Alona iheringula Sinev & Kotov, 2004 (Crustacea, Anomopoda, Chydoridae, Aloninae): Life Cycle and DNA Barcode with Implications for the Taxonomy of the Aloninae Subfamily

Knowledge of reproductive rates and life cycle of the Cladocera species is essential for population dynamic studies, secondary production and food webs, as well as the management and preservation of aquatic ecosystems. The present study aimed to understand the life cycle and growth of Alona iheringula Kotov & Sinev, 2004 (Crustacea, Anomopoda, Chydoridae), a Neotropical species, as well as its DNA barcoding, providing new information on the Aloninae taxonomy. The specimens were collected in the dammed portion of the Cabo Verde River (21°26′05″ S and 46°10′57″ W), in the Furnas Reservoir, Minas Gerais State, Brazil. Forty neonates were observed individually two or three times a day under controlled temperature (25±1°C), photoperiod (12 h light/12 h dark) and feeding (Pseudokirchneriella subcapitata at a concentration of 105 cells.mL−1 and a mixed suspension of yeast and fish feed in equal proportion). Individual body growth was measured daily under optical microscope using a micrometric grid and 40× magnification. The species had a mean size of 413(±29) µm, a maximum size of 510 µm and reached maturity at 3.24(±0.69) days of age. Mean fecundity was 2 eggs per female per brood and the mean number of eggs produced per female during the entire life cycle was 47.6(±6.3) eggs per female. The embryonic development time was 1.79(±0.23) days and the maximum longevity was 54 days. The species had eight instars throughout its life cycle and four instars between neonate and primipara stage. The present study using molecular data (a 461 bp smaller COI fragment) demonstrated a deep divergence in the Aloninae subfamily.


Introduction
Cladocera are important for energy transference in the food chain in natural ecosystems. These organisms are used as a food source for larvae and juvenile fish in aquaculture due to their high nutritional value and short development time [1,2].
The family Chydoridae Dybowski & Grochowski, 1894 emend. Frey, 1967 inhabit the littoral region of water bodies and normally lives associated with macrophytes, periphyton or sediment, and are represented by substrate scraper organisms [3,4,5]. The littoral region plays an important role in productivity and nutrient cycling in aquatic ecosystems. Environments colonized by macrophytes have high environmental heterogeneity because they have a high diversity of ecological niches and species biodiversity [6].
Studies on the reproductive rates and life cycle of Cladocera species allow a better understanding of the role each plays in zooplankton communities. They also provide data for other studies, such as secondary production determination and toxicity tests, and contribute to many ecological studies, such as energy flow, population dynamics and functional groups [7,8,9]. Cladocera reproduce mainly by parthenogenesis, but under unfavorable conditions, the birth of males and production of resting eggs may occur, which contribute to the spread and propagation of these species [10,11]. Males are little known or unusual in some species of Cladocera and probably associated with specific environmental changes. These changes can modify the development rates and the life cycle of species of Cladocera [12,13].
Due to the high richness of the chydorids found in freshwater, the number of species whose life cycle has been studied is still very small, compared to other families of Cladocera, covering approximately 18.31% of the known species. Therefore, it is necessary to extend such studies to a larger number of species.
Alona iheringula Kotov & Sinev, 2004 belongs to the Aloninae subfamily. It is a Neotropical species and belongs to the Alona costata Sars, 1862 [24], species complex. It was considered as a synonym of Alona rustica Scott, 1895 [25] for some time, but Sinev [26] showed clear differences and considered it to be Alona iheringi Sars, 1901. Later, it was renamed as Alona iheringula by Kotov and Sinev [24].
The region known as the DNA barcode is based on a segment of the mitochondrial gene cytochrome c oxidase I subunit (COI) and has demonstrated to be an important molecular marker in taxonomic classification [27].
For the Subphylum Crustacea and especially for the Chydoridae family, the COI region was demonstrated to be effective in separating taxa [28,29] and detecting crustaceans as prey in ecological studies [30] and cladocera aquatic invasions [31]. Important COI studies were made for Simocephalus [32], Eurycercus [33], Leberis chihuahuensis from the northern Mexican semi-desert [34]. Studies using Branchiopoda species diversity in Canada [35] and Cladocera species from Guatemala and Mexico [36] have highlighted the DNA barcode as a useful tool for many applications. We sequenced the COI from A. iheringula with the aim of clarifying its specific status and taxonomic position.

