Populations of Flavalona setigera ( Brehm , 1931 ) in Brazil belong to a new species : Flavalona asymmetrica ( Cladocera : Chydoridae : Aloninae )

Flavalona asymmetrica sp. nov. belongs to the costata-clade. Th is species has two main head pores, a feature shared only with Flavalona setigera (Brehm, 1931). Morphological differences between them are related to main and lateral head pores, armature of scrapers on the second limb, and proportion of setae on the third and fourth limbs, number of denticles on the postabdomen. Males of F. asymmetrica sp. nov. has genital process about ten times shorter than length of postabdominal claw, which is an exclusive morphological trait. Flavalona asymmetrica sp. nov. is a Neotropical species, so far, it occurs only in Brazil and can be found together with Flavalona iheringula (Sinev & Kotov, 2004) and Flavalona margipluma (Sousa, Santos, Güntzel, DinizFilho, de Melo-Júnior & Elmoor-Loureiro, 2015). key words Amphi-Pacifi c distribution, Biapertura, main head pores, postabdomen.

Flavalona setigera was reported for the first time in the Neotropics by Santos-Wisniewski et al. (2001) from samples collected in São Paulo state, Brazil (Santos-Wisniewski et al., 2002;Rocha et al., 2011).Later, it was reported from other localities in southeastern and central Brazil (Sousa and Elmoor-Loureiro, 2012;2013;Sousa et al., 2013;2014).However, the validity of Neotropical populations of F. setigera is questionable (Sinev and Dumont, 2016) because it is an Australasian species and this fact does not agree with the concept of non-cosmopolitanism in cladocerans (Frey, 1982).Thus, the aim of this study is to evaluate putative F. setigera specimens from Brazil and to describe a new species of Flavalona.

