Stream macroalgal flora from Parnaíba River Basin, Brazil: reducing Wallacean shortfall

Abstract: The global biodiversity loss is a consensus. The biodiversity conservations shortfalls make conservation of biological diversity even more challenging. For many taxa, the knowledge about their distributions is deficient, and this is called the Wallacean shortfall. This situation is no different within algae biodiversity, especially in Brazil. There is still an enormous inequity of sample effort, as is the case of the Parnaíba River Basin (Northeast region), which had only 10 algal species in published (the lowest number of algal species reported among the main Brazilian basins). The present work had the objective of increasing the knowledge of algal flora in Brazil by conducting a taxonomic study of the stream macroalgal species of the Parnaíba River Basin. The sampling of macroalgae was carried out in 21 segments of streams from the Middle and Lower Parnaíba Basin, in the Piauí and Maranhão states. Macroalgae were manually removed and preserved in 4% formaldehyde. Environmental characteristics of each segment were measured in order to describe the sampling sites. The taxonomic survey of the macroalgal communities resulted in the identification of 38 taxa in total, of which 32 at a specific level; three vegetative groups; two sporophytic stages of red algae and one unidentified species. Among the 38 species recorded, 37 are new records for the Parnaíba River Basin. Following the same pattern, 23 species are new records for the Brazilian Northeast region, and Microcoleus lacustris represented the first report in Brazil. Despite the fact that it remains the Brazilian basin with the lowest number of algal species documented, this study contributed to the increase of almost five times the number of species sampled in the Parnaíba River Basin (from only 10 to 47 species). These data reinforce that the differences presented in algal diversity in the Brazilian regions are more related to the sampling effort than other factors (e.g. environmental characteristics, geographic distribution, biomes, among others). In addition, this survey illustrates not only the group's lack of information in the region but also shows the importance of this type of study as a tool for expanding the knowledge about biodiversity and its conservation.


Introduction
It is a consensus that global biodiversity is threatened and the species loss is taking place at an accelerated level (Whittaker et al. 2005). The main factors associated with this loss of biodiversity have an anthropogenic origin (Brown & Lomolino 1998). In addition to the anthropic impacts, the Biodiversity Conservations Shortfalls make conservation of biological diversity even more difficult (Mace 2004). For many taxa, the knowledge about their global, regional, or even local distributions is insufficient, a problem that Lomolino (2004) has named the Wallacean shortfall. As a result of this lack of knowledge that many areas of the globe still remain poorly sampled, which for most taxa results in scarce basic information (Whittaker et al. 2005). In contrast, this gap could be circumvented, or at least mitigated, by investing in biodiversity inventories (Whittaker et al. 2005, Bini et al. 2006). This scenario is no different when the algal biodiversity is approached, especially in Brazil. Despite the total number of described algae for the whole Brazilian territory (4700 species, Flora do Brasil 2020 under construction, 2018) be close to the estimated number of 5600 species in the country (Menezes et al. 2015), no doubt there's still an enormous inequality of sampling effort, with huge regions without any type of surveys. This fact not only makes it impossible for adequate management and the proposal of local conservation strategies but also creates an illusion that different regions present more species richness than others. In terms of Brazilian Hydrographic Basins (sensu Agência Nacional de Águas 2015), most surveys with this algal group cover only two basins: Southeast Atlantic (ASe) and Paraná (Pr). Both basins, according to Menezes et al. (2015), are the richest in number of species in Brazil, reaching values that exceed a thousand species. In contrast, little sampled Hydrographic Basins have a modest number of recorded species, as is the case of the Parnaíba River Basin (Pnb), which has only 10 species of algae (Menezes et al. 2015, Flora of Brazil 2020under construction, 2018 (Figure 1). Among these species, seven are planktonic microalgae (four euglenoids, two diatoms and one cyanobacteria) and three are filamentous algae (Chara martiana, Cladophora glomerata and Hapalosiphon pumilus).