Materials and Methods
Some females were collected in the littoral region of a dammed portion of the Cabo Verde River/Furnas Reservoir (21u 269050 S, 46u 109570 W), in the southern region of Minas Gerais State, Brazil, by vertical and horizontal hauls using a zooplankton net of 68 mm mesh size. It is a public area and no special permission is necessary to authorize access. The species involved are not endangered or protected. Therefore, there are no restrictions on collecting. The sample was collected near macrophytes in February 2009.
In the laboratory, parthenogenetic females of Alona iheringula (Fig 1A-C) were isolated and placed in 2 L beakers containing reconstituted water, according to ABNT [37]. The culture media and food suspension were renewed every two days. Specimens were acclimated for about 10 generations (30 days). The pH of the culture media was 7.4; the electric conductivity was 180 mS.cm 21 and the hardness was 46 mg.L 21 CaCO 3 . Experimental cultures were maintained in germination chambers with a photoperiod of 12h-light/12h-dark and temperature of 25(61)uC (a mean temperature observed in the sampling area when the organisms were collected). The organisms were fed with Pseudokirchneriella subcapitata algae, cultured in Chu 12 medium and cropped in the exponential phase, with 10 5 cells per individual and a suspension of mixed suspension (fish-food and biological yeast), supplied in equal proportions [37,38].
Twenty females were isolated and maintained until the production of neonates. The 40 neonates of less than 24 hours old were placed in 100 mL of culture medium in polypropylene bottles and kept in a germination chamber with temperature, light and feeding conditions as specified above. The isolated organisms were observed two or three times a day under the stereoscopic microscope to obtain life cycle data (development times, longevity and fecundity). Individual body growth was measured daily under optical microscope using a micrometric grid and 406 magnification.
In the same collecting coordinates, qualitative samples of the zooplankton community were collected (near the macrophytes) using a net of 68 mm mesh size, every two months, from June 2008 to October 2009. The samples were fixed in formalin 4% and kept in the Coleção do Laboratório de Limnologia (sample CL3053), at the Universidade Federal de Alfenas, MG (UNIFAL-MG). From Secchi disk and chlorophyll-a data, obtained from the same place, the Trophic State Index was calculated by Carlson methodology, adapted by Toledo et al [39]. The chlorophyll concentration was determined using the extraction method with 90% acetone as described in Golterman et al [40].
For the DNA barcode analysis, the specimens were fixed with 90% EtOH and placed into pure water for 12 h for cleaning. Genomic DNA was extracted using phenol extraction and ethanol precipitation [41]. To amplify the mitochondrial COI gene, the internal primers Chy-f 59-TTG GGG ATG ATC AAATTT ATA ATG T-39 and Chy-r 59-AGA GGT ATT CAG ATT TCG ATC TGT CA-39 [42] were used. PCR reactions had a total volume of 25 mL and were performed according to Ivanova et al [43] using Platinum Taq (Invitrogen) as the enzyme. The PCR conditions were 94uC for 3 min as initial denaturation and 40 cycles of 94uC for 30 s, 50uC for 30 s and 72uC for 90 s. Direct DNA sequencing was done using purified Exo-SAP (Fermentas) PCR products, carried out in a 3130xl Genetic Analyzer (Life Technologies, Carlsbad/CA/USA) automated DNA sequencer, following the manufacturer's instructions. The sequences were obtained bidirectionally for accurate reading (Genbank access number KF383284).

Alona iheringula culture and life cycle
The sampling area in the dammed portion of the Cabo Verde River was characterized as a shallow (3-9 m depth) mesotrophic environment (Trophic State Index of 47), with macrophytes (Myriophylum aquaticum, Eichornia azurea, Typha domingensis and Pistia stratiotes). The water had a mean pH of 5.2 (60.6) (slightly acidic), temperature ranged from 20 to 25uC, dissolved oxygen concentration from 4.5 to 8.0 mgL 21 and low medium conductivity, 41.7 (69.5) mS.cm 21 .
The neonate had an average size of 288 (620) mm, reaching maturity after 3.3 (60.7) days and 413 (629) mm in mean size. The maximum size (510 mm) was reached in 11 days. The mean individual growth curve of Alona iheringula is shown in figure 2.
The main results for the life cycle of Alona iheringula are presented in Table 1. This species has a maximum longevity of 54 days and mean longevity of 46 (66) days. The mean of embryonic development time was 1.79 (60.24) days. The mean fecundity of parthenogenetic females was 2.0 eggs per female per brood, producing a mean of 47.6 (66.3) eggs per female during the entire life cycle. The species had eight instars throughout its life cycle and four instars from neonate to primipara stage.