Morphological analyses
The animals selected for this study were taken to a binocular stereomicroscope, where they were put in glycerin slides and studied under a phase contrast Olympus BX41 microscope.Several individuals were dissected for the observation of the appendages.The organization of morphological structures followed suggestions from Van Damme (2016).To enumerate the limb setae, we adopted the homology criteria of Kotov (2000a;2000b), which exhibited stability when tested in different groups of Cladocera (Kotov et al., 2010).All drawings were made in a camera lucida.
Material examined.Holotype: Undissected, adult parthenogenetic female in a tube with 92%   Etymology.The epithet comes from joining the Greek prefix "a" (absence) and Latin word "symmetria" (symmetry).The name makes reference to the rim around the posterior pore, which is broader than the connection around the anterior one.
Differential diagnosis.Flavalona asymmetrica sp.nov.has two main head pores, differently from Flavalona costata (Sars, 1862), Flavalona cheni (Sinev, 1999), Flavalona natalensis (Sinev, 2008), F. margipluma, Flavalona weltneri (Kilhack, 1905), Flavalona rustica (Scott, 1895), Flavalona bicolor (Frey, 1965), F. hudeci, F. iheringula andFlavalona sphagnophila (Van Damme &Eggermont, 2011).Regarding the females, F. asymmetrica sp.nov. is differentiated from F. setigera because it has the rim around the posterior pore broader than anterior one, PP about 0.7 IP, deep and elliptic sacs about 0.6 of length of the lateral pores; long seta 1 of IDL on the first limb; second limb with scrapers 7-8 armed with denticles; third limb with first exopodite seta about 0.7 of length of the second seta; fourth limb with exopodite setae 4-5 of similar lengths; epipodite of the fifth limb with long projection; sixth limb about 2.4 times longer than wide with setulae on the margins; postabdomen armed with up to nine marginal denticles.Regarding the males, F. asymmetrica sp.nov. is differentiated from F. setigera because it has seta of the copulatory brush markedly shorter than setae 2-3 of IDL, postabdomen armed with marginal setulae on the distal and anal margins not separated in groups, basal spine present and genital process about ten times shorter than length of postabdominal claw.
Description of parthenogenetic female.General Habitus (Fig. 1A-C).Body more elongate than spherical, length 0.37-0.45mm, maximum height at middle of body, height/length ratio about 0.6; dorsal margin arched, without dorsal keel or lateral projection; in dorsal and ventral view body not laterally compressed.
Carapace (Fig. 1A-F).With slight longitudinal lines; ventral margin almost straight; each valve with 49-52 plumose setae and relatively long setulae between them; setae arranged in three groups, anterior and posterior groups longer than median group; internal spinulae of the posterior part not arranged in groups, proximal spinulae exceeding the line of valves.
Cephalic structures (Fig. 1G-N).Ocellus shorter than eye; rostrum short and not sharp in lateral view.Head shield (Fig. 1G-J).About 1.6 times longer than wide, posterior part (posterior to mandibular articulations) about 0.52 of length of the anterior part, posterior margin strongly arched; rostrum short with margin rounded; two connected main head pores, rim around the posterior pore about 1.3 times broader than the anterior one; PP about 0.7 IP; transverse lateral head pores about 0.7-0.9IP and located at the level of posterior main head pore or between posterior and anterior main head pores, deep and elliptic sacs underneath about 0.6 of length of the lateral pores.Labrum (Fig. 1K).Of moderate size; keel without a notch, anterior margin convex, posterior margin with two clusters of setulae.Maxilla (Fig. 1L).Well developed, with two long setulated setae.Antennule -A1 (Fig. 1M).Antennular body length about 2.2 times the width, does not exceeding tip of rostrum, with three rows of setulae; antennular sensory seta slender, about three times shorter than antennular body, inserted at the second third of the antennular body; nine aesthetascs projecting beyond the tip of rostrum, two aesthetascs about 1.4 times longer than the others.Antenna -A2 (Fig. 1N).Coxal setae of similar length; basal segment thick with a short spine; first exopodite segment slightly longer than first segment of endopodite; second exopodite segment armed with long setulae on the tip and a seta about 0.45 of the length of longest apical seta; first endopodite segment armed with a spine slightly shorter than second segment; apical spines longer than length of apical segments.Antennal formula (exo/endo): spines 001/101, setae 113/003.Thoracic limbs (Fig. 2A-K).Six pairs of thoracic limbs.First limb (Figs.2A, B).Epipodite with long projection; ODL bears a thin seta armed with fine A new species of Flavalona from Neotropics Nauplius, 26: e2018003 setulae, similar in length to IDL setae; IDL (en 4) with three setae, first seta about 3.5 times shorter than setae 2-3, setae 2-3 bisegmented and armed with hard setulae; endite 3 with four setae, seta 1 markedly shorter than posterior setae (a-c), setae (a-b) of similar length and slightly longer than seta (c); endite 2 with a row of long spinulae, three posterior setae present (d-f), setae (d-e) of similar length and about 0.8 of the length of seta (f), setae (e-f) bearing spinulae on the lateral face, anterior stiff seta short, about 0.16 of setae (d-e); endite 1 with three posterior setae (g-i), two being bisegmented and densely setulated from to distal part (g-h), a short flat plumose seta (i), anterior stiff seta about 0.24 of setae (d-e); ejector hooks of different length and armed with short spines; ventral face of the limb with seven groups of setulae organized in clusters, decreasing in length towards the distal portion; gnathobase elongated, apex sharp and setulated.Second limb (Fig. 2C, D).Exopodite elongated with a row of short setulae, seta on the exopodite about 0.57 of length of exopodite itself; inner limb portion armed with eight scrapers; scrapers 1-2 of similar length and armed with fine and short spinulae, scrapers 3-8 decreasing in length towards to gnathobase; scraper 3 armed with spines; scraper 7 armed with strong denticles, about 1.4 times shorter than scraper 6; scraper 8 armed with strong denticles, about 1.8 shorter than scraper 6; proximal portion of gnathobase wide and short, armed with short setulae; distal portion armed with four elements, first as a short sensillum, second and third elements with blunt distal portion, fourth element short and obtuse; filter comb with seven setae, first one short and densely setulated, others long and slightly setulated.Third limb (Fig. 2E, F).Epipodite elongated, without projection; exopodite relatively large, subquadrangular, with five distal and two lateral setae; sixth and seventh setae setulated, short and of similar length; fifth seta about 1.8 times longer than first seta and 1.4 times longer than second seta; fourth seta slightly longer than third seta; First seta about 0.7 of length of the second, both naked; distal endite with three setae (1-3), seta (1-2) scraper-like, seta (3) curved and armed with many setulae bilaterally implanted; four long and plumose posterior setae increasing in length toward to posterior part of the endite (a-d); basal endite with four soft anterior setae slightly increasing in length towards the gnathobase (4-7); gnathobase armed with three elements, the first being a cylindrical sensillum, the second a geniculated and relatively short seta, third and fourth elements naked and with acute tip; filter comb with seven setae.Fourth limb (Fig. 2G-I).Pre-epipodite rectangular, densely setulated; epipodite oval with a long projection; exopodite subquadrangular with six marginal setae; setae 3-6 plumose; sixth seta about two times longer than the third and slightly longer than fourth and fifth setae; third seta about 0.53 of length of the fourth and fifth setae and about 0.7 of length of the second seta; first seta markedly shorter than second seta; distal endite with four setae (1-4), one scraper-like (1), three flaming-torch-like (3-4); first flaming-torch ticker than the others and armed with long setulae; gnathobase armed with a bottle-shaped sensillum and a setulated seta shorter than width of endite; filter comb with five setae.Fifth limb (Fig. 2J).Pre-epipodite short and rounded, densely setulated; epipodite oval with long projection; exopodite clearly divided in two lobes; four plumose setae; third seta about 0.9 of length of the fourth seta; second seta about 0.75 of length of the fourth seta; first seta about 0.46 of length of the second seta; internal lobe elongated, relatively oval and armed with many setulae, two setulated setae of different length on inner face of the lobe, seta 1 about 0.9 of length of the seta 2; gnathobase armed with two elements; filter comb with three setae about two times shorter than seta 1 of internal lobe.Sixth limb (Fig. 2K).An elongated lobe, about 2.4 times longer than wide; apical and lateral margins armed with long setulae.