Particularly for stream macroalgae (sensu Sheath & Cole 1992), the floristic surveys have focused basically only the Southeastern (Branco & Necchi Júnior 1996, Pereira & Branco 2010, Almeida et al. 2011 and Southern regions of Brazil , 2009. Although these efforts have contributed profoundly to the knowledge of Brazilian flora, there is a huge gap in the knowledge of the distribution of organisms throughout the territory, thus clearly contributing to the Wallacean shortfall in the group. Studies in different regions and biogeographic provinces are essential, not only providing occurrence information but also allowing an increase of reference material in herbaria. The increase of reference collections constitutes a valuable asset (Pyke and Ehrlich, 2010), also making possible future studies re-evaluating species and taxonomic positions.
In this context, the present investigation had the objective of increasing the knowledge of algal flora in Brazil by conducting a taxonomic study of the stream macroalgae species of the Parnaíba River Basin. This basin comprises a very interesting biogeographic region with the contact of three major Brazilian biomes (Cerrado, Caatinga, Amazônia, and its ecotones) and has a wide land use gradient. Moreover, as this basin presents the lowest number of algal species recorded so far (among the main Brazilian basins), it is considered as of greater relevance and a valuable contribution to the reduction of the Wallacean shortfall.

Material and Methods
The Parnaíba River Basin (Agência Nacional de Águas 2015) is located in the northeastern region of Brazil between latitudes 02º21'S and 11º06'S and longitudes 47º21'W and 39º44'W, encompassing the states of Ceará (4.1% of the total area of the basin), Maranhão (19.8%) and Piauí (75.3%) (MMA 2006). The region is an ecotone between the Cerrado and Caatinga biomes, also influenced by Amazonian and coastal vegetation (MMA 2006). It presents a well defined seasonal water regime (rainy and dry period) with several streams being intermittent (MMA 2006). Most of the selected streams are placed in areas disturbed by the extensive farming use, typical of this region. Nevertheless, some sampled streams are inserted in low disturbed landscapes while others are inserted in very disturbed areas with an urban influence.
The sampling of macroalgae was carried out in 21 segments of streams belonging to Parnaíba River Basin, in the Piauí and Maranhão states ( Figure 2, ICMBio License number 58717-3), during a period of drought and after a period of regular precipitation. The selected segments are 1st to 3rd stream order and comprised microbasins in different parts of the Middle and Lower Parnaíba River Basin. The collection and preservation of the algal samples followed the standard procedures described by Necchi Júnior et al. (1991). Each sampling unit consisted of a stream segment of 10 meters long. Using a transparent underwater viewer it was possible to observe and remove macroalgae, which were preserved in 4% formaldehyde. Environmental characteristics were taken in field, such as water temperature (Temp.) using a thermometer Akso TM , pH using a pHmeter Akso TM , conductivity (Cond.) using a Rio Parnaíba. Seguindo o mesmo padrão, 23 espécies são novos registros para a região Nordeste, e Microcoleus lacustris representou o primeiro registro para o Brasil. Apesar do fato de permanecer como a bacia brasileira com o menor número de espécies de algas documentadas, este trabalho contribuiu para o aumento de quase cinco vezes o número de espécies amostradas na Bacia do Rio Parnaíba (de apenas 10 para 47 espécies). Estes dados reforçam que as diferenças apresentadas na diversidade algal nas regiões brasileiras estão mais relacionadas com o esforço amostral do que com outros fatores (e.g. características ambientais, distribuição geográfica, biomas, entre outros). Além disso, este trabalho ilustra não só a falta de informação do grupo na região, mas também mostra a importância deste tipo de estudo como ferramenta para expandir o conhecimento sobre a biodiversidade e conservação. Palavras-chave: região Nordeste, Piauí, Maranhão, algas filamentosas, taxonomia. conductivimeter Akso TM , predominant substrate type (Subst.) by visual observation, according Gordon et al. (1992) classification, average current velocity (Vel.) using the floating object method, average width and depth of each segment using a measuring tape. The range and mean of these variables are presented for each species. In addition to these variables, the geographic coordinates and the estimate of incident irradiance (Shading) were taken by canopy photos in each segment, which were analyzed through ImageJ TM (software that allows quantifying pixel area with presence or absence of canopy cover) and expressed as a percentage to describe each sampling site.
