A new species of freshwater crab genus Fredius Pretzmann, 1967 (Crustacea: Brachyura: Pseudothelphusidae) from a naturally isolated orographic forest enclave within the semiarid Caatinga in Ceará, northeastern Brazil

Procedures with material examined

The specimens were collected using license permission from the Sistema de Autorização e Informação em Biodiversidade (SISBIO #29615) of the Brazilian Ministry of Environment (MMA). The studied specimens are deposited in the collections of the INPA (Instituto Nacional de Pesquisas da Amazônia, Manaus), MZUSP (Museu de Zoologia, Universidade de São Paulo, Brazil) and LACRUSE (Laboratório de Crustáceos do Semiárido). Other acronyms: SMF (Naturmuseum Senckenberg) and CCDB (Coleção de Crustáceos do Departamento de Biologia da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto). Measurements: cl (carapace length, taken along the carapace axis to the posterior median margin) and cw (carapace width, taken at the widest point), in millimeters (mm). Dates are written in the format day.month.year, with months in lower-case Roman numerals. Abbreviations are as follows: G1, G2, first and second gonopods, respectively. Mxp3, third maxilliped. The terminology used in the description of the G1 essentially follows (Rodríguez & Pereira, 1992; Rodríguez & Campos, 1998) (Fig. 2).

Molecular data analysis

DNA extraction, amplification and sequencing: muscle tissue samples were obtained from the pereopods or pleon of Fredius ibiapaba n. sp., F. buritizatilis Magalhães & Mantellato in Magalhães et al., 2014, and Prionothelphusa eliasi Rodriguez, 1980. At the Laboratório de Biologia Molecular da Universidade Estadual do Sudoeste da Bahia- LBM/UESB a small region of the 16S rDNA gene was extracted with Wizard^® Genomic DNA Purification Kit (Promega), amplified in a 12,5 µl final volume reaction with 2,5 mM de MgCl2 (Invitrogen), 0,05 mM de dNTP (Invitrogen), buffer 1x (Invitrogen –10xPCR Buffer: 200mM Tris-HCl (pH 8.4), 500mM KCl), 1U de taq platinum (Invitrogen) and 0,3µM of each primer. The PCR conditions were: one cycle at 94 °C, 60 s; five cycles at 94 °C, 60 s; 45 °C, 40 s and 72 °C, 60 s; and 35 cycles at 94 °C, 60 s; 51 °C, 40 s and 72 °C; 60 s; a final extension of five minutes at 72 °C was performed. The primers used were 16Sar (5′-CCGGTCTGAACTCAGATCACGT-3′) and 16Sbr (5′-CGCCTGTTTATCAAAAACAT-3′) (Palumbi et al., 1991). PCR products were purified using a polietilenoglicol (PEG) 20% and sequenced in an ABI Prism 3100 Genetic Analyzer^® (Applied Biosystems) at the Departamento de Tecnologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Jaboticabal. Sequencing reaction was performed with Big Dye v3.1 (Applied Biosystems), prepared with 4,75 µl ultrapure water, 1,5 µl BigDye 5×buffer, 0,75 µl BigDye terminator Mix, 2 µl primer (0,8 pmol) and 1 µl of Purified PCR product. Sequence conditions were: one minute at 96 °C; 35 cycles of 15 s at 96 °C; 15 s at 50 °C and 2 min at 60 °C. Both, forward and reverse sequence strands were obtained and the consensus generated by the software BioEdit 7.0.5 (Hall, 2005). The identities of the final sequences were confirmed with a BLAST (Basic Local Alignment Search Tool) on GenBank database. Additional comparative sequences were retrieved from GenBank (Table 1).

Phylogenetic analyses: substitution saturation in 16S rDNA was tested using the saturation index implemented in DAMBE 5 (Xia, 2013). The sequences were grouped and edit in BioEdit and aligned using the ClustalW interface (Thompson, Higgins & Gibson, 1994). Prionothelphusa eliasi (Pseudothelphusidae) and Trichodactylus dentatus H. Milne Edwards, 1853 (Trichodactylidae) were chosen as outgroups. The best-fit model HKY + G was selected using jModeltest 2.1.7 (Darriba et al., 2012). This model was used to generate Maximum Likelihood gene trees in MEGA 6.06 (Tamura et al., 2013). Branch support values were calculated using bootstrap analyses with 1,000 replicates (Felsenstein, 1985). Only nodes with bootstrap support greater than 50 are shown on the phylogenetic tree. Nucleotide divergence estimated from pairwise distance was calculated in MEGA 6.06 with the same best-fit model (Table 2).