Abdominal and postabdominal structures.
Abdomen (Figs. 1A, 3J).Shorter than thorax, armed with three rows of abdominal setae.Postabdomen (Fig. 3A).About 3 times as long as wide, ventral margin almost straight with two rows of short spinulae; preanal margin slightly longer than anal margin; anal margin armed with 4-5 groups spines; postanal margin about 1.3 times longer than preanal and anal margin, straight or slightly concave, distal angle slightly projected, a short incision is present near to base of postabdominal claw; eight-nine merged marginal denticles with width at base/height ratio about 0.3; eight-ten lateral fascicles armed with thin spinulae which do not exceed the margin of postabdomen; postabdominal seta about 2.2

A new species of Flavalona from Neotropics
Nauplius, 26: e2018003 times shorter than postabdomen length.Postabdominal claw (Fig. 3A, B).About 0.3 of the length of the postabdomen and longer than anal margin, base armed with short spinulae; inner pecten with distalmost spinulae longer than proximalmost ones; outer pecten with spinulae of similar length.Basal spine (Fig. 3A,  B).Naked, not inserted on the base of postabdominal claw, about 0.1 of length of the postabdominal claw, similar to width of postabdominal claw at its base.
Description of ephippial female (Fig. 4B).Length 0.37-0.46mm, maximum height at middle of body, height/length ratio about 0.6; dorsal margin arched, without dorsal keel, not laterally compressed; carapace on the region of ephippium covered by longitudinal lines, yellow-brown.
Description of male.General Habitus and Carapace (Fig. 3C, D).Lower than female, length up 0.35 mm, about 1.7 times longer than height; maximum height near to middle of body; without dorsal keel; not laterally compressed; carapace with slight longitudinal lines; ventral margin almost straight; each valve with 40-44 plumose setae and relatively long, setulae between of them; setae arranged in three groups, anterior and posterior groups longer than median group.
Cephalic structures (Fig. 3E-G).Ocellus shorter than eye; rostrum short and not sharp in lateral view.Head shield (Fig. 3E).Two connected main head pores, rim around the posterior pore about 1.3 times broader than the anterior one; PP about 0.5 IP; transverse lateral head pores about 0.62 IP and located at the level between posterior and anterior main head pores, deep and elliptic sacs underneath of similar length to the lateral pores.Labrum (Fig. 3F).Shorter than in female; keel more rounded than in female, without a notch, anterior margin convex, posterior margin with two clusters of setulae.Antennule -A1 (Fig. 3G).Antennular body length about two times width, does not exceeding tip of rostrum; antennular sensory seta slender, about two times shorter than antennular body, inserted at the last third of the antennular body; twelve aesthetascs projecting beyond the tip of rostrum; male seta about 3 times shorter than antennular body.
Thoracic structures (Fig. 3H, I).First limb with copulatory hook U-shaped; arms of different lengths, not projected one over the other, scales present on the tip; seta of the copulatory brush about 0.8 of the length of the male seta on the IDL (en 4); seta of the ODL about 1.3 times longer than IDL setae (2-3); IDL with two setae (2-3) of similar length and armed with short hard setulae; male seta about 0.7 of length of the IDL setae; five clusters of long setulae inserted on the body of the limb.
Abdominal and Postabdnominal structures (Figs.3C, 3J).Abdomen (Fig. 3C).Shorter than thorax, armed with three rows of abdominal setae.Postabdomen (Fig. 3J).About 2.9 times as long as wide, narrowing distally; anal margin about 1.3 times longer than postanal; anal and postanal margin armed with short setulae not arranged in groups; nine lateral fascicles armed with thin spinulae; genital process about ten times shorter than length of postabdominal claw; postabdominal seta about 1.4 times shorter than postabdomen length.Postabdominal claw (Fig. 3J).Smaller and more robust than in the female, about 0.2 of the length of the postabdomen, slightly shorter than preanal margin; inner pecten with distalmost spinulae longer than proximalmost ones; outer pecten with spinulae of similar length.Basal spines (Fig. 3J).Very short and thin.
Distribution.So far, Flavalona asymmetrica sp.nov.was observed from Southeast to Central Brazil (Distrito Federal, Goiás, Minas Gerais and São Paulo).A wider distribution in Neotropics is expected.To distribution on the hydrographic regions, see Fig. 5.