The identification of the macroalgae was carried out with a Zeiss trinocular microscope (model AxioLab.A1 TM ) equipped with an image capture system (AxioCam). Using a software (ZEN), photomicrographs were obtained to perform ten randomized morphometric measurements of each structure of the populations. For the algae with larger structures, the photomicrographs were obtained with a stereomicroscope (model Zeiss Discovery V.12 TM ). Macroalgae were identified at the specific level whenever possible, and appropriate references were used for each taxonomic group (e.g. Senna & Ferreira 1987, Necchi Júnior 1990, Azevedo et al. 1996, Necchi Junior et al. 2001   Peres . The samples were kept in formaldehyde 4% and deposited in the Herbarium of the Universidade Estadual do Oeste do Paraná (UNOP). New records of the species are marked before the name of each species, single asterisk (*) represent a new record for the Rio Parnaíba basin, two asterisks (**) mean that the species is a new record for the northeastern region of Brazil, and three asterisks (***) show that the species is a new record for Brazil.
The following informations are presented: i) descriptions of the species; ii) photomicrographs of the main structures; iii) location of the sampling points; iv) environmental information for each taxon and; v) taxonomic comments (when applicable). The classification system was based in Guiry & Guiry (2018).

Results
The taxonomic survey of the macroalgal communities from the Parnaíba River Basin resulted in the identification of 38 taxa in total, of which 32 at a specific level; three vegetative groups of: Spirogyra, Oedogonium and Batrachospermum; two sporophytic stages of red algae: 'Chantransia' macrospora and 'Chantransia' pygmaea and one unidentified species of the genus Phormidium. The best represented taxonomic group was Chlorophyta with 12 taxa (31.6% of the total), followed by Cyanobacteria with 10 taxa (26.3%), Bacillariophyta with nine taxa (23.7%), Rhodophyta with six species (15.8%) and finally Charophyta with only one taxon sampled (2.6%).  Cond. 49-51 µS.cm -1 (x = 50 µS.cm -1 ); pH 6.6-7.3 (x = 6.9); Vel. 0.8-1.2 m.s -1 (x = 1 m.s -1 ); Depth 11.7-25.2 cm (x = 18.4 cm); Width 3.0-12.1 m (x = 7.5 m); Subst. bedrock and boulders; Shading 32-86% (x = 59%). Taxonomic comments: the population presented slightly smaller width than described by Costa et al. (2017) and Metzeltin & Lange-Bertalot (1998), but polymorphism of the species is widely recognized.  Pseudo-filamentous colonies formed by a connection between frustules by the valvar face; lanceolate valves with sharp to slightly rounded ends, 20.2-24.4 μm long and 6.7-9.7 μm in wide; filiform raphe and rounded central nodule; cellular content yellowish-green. Taxonomic comments: the population showed a great variation in length, being possible the existence of another species of Terpsinoe co-occurring in the sample. However, it was not possible to clearly define it using optical microscopy. Plants 9-12 cm high; internodes equal to or longer than branchlets, 1-1.6 cm long; 5-6 monomorphic whorled branchlets, 2-4 furcated, 0.9-1.5 cm long; 1-3 dactyls, 1-2 celled, abbreviated or elongated, 241.5-801.7 µm long and 84.4-100.9 µm diam., L/D 4.1-8.5; gametangia sessile, present in first and/or second branchlets furcation; 1-2 nucules, 8 convolutions, 328.6-533.8 µm long and 274.2-407.9 µm diam., L/D 0.9-1.3; globules scarce, 208.9-218.7 µm long and 204.9-214.9 µm diam., L/D 1.0-1.1. Heterotrichous tufts formed by branched filaments immersed in a gelatinous matrix relatively firm, pale green, spherical or hemispherical; basal system composed by prostrate branched filaments with cylindrical cells, producing erect filaments and rhizoids; erect filaments di-or tricotomically branched; lateral branches lax and sparse, fasciculated near the apex; cells of erect system cylindrical, 25.1-63.3 µm long and 4.5-9.1 µm diam., L/D 3.3-9.1; terminal branches densely grouped, tapering at the towards the apex or ending abruptly at an acute apex. Distribution in the Parnaíba River Basin (n=1) Figure D. Basionym: Rivularia pisiformis Roth 1802. Heterotrichous tufts formed by branch filaments immersed in a gelatinous matrix relatively firm, pale green, spherical or hemispherical; basal system composed exclusively by rhizoids which arise from basal cells of erect filaments or rarely from median region of filaments; erect filaments di-or tricotomically branched; lateral branches lax and sparse, non fasciculated near the apex; cells of erect system cylindrical, rarely     Figure B. Entangled unbranched filaments, non or constricted in septa with non-evident H pieces; cylindrical cells, 9.1-178 µm long and 9.3-11.9 µm diam., L/D 0.  Taxonomic comments: According to Komárek & Anagnostidis (2005), L. majuscula is a typical species of marine environments. However, the identification of this material is consistent with the description proposed by the same authors. In addition, other authors have already described the species in freshwater environments (e.g. Desikachary 1959) and, for this reason, we maintained the identification. In spite of this, we consider that the environmental variation between freshwater Taxonomic comments: the identification agrees with the description of P. tergestinum by Komárek & Anagnostidis (2005), however, according to these authors, this concept is broad, being likely to correspond to a set of species.

ORDER EUPODISCALES
10.*** Phormidium sp. Plate 4. Figure H. Mats of filaments, dark green; sheath thin, hyaline, generally absent, not lamellated; trichomes unconstricted, not granulated in septa; intercalar cells wider than long, 4.4-8.9 µm long and 9.6-11.8 µm diam., L/D 0.4-0.8; apical cell truncate or rounded, 5.9-11.0 µm long and 10.0-11.9 µm diam., L/D 0.6-1.0; cellular content homogeneous, blue-green, with few small granules. Taxonomic comments: although the cellular dimensions are within the predicted in the description of P. retzii proposed by Komárek & Anagnostidis (2005), the other characters (e.g. coloration and granulation) present considerable differences with the populations of P. retzii identified here. On the other hand, the characteristics of the material do not allow identification in other species. Thus, it is quite likely that this is a new species to be confirmed by future studies including ultrastructural or molecular characters.

Discussion
The total number of taxa found in the present study (38) can be considered high when compared to studies carried out in other Brazilian regions that used similar methodology, namely: i) 13 taxa (Pereira & Branco 2010) and 21 taxa (Almeida et al. 2011) in the northwest of the São Paulo State; ii) 23 taxa ) and 34 taxa , in the central-southern region of the Paraná State; iii) 24 taxa (Branco et al. 2009) in the mid-western region of the Paraná State, and; iv) 19 taxa  reported to the eastern region of Paraná State. Likewise, when considering the relative richness, found 1.81 taxa per sampling point, which is also higher when compared to what was reported in those other regions (taxa per sampling point and respective studies: 1.09 in Branco et al. 2009, 1.23 in Almeida et al. 2011, 1.35 in Peres et al. 2008, 1.78 in Krupek et al. 2008, 2.09 in Branco et al. 2008. Studies in tropical streams have shown that biomes without a dense canopy cover can sustain richer macroalgae communities (Necchi Júnior et al. 2003. Apparently, a larger radiation area in the drainage basin (which is typical in the Caatinga and Cerrado biomes, here sampled) may provide support for a bigger regional species pool that enables greater local richness (as demonstrated for green algae in Peres et al. al. 2017).
Although Bacillariophyta was not the richest group, in this study a bigger number of diatom species was found than conventionally for stream macroalgae studies (e.g. Necchi et al. 2000, 2009. Even though unicellular, diatoms can form monospecific macroscopic colonies (Biggs & Kilroy 2000, as shown in the Plate 1, Figure A in this study) that can dominate the streambed -hence macroalga, sensu Sheath & Cole (1992).
The results of this work make very clear the importance of floristic surveys to the reduction of the algae biodiversity conservation shortfalls, specially the Wallacean Shortfall (Whittaker et al. 2005). Of the 38 species recorded by this taxonomic survey, 37 are new records for the Parnaíba River Basin where only Cladophora glomerata was already registered for the basin. Following the same pattern, 23 species are new records for the Brazilian Northeast region (60.5% of the total) and Microcoleus lacustris represented the first record for the Brazilian territory. Despite the fact that it remains the Brazilian watershed with the lowest number of algal species documented, this work contributed to the increase of almost five times the number of species sampled in the Parnaíba River Basin (from only 10 to 47 species, Flora do Brasil 2020 under construction 2018). These data reinforce that the differences presented in the algal diversity in the Brazilian regions are much more related to the sampling effort than to environmental differences. In addition, this study illustrates not only the group's lack of information in the region but also shows the importance of this type of study as a tool for expanding knowledge about biodiversity and its conservation.