Registration of nomenclatural act

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: [urn:lsid:zoobank.org:pub:0925982D-7441-120 4256-9856-A553987956A6]. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Phylogenetic analysis

The mitochondrial loci 16S was successfully amplified and sequenced for Fredius buritizatilis, F. ibiapaba n. sp., and Prionothelphusa eliasi. Additional sequences used were retrieved from GenBank (Table 1). Bootstrap support values are shown on nodes of the phylogenetic tree (Fig. 8). The sister species relationships between Fredius reflexifrons and the new species is well supported by high bootstrap value. The close morphological similarity between the two species also supports such relationship.

The divergence rates between Fredius reflexifrons and F. ibiapaba n. sp. (4%) is higher than between F. estevisi x F. stenolobus, F. platyacanthus x F. stenolobus and F. platyacanthus x F. estevisi all with 2% of divergence (Table 2). Morphology and molecular data hence provide evidences for the differentiation between F. ibiapaba n. sp. and F. reflexifrons.

A survey of the pseudothelphusids described from 1840 to 2004 (Yeo et al., 2008) showed that the curve of described species is still far from being asymptotic. And indeed, new species are still being discovered either by collecting in new biomes (e.g., F. buritizatilis from a palm swamp known as “buritizal”), or by revisiting the taxonomy of widely disjunct species for testing as to their conspecific identity, such as F. ibiapaba n. sp. and F. reflexifrons.

Zoogeographical notes

Fredius currently consists of 14 species (Table 3), distributed over a vast territory, which encompass five main river basins (Rodríguez & Campos, 1998; Magalhães et al., 2014): (1) the Orinoco River basin; (2) the Essequibo-Cuyuni River basin; (3) the Amazon River basin; (4) the Madeira River basin and its tributary (Machado River); and (5) the Atlantic rivers basin, a coastal drainage of small rivers in northern South American (Guyana, Suriname and French Guiana) discharging directly into the Atlantic Ocean.

Rodríguez & Pereira (1992) performed a cladistic analysis of Fredius and suggested that F. reflexifrons and F. adpressus were sister species. The purported clade F. reflexifrons / F. adpressus was presumably supported by three putative synapomorphies: (1) [G1] mesial lobe attached to back of auricular lobe; (2) basal denticle of mesial lobe present; and (3) subapical bulge well developed.

Later, however, Rodríguez & Campos (1998) reviewed the previous data and performed a new analysis in which they decided that character 1 (mesial lobe attached to back of auricular lobe) was no longer tenable and hence was eliminated from the new analysis. They also realized that the basal denticle of the mesial lobe was indeed present in F. adpressus (character 2), but was absent in all other Fredius species. They further concluded that the subapical bulge was actually “reduced” in F. adpressus and “strongly developed” in F. granulatus, F. reflexifrons, F. fittkauii, and F. denticulatus, so that these latter two characters were also removed from the new analysis. Therefore, the putative sister taxon relationship between F. reflexifrons and F. adpressus dissolved. Rodríguez & Campos (1998) put forward, instead, the hypothesis that F. reflexifrons was sister to F. fittkauii, not to F. adpressus, based on the assumption that F. reflexifrons and F. fittkauii synapomorphically share the cephalic lobe distal margin armed with several spinules. However, as found here, this character is more widely distributed being also found in F. ibiapaba n. sp. and, therefore, cannot be used to argue for the sister taxon relationship between F. reflexifrons and F. fittkauii.

Magalhães et al. (2014) performed a distance analysis based on 16S rRNA, in which F. reflexifrons was recovered as the sister taxa to (F. fittkauii (F. denticulatus (F. granulatus (F. buritizatilis (F. platyacanthus (F. denticulatus (F. stenolobus))))))). The discovery of F. ibiapaba n. sp. revealed, however, that it is actually the sister taxa of F. reflexifrons, as shown by a comparative 16S rDNA sequencing used to infer the phylogenetic placement of Fredius ibiapaba n. sp., with F. fittkauii recovered as the sister taxa to the remaining species (Fig. 8).

