M. brasiliensefrom 0.0 to 4.3% ( Table 2) and 0.0 to 2.5% for COI. The diversity among the individuals from the São Francisco river basin in the state of Minas Gerais(  M. veredensis  sp. nov.) ranged from 0.0 to 1.70% for 16S ( Table 2) and 0.0 to 0.08% for COI. The nucleotide divergence between sequences from  M. veredensis  sp. nov.and  M. brasilienseranged from 7 to 8.5% for 16S and 11.8 to 12.5% for COI. All these individuals and congener species (  M. amazonicum, M. aracamuni,  M. borelli, M. ferreirai,  M. iheringi, M. inpa,  M. potiunaand  M. nattereri) ranged from 6.8 to 14.3% for 16S (see details in Table 2). Similar topologies were achieved on ML and BI analyses for 16S data set. Therefore, the final tree obtained by ML with bootstrap values and posterior probability (BI) more than 50 were added ( Fig. 4). The topologies obtained by both analyses revealed that  M. veredensis  sp. nov.and  M. brasilienserepresent monophyletic clades with high values to support (bootstrap 75 and posterior probability 80). Individuals herein considered as from São Francisco river system in central-western Minas Gerais,  M. veredensis  sp. nov.is clustered as sister-group of  M. brasiliense. Moreover, our phylogenetic hypothesis showed distinctive phylogroups with similar morphology in M. brasiliense, as well as in  M. potiuna, which had already considered a possible cryptic species group ( Carvalho et al.2013).   Macrobrachium aracamuni, M. ferreiraiand  M. inpawere positioned in another clade, which is sister-group of  M. nattereri.  Macrobrachium iheringis clustered as sister-group of  M. potiunaclade.  Macrobrachium borelliiis sister-group of the clade with outgroup species. The phylogenetic tree was rooted with  M. amazonicumclade ( Fig. 4).   FIGURE 4.Left: Phylogenetic tree obtained from maximum likelihood analysis (ML) of 16S sequences of  Macrobrachium rasiliense( Heller, 1862)and  Macrobrachium veredensis  sp. nov.Highlighted numbers on nodes represent the posterior probabilities obtained by Bayesian Inference analysis of 16S sequences and numbers without asterisk are bootstrap obtained by ML. Right: Haplotype network based on Median-Joining analysis of COI sequences, indicating the distribution of each haplotype found in both species. The size of the circles is proportional to the haplotype frequency. The haplotype identification is below each circle. Different colors represent populations from different states of Brazil: Pará (PA), Tocantins (TO), Mato Grosso (MT), São Paulo (SP), Minas Gerais (MG). The length of the lines between circles is proportional to mutational steps among the haplotypes, except between haplotypes from Minas Gerais (MG) (  Macrobrachium veredensis  sp. nov.) and the others (  Macrobrachium brasiliense), where three small traces represent a lot of the mutational step. Yellow star indicates median vector. Fourteen haplotypes (H) with high diversity (0.98) were recognized based on the COI fragment of sixteen unambiguous sequences, with 581bp, without pseudogenes ( Table 3). Twelve haplotypes (85.71%) represented single individuals, and two (14.29%) were polymorphic. The frequencies of haplotypes in different localities were heterogeneous ( Table 3). The sixth haplotype (H6) was shared between two individuals from northern São Paulo(BR-SP). The tenth (H10) was shared between two individuals from Pará(BR-PA). Networks haplotypes were similar based on the statistical parsimony method (data not shown) and Median-Joining method ( Fig. 4).   TABLE 3.Distribution of COI haplotypes detected in  Macrobrachium brasiliense( Heller, 1862)from different localities and  Macrobrachium veredensis  sp. nov.from Minas Gerais.    Locality Haplotypes N Hd±Sd  Mato Grosso H1, H2, H3 3 1.0±0.2   Minas Gerais  H7, H8, H9, H12, H13, H14  6  1.0±0.9  Pará H10, H11 3 0.6±0.3  São Paulo H6 2 0.0±0.0  Tocantins H4, H5 2 1.0±0.5 Analysis of molecular variance (AMOVA) indicated that specimens within the  M. brasiliensepopulations have the lowest percentage of variation (5.4% without hierarchical structure— i.e.,  M. veredensis  sp. nov.included—and 3.7% with hierarchical structure), whereas the variation among populations was higher (94.6% without hierarchical structure and 12.6% with hierarchical structure). When populations were structured according to the Minas Gerais region(  M. veredensis  sp. nov.) and other Brazilian populations (  M. brasiliense), the variations among groups were very high (83.7%) and the values obtained by AMOVA based on haplotypes frequencies with and without hierarchical structure were significant ( Table 4). 2571581879 2011-07-14 MZUSP Carvalho, F. C. & Souza-Carvalho, E. Brazil Male -15.1495 Grande Sertao Veredas National Park 13 -45.752666 Chapada Gaucha 5 182 MZUSP 39527 1 Minas Gerais holotype 2571581878 [199,1080,1653,1681] MZUSP Brazil Minas Gerais state 5 182 MZUSP 39528 1 1 Minas Gerais paratype 2571581875 2011-07-14 CCDB Carvalho, F. C. & Souza-Carvalho, E. Brazil Male -15.1495 Grande Sertao Veredas National Park 13 -45.752666 Chapada Gaucha 5 182 CCDB 535 5 5 Minas Gerais state paratype 2571581880 2011-07-13 CCDB Carvalho, F. C. & Souza-Carvalho, E Cariranha river -15.120833 Chapada Gaucha 13 -45.7405 Grande Sertao Veredas National Park 5 182 CCDB 543 9 9 2571581873 2011-07-13 CCDB Carvalho, F. C. & Jacobucci, G. & Carvalho, E Brazil Black river -15.182 Chapada Gaucha 13 -45.7695 Grande Sertao Veredas National Park 5 182 CCDB 464 2 2 2571581871 2014-02-12 CCDB Bueno, A Brazil -15.512777 Povoado de Pandeiros 1 -44.753445 5 182 CCDB 5456 3 3 2571581874 2011-07-12 CCDB Comunidade S. Joaquim & Carvalho, F. C. & Souza-Carvalho, E Brazil Chapada Gaucha Jabuticaba river Januaria 5 182 CCDB 534 1 1 2571581876 2011-07-12 CCDB Carvalho, F. C. & Souza-Carvalho, E Brazil -15.556 Corrego do Bom Jantar Januaria 5 182 CCDB 922 2 2 2571581870 2000-11-25 2013-05-19 2000-11-25 CCDB Magalhaes, C. & Mantelatto, F. L. Brazil Amazonas Jau National Park Jau National Park 6 183 CCDB 2587, CCDB 2588, CCDB 6204 4 4 Tocantins 2571581877 2005-01-15 CCDB Brazil Amazonas 6 183 CCDB 2130 1 1 2571581872 [197,1392,332,359] 2013-05-19 CCDB Mantelatto, F. L. Brazil Tocantins state 6 183 CCDB 5023 2 2 Tocantins 2571581869 Brazil Grande Sertao Veredas National Park -15.1495 Chapada Gaucha 13 -45.752666 Sertao Veredas 9 186 1 Minas Gerais