From qualitative sample analysis, during the collecting period, A. iheringula was only registered in February, August, and October 2009, in a sample collected near macrophytes. Chydoridae and Daphnidae families were the most representative, with 42% and 41% of total species. Bosminidae, Sididae and Ilyocryptidae families had a lower representation, with 11%, 3% and 2% of total species found.
As this size using the Chy-f and Chy-r primers [42] is smaller than those usually obtained with the universal primers HCO2198 and LCO1490 of 658pb [46], a test using all COI sequences for Alona and Leberis species found at BOLD (http://www. barcodinglife.org) was performed using the 658 bp and the 461 bp smaller fragment corresponding to the aforementioned isolated region for A. inheringula, in order to verify if a smaller COI sequence could modify the taxonomic status. The identification tree obtained using both the 658 bp and the 461 bp fragments provided similar results (data not shown), not changing the taxonomic status among all the 14 sequences (not including the A. iheringula sequence).
A genetic divergence ranging from 18.5% to 23.1% among Alona iheringula (GenBank access number KF383284) and other species of the genus Alona was found ( Table 2). Among the 15 COI analyzed sequences, the identification tree (Fig. 3) shows Alona iheringula as closely related to: Alona sp1 (JN233808) and Alona sp2 (JN233811), both from Manitoba, Canada. A. glabra (EU701990 and EU701994, from Chihuahua/Mexico) and A. setulosa (DQ889138 and DQ310646, with undetermined location) were mixed together as a well supported group exhibiting an 89% bootstrap value. However, A. cf glabra (EU701966 and EU701967) separate as another clade, also with the Alona sp (EU701999) as a separate clade (Fig. 3). A. dentifera and A. cf dentifera cluster together with the two Leberis species with a poorly supported bootstrap value of 40% (Fig. 3).

Discussion
In Brazil, there are records of occurrence of A. iheringula in Pará, Mato Grosso do Sul, Maranhão, Goiás, Distrito Federal, Minas Gerais, Rio de Janeiro, São Paulo and Rio Grande do Sul states [47,5,48,49]. In Minas Gerais state, it was recorded in the Rio Doce Basin, San Francisco Basin [50,51] and in a dammed portion of the Cabo Verde River/Furnas Reservoir (present study). The sampling area had a slightly acidic pH. Tolerance to acidic environments is a typical physiological adaptation of the Alona rustica group, which is the closest to A. iheringula, and the occurrence of A. rustica Palearctic is known in acid systems [52]. The mean water temperature was close to that used in the experiment (2561uC) and had good oxygenation and low electrical conductivity values compared to other reservoirs [53,54,55].
The mean size of the A. iheringula neonate (288619 mm) was lower than that found by Melão [13] for Chydorus dentifer (302 mm) and by Sharma and Sharma [20] for Alonella excisa (368 mm). According to Kotov [19], some species have a special molt, immediately after being released from the mother's pouch, corresponding to the first juvenile stage. The first measurement of this study probably corresponds to this stage. The maximum size reached by the A. iheringula (510 mm) was similar to that found by other authors and species of the subfamily Aloninae. Kotov and Sinev [24] recorded a mean size from 380 to 450 mm for parthenogenetic females of A. iheringula, in São Paulo State. Sinev [56] found a mean size of 500 mm for Alona costata, in a European study.
Temperature influences the species development, when maintained in laboratory under non-limiting food conditions, and there is a negative relationship between temperature and development time and longevity [57,58,7]. The mean longevity of A. iheringula (4666 days) was similar to that obtained for Leydigia ciliata at temperatures of 28 and 30uC [18]. Martínez-Jerónimo and Gómez-Díaz [21] also found differences in development times associated with temperature for Leydigia louisi mexicana species.  Bottrell [15] found 74 days at 10uC for Acroperus harpae, showing the temperature dependency for development time and longevity.
The quality, diversity and quantity of food also influences longevity [59,58]. Melão [13] found shorter longevity for Acroperus harpae than in the present study, using only algae as food, in otherwise similar conditions. Although food was offered ad libitum, the less diverse food offered probably influenced longevity, which decreased.
According to Smirnov [3], the duration of the life cycle of the chydorid species ranges from 5 to 94 days and, according to Lynch [60], from 24 to 42 days. Therefore, the maximum longevity recorded for A. iheringula is within the range established by Smirnov [3].
The age of the primipara of A. iheringula (3.2460.69 days) was similar to that found for Leydigia acanthocercoides (3 days) at temperatures of 28 to 30uC [16] and Alonella excisa (3.17 days) at 19 and 23uC [20]. Santos-Wisniewski et al [22] recorded the primipara age of 2.4 days for Chydorus pubescens, which is lower than registered for A. iheringula.
The embryonic development time (1.7960.24 days) was close to that found by other authors for some Chydoridae species [13,22] at the same temperature. Embryonic development depends on several factors. The influence of temperature and quality of food in embryonic development of the Cladocera species has been observed by several authors [61,58,7,22,21]. Embryonic development times may also be related to egg sizes from species of   Table 2. K2P Genetic distance between COI sequences of A. iheringula and other Alona and Leberis species. Genbank access numbers are located after each species name.  Cladocera [13,62]. The species in this study belongs to the subfamily Aloninae, which has characteristics such as a more elongated body, larger eggs and longer embryonic development time than species that belong to the subfamily Chydorinae, which has a small body, small eggs and short development time [22]. The mean fecundity (2 eggs per female per brood) and total fecundity (47.666.3 eggs per female) were similar to that found by other authors [16,18,20,22,21] for other species in the Chydoridae family. This feature may be less variable among species. According to Kotov [63], the Chydoridae family has a laterally compressed body, which is a morphological adaptation to benthic environments. Other families like Daphnidae, Moinidae and Sididae, for example, have rounded bodies, which are adapted to living free in a limnetic environment. Therefore, different body shapes of Cladocera species are likely to influence the number of eggs produced per brood, since their flattened bodies have a smaller incubator chamber and thus reduce the number of eggs produced.
Four juvenile stages were recorded for A. iheringula. According to Bottrell [15] the number of instars ranges from 3 to 8 stages for the Cladocera species. The number is generally constant for all species, although it may increase under limited food conditions [18,19,20]. Smirnov [3], Lynch [60] and Venkataraman [18] registered three instars for Chydoridae species and showed that the range is lower than observed for other Cladocera families, which may have anywhere from 2 to 7 juvenile stages. For adult stages, A. iheringula presented 8 stages, fewer than those found by other authors [3,16], probably because this is an intrinsic characteristic of this species.
A. iheringula is characteristic of the littoral region, where it is associated with aquatic macrophytes, and consequently it was only recorded in qualitative samples collected near macrophytes. The presence of macrophytes provides greater species diversity because they provide shelter and a food source for many animal species and produce high environmental heterogeneity [64].
Collections in the macrophyte region need to be intensified. On the days that A. iheringula was found, there was a high abundance of macrophytes (Myriophylum aquaticum, Eichornia azurea, Typha domingensis and Pistia stratiotes) nearby and greater frequency and representativeness of Chydoridae species.
The knowledge of the life cycle and laboratory culture facilitates the molecular studies of the Cladocera species as a large number of specimens are derived from the same clonal population, which is very interesting as a large amount of DNA can be obtained for different molecular studies.
This study provides new information regarding the subfamily Aloninae, stressing that it is a group whose taxonomy is complex and still not very well defined. In recent years several species of the subfamily Aloninae have been described and redescribed [65], emphasizing the need for taxonomic revision of the group, which is underestimated.
The percentage found for A-T (62.5%) is similar to the 60% A-T percentage for COI of Chydoridae [42,28].
As shown in the identification tree (Fig. 3) the classification and taxonomy can be obtained with a smaller fragment of COI (461 bp), amplified with Chy-f and Chy-r [42], given the difficulty of amplifying this species with universal primers [46], thus showing that this smaller sequence is sufficient for molecular taxonomy for the Cladocera species analyzed.
The determination of the A. iheringula COI added new information to daunting challenge of elucidating the still unclear taxonomy of the Aloninae subfamily. Here we found a strong genetic differentiation between the alonine clades. The present study using molecular data (a smaller COI fragment of 461 bp) also corroborates the polyphyletic nature of the Aloninae group as already discussed using morphological [66] and molecular data (using COI, 18S rRNA and 16S rRNA, [28]).
The use of molecular depth studies as the present one and morphological characters with a large number of species from different localities can resolve some problems of taxonomic issues and further establish the most probable phylogeny of the Aloninae subfamily. This will help better establish taxonomic status for the already described and the new morphospecies, as important aspects for detection of distinct possible ecological niches and different needs for preservation studies on plankton biodiversity and biotechnological purposes, including determination of new model species for biomonitoring experiments and xenobiotic degradation pathways. Also, efforts should be directed towards the growth of global databases such as BOLD and Genbank, in order to help future identifications and phylogenetic studies.