The distribution range of Fredius ibiapaba n. sp. is very narrow and currently restricted to a humid enclave, a small mid-altitude forested patch in Ipú (Ceará, northeastern Brazil, Figs. 1A–1E), nested within the vast semiarid Caatinga domain (Figs. 1F, 1G). The orographic forest enclaves, such as Ipú, are typically located along the slopes of plateaus, between 600 and 1,100 m, hence high enough to receive rainfall of more than 1,200 mm year⁻¹ of Atlantic origin (Tabarelli & Santos, 2004 and references therein). These enclaves are regionally known as “Brejos” (or “Brejos de altitude” or even “Brejos nordestinos”) (Andrade-Lima, 1982; Silva & Casteletti, 2003; Tabarelli & Santos, 2004). Fredius ibiapaba n. sp. inhabits the mid-highlands of the Ibiapaba plateau, between about 635 to 782 m, where it digs burrows among the leaf litter, alongside little streams and water ponds inside forest stands or directly on the humid forest floor (Fig. 1E). In contrast, F. reflexifrons is widely distributed in the Amazon basin’s lowlands (<100 m) from as far west as Peru (Ampyiacu River, a tributary of the Amazonas River) to as far east as the Atlantic basin (French Guiana) (Magalhães, 2003). It is found in burrows alongside the “igarapés” (streams) or digs its burrows on the humid forest floor (Magalhães & Rodríguez, 2002). Magalhães et al. (2005) misidentified the specimens from the mid-highlands of the Ibiapaba plateau with F. reflexifrons and explained its presence in Ibiapaba by a migration “…eastwards as far as Serra de Ibiapaba” during the expansion of the humid tropical forest.

Fredius ibiapaba n. sp. and F. reflexifrons are highly dependent upon humidity and our view is that they most probably were once part of an ancestral population living in a wide humid territory. The shrinking humid forests during several dry periods of the Tertiary and Quaternary (Katzer, 1933; Andrade-Lima, 1953; Bigarella, Andrade-Lima & Riehs, 1975; Ab’Saber, 1977; Bigarella & Andrade-Lima, 1982; Andrade-Lima, 1982; Clapperton, 1993; Thomas, 2000; Haffer, 2001; Haffer & Prance, 2002) likely have resulted in the fragmentation of the ancestral humid area and hence of the ancestral crab population, which was split into two sister species. Fredius reflexifrons evolved and spread in a lowland, humid river basin and is now widely distributed, whilst F. ibiapaba n. sp. evolved isolated on the top of a humid plateau (Figs. 1A–1E). The two species are now separated by a vast intervening area occupied by the semiarid Caatinga (Figs. 1F, 1G).

The expansion and shrinkage of mountain, floodplain, and gallery forests, associated to complex topography are known to have affected flora and fauna (Vanzolini, 1970; Vanzolini & Williams, 1970; Vuilleumier, 1971; Andrade-Lima, 1982; Teixeira, Nacinovic & Tavares, 1986; Haffer, 1969; Haffer, 2001; Haffer & Prance, 2002; Santos et al., 2007; Leite et al., 2016). Andrade-Lima (1982) provided a number of examples of plant species that are now confined to the Brejos, isolated from the surrounding, widely distributed Caatinga. He found two floristic components in these refuges on the top of hills, one whose species and genera have mostly originated from the southeastern flora, lies further inland in the states of Alagoas and Rio Grande do Norte; and a second one in the humid mid highlands closer to the coast, especially between Pernambuco and the border of Ceará and Piauí states (referred to as the Pernambuco Centre by Santos et al. (2007)), in which the Amazonian flora are better represented (Andrade-Lima, 1982). Santos et al. (2007) found strong bootstrap support for a close floristic relationship between the Pernambuco Centre and Amazonian localities.

It has long been known that a number of freshwater fish species inhabiting the Brejos have their closest relationships with those from the Amazonian Basin (Géry, 1969; Paiva, 1978; Weitzman & Weitzman, 1982; Ploeg, 1991; Vari, 1991; Menezes, 1996; Rosa & Groth, 2004). More recently, Pinheiro & Santana (2016) described a new species of freshwater crab genus Kingsleya Ortmann, 1897 (also a Pseudothelphusidae), from a Brejo about 750 m in Arajara district, municipality of Barbalha, Ceará. Previously to their discovery Kingsleya was known from nine species inhabiting the Amazonian lowlands (Pedraza & Tavares, 2015).