discussion
When revising the costata-group of Hexalonabranch, Sinev and Dumont (2016)  morphology of lateral head pores and postabdomen of both females and males.Flavalona asymmetrica sp.nov. shares many important morphological traits with the costata-clade: (i) valves with pronounced linear sculpture, (ii) very elongated lateral head pores with length over 0.5 IP, (iii) distalmost setulae of each fascicle of the postabdomen thicker than others, (iv) genital process of the postabdomen male is short.The costata-clade is also composed of F. costata, F. cheni, F. natalensis, F. margipluma, and F. weltneri which are easily differentiated from F. asymmetrica sp.nov. in the morphology of main head pores.Flavalona asymmetrica sp.nov.has two main head pores while the aforementioned species have three (see Sinev and Dumont, 2016).
The antiquity of Cladocera taxa of different ranks (Frey, 1987;Kotov and Taylor, 2011;Van Damme and Kotov, 2016) suggest that important morphological differences are expected between groups with divergence related to large-scale biogeographical pattern, for instance, Neotropical Magnospina dentifera (Sars, 1901) versus South-East Asian Magnospina siamensis (Sinev & Sanoamuang, 2007) (Sinev et al., 2004;Sinev and Sanoamuang, 2007;Sousa et al., 2016b).In the case of F. asymmetrica sp.nov.and F. setigera, morphological differences are observed in many structures and might be in part explained by biogeographical divergence.The rim of posterior main head pore about 1.3 times broader than anterior, PP about 0.7 IP, deep and elliptic sacs about 0.6 of length of the lateral pores (Fig. 1G, H).In F. setigera the main head pores has similar morphology, PP about 1.1-1.3IP, semicircular sacs with length similar to lateral pores (Sinev and Dumont, 2016).The most important differences in the limbs of F. setigera and F. asymmetrica sp.nov.are related to length of seta 1 of IDL on the first limb, armature of scraper 3, 7-8 on the second limb, and ratio between first and second setae of third limb exopodite (Tab.1).The main difference observed in the postabdomen of F. asymmetrica sp.nov. is the presence of eight to nine marginal denticles, while F. setigera has 10-14 marginal denticles (Sinev, 1999a;Sinev and Dumont, 2016).

Figure 1 .Figure 2 .
Figure 1.Flavalona asymmetrica sp.nov.from Sempre Vivas National Park, Minas Gerais, Brazil, parthenogenetic female.A, Habitus of parthenogenetic female; B, Dorsal view of carapace; C, Ventral view of carapace; D, Anterior portion of ventral margin of carapace; D, Median portion of ventral margin of carapace; E, Median portion of carapace; F, Posterior portion and posteroventral corner of carapace; G, Head shield; H-I, Head pores of adult; J, Main head pore of juvenile; K, Labrum; L, Maxilla; M, Antennules; N, Antenna.Scale bars = 50